ANALYSIS OF THE EFFECTS OF LEPTOMYCIN B ON CELLS EXITING

MITOSIS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor

of Philosophy in the Graduate School of The Ohio State University

By

Gin-Yun Liu, B.S.

*******

The Ohio State University

2006

Dissertation Committee:

Dale D. Vandré, Ph.D., Advisor Approved by

John M. Robinson, Ph.D.

Sissy Jhiang, Ph.D.

Doug Kniss, Ph.D. ______

Advisor

Graduate Program in Molecular, Cellular, and Developmental Biology

ABSTRACT

The to interphase transition in higher eukaryotic cells is characterized by

reassembly of the nuclear envelope, decondensation of the , and

reformation of the nucleolus. Coincident with reassembly of the nuclear envelope,

nucleocytoplasmic trafficking is also restored, which allows for reestablishment of the

nuclear and cytoplamsic compartments. To determine whether restoration of nuclear

export is critical for completion of the interphase transition and formation of the

interphase nucleus, we examined the effect of leptomycin B (LMB) when applied to cells

during exit from mitosis. LMB is a metabolite that inhibits chromosomal

maintenance region 1 (CRM 1) mediated nuclear export. Cells treated with LMB during

the mitosis to interphase transition underwent apoptosis within 8 hours, whereas cells

treated with LMB 2 hours after release from the mitotic block remained viable. In

addition, interphase cells treated with LMB also showed little or no induction of

apoptosis. LMB treatment had no apparent effects on nuclear envelope reconstruction, decondensation, swelling of the nucleus, or establishment of cytoplasmic microtubule arrays. However, microscopic examination of cells prior to apoptosis revealed defective nucleolar morphology. Immunostaining of the nucleolar B23,

C23, and fibrillarin showed colocalization to the nucleolus in untreated cells, interphase cells treated with LMB, and cells treated with LMB 2 hours after release from mitosis.

ii However, in cells treated with LMB during the mitosis to interphase transition, B23 appeared to be distributed throughout the nucleoplasm, while C23 and fibrillarin were observed to localize small to phase dense structures within the nucleus. These results indicate that LMB inhibits the nuclear export of a critical factor(s) from the newly reformed nucleus that inhibits nucleologenesis, which will be referred to as a nucleologenesis licensing factor. This licensing factor(s) may become trapped within the nucleus during nuclear envelope reassembly or imported into the nucleus during early stages of nuclear reformation. Furthermore, a comparison of other known nucleolar inhibitors showed the nucleolar disruption effects observed in LMB treated cells are specific and unique effects. Therefore, we conclude that reestablishment of CRM1- mediated nuclear export immediately after mitotic release is a critical mechanism required for nucleologenesis and cell survival.

iii ACKNOWLEDGEMENTS

This is a turning point in my life as I am about to graduate from OSU and obtain

my Ph.D. degree. This has been a long journey that at various moments in my life, I did

not think I would ever see the end of it. I continued to work hard despite the mistakes

and problems that I had encountered with in many of my experiments. The many “bad”

days made me want to cry and shout out “why is this happening?” or “why didn’t the

PCR work?” However, there were always “good” days that made all the “bad” days

seemed like a distant memory. This glimmer of hope had brought joy and happiness to my life, especially when the experiments worked, or the immunofluorescent pictures had very little background. It is amazing of how one small experiment can instantly change a person’s day from “bad” to “good”. The graduate school experience has taught me to be persistent when encountering a problem, and tackle challenges from all angles rather than just from one side.

There are many people who have shared the triumph, the happiness, the sadness, the frustration, the anger, and all other emotions that I can not describe in one word throughout my graduate career. First of all, I would like to thank Dr. Vandre for allowing

me to join his lab, to teach me, to guide me, and to help me mature into the scientist that I

am today. Thank you! I could not have done this without your help, and I could not have

asked for a more helpful advisor!

iv Next, I would like to thank the present and past post docs in the lab, Hai Yang,

Fred, Jeff, and Arun. Thank you so much for your support, your answers to the many

questions that I had, your advice, and your detailed explanations on the techniques used.

Your suggestions and advice has helped me tremendously, especially I encountered problems in my experiments.

To Rodger, Selvi, and Pam, thank you so much for being there for me. Rodger and Pam, thank you for having lunch with me every week. I enjoy the many “silly” conversations that we had together. Selvi, you are a great workout partner. Thanks for being my gym partner so that I can release my stress.

To my parents, Viv, Christy, Emily, Lyndsie, Ebony, Brittan, Anita, Jeff, Matt, and Chiang , you guys are the best supporters in the world. You worried about my safety

when I have those crazy late night time point experiments at the lab, even though you knew that I would be safe. You cheered me up when I was frustrated about things and tell me that everything will be alright. You celebrated with me whenever I have good

days at the lab. Words just cannot describe how much I appreciate each and every one of

you.

To Mike, my wonderful soon to be husband, you went above and beyond what a

boyfriend/fiancé would do. You went with me when I had the late night time point

experiments at the lab, even though you were tired. You listened to me complain about

the crazy margins for this thesis. You held me when I cried about losing a portion of my

unsaved dissertation. Best of all, you bought me a professional massage session to help

release my stress. Your kindness, patience, supports, and love has helped me through this long path. Thank you so much for everything you have done for me.

v VITA

January 13, 1977…………………………………………………. Born in Taipei, Taiwan

June, 1996………………………………………………………... Graduate from Hilliard High School

June, 2000………………………………………………………... Bachelor of Science in Molecular Genetics from The Ohio State University

2000-present………………………………………………………Graduate Research Associate, The Ohio State University

PUBLICATIONS

Journal of Cell Science Submitted in July, 2006 Paper title: “Induction of Apoptosis in Cells After Treatment with Leptomycin B During the Mitosis to Interphase Transition is Preceded by a Failure of Nucleologenesis”

Experimental Cell Research Submitted in July, 2006 Paper title: “The Effects of LMB on Nucleolar Structures are Distinct from Other Nucleolar Inhibitors”

FIELDS OF STUDY

Major Field: Molecular, Cellular and Developmental Biology

vi TABLE OF CONTENTS Page

Abstract…………………………………………………………………………… …..ii

Acknowledgements………………………………………………………………...... iv

Vita……………………………………………………………………….………...... vi

List of Tables……………………………………………………………………...... ix

List of Diagrams…………………………………………………………………...... x

List of Figures……………………………………………………………...……...... xi

Introduction………………………………………………………………………...... 1

Nuclear membrane………………………………………………………………...2 Nuclear Envelope……………………………………………………………..…...5 Nuclear Reassembly…………………………………………………………..…...7 Nuclear Transport……………………………………………………………. …...9 Transport Receptors…………………………………………………………..….11 Mechanisms of Nuclear Transport……………………………………………….16 Leptomycin B………………………………………………………………….....20 The Nucleolus………………………………………………………………...... 22 Types of Nucleolar Organization……………………………………………..….25 Ribosome Biogenesis…………………………………………………………….27 Nucleolar Proteins…………………………………………………………….….32 Fibrillarin…………………………………………………………….. ….33 C23……………………………………………………………………….35 B23……………………………………………………………………….38 Nucleologenesis……………………………………………………………… ….42

vii

Chapters: 1. Inhibition of Nuclear Export During M to G1 Transition Induces Apoptosis Introduction…………………………………………………………………………..68 Materials and Methods…………………………………………………………….....72 Results………………………………………………………………………………..75 Discussion………………………………………………………………………….. ..80 Figures and Tables…………………………………………………………………. ..85

2. The Effects of LMB Are Specific for M to G1 Transition, Which Correlates to Nucleologenesis, a Specific Process to the M to G1 Transition

Introduction…………………………………………………………………...... …97 Materials and Methods…………………………………………………………… ..102 Results…………………………………………………………………...... 105 Discussion…………………………………………………………………………..113 Figures and Tables………………………………………………………...... 118

3. The Effect of LMB on Nucleolar Structures are Distinct from Other Nucleolar Inhibitors

Introduction…………………………………………………………………...... 144 Materials and Methods………………………………………………………...... 151 Results……………………………………………………………………………....153 Discussion…………………………………………………………………………..162 Figures and Tables………………………………………………………...... 168

4. Summary………………………………………………………………………. .254

References……………………………………………………………………...... 270

viii LIST OF TABLES

Table Page

1 Examples of transport receptors of the importin β superfamily………………..….51 2 List of acronyms used in the thesis……………………………………………. ….52

ix LIST OF DIAGRAMS

Diagram PAGE

1. Diagram of the complex………………………………………………..53 2. The Ran-GTP cycle…………………………………………………………………..54 3. Nuclear import model with importin β receptor…………………………………… ..55 4. Nuclear import model with importin α/β receptors………………………………... ..56 5. Nuclear transport model with Ran-GTP…………………………………………… ..57 6. Model of CRM1-mediated nuclear export…………………………………………...58 7. Chemical structure of LMB………………………………………………………... ..59 8. Ultrastructure of nucleoli…………………………………………………………... ..60 9. Types of the nucleolar organization……………………………………………….....61 10. General scheme of rRNA processing and modification…………………………… ..62 11. Schematic model the electron micrograph view of the rDNA transcription………. ..63 12. Ribosome Biogenesis Model………………………………………………………. ..64 13. The domains of the nucleolar proteins, fibrillarin, C23, and B23…………………. ..65 14. Nucleologenesis model…………………………………………………………….. ..66 15. Temporal kinetics of nucleolar reassembly model………………………………… ..67 16. Venn diagram o f the nucleolar localizations in CHO control cells vs. drug treated cells during M to G1 transition…………………………………………….. 264 17. Venn diagram o f the nucleolar protein localizations in CHO control cells vs. drug treated cells after 2 hours of release from mitosis………………….………..…….. 265 18. Venn diagram o f the Nucleolar Protein Localizations in CHO Control Cells vs. interphase cells treated with nucleolar inhibitors………………………………….. 266 19. Proposed model of nucleologenesis involving CRM1-mediated nuclear export…...267 20. Proposed model of inhibition of nucleologenesis in the presence of LMB………...268 21. Evolution of the Nucleolar Compartments………………………………………… 269

x LIST OF FIGURES

Chapter 1 Figures: Page 1.1 Mitotic release index and apoptotic index in cells treated with LMB…………... .85 1.2 Flow Cytometry Analysis of CHO Cells Released from Mitosis……………..….87 1.3 Flow Cytometry Analysis of CHO Cells Released from Mitosis and into LMB for 6, 12, 18, and 24 hours…………………………………………………..…... .89 1.4 Flow Cytometry Analysis of CHO Cells Released from Mitosis for 2 Hours Prior to the Addition of LMB for 6, 12, 18, and 24 hours………………..…...... 91 1.5 Flow Cytometry Analysis of CHO Interphase Cells Incubated with LMB for 8, 12, 18, and 24 hours……………………………………………………………....93 1.6 Western Analysis of CHO Mitotic Cells Released into LMB and Interphase Cells Incubated with LMB………………………………………………………..95

Chapter 2 Figures: Page 2.1 The effects of LMB on the localization of cyclin B in HeLa cells……………...118 2.2 Localization of C23 and B23 in HeLa control cells vs. mitotic cells released into LMB………………………………………………………………………. .120 2.3 Localization of C23 and B23 in CHO control cells vs. mitotic cells released into LMB………………………………………………………………………. .122 2.4 Localization of B23 and Fibrillarin in CHO control cells vs. mitotic cells released into LMB……………………………………………………………... .124 2.5 Localization of C23 and Fibrillarin in CHO control cells vs. mitotic cells released into LMB……………………………………………………………... .126 2.6 Ultrastructure of the nucleolus in control HeLa and CHO control cells vs. mitotic cells released into LMB……………………………………………….. .128

xi 2.7 Localization of C23 and B23 in CHO cells released from mitosis 2 hours prior to the addition of LMB…………………………………………………....130 2.8 Localization of B23 and Fibrillarin in CHO cells released from mitosis 2 hours prior to the addition of LMB……………………………………………. .132 2.9 Localization of C23 and Fibrillarin in CHO cells released from mitosis 2 hours prior to the addition of LMB……………………………………………. .134 2.10 Localization of C23 and B23 in CHO interphase cells treated with LMB……. .136 2.11 Localization of Fibrillarin and B23 in CHO interphase cells treated with LMB.138 2.12 Localization of C23 and Fibrillarin in CHO interphase cells treated with LMB.140 2.13 A Model of the Effects of LMB on Temporal Sequence of PNBs to the NOR.. .142

Chapter 3 Figures: Page 3.1. Chemical Structure of the Nucleolar Disruption Agent………………………....168 3.2. Localization of C23 and B23 in CHO Mitotic Cells Released into Ratjadone.....170 3.3. Localization of C23 and B23 in CHO Mitotic Cells Released into LMB…….. ..172 3.4. Localization of C23 and B23 in CHO Control Cells………………………….. ..174 3.5. Localization of Fibrillarin and B23 in CHO Mitotic Cells Released into Ratjadone……………………………………………………………………… ..176 3.6. Localization of Fibrillarin and B23 in CHO Mitotic Cells Released into LMB...178 3.7. Localization of Fibrillarin and B23 in CHO Control Cells…………………… ..180 3.8. Localization of C23 and Fibrillarin in CHO Mitotic Cells Released into Ratjadone……………………………………………………………………… ..182 3.9. Localization of C23 and Fibrillarin in CHO Mitotic Cells Released into LMB...184 3.10. Localization of C23 and Fibrillarin in CHO Control Cells…………………… ..186 3.11. Localization of C23 and B23 in CHO Mitotic Cells Released into Actinomycin D…………………………………………………………………..188 3.12. Localization of Fibrillarin and B23 in CHO Mitotic Cells Released into Actinomycin D…………………………………………………………………..190 3.13. Localization of C23 and Fibrillarin in CHO Mitotic Cells Released into Actinomycin D…………………………………………………………………..192

xii 3.14. Localization of C23 and B23 in CHO Mitotic Cells Released into Roscovitine….………………………………………………………………… ..194 3.15. Localization of Fibrillarin and B23 in CHO Mitotic Cells Released into Roscovitine….………………………………………………………………… ..196 3.16. Localization of C23 and Fibrillarin in CHO Mitotic Cells Released into Actinomycin D…………………………………………………………………..198 3.17. Localization of C23 and B23 in CHO Mitotic Cells Released from Mitosis 2 Hours Prior to the addition of LMB……………………………………………..200 3.18. Localization of Fibrillarin and B23 in CHO Mitotic Cells Released from Mitosis 2 Hours Prior to the addition of LMB…………………………………..202 3.19. Localization of C23 and Fibrillarin in CHO Mitotic Cells Released from Mitosis 2 Hours Prior to the addition of LMB…………………………………..204 3.20. Localization of C23 and B23 in CHO Mitotic Cells Released from Mitosis 2 Hours Prior to the addition of Ratjadone………………………………………..206 3.21. Localization of Fibrillarin and B23 in CHO Mitotic Cells Released from Mitosis for 2 Hours Prior to the Addition of Ratjadone………………………. ..208 3.22. Localization of C23 and Fibrillarin in CHO Mitotic Cells Released from Mitosis for 2 Hours Prior to the Addition of Ratjadone………………………. ..210 3.23. Localization of C23 and B23 in CHO Mitotic Cells Released from Mitosis for 2 Hours Prior to the Addition of Actinomycin D……..………………………. ..212 3.24. Localization of Fibrillarin and B23 in CHO Mitotic Cells Released from Mitosis for 2 Hours Prior to the Addition of Actinomycin D…………………...214 3.25. Localization of C23 and Fibrillarin in CHO Mitotic Cells Released from Mitosis for 2 Hours Prior to the Addition of Actinomycin D…………………...216 3.26. Localization of C23 and B23 in CHO Mitotic Cells Released from Mitosis for 2 Hours Prior to the Addition of Roscovitine…………………………………...218 3.27. Localization of Fibrillarin and B23 in CHO Mitotic Cells Released from Mitosis for 2 Hours Prior to the Addition of Roscovitine…………………….. ..220 3.28. Localization of C23 and Fibrillarin in CHO Mitotic Cells Released from Mitosis for 2 Hours Prior to the Addition of Roscovitine…………………….. ..222

xiii 3.29. Localization of C23 and B23 in CHO Interphase Cells Treated with LMB….....224 3.30. Localization of Fibrillarin and B23 in CHO Interphase Cells Treated with LMB……………………………………………………………………………..226 3.31. Localization of C23 and Fibrillarin in CHO Interphase Cells Treated with LMB……………………………………………………………………………..228 3.32. Localization of C23 and B23 in Interphase Cells Treated with Ratjadone…… ..230 3.33. Localization of Fibrillarin and B23 in Interphase Cells Treated with Ratjadone……………………………………………………………………… ..232 3.34. Localization of C23 and Fibrillarin in Interphase Cells Treated with Ratjadone……………………………………………………………………… ..234 3.35. Localization of C23 and B23 in CHO Interphase Cells Treated with Actinomycin D…………………………………………………………………..236 3.36. Localization of Fibrillarin and B23 in CHO Interphase Cells Treated with Actinomycin D…………………………………………………………………..238 3.37. Localization of C23 and Fibrillarin in CHO Interphase Cells Treated with Actinomycin D…………………………………………………………………..240 3.38. Localization of C23 and B23 in CHO Interphase Cells Treated with Roscovitine……………………………………………………………………. ..242 3.39. Localization of Fibrillarin and B23 in CHO Interphase Cells Treated with Roscovitine……………………………………………………………………. ..244 3.40. Localization of C23 and Fibrillarin in CHO Interphase Cells Treated with Roscovitine……………………………………………………………………. ..246 3.41. Localization of C23 and B23 in CHO LMB Reversibility Experiment………. ..248 3.42. Localization of Fibrillarin and B23 in CHO LMB Reversibility Experiment…..250 3.43. Localization of C23 and Fibrillarin in CHO LMB Reversibility Experiment…..252

xiv INTRODUCTION

Successful progression of cells exiting mitosis and entering G1 phase requires

functional nuclear transport. In vertebrate cells, the nuclear envelope disassembles

during late prophase to allow the mitotic spindle to form and attach to the condensed

chromosomes at the kinetochores. The absence of a barrier between the cytoplasm and

nucleoplasm allows for the cytoplasmic and nuclear compartments to be mixed. After

reformation of the nuclear envelope at the end of mitosis, some cytoplasmic proteins are found in the nucleus, while some nuclear proteins are distributed throughout the cytoplasm. These proteins need to be recompartmentalized to their correct location.

Inhibiting nuclear transport at this critical cell cycle transition results in cells that maintain a telophase-like configuration; chromosomes remain condensed, and no inter- chromatin material or nucleoli form. The nucleolus is the site of ribosome biogenesis, thus, in the absence of nucleoli, cells are unable to produce proteins for continued cell

growth. Similar to the nuclear envelope, the nucleolus disassembles prior to mitosis.

Therefore, reassembly of the nucleolus at the end of cell division also requires the import of nucleolar proteins distributed in the cytoplasm via the nuclear pore complex. Unlike vertebrate cells, yeast cells do not disassemble the nuclear membrane during mitosis; the

1 requirement for recompartmentalization of proteins after mitosis is not required in those cells. Thus, recompartmentalization is an essential event that occurs after cell division but only in higher eukaryotic cells.

Nuclear Membrane

In interphase eukaryotic cells, the double-membrane nuclear envelope (NE) spatially separates the cytoplasm from the cell nucleus. It is composed of two lipid bilayer membranes, nuclear pore complex (NPC), and the nuclear lamina. The outer nuclear membrane (ONM) is continuous with the rough endoplasmic reticulum (ER) and is covered with ribosomes. The inner nuclear membrane (INM), however, is ribosome free and associates with chromatin at the nuclear periphery (Salina et al. 2001;

Fahrenkrog and Aebi 2003; Gruenbaum et al. 2005; Margalit et al. 2005). There are several proteins localized to the INM such as LAP, LBR, Emerin, Nurim, and MAN1

(Salina et al. 2001). These integral membrane proteins associate with the nuclear lamina to maintain nuclear shape.

Underneath the NE is a meshwork of nuclear lamina proteins, which primarily consists of a polymeric assembly of intermediate filament-type-V proteins called nuclear lamins (Gruenbaum et al. 2005). There are two classes of lamin proteins, which are grouped on the basis of their biochemical properties and behavior during mitosis: A-type and B-type lamins (Salina et al. 2001; Gruenbaum et al. 2005; Margalit et al. 2005). In mammalian somatic cells, lamins A, B1, B2 and C have been identified (Salina et al.

2001). Lamins B1 and B2 are translated from separate (LMNB1 and LMNB2), and 2 lamins A and C arise from the same , but are spliced alternatively from the LMNA

gene (Salina et al. 2001). Both type A and B lamins are post-translationally modified by

isoprenylation, a covalent attachment of a hydrophobic isoprenyl moiety to proteins,

which allows them to attach to nuclear membranes throughout the cell cycle. However, a cleavage after isoprenylation modification removes the last 15 residues from A-type lamins allowing them to remain soluble during mitosis (Margalit et al. 2005). B-type lamins are expressed in all somatic cells, whereas A-type lamins are absent in some nonterminally differentiated cells (Steen and Collas 2001). Lamins range in size from 60 to 70 kDa and have a small N-terminal ‘head’, a coiled-coil ‘rod’ and a globular C- terminal ‘tail’ (Cohen et al. 2001).

Nuclear lamins are involved in various functions. They provide a structural framework for the NE by associating either directly or indirectly to the integral membrane proteins, such as lamin-B binding receptor (LBR), and emerin (Gruenbaum et al. 2005). The nuclear lamina also provides structural support for chromosomes. A large

proportion of transcriptionally silent heterochromatin localizes near the nuclear

periphery. This allows lamins, which are known to bind to DNA and core histones via their rod and tail domains, respectively, to bind to the chromosomes (Margalit et al.

2005).

Embedded in the NE are nuclear pore complexes (NPCs) that are composed of

50-100 proteins termed nucleoporins. It is one of the largest types of macromolecular complexes in the cell and has an estimated molecular mass of 125 MDa in vertebrates

and 66 MDa in yeast (Allen et al. 2000; Adam 2001; Salina et al. 2001; Fahrenkrog and

3 Aebi 2003). There are about 2,000 NPCs in vertebrate cells, whereas yeast cells only

have approximately 200 NPCs (Adam 2001). The increase in the NPC number may

reflect the complexity of vertebrate cells compared to yeast cells. The yeast and

vertebrate NPCs have similar core structural elements, as well as nucleoporins, which share sequence similarity of ~ 25% suggesting that they share a common translocation

mechanism (Allen et al. 2000). The basic framework of the vertebrate NPC consists of a

three-layered structure that forms the eight-fold radial and two fold mirror symmetry of

the pore, including the cytoplasmic ring and nuclear ring domains that surround a central

spoke domain (Figure 1). The eight-fold radial symmetry indicates that every

nucleoporin is present at a copy number of either eight or an integer multiple of eight.

The central transporter is suspended in the center of the spoke complex. The radial arms

of the spoke complex penetrate the pore membrane and project into the lumen of the

nuclear envelope. The cytoplasmic coaxial ring and the nucleoplasmic coaxial ring lie

above and beneath the spoke complex, respectively. Extending from the cytoplasmic ring

are eight filaments, each of 2-3 nm in diameter and approximately 50nm in length. The

nucleoplasmic side of the NPC is comprised of eight 100nm filaments that join at a

smaller ring structure, forming a basket-like structure emanating from the nuclear ring

(Adam 2001) (Figure 1). Nup358 and CAN/Nup214 are two of the major cytoplasmic

filaments that provide the initial docking site for active nuclear import by binding to Ran-

GDP and importin β, respectively, whereas the nucleoplasmic filaments are the initial

docking sites for nuclear export (Allen et al. 2000).

4 Nuclear Envelope Breakdown

In higher eukaryotes, the NE completely disassembles during mitosis in a process

called “open mitosis”. The nuclear membranes, nuclear lamina, and NPC disassemble

between prophase and prometaphase to allow for the association between the cytoplasmic

mitotic spindle and condensed chromosomes. In contrast, single-cell eukaryotes, such as

S. cerevisiae, have “closed mitosis” where the spindle poles are embedded in the NE and

the different components of the NE probably remain intact during mitosis. Thus,

formation of spindle microtubules in yeast cells requires tubulin proteins to be imported

into the nucleus (Margalit et al. 2005). Higher eukaryotic cells are on the opposite ends

of the spectrum displaying complete disassembly of the NE and intact NE during mitosis,

respectively. However, eukaryotic cells such as Aspergillus nidulans have partial NE

breakdown during mitosis, while in C. elegans and Drosophila, NE disassembly occurs

only at mid-late anaphase, leaving nuclear envelope intact during most stages of mitosis

(Margalit et al. 2005). This suggests that the process of NE breakdown has probably co-

evolved with the increased complexity of the nuclear scaffold and lamina.

NE breakdown involves the sequential disassembly and dispersal of all of the major NE structural components (Lee et al. 2000). The nuclear lamina is depolymerized,

followed by NPCs disassembly into subunits, while the components of the nuclear

membrane are dispersed into the cytoplasm (Salina et al. 2001). The mechanism triggering NE breakdown remains somewhat elusive. The first model proposes that NE breakdown is triggered by phosphorylation (Yang et al. 1997; Salina et al. 2001; Gonczy

2002; Margalit et al. 2005). Cyclin B1-Cdk1 (MPF: mitosis promoting factor) shuttles

5 between the nucleus and the cytoplasm during G2 phase of the cell cycle. At the end of

G2, Polo-like kinase 1 (Plk1) phosphorylates cyclin B at its amino terminal cytoplasmic

retention sequence, causing its accumulation in an active form in the nucleus (Pines and

Rieder 2001). Once inside the nucleus, activated Cdk1 phosphorylates nucleoporins, lamins, and integral proteins of the INM. The phosphorylation of lamin proteins triggers

depolymerization of the lamin filaments and eventually NE breakdown (Margalit et al.

2005). This was shown with mutations in lamin proteins that blocked Cdk1

phosphorylation, which prevented lamina disassembly, NEBD, and induced cell cycle

arrest (Heald and McKeon 1990). However, in some systems, such as frog and chicken

meiotic cells, the nuclear lamins can become solubilized while the other components of

the NE remain intact (Stick and Schwarz 1983). Furthermore, there is evidence that the

lamin B1 is stably bound to the NE until the time of NEBD (Beaudouin et al. 2002). This

suggests that depolymerization of the lamin proteins is not a prerequisite for NEBD.

The second model proposes that the spindle microtubules cause NE folds and

invaginations in both the INM and the lamina up to 1 hour before NEBD (Beaudouin et

al. 2002; Salina et al. 2002). One of the earliest morphological changes accompanying

entry into mitosis is the invagination of the NE close to the centrosomes (Robbins and

Gonatas 1964). In the presence of nocodazole, however, NE invaginations were

completely abolished within 2 minutes demonstrating that they were dependent on

dynamic microtubules (Beaudouin et al. 2002). Mechanical tension produced by the

spindle microtubules and associated dynein motors tear holes in the nuclear lamina and

nuclear membranes (Beaudouin et al. 2002; Salina et al. 2002). The formation of holes,

or tearing, in the NE occurs distally from the centrosomes, at the site of maximal tension 6 (Beaudouin et al. 2002). The holes then expand, and the fragmented NE is pulled toward

the centrosome in a vectorial manner by the microtubule minus end motor, dynein, and

the dynactin component p62 (Beaudouin et al. 2002; Salina et al. 2002). Prior to the formation of the centrosome-containing invaginations, there is evidence of cytoplasmic dynein and dynactin complex components localizing on the NE, which supports the movement of the NE fragments towards the centrosome (Salina et al. 2002). Recent studies suggest that lamin B1 polymers form solid-like and resilient structures that display a strain-hardening behavior (Panorchan et al. 2004). It was concluded from these studies that lamin B1 can sustain tension significantly greater than the deformation caused by spindle microtubules impinging on the NE in prophase (Panorchan et al. 2004).

This suggests that both models are involved in NEBD; first phosphorylation of lamin proteins to weaken the lamin network, followed by the physical tearing of the NE by the microtubules.

NE Reassembly

NE reassembly after mitosis occurs during late anaphase as the chromatids arrive at the spindle poles and prior to the initiation of cytokinesis. NE components are recruited sequentially to the reforming nuclei around the segregated condensed chromosomes. The first event is the targeting of individual NE integral proteins to the chromosomal surface. INM proteins, such as LBR, are one of the earliest proteins that are observed to locate to the chromosomes ~5 minutes after the metaphase-anaphase transition (Haraguchi et al. 2000). Recruitment of certain INM proteins to the chromosomes initiates the next NE reassembly event; membrane recruitment and fusion.

7 The fate of nuclear membranes during mitosis is controversial. There is evidence to

suggest that nuclear membrane forms vesicles, and they are reassembled around the

segregated chromatin at the end of mitosis (Margalit et al. 2005). There are two types of

membrane vesicles formed during NEBD, one with the capacity to bind to chromatin, and

the other vesicle type is required for fusion (Drummond et al. 1999). However, other

evidence suggests that after NEBD, NE membranes are fused to the ER network

(Ellenberg et al. 1997). Components of the NE will form membrane subcompartments

within the ER and diffuse laterally to continue the formation of the newly formed NE. In

either situation, both models will form a continuous double membrane around the

chromatin (Margalit et al. 2005).

Formation of a functional NPC occurs after the assembly of a continuous

membrane. One of the earliest NPC recruitment events is Nup107-160 complex,

composed of nine nucleoporins, associating with the chromatin (Walther et al. 2003).

This association allows the complex to be incorporated into the NE by direct or indirect

interaction with one or more integral membrane proteins, gp210 and POM121, in the NE

vesicle fraction (Walther et al. 2003). Assembly of post-mitotic NPC requires both the

dephosphorylation of nucleoporins and RanGTP (Walther et al. 2003). The last step of

NE reformation is the transport of the bulk of lamins to the nucleus through the newly

formed NPCs to form the nuclear lamina (Margalit et al. 2005). A-and B- type lamins have different assembly method after mitosis. B-type lamins are found near the chromosomes during the early stages of NE reformation and it is assembled into the lamina at the inner surface of the membrane. A-type lamin proteins, however, are imported into the nucleus and redistributes to the nuclear interior (Margalit et al. 2005). 8 The regulation of B-type lamins assembly involves dephosphorylation by protein phosphatase 1 (PP1) (Steen et al. 2000). The recruitment of PP1 to the NE involves interaction with A-kinase anchoring protein 149 (AKAP149), a membrane protein that is localized to the ER as well as the NE (Steen et al. 2000). After reassembly of the NE, functional nuclear transport through the NPC allows the nucleus to grow in volume as well as allowing the chromosomes to decondense.

Nuclear Transport

In eukaryotic cells, the presence of the nuclear membrane allows the separation and compartmentalization of cytoplasmic proteins from the nuclear proteins. In cells with “open” mitosis, this boundary of spatial separation is abolished resulting in mixing of nuclear and cytoplasmic proteins during mitosis. Functional nuclear transport through the NPC is critical for cells to increase nucleoplasm volume, chromosome decondensation, and reformation of the nucleoli. It was observed that when nuclear transport was inhibited by injecting wheat germ agglutinin (WGA) into mitotic cells, the chromosomal packing density in cells that completed mitosis remained, was comparable to that of metaphase chromosomes. In addition, the cells had small nuclei, little or no interchromatin material, and the absence of nucleoli (Benavente et al. 1989; Benavente et al. 1989). WGA is a lectin that binds to N-acetylglucosamine (GlcNAc) carbohydrate residues that are found on some of the nucleoporins of the NPC. Binding of WGA to the

NPC interferes with the translocation of proteins (Benavente et al. 1989; Benavente et al.

1989). The presence of a telophase-like nucleus in daughter cells suggests that proteins

9 need to be recompartmentalized via NPC mediated trafficking after the reformation of the

NE so that cells can fully enter G1 phase.

All nuclear transport events occur through the NPC. It allows passive diffusion of molecules smaller than 9nm or 60kDa, as well as nuclear transport of larger proteins that

require receptors and aid through the NPC. Molecules that are destined for nuclear

import or export are referred to as cargos, which include proteins, various types of RNA,

and RNPs. The continuous movement back and forth across the NE is termed

nucleocytoplasmic shuttling (Michael 2000). The majority of cargos are identified to the

nucleocytoplasmic transport receptors by the classical nuclear localizing signals (NLS)

and/or (NES). However, there are suggestions that

nucleocytoplasmic shuttling can be controlled by other signals, such as

nucleocytoplasmic shuttling signals (NS) which can also direct both nuclear import and

nuclear export (Michael 2000). The NS signals are neither NES nor NLS and are

speculated to be involved in mRNA nuclear export because they are found on proteins

that interact with mRNA (Michael 2000). For instance, hnRNP A1 is a pre-

mRNA/mRNA binding protein that contains a glycine-rich sequence of 38-amino-acids

termed M9 domain which controls its nucleocytoplasmic trafficking (Michael 2000).

Proteins that are imported into the nucleus require a specific signal. Two major types of NLS have been identified: SV40 monopartite and bipartite type. The monopartite NLS was discovered in the SV40 large T antigen and contains a PKKKRKV sequence (Weis 1998; Macara 2001). This type of signal is characterized by a few consecutive basic residues and/or a proline residue. The bipartite NLS was first

10 identified in Xenopus nucleoplasmin containing KRxxxxxxxxxxKKKK (Weis 1998;

Macara 2001). The bipartite sequence has two basic residues (highlighted in bold at the

N-terminus of the sequence), a 10 amino acid spacer region, and another basic region of

3-5 residues (highlighted in bold at the C-terminus) (Weis 1998).

Similar to nuclear import, proteins that are exported out of the nucleus require a specific signal as well. One of the well known NES was first discovered in the human immunodeficiency virus (HIV) Rev protein, and in the small cAMP-dependent protein kinase inhibitor PKI (Weis 1998; Macara 2001). It is characterized by a short stretch of hydrophobic amino acids that is rich in leucine, L-X2-3-Y-X 2-3-L-X-L/I, where X

represents any amino acid and Y represents Leu/Ile/Phe/Val or Met (Bogerd et al. 1996;

Weis 1998; Macara 2001). This type of NES is often referred to as Rev-like NES or the

leucine-rich NES which are found in several proteins, including the transcription factor

TFIIA (Fridell et al. 1996), cyclin B, involved in 5S rRNA export and ribosomal proteins.

Other NES that are not leucine-rich are also found in proteins, such as the NFAT transcription factor with the sequence of IVAAINALTT (Macara 2001).

Transport receptors

Cargos with NLS and/or NES are required to associate with receptors in order to complete the translocation through the NPC. Most of the receptors are members of the importin β /karyopherin β superfamily of RanGTP binding proteins, such as

karyopherins, p97, PTACs, importins, transportins, and exportins (Table 1) (Gorlich and

Kutay 1999; Nakielny and Dreyfss 1999). Transport receptors are generally large acidic

11 proteins with an N-terminal RanGTP-binding domain, a C-terminal cargo-binding domain and the capacity to bind to the components of NPC (Nakielny and Dreyfss 1999).

Import cargos bind to the transport receptors either directly or binding is mediated by a specific adaptor such as importin α, one of the best known adaptor proteins, to cargos with classical NLS (Pemberton et al. 1998; Weis 1998; Gorlich and Kutay 1999).

Importin α, a 55 kDa protein, provides the NLS-binding site (an armadillo repeat domain) and interacts via its basic N-terminal importin β-binding domain (IBB domain) with the

β-subunit to form the importin α/β heterodimer (Gorlich et al. 1996; Weis et al. 1996). In cells from multicellular organisms, there are at least six distinct importin α family members, whereas in yeast, importin β can bind to only one single adapter molecule,

Srp1p, the yeast importin α (Gorlich and Kutay 1999). There are three subclasses of importin α that are conserved > 75% between subclasses, and ~50% conserved within subclasses in vertebrates, insects, nematodes, and plants, indicating that these three branches diverged very early in metazoan evolution (Gorlich and Kutay 1999). The existence of more receptors and adaptors in multicellular organisms than in S. cerevisiae shows the evolved complexity of the higher eukaryotes. Other α –like adaptor proteins containing IBB domains such as Snurportin, act as a mediator for snRNP import, and

RIP α adaptor protein mediates replication protein A import (Gorlich and Kutay 1999;

Jullien et al. 1999; Nakielny and Dreyfss 1999; Gama-Carvalho and Carmo-Fonseca

2001). Importin β itself can also function without an adaptor in the import of some ribosomal proteins, the HIV-1 Rev and Tat proteins, and probably cyclin B1 (Henderson and Percipalle 1997; Truant and Cullen 1999; Gama-Carvalho and Carmo-Fonseca

2001). There are importin β-like proteins that also do not require a separate adapter 12 protein. The β-like proteins are also members of the large family of Ran-GTP binding

proteins exhibiting limited sequence similarity with the Ran binding domain of importin

β (Gorlich et al. 1997). For example, the import receptor for the M9 sequence of hnRNP

A1 is a β-like protein called Transportin in humans (Fridell et al. 1997).

For export out of the nucleus, cargos with Rev-like NES (leucine-rich NES) have now been identified which bind to CRM1 (Chromosomal Region Maintenance 1) receptor (Fornerod et al. 1997; Fukuda et al. 1997; Ossareh-Nazari et al. 1997). CRM1 was originally identified in the fission yeast Schizosaccharomyces pombe as a gene that is required for chromosome maintenance (Adachi and Yanagida 1989). However, further examination of the CRM1 protein sequence showed that it shares sequence in its N-terminus with importin β family members as well as with the Ran-guanosine triphosphate (GTP)-binding domain of the Ran-GTP-binding family. This indicates that

CRM1 is a potential transport receptor. Evidence that CRM1 is a transport receptor came from inhibition studies with the antifungal drug leptomycin B (LMB). The first evidence came from studies with S. pombe, where resistance to LMB was mapped to the CRM1 gene (Nishi et al. 1994). This was followed by an observation that LMB blocks HIV

(Human Immunodeficiency Virus) Rev-mediated nuclear export in mammalian cells and

Xenopus (Fornerod et al. 1997; Fukuda et al. 1997; Wolff et al. 1997). Concurrently,

Ossareh-Nazari et al. showed that CRM1 forms a complex with the NES, and this

complex formation is completely blocked in the presence of LMB (Ossareh-Nazari et al.

1997).

13 CRM1, similar to importin β, can bind to NES-cargos directly or it may rely on

adapter proteins to bridge the interactions between transport receptors and cargos.

Nuclear export of both ribosomal subunits has been shown to require nuclear export

receptor CRM1 and a functional RanGTPase system (Hurt et al. 1999; Ho et al. 2000).

The binding of CRM1 to pre-ribosomal subunits requires bridging by adaptor proteins.

Recently, yeast protein, Nmd3p has been suggested to serve as an NES-containing export

adaptor for the ribosomal 60S subunit (Ho et al. 2000). Nmd3p is an essential 59kDa

shuttling protein that contains a NLS and a leucine-rich NES in its C-terminus domain

(Ho et al. 2000). Similar to the yeast protein, human homologue of Nmd3p (hNMD3)

also contains a conserved NES in its C-terminal region (Thomas and Kutay 2003).

Several experiments showed that Nmd3p (hNMD3) is required for exporting the 60S

ribosomal subunit to the cytoplasm. Deletion of the NES from Nmd3p led to the retention of Nmd3p as well as the 60S subunit in the nucleus (Ho et al. 2000). The presence of 60S subunits accumulating in the nucleus was further supported by inhibiting

CRM1 mediated nuclear export with LMB (Ho et al. 2000; Thomas and Kutay 2003).

Nmd3p (hNMD3) was shown to bind directly to CRM1 using pulldown and co- sedimentation assays (Thomas and Kutay 2003). These results suggest that NMD3 acts as an adaptor to CRM1 to export the 60S ribosomal subunit. As of now, no adaptor proteins have been discovered for the 40S ribosomal subunit.

There are other proteins that are involved in CRM1-mediated export. For instance, proteins such as RanBP3 acts as an accessory protein to stabilize and enhance

cargo binding affinity to the CRM1-RanGTP complex as well as stimulating export in

vitro (Englmeier et al. 2001). The affinity of NES substrates for CRM1-RanGTP is 14 relatively low. For example, exportins such as CAS and Exportin-t (exports tRNA) bind

to their substrates with affinities in the low-nanomolar range, however, NES proteins have 100 to 500-fold lower affinities for CRM1 (Reviewed in (Kutay and Guttinger

2005)). Nxt1, RanBP1, and possibly eIF-5A are other cofactors that help the CRM1-

cargo complex bind to nucleoporins in the NPC and stimulate cargo unloading in the

cytoplasm (Kehlenbach et al. 1999; Black et al. 2001).

Of all the characterized vertebrate exportins, export receptor CRM1 has the

broadest known substrate range. This allows CRM1 to be involved in a number of

cellular processes, such as cell cycle control and centrosome duplication. Activation of

the cyclinB/Cdk1 kinase complex triggers entry into mitosis. During interphase, CRM1- mediated nuclear export maintains the high levels of cyclin B in the cytoplasm (Hagting et al. 1998; Toyoshima et al. 1998; Yang et al. 1998). Cyclin B contains both nuclear

import and export sequences, and continuously shuttles between the cytoplasm and nuclear compartments. At prophase, however, cyclin B is rapidly accumulated in the nucleus when the cytoplasmic retention sequence, which contains its nuclear export sequence, is phosphorylated to prevent its interaction with CRM1 (Hagting et al. 1999).

The inability of cyclin B to export out of the nucleus allows for the cyclin B/cdk 1 kinase complex to phosphorylate nuclear targets required for both NEBD as well as chromatin condensation.

In addition to CRM1 involvement in cyclin B localization during the cell cycle, it is also involved in centrosome duplication. Recently, studies have indicated that a nucleolar protein, nucleophosmin (B23), is involved in centrosome duplication (Wang et

15 al. 2005). Sequence analysis indicates that B23 contains a putative hydrophobic leucine-

rich NES motif, suggesting that the CRM1-RanGTP complex may modulate centrosome

duplication by regulating NPM transport. Addition of LMB completely blocked B23

transport to the centrosome (Wang et al. 2005). The significant increase in the number of

cells with a single centrosome indicates that the CRM1-GTP complex is an important

factor in centrosome duplication.

Mechanism of Nuclear Transport

RanGTP has been implicated to play a key role in nuclear transport. Ran proteins

make up a distinct subset of small GTPases that belong to the Ras superfamily. Both Ran

and Ras have conserved domains for guanine nucleotide binding, however, Ran has an

acidic C-terminus and is not modified by prenylation (reviewed in (Stochaj and Rother

1999)). Ran is an abundant protein that can switch between GDP- and GTP-bound states

by nucleotide exchange (Figure 2). Since Ran proteins have very low nucleotide

hydrolysis and exchange activities by themselves, their activities are stimulated by

regulatory proteins, GTPase –activating protein (RanGAP) and Guanine nucleotide

exchange factor RCC1 (RanGEF/RCC1) (reviewed in (Moore 1998; Nakielny and

Dreyfss 1999; Stochaj and Rother 1999)). The RanGAP stimulates the intrinsic GTPase

activity of Ran and triggers the conversion of RanGTP into RanGDP with the help of another cofactor, Ran binding protein 1 (RanBP1) (Coutavas et al. 1993). When importin

β binds to RanGTP, it prevents the access of RanGAP to RanGTP. However, in the presence of RanBP1, it relieves the block of GTPase activation by binding to RanGTP and releasing importin β from RanGTP (Bischoff and Gorlich 1997; Gorlich et al. 1997).

16 This allows RanGAP to convert RanGTP into RanGDP. RanGEF-mediated nucleotide exchange is not specific for either GDP or GTP, though it will promote the exchange of

Ran-bound GDP to GTP due to the higher GTP concentration than GDP in the nucleus

(reviewed in (Izaurralde and Adam 1998; Gorlich and Kutay 1999)). Both RanGAP and

RanBP1 are predominantly cytoplasmic proteins, whereas RanGEF/RCC1 is a nuclear

protein that is bound to chromatin (reviewed in (Izaurralde and Adam 1998)). The

asymmetric localization of the cofactors suggests that Ran is hydrolyzed from GTP to

GDP in the presence of RanGAP/RanBP1 in the cytoplasm, whereas, GDP is exchanged

with GTP to form RanGTP when it encounters RanGEF in the nucleus (Figure 2).

Nuclear transport is a complex mechanism that is regulated by Ran. The NLS of

a substrate is recognized either by importin β, or by importin α, which the latter then

complexes with importin β, for nuclear import (Izaurralde and Adam 1998; Gorlich and

Kutay 1999; Allen et al. 2000). Importins bind to substrates without the physical binding

of RanGDP to importins, however, the high concentrations of RanGDP in the cytoplasm

promotes the formation of import complexes (Allen et al. 2000). Once the cargo complex

is formed, it is then translocated through the NPC into the nucleus, where the cargo is

dissociated from the importins by RanGTP (Figure 3 and 4). The binding of RanGTP

causes structural conformational changes to importin β, or the α/β complex, and releases

the cargo in the nucleus (Gorlich et al. 1996). RanGTP binds to importin β and recycles

back to the cytoplasm, where GTP hydrolysis of Ran is stimulated by the RanGAP, and

releases importin β for the next round of import (Figure 3 and 4). The constant nuclear

export of RanGTP would eventually deplete the concentration of RanGTP in the nucleus.

17 To overcome that, new RanGDP is shuttled back into the nucleus by NTF2, where

RanGEF will undergo nucleotide exchange into RanGTP for the next round of transport

(Figure 5) (Ribbeck et al. 1998).

All known transport receptors shuttle between the cytoplasm and the nucleus.

Importin β and importin α have separate methods of returning to the cytoplasm after completing a cycle of nuclear import. Since importin β has the ability to bind to nucleoporins, it can shuttle to the cytoplasm without an additional receptor (Gorlich et al.

1995; Moroianu et al. 1995). Unlike importin β, importin α requires an export receptor for it to shuttle out of the nucleus and into the cytoplasm. CAS, a member of the β importin family of proteins, has been reported to catalyze the re-export of importin α into the cytoplasm (Figure 4) (Kutay et al. 1997). Binding of importin α to CAS is regulated by RanGTP; RanGTP dissociates importin α from importin β and increases CAS affinity for importin α by ~ 300 fold (Kutay et al. 1997). Once the importin α/CAS/RanGTP complex has formed, it will translocate across the NPC and into the cytoplasm. This complex is disassembled in the cytoplasm when RanBP1 binds to RanGTP, causing

RanGAP to hydrolyze GTP and triggers the release of importin α from CAS (Bischoff and Gorlich 1997). CAS can shuttle back into the nucleus to pick up an importin α for another round of export, whereas, importin α can bind to NLS cargo and complex with importin β for another cycle of nuclear import.

CRM1-mediated nuclear export is also regulated by RanGTP. CRM1 binds cargo and Ran-GTP cooperatively in the nucleus prior to translocation through the

NPC(Fornerod et al. 1997; Floer and Blobel 1999). The association of Ran-GTP with

18 CRM1 enhances their affinity for proteins containing leucine-rich NES (Fornerod et al.

1997; Kutay et al. 1997). Once the export complex enters the cytoplasm, it is

disassembled by RanBP1 and RanGAP (Bischoff and Gorlich 1997; Gorlich et al. 1997;

Askjaer et al. 1999; Floer and Blobel 1999). This allows RanGAP to hydrolyze RanGTP

into RanGDP, and the release of the NES cargo (Figure 6). RanGDP and CRM1 are then shuttled back into the nucleus for another round of nuclear export.

The translocation mechanism through the NPC has been poorly understood due to the complexity of NPCs. Thus, models such as the Brownian affinity gating model,

Brownian affinity-gradient model, selective-phase model, and mechanistic transport model have been proposed to explain the translocation mechanism (Reviewed in

(Fahrenkrog and Aebi 2003)). However, none of the models provide a complete description of nucleocytoplasmic transport. The current understanding of the translocation mechanism is that binding of the transport receptors with FG-repeat

(phenylalanine and glycine) domains of nucleoporins is essential. For instance, CRM1

has been shown to interact with the FG-repeat domain on nucleoporin CAN/Nup214 and

Nup88 when exporting NES-cargos to the cytoplasm (Fornerod et al. 1997). Since

CAN/Nup214 and Nup88 are found on the cytoplasmic side of the NPC, this suggests that the association of CRM1 with CAN/Nup214 and Nup88 may be the termination site of NES export (Fornerod et al. 1997). There are reports that many of the transport receptors have multiple FG-repeat binding domains; however, it does not further increase the transport efficiency (Reviewed in (Fahrenkrog et al. 2004). Initial docking of the import complex occurs at the nucleoporins, RanBP2/Nup358, located at the tips of the cytoplasmic fibrils that emanate from the NPC (Wu et al. 1995). The cargo–receptor 19 complex is then transferred to the extended FG-repeat domain of a nucleoplasmic

nucleoporin, p62 complex, in the center of the NPC. Next, the cargo-receptor complex is

moved to the nuclear side of the NPC and interacts with Nup153, where the binding of

RanGTP to the receptor induces a conformational change in the receptor. The change in

the structure of the receptor leads to the release of the cargo into the nucleus (Reviewed

in (Fahrenkrog and Aebi 2003)). The exact mechanism of the translocation is yet

unknown. One of the models suggests that receptor-cargo complexes move through the

NPC by binding to the nucleoporins in an affinity gradient manner. In other words, cytoplasmic nucleoporins have about 20-fold lower affinity for importin β than

nucleoporins, such as Nup153, located on the nuclear side of the NPC (Reviewed in

(Fahrenkrog and Aebi 2003)). The cargo-transport receptor complex is targeted to the

NPC via the FG-repeat binding domain on the receptors. However, since the affinity

between the receptor and the FG-repeat nucleoporins on the cytoplasmic side are not as

strong, the receptor-cargo complex will release from the cytoplasmic nucleoporins and

bind to the nuclear nucleoporins with a higher binding affinity.

Leptomycin B

Leptomycin B (LMB) has been an important tool in the elucidation of the role of

CRM1 in the nuclear export process. LMB (C33H48O6) is composed of an unsaturated

fatty acid branched chain with a terminal lactone ring (Figure 7). It was originally

discovered as an antifungal from Streptomyces sp. strain ATS1287 (Hamamoto

et al. 1983; Hamamoto et al. 1983). Earliest studies showed that LMB caused inhibition

of the eukaryotic cell proliferation with abnormal nuclear structures (Hamamoto et al.

20 1985). This was supported by later studies on mammalian cells and fission yeast cells

showing that they arrested in G1 and G2 phase in the presence of LMB (Yoshida et al.

1990). In addition, LMB has also been shown to be an effective anti-tumor agent against

experimental tumors in vivo (Komiyama et al. 1985). Investigation of LMB-resistant

mutants in the fission yeast Schizosaccharomyces pombe, led to the identification of a

LMB resistance gene that encodes for an essential nuclear protein CRM1, which is

required for maintaining higher order chromosome structure, correct and

cell growth (Nishi et al. 1994). The understanding of CRM1 as a leucine-rich NES receptor first came from the localization of HIV-Rev (Human Immunodeficiency Virus)

protein in the presence of LMB. Rev is a regulatory protein that is required for viral replication. Rev promotes the nuclear export of unspliced and single spliced HIV mRNA

species, which are specified by the presence of a complex RNA structure, the Rev

response element (RRE). Rev protein has the ability to shuttle between the nucleus and

the cytoplasm due to the presence of both an NLS and a leucine-rich NES. It was

reported that in the presence of LMB, the export of Rev protein to the cytoplasm was inhibited however, the nuclear import of Rev was not affected (Wolff et al. 1997). This

suggests that LMB only inhibits nuclear export and not the import. CRM1 was later

shown to bind to Rev in the absence of Ran, implying a direct association between the

two proteins (Askjaer et al. 1998). Identification of the LMB binding site on CRM1 was first discovered in a S. pombe CRM1 mutant, which showed an extreme resistance to

LMB. Further analysis indicated that the mutant strain contained a base change that caused an amino acid change from -529 to Serine in the central control region

(CCR) of CRM1 (Kudo et al. 1999). Sequence analysis of CRM1 from other species

21 showed that Cysteine-529 in CRM1 is conserved in LMB-sensitive organisms such as humans, but it is not conserved in Sachromyces cerevisiae, which contains Threonine-

539 and is LMB-insensitive (Kudo et al. 1999). Kudo et. al went on to show that LMB covalently binds to the sulfhydryl group of the Cysteine amino acid via its terminal lactone ring and that LMB was not able to bind to CRM1 mutant (Cysteine-529 changed to Serine) nor CRM1 from S. cerevisiae (which contains Threonine-539) (Kudo et al.

1999). Thus, Cysteine-529 on CRM1 is an essential binding site for LMB and is required for LMB inhibitor of nuclear export of proteins containing leucine-rich NES sequences.

The Nucleolus

The nucleolus was first described as an oval-shaped body with a central spot in animal cell nuclei by F. Fontana in 1781 (Reviewed in (Thiry and Goessens 1996)).

However, the term “nucleolus” was not introduced until 1839 by Valentin (Reviewed in

(Thiry and Goessens 1996)). Several subsequent reports identified the nucleolus as a dynamic, membraneless organelle that was not uniform in its composition (Reviewed in

(Thiry and Goessens 1996)). In 1898, Montgomery noted that there are variability in the nucleolar number and size and that the organelles disappear and reappear during mitosis

(Reviewed in (Leung and Lamond 2003; DiMaro 2004)). Although several groups suggested that there was an association between the nucleoli and the chromosomes as well as the RNA, the function of nucleoli was not established until the 1960s when evidence of the presence of ribosomal proteins and the assembly of nascent ribosomes emerged to indicate that the nucleolus is the site of ribosomal RNA synthesis and ribosome assembly (Reviewed in (Thiry and Goessens 1996)).

22 The nucleolus is not a stable structure. Rather it’s structure, size and organization

depends on ribosome biogenesis. Cells with higher protein synthesis have larger, irregular shaped nucleoli, whereas cells arrested in G0 phase, with no protein synthesis, have smaller round shaped nucleoli. Mammalian nuclei contain one or a few nucleoli.

For instance, HeLa cells contain 1 to 6 nucleoli with considerable size variability between them. Often times, the shape of nucleoli can differ within a single cell. Interphase nucleoli are composed of three major components: the granular component (GC), the

dense fibrillar component (DFC), and the fibrillar component (FC) (Figure 8 and Table 2

for acronyms).

The term fibrillar center was proposed by Recher et al. (1969) to designate the

loose fibrillar material and the central location it occupies within the dense fibrillar

component (Reviewed in (Goessens 1984)). FCs contains nucleolar proteins (such as

C23 and B23) that are involved in rDNA (ribosomal DNA) transcription, and non-

nucleolar proteins such as RNA polymerase I, and transcription factors (UBF: upstream

binding factor, and DNA topoisomerase I) (Reviewed in (Goessens 1984)). The presence

of nucleolar proteins and non-nucleolar proteins that are involved in transcription

suggests that FC is involved in rDNA transcription. A main morphological characteristic

of FCs is that they are surrounded by DFC.

DFC consists of dense fibrils that are packed into strands associated with FC

suggesting a close functional relationship between the two components (Reviewed in

(Goessens 1984)) (Figure 8 and Table 2 for acronyms). DFC can assume various

morphologies that reflect the intensity of ribosome biosynthesis (Reviewed in (DiMaro

23 2004)). For instance, in cells with maximal ribosome synthesis, DFC has a reticulated

appearance. In electron micrographs, DFC appears phase dense because it contains

relatively large amounts of metals such as Ca, Zn, Cu, Mn, Cr and Ni and the high

affinity for heavy metals during fixation and staining methods (Reviewed in (Goessens

1984; DiMaro 2004)). Similar to FC, DFC contains many nucleolar and non-nucleolar proteins that are involved in rDNA transcription, translation, pre-rRNA maturation and ribosome assembly, such as snoRNP (small nucleolar Ribonucleoprotein, which are

important for processing the pre-rRNA into mature rRNA), nucleolar proteins that are

involved in pre-rRNA processing (such as C23, B23, fibrillarin), and rDNA (Reviewed in

(Goessens 1984; DiMaro 2004)). Furthermore, RNase digestion and pulse-labeling

experiments showed that RNA is present in the DFC and that it is the site of initial pre-

rRNA processing and early ribosome assembly (Reviewed in (Goessens 1984; DiMaro

2004)). DNA is also present in the DFC, which suggests that the DFC is involved in

rDNA transcription. As a result, there is controversy as to the exact location of rDNA

transcription. Based on the data presented in various studies, the general consensuses

among researchers is that rDNA transcription occurs somewhere near to or between the

FC and DFC boundary.

The GC is the most peripheral zone of the nucleolus and is normally situated

around the DFC (Figure 8). It is composed of loosely packed granules approximately

15nm in diameter (Reviewed in (Goessens 1984)). It has been shown that the DFC is initially labeled with tritiated uridine using pulse-labeling methods and the GC is only labeled after longer periods of incubation (Reviewed in (Goessens 1984)). This illustrates the migration of a newly synthesized nucleolar RNA from the DFC into the 24 GC. The newly synthesized rRNA will then be assembled with other ribosomal proteins to make the 40S and 60S ribosomal particles and travel towards the NE for export into the cytoplasm. Similar to FC and DFC, there are many nucleolar proteins, such as B23 and some C23, localized to the GC. The GC appears to be mainly involved in pre-rRNA maturation and ribosomal assembly.

Interphase nucleoli frequently contain spherical light areas of lower density than the surrounding GC termed nucleolar interstices. The morphology, size, and number of the nucleolar interstices are variable within each cell. Cytochemical studies of granules found in the interstices showed that it contains a material which exhibits a texture and an electron density matching those of the surrounding nucleoplasm to suggest continuity between the GC and the interstices (Reviewed in (Goessens 1984)). Furthermore, other studies suggested that there are similarities between the granules in the interstices and the preribosomal particles, leading them to identify interstices as mature preribosomal particles (Reviewed in (Goessens 1984)). Often times, nucleolar interstices also contain clumps of condensed chromatin connected to intranucleolar masses of condensed chromatin scattered throughout the GC.

Types of Nucleolar Organization

Nucleolar number, size, and shape vary among organism, cell type, and physiological state of the cell. For instance, nucleoli of active protein synthesizing cells are larger than those with less active protein synthesis. The decrease in the size of the nucleolus correlates to the decrease in the protein synthesis of the cell. On the basis of electron microscope studies, four main categories of mammalian nucleoli have been 25 classified according to the organization of the components: reticulated, compact,

segregated, and ring-shaped nucleoli (Hernandez-Verdun and Junera 1995; Thiry and

Goessens 1996). Compact and reticulated nucleoli are found in cells with active

ribosome synthesis, whereas segregated and ring-shaped nucleoli are found in quiescent

cells or cells treated with transcription inhibition drugs (Figure 9). In the reticulated

nucleoli, the FC are often very small and sometimes not visible (Figure 9A). The sponge-like DFC covers the entire nucleolus along with patches of GC, interstices and condensed chromatin within it. Reticulated nucleoli are often found in very active cells, such as cancer cells. Compact nucleoli are characterized by the compact organization of the GC in which some interstices can be seen (Figure 9B). This type of nucleoli contains several FC of various size surrounded by DFC. Compact nucleoli are often found in active cell lines in culture. Ring-shaped nucleoli are characterized by a single FC surrounded by a small amount of DFC, which in turn is surrounded by GC (Figure 9C).

This type of nucleoli is typically small and is found in cells with a low rate of ribosome biogenesis. The last nucleolus model is that of the segregated nucleoli (Figure 9D). It is characterized by separation of the nucleolar components, which remain superimposed but no longer intermingled. Segregated nucleoli are often found in cells in which the RNA synthesis is blocked by inhibitors or other drugs.

Although the nucleolar models that were just presented show the variation of FC,

DFC and GC arrangement, there are some cells where the nucleoli are only composed of two components: DFC and GC. Higher eukaryotic cells, such as humans and birds have all three components, whereas lower eukaryotes contain only DFC and GC. The reason

26 for some cells lacking FC is unknown; however, it may suggest the evolved complexity

of higher eukaryotes compared to the lower eukaryotes.

Ribosome Biogenesis

Higher eukaryotic cells contain approximately 400 copies of ribosomal genes and

in HeLa cells, they are redistributed and distributed on chromosomes 13, 14, 15, 21, and

22 (Reviewed in (Raska et al. 2004)). Ribosome synthesis is a complex process and

starts with the transcription of rDNA at the FC and DFC border. The ribosomal genes are

tandemly repeated and form arrays in one or several ribosomal genes (Reviewed in

(Raska et al. 2004)). The chromosomal gene clusters are identified in mitotic

chromosomes as the nucleolar organizer region (NOR), because the nucleolus reforms

around this rDNA after mitosis.

The primary ribosomal gene transcript (mammalian: 47S pre-rRNA, yeast: 35S

pre-rRNA) consists of internal and external transcribed spacers (ITS, ETS) and external non-transcribed spacers (NTS) (Reviewed in (Hernandez-Verdun and Junera 1995; Raska et al. 2004)) (Figure 10). Transcription of rRNA genes requires a number of essential proteins. Similar to mRNA transcription, rRNA transcription requires a promoter region that contains an upstream control element (residues -186 to -107 on human rDNA) and a core promoter binding sequence (residues -45 to +20 on human rDNA) to allow the formation of a transcriptionally competent complex (Reviewed in (DiMaro 2004; Raska et al. 2004)). In mammals the complex contains the multisubunit RNA Pol I, upstream binding factor (UBF), and selectivity factor protein complex (SL1), transcription initiation factors (TIF-IA/C) (Pikaard et al. 1989; Schnapp et al. 1990; Schnapp et al. 27 1994; Heix et al. 1997; Voit et al. 1997) (Figure 11). The SL1 (also known as TIF-IB in

mouse) consists of TATA-binding protein (TBP), and three transcription activating

factors (TAFs). During interphase, the UBF binds the upstream element and recruits

SL1/TIF-IB that binds the core element of the promoter. RNA Pol I then binds to form

the preinitiation complex, and proceeds to start rRNA transcription (Figure 10 and 11)

(Reviewed in (DiMaro 2004)). rRNA are transcribed at a very high rate, and the tandem

arrays of rRNA genes can be easily seen in electron microscopy. Images of active

ribosomal genes were first observed by Miller and collaborators in the form of a

Christmas tree (Figure 11) (Reviewed in (Hernandez-Verdun and Junera 1995; Raska et

al. 2004)). The rDNA forms the axis of the transcription units and the nascent rRNAs

constitute the lateral fibrils of different sizes attached to the rDNA (axis). The tip of the

“tree” represents the point on the DNA at which transcription begins and where the

transcripts are thus shortest. The 5’ ends of the nascent pre-rRNA fibrils, contain

terminal knobs that are rRNA processing complexes, RNP (ribonucleoproteins) (Mougey

et al. 1993). The presence of the processing complexes on the newly transcribed

transcripts still attached to the rDNA suggests that the processing of pre-rRNA into

mature rRNA occurs concurrently with rDNA transcription.

From yeast to human, eukaryotic ribosomes possess conserved rRNA and

ribosomal proteins (r-proteins), as well as conserved non-ribosomal factors that associate

transiently with the precursor. Thus, many components involved in the complex steps of ribosome formation have been identified and characterized in the yeast S. cerevisiae.

Processing rRNA and the assembly of rRNA with ribosomal proteins occurs in the GC

and requires a number of nucleolar proteins, nucleases, and small nucleolar 28 ribonucleoprotein particles (snoRNPs), which contain snoRNAs (small nucleolar RNA).

The snoRNAs within the snoRNP complex act as a guide to chemically modify the

35S/47S pre-rRNA through methylation (CH3) of the ribose moiety at the 2’hydroxy

group and through the conversion of the uridines into pseudouridines (ψ) prior to cleavage steps (Reviewed in (Kiss 2001; Decatur and Fournier 2002)). The 35S/47S pre- rRNA then undergoes a series of cleavages that will eliminate ETS (5’ETS and 3’ETS),

ITS1, and ITS2 to give the mature 18S (1900bp), 5.8S (160bp), and 25S/28S (~5100bp) ribosomal RNAs (Figure 10). Nucleolar proteins such as fibrillarin and nucleolin (C23)

associate with U3 snoRNA and play a role during the first steps of rRNA processing.

Endoribonuclease activities of B23 and U8 snoRNA are involved in cleaving the ITS2 of pre-rRNA (Savkur and Olson 1998; Michot et al. 1999). Recently it was found that there is a clear distinction between the 18S synthesis pathway, which involves four successive endonuclease cleavages, and the pathway involved in 5.8S and 25S/28S synthesis, which is more complex and requires endonuclease cleavage followed by multistep exonuclease digestion (Figure 10) (Reviewed in (Fatica and Tollervey 2002)). The 18S rRNA and r- proteins will form the 40S small ribosomal subunits, while the 25S/28S rRNA, 5.8S rRNA, the nucleoplasmic transcribed 5S (120bp) rRNA, and r-proteins assemble together to form the 60S large ribosomal subunits.

Assembly of the small and large ribosomal subunits is a complicated process that starts with the synthesis of r-proteins (ribosomal proteins) in the cytoplasm, which are subsequently imported into the nucleolus. Following the transcription of rDNA and assembly of r-proteins onto the new transcript, the processing and modification of the pre-rRNA occurs in a series of steps. The earliest evidence of pre-ribosomal particles 29 was identified by Warner and Planta, which were termed 90S particles due to their sedimentation characteristics on sucrose gradients (Reviewed in (Tschochner and Hurt

2003)). The 90S particles contain 35S/47S pre-rRNA, many r-proteins, non-ribosomal proteins (such as helicases, GTPases), processing components (such asU3 snoRNP, methlytransferases), and factors that are required for 18S RNA synthesis (Reviewed in

(Tschochner and Hurt 2003)). Further studies on the 90S particles illustrated that it contained U3 snoRNP particles and other factors specifically required for the 40S subunit synthesis, whereas the components that are required for the synthesis of the 60S subunit

were absent (Grandi et al. 2002). This suggests that the 90S pre-ribosomes are formed through the early assembly of the pre-40S factors onto the 35S/47S pre-rRNA before the pre-60S factors are recruited. After the recruitment of the pre-40S factors and 5S rRNA to the 35S/47S pre-rRNA to form the 90S pre-ribosomes, the 35S/47S pre-rRNA is modified by methylation (CH3), pseudouridylation (ψ), and a series of cleavages (Figures

10 and 12). The first few cleavages of the 35S/47S pre-rRNA resulted in the separation

of the pre-40S subunit (with pre-18S rRNA) from the pre-60S subunit (with 5S rRNA

and pre-rRNA transcript that contains 5.8S and 25S/28S rRNA) (Figure 12) (Reviewed in

(Tschochner and Hurt 2003)). The pre-60S factors are recruited onto the pre-60S particle

and continue the series of cleavages that are required for maturation the 25S/28S and

5.8S rRNA within the 60S particle (Figure 12). The final maturation steps for 40S and

the 60S ribosomal subunits occur after they are exported to the cytoplasm via receptor-

mediated process to prevent the premature translation of mRNA in the nucleus. Once the

30 last processing steps for maturing 40S and 60S ribosomal subunits are complete, the two

subunits will associate with each other to form a functional ribosome for the translation process.

The mechanism underlying nuclear export of ribosomal subunits has been somewhat elusive for years; however, recent studies showed that export of the ribosomal subunits requires a receptor-mediated process that is energy-dependent. The export receptor for both subunits is CRM1, however, the large subunits do not interact directly with CRM1 (Johnson et al. 2002). Transport factor, NMD3, has been shown to have an

NES and has the ability to bind to a ribosomal protein, Rp110p, within the 60S pre- ribosome as well as CRM1 (Ho et al. 2000; Gadal et al. 2001; Johnson et al. 2002; Nissan

et al. 2002). Thus, NMD3 serves as an adaptor between CRM1 and the 60S pre-

ribosome. The export of the 40S subunit also uses CRM1 as an export receptor, probably

through an adaptor similar to NMD3, however, the adaptor protein has yet to be

identified.

For many years, the main function of nucleolus has been understood to be

ribosome biosynthesis. Recent evidence suggests that the nucleolus is also involved in

cell cycle regulation and apoptosis (Carmo-Fonseca et al. 2000; Visintin and Amon

2000). The nucleolus has also been described as serving as a sequestering compartment

for regulatory complexes, thus preventing them from reaching their targets (Carmo-

Fonseca et al. 2000; Visintin and Amon 2000). In yeast, a nucleolar protein, Net1 (Cfi1)

recruits the repressor protein Sir2 to rDNA as well as associating with the Cdc14

phosphatase that has a critical role in exiting cells from mitosis (Straight et al. 1999; 31 Visintin et al. 1999). During G1 phase, inactive Cdc14 is sequestered in the nucleolus by

Net1/Cfi1. When Cdc14 is released from Net1/Cfi1 during mitosis, it becomes active

and leaves the nucleolus to dephosphorylate proteins that promote exit from mitosis

(Visintin et al. 1999). There are some reports that suggest the nucleolus is also involved

in apoptosis regulation. The oncogene Mdm2 has an important role in regulating

protein levels and activity. The binding of Mdm2 to p53 prevents p53 from becoming an

active transcription factor, and leads to its exports out to the cytoplasm for protein

degradation (Momand et al. 1992; Roth et al. 1998). This prevents the level of p53 from

increasing when there is no DNA damage. Recently, an oncoprotein that localizes to the

nucleolus, p19ARF, has been shown to stabilize p53 by sequestering Mdm2 to the

nucleolus in response to DNA damage (Tao and Levine 1999). Retention of Mdm2 to

the nucleolus prevents p53 from being exported to the cytoplasm for degradation, and

thus p53 becomes active as a transcription factor for genes that bring about cell cycle

arrest or apoptosis (Prives 1997; Tao and Levine 1999).

Nucleolar Proteins

In human cells, the size of the nucleoli varies from less than 0.5µm in diameter in

mature lymphocytes to 3-9µm in proliferating cells ((Hernandez-Verdun and Junera

1995). Several groups carried out a proteomic analysis of the nucleolus and found that

there are about 350 different nucleolar proteins (Andersen et al. 2002; Scherl et al. 2002).

In their studies, they concluded that the nucleolus consists of about 15% ribosomal proteins, 23% proteins involved in ribosome biogenesis, 2-3% translation factors, 3-5%

chaperon proteins, 2% of proteins involved in chromatin structure, and about 25-28%

32 proteins with unpredictable functions (Andersen et al. 2002; Scherl et al. 2002). Of the

known nucleolar proteins, we chose C23, B23 and fibrillarin as markers for nucleolar

structure since each of these proteins have been characterized in some detail.

Nucleolar Protein: Fibrillarin

Fibrillarin is highly conserved protein (Figure 13). For instance, human fibrillarin is 67% identical to its yeast homolog, Nop1p, and 81% identical to the Xenopus protein

(Henriquez et al. 1990; Aris and Blobel 1991). The proteins are also functionally

conserved. A fibrillarin double knockout in yeast is lethal but can be rescued when human or Xenopus fibrillarin is added to the knockout yeast cells (Jansen et al. 1991).

Fibrillarin double knockout in mouse embryos also resulted in early embryonic cell death as shown by positive TUNEL staining, which is diagnostic of apoptotic cell death

(Newton et al. 2003). Both of these experiments suggest that fibrillarin is an essential protein that is involved in early stages of development. Fibrillarin is located in the DFC, where pre-rRNA processing occurs, and it is involved in the early stages of pre-rRNA processing, pre-rRNA modification, and ribosome assembly (Tollervey et al. 1993). It is associated with several snoRNAs required for pre-rRNA cleavage (U3, U8, U14, and

U22 snoRNA), and methylation of rRNA (U14, U18, U24-U63 snoRNA) (Cavaille et al.

1996; Tollervey 1996). Fibrillarin, snoRNAs, and other proteins form complexes known

as small nucleolar ribonucleoproteins (snoRNPs) that act as guides for the methylation

and pseudouridylation process (Weinstein and Steitz 1999).

snoRNAs are classified into three major groups (Reviewed in (Tollervey and Kiss

1997; Weinstein and Steitz 1999)). The first and largest group of snoRNAs shares two 33 short conserved sequence elements, box C (RUGAUGA, where R is any purine) and box

D (CUGA) at either the 5’ or the 3’ ends (Smith and Steitz 1997; Tollervey and Kiss

1997). The box C/D sequence elements are essential for the stable accumulation of the

snoRNAs and for binding of fibrillarin (Baserga et al. 1991; Huang et al. 1992). The second group of snoRNAs are defined by an evolutionarily conserved “hairpin-hinge- hairpin-tail” structure that contain box H (ANANNA, where N stands for any nucleotide) and ACA (Reviewed in (Tollervey and Kiss 1997; Weinstein and Steitz 1999)). Similar to box C/D, box H/ACA sequence elements are required for the synthesis or stability of this group of snoRNAs. The third group of snoRNAs is defined by its sole known member, the RNA component of the endonuclease RNase MRP, which is closely related to RNase P (reviewed in (Tollervey and Kiss 1997; Weinstein and Steitz 1999)). The majority of the box C/D and box H/ACA snoRNAs function in the selection of sites of

2’-O methylation and pseudouridylation in the pre-rRNAs, respectively. All snoRNAs are associated with specific proteins in the snoRNPs. The H/ACA snoRNAs are associated with an essential nucleolar protein, Gar1p and C/D snoRNAs are associated with fibrillarin and Nop58p (Schimmang et al. 1989; Lafontaine and Tollervey 1999).

Analyzing point mutations in the putative methyltransferase domain of the yeast fibrillarin, Nop1p, Tollervey et al. (1993) showed that ribose methylation of rRNAs was inhibited (Tollervey et al. 1993). This suggests that fibrillarin is the methyltransferase in box C/D snoRNPs. Sequence analysis of the fibrillarin gene showed that it contains an amino-terminal domain that is rich in glycine and arginine residues (termed the GAR domain), a central RNA-binding domain comprising an RNP-2-like consensus sequence, and a C-terminal α-helical domain (Figure 13) (Aris and Blobel 1991).

34 Nucleolar Protein: C23

C23, also called nucleolin, is a ubiquitous, nonhistone nucleolar phosphoprotein present in exponentially growing eukaryotic cells. The C23 protein is present in the abundance in fibrillar center, dense fibrillar component and granular regions of the nucleoli. One of its major functions is its involvement in ribosome biogenesis; rDNA transcription, rRNA maturation and processing, and ribosome assembly (Reviewed in

(Srivastava and Pollard 1999)). In addition to ribosome biogenesis, C23 has been implicated in other functions such as chromatin decondensation, cytoplamsic nucleolar transport of ribosomal components and preribosomal particles, DNA/RNA helicase activity, and transcriptional repressor activity (Borer et al. 1989; Erard et al. 1990; Roger et al. 2002). In Chinese Hamster Ovary (CHO) cells, it was determined that C23 makes up 10% of the nucleolar proteins (Bugler et al. 1982). C23 was first described by Orrick et al. in 1973 and was called C23 because of its mobility on a two-dimensional gel

(Reviewed in (Ginisty et al. 1999)). It is composed of 707 amino acids and analysis of the amino acid sequence reveals that there are three different structural domains (Figure

13). The N-terminal domain is made up of highly acidic regions interspersed with basic sequences and contains multiple phosphorylation sites (Rao et al. 1982; Bourbon et al.

1983). Because of the high content of negatively charged amino acids in the N-terminal domain, C23 runs in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE) gels with a molecular mass of 105kDa, even though its calculated mass is about

77kDa from the cDNA sequence (Lapeyre et al. 1987). C23 is a substrate for several kinases such as casein kinase II (CK2), Cdc2, and protein kinase C-ξ (PKC-ξ)

(Caizergues-Ferrer et al. 1987; Belenguer et al. 1990; Peter et al. 1990; Zhou et al. 1997). 35 CK2 phosphorylates C23 in vitro and in vivo at serine residues found predominantly in the acidic region, whereas Cdc2 phosphorylates the threonine residues with the basic region of the N-terminal domain (Caizergues-Ferrer et al. 1987; Belenguer et al. 1990;

Peter et al. 1990). Phosphorylation by CK2 occurs in interphase and phosphorylation by

Cdc2 occurs during mitosis (Belenguer et al. 1990; Peter et al. 1990). This suggests that

phosphorylation of C23 is highly regulated during the cell cycle. A basic motif TPXK

(where X is a non-polar residue) that lies between the first two acidic domains of C23 has

been proposed to bind to histone H1 and induces chromatin decondensation (Erard et al.

1988). The TPXK motif is reminiscent of sequences present in the amino-and carboxy-

domains of histone H1 (Erard et al. 1990). Furthermore, Cdc2 phosphorylation occurs on

threonine residues within the same basic motif, TPXK (Belenguer et al. 1990). Since the

basic TPXK motif on C23 is identified by both Cdc2 and histone H1, this suggests that

C23 could modulate DNA condensation. In addition to binding to histone H1, the acidic

domain has also been involved in protein-protein interactions with U3 snoRNP and other

ribosomal proteins (Bouvet et al. 1998; Ginisty et al. 1998; Sicard et al. 1998).

In the central part of C23, sequences containing four RNA-binding domains

(RBD1, RBD2, RBD3, RBD4) have been identified (Figure 13). These domains are also

known as RNA recognition motif (RRM). A typical RBD contains 80-90 amino acid

residues with two highly conserved sequences, the RNP-1 octapepetide

(R/K)G(F/Y)(G/A)(F/Y)VX(F/Y) and the RNP-2 (L/I)(F/Y)(V/I)(G/K)(G/N)L

hexapeptide motifs (Query et al. 1989; Birney et al. 1993). There are more than 200

RDB-containing proteins with various functions in RNA splicing, processing or

translation. Since C23 is found in the DFC where pre-rRNA processing occurs, it was 36 suggested that C23 may have interactions with rRNA. Ghisolfi-Nieto et al. illustrated that C23 interacts specifically with the RNA sequence UCCCGA that is found within an eight-nucleotide hairpin loop (Ghisolfi-Nieto et al. 1996). The 5 sequence and the 7-10nt hairpin loop are known as the nucleolin recognition element (NRE) motif,

which is found all along the pre-rRNA. Biochemical and genetics studies later showed

that C23 RNA-binding specificity to NER is the result of cooperation between RBD1 and

RBD2 and not RBD3 nor RBD4 (Serin et al. 1997). In addition to the presence of the

NER motif in the pre-rRNA, the linker connecting the two RBDs (RBD1 and RBD2) has also been found to contribute to the specificity of the C23 RBD1/2 NRE interaction

(Finger et al. 2004). The RBD domains also bind to another motif on the pre-rRNA.

This motif is the evolutionary conserved motif (ECM), which is located in the 5’-ETS of the pre-rRNA (Ginisty et al. 2000). The ECM is a short evolutionary conserved 11

nucleotide sequence found 5 nucleotides downstream of the 5’ETS cleavage site, the early pre-rRNA processing events. Deletion and mutational analysis of the RBD

domains illustrated that all four RBD domains are involved in the interaction with the

ECM motif (Ginisty et al. 2001). Therefore the interaction between C23 and the ECM of

the pre-rRNA via the four RBD domains of C23 represent the involvement of C23 in the

early processing of pre-rRNA.

The C-terminal domain of C23, termed the GAR/RGG domain, is involved in

many functions. This domain is defined by Arg-Gly-Gly (RGG) repeats interspersed

with other amino acids. One of the functions of this domain is to control unstacking of

bases and the unfolding of RNA secondary structures (Ghisolfi et al. 1992). Further

studies indicated that the RGG domain interacts with a subset of ribosomal proteins, such 37 as L3 protein (Bouvet et al. 1998). Since C23 has been shown to shuttle between the

cytoplasm and the nucleus, it was speculated that C23 transports ribosomal proteins from

the cytoplasm to the nucleus and nucleolus for assembly of the large and small ribosomal

subunits. Another function of the RGG domain is DNA annealing (Hanakahi et al.

2000). This requires the RGG domain and the RBD3 and RBD4 domains to accelerate

the DNA annealing activity. However, the annealing activity was not always perfectly

base-paired duplexes (Hanakahi et al. 2000). The observation of the C-terminal domain’s

ability to anneal rDNA suggests that C23 may be involved in maintaining homogeneity

within a repeated gene family by pairing DNA mismatches and targeting them for subsequent repair. The GAR domain is required, but not sufficient, for efficient

localization to the nucleolus. Deletion of the GAR domain on C23 resulted in B23,

another nucleolar protein (see below), redistributed to the nucleoplasm, while localization

of other nucleolar protein such as fibrillarin were not affected (Pellar and DiMaro 2003).

Nucleolar Protein: B23

Protein B23 (nucleophosmin/NO38/Numatrin) is a major nucleolar

phosphoprotein which is significantly more abundant in tumor cells and proliferating

cells than in normal resting cells (Feuerstein and Mond 1987; Schmidt-Zachmann et al.

1987; Chan et al. 1989). B23 has been shown to be involved in pre-rRNA processing

and ribosome assembly. First, immunoelectron microscopic studies have shown that B23

is localized primarily in the DFC and GC regions of the nucleolus, which is where pre-

rRNA processing and ribosome assembly occurs (Spector et al. 1984). Then, using

biochemical methods, B23 was found to be associated with maturing pre-ribosomal RNP

38 particles (Olson et al. 1986). In the presence of inhibitors of pre-rRNA synthesis, or

inhibitors for pre-rRNA processing, B23 was shown to translocate from the nucleolus to

the nucleoplasm, suggesting that B23 interacts with the processing machinery. Lastly,

B23 was shown to be an endonuclease that cleaves pre-rRNA preferentially at the ITS2

(~250 nt downstream from the 3’-end of 5.8SrRNA) but not at the 5’ETS nor ITS1

(Savkur and Olson 1998). Thus, these experiments show that B23 is involved in pre-

rRNA processing and ribosome assembly.

Protein B23 is expressed as two isoforms, B23.1 and B23.2, coded by a single

gene (Chang and Olson 1989). The two proteins are identical except that the C-terminal

35 amino acids observed in B23.1 are absent in B23.2. B23.1 is the major form and is

predominantly located in nucleoli, while B23.2 is in the cytosol. Sequence analysis of

B23 showed that it has several functional domains; the N-terminal region has a relatively high density of hydrophobic residues, the central region contains two highly acidic segments and a bipartite NLS, and the C-terminal of the transcript carries a net positive charge (Figure 13) (Hingorani et al. 2000). Hingorani et al. determined that the aromatic and basic segments within the C-terminal 76 residues are required for nucleic acid binding. Deletion of the nonpolar region in the N-terminal domain and/or removal of the acidic region in the center of B23 resulted in a nearly complete abolition of chaperone activity (Hingorani et al. 2000) (see below). They also observed that B23 deletion mutants that still had chaperon activity were able to oligomerize, whereas deletion mutants that had only 10% of chaperone activity, were in a trimer or monomer form

(Hingorani et al. 2000). This suggests that chaperone function of the B23 requires oligomerization of the protein as well as both the N-terminal and the C-terminal domains. 39 The ribonuclease activity domain requires segments from the central and the C-terminal

regions of B23, which overlaps with the chaperone function domains.

B23 has been shown to have molecular chaperone activities that prevent aggregation of molecules (Szebeni and Olson 1999). The nucleolus contains a high concentration of proteins, especially during ribosome synthesis, which could cause proteins to aggregate and be immobilized during ribosome synthesis. One of the earlier pieces of evidence that showed B23 to be a putative chaperone molecule was its ability to bind to HIV-Rev protein (Borer et al. 1989). Human Immunodeficiency Virus type 1

(HIV)-Rev protein has a marked tendency to aggregate under normal physiology

conditions. B23 acted as a molecular chaperone to maintain the solubility of Rev, and

stimulate Rev import into the nucleus at a 1:1 molar ratio of the two proteins (Wingfield

et al. 1991; Szebeni and Olson 1999). In addition to preventing proteins from

aggregating in a temperature dependent or independent environment, B23 can also

promote the renaturation of chemically denatured proteins, suppress protein aggregation

due to thermal denaturation, and protect enzymes against activity loss during thermal

denaturation (Szebeni and Olson 1999). These findings all suggests that B23 has several

activities that are typically found in chaperone proteins. B23 contains both a bipartite

NLS and a NES, and has the ability to bind to proteins with NLS. This allows B23 to form specific complexes with other proteins, such as ribosomal proteins, HIV-Rev proteins or nonribosomal proteins and prevent aggregation of the molecules as well as act as a transport vehicle that shuttles proteins between the cytoplasm, the nucleus, and the nucleolus (Borer et al. 1989; Fankhauser et al. 1991; Adachi et al. 1993).

40 During interphase, B23 and other nucleolar proteins such as C23 and fibrillarin,

all are located in the nucleolus. These nucleolar proteins are translocated from the

nucleolus and dispersed throughout the cell during mitosis and regroup back to the

nucleolus at the start of mitotic exit (Ochs et al. 1983; Dundr et al. 1997). The majority

of the nucleolar proteins aggregate in NDF particles during telophase (see

nucleologenesis section below), however, some B23 has shown to localize to the

centrosome suggesting that B23 may be involved in centrosome duplication (Zatsepina et

al. 1999; Okuda 2002). Using various fixation methods, Zatsepina, et al. showed that

B23 is located to the mitotic spindle poles starting from early prometaphase (Zatsepina et

al. 1999). Centrosome duplication occurs concomitantly with the initiation of DNA

synthesis and is induced by CDK2/cyclin E kinase complex (Ohtsubo and Roberts 1993;

Van de Heuvel and Harlow 1993; Lacey et al. 1999; Matsumoto et al. 1999). B23 has

recently been found to be one of the targets of CDK2/cyclin E during the initiation of

centrosome duplication (Okuda et al. 2000; Tokuyama et al. 2001). Furthermore, B23

was found to specifically associate with unduplicated centrosomes, but not with

centrosomes after duplication. Phosphorylation of B23 on threonine 199 by CDK2/cyclin

E, and the subsequent dissociation of B23 from the centrosomes is essential for

centrosome duplication (Okuda et al. 2000; Tokuyama et al. 2001). Since cyclin E is

intrinsically unstable, CDK2/cyclin E activity becomes minimal during S phase.

However, the level of cyclin A is low in late G1 phase, but increases during S and G2 phases (Koff et al. 1992; Marraccino et al. 1992). Furthermore, it was shown that

CDK2/cyclin A also phosphorylates B23 on threonine 199 (Tokuyama et al. 2001).

Thus, B23 is first phosphorylated by CDK/cyclin E during S phase, and subsequently

41 phosphorylated by CDK2/cyclin A to prevent re-association of any B23 to centrosomes during S and G2 phases. B23 then re-associate with the centrosomes during mitosis

(prometaphase) to prevent premature centrosome duplication prior the start of the next cell cycle (Zatsepina et al. 1999; Okuda et al. 2000).

Nucleologenesis

The nucleolus is not a stable organelle. Its structure, size and organization depend on ribosome biogenesis. During cell division when transcription is inhibited, nucleoli are dismantled and are not reassembled until late telophase. The process of reforming the nucleoli after mitosis is termed nucleologenesis. Nucleolar disassembly takes place during prophase. Transcription is completely repressed by the end of prophase, and results in the absence of visible nucleoli (Olson and Dundr 2005). During mitosis, inhibition of rDNA transcription is regulated at the level of initiation, where the RNA Pol

I transcription factor, SL1 and the UBF are inactivated by Cdc2-Cylin B phosphorylation

(Heix et al. 1998; Kuhn et al. 1998; Klein and Grummt 1999). Reactivation of rDNA transcription requires the dephosphorylation of SL1 and UBF, as well as rephosphorylation of UBF at a different site (Voit et al. 1999). Nucleolar breakdown starts with the loss of the FC before the onset of the NE breakdown (Leung et al. 2004).

This is followed by subsequent loss of some pre-rRNA processing machineries from the

DFC and GC, which coincides with NE disassembly (Leung et al. 2004; Olson and Dundr

2005). Transcription factors such as UBF and RNA Pol I remain bound to the mitotic

NORs when rRNA transcription ceases in prophase (See Table 2: Acronyms) (Roussel et al. 1993; Suja et al. 1997). However, recent reports using live cells indicate that RNA

42 Pol I subunits, RPA16, RPA 20, RPA39, and RPA194 dissociate from the NOR during a

brief period within metaphase, and reappear on the NOR at late anaphase (Leung et al.

2004; Olson and Dundr 2005).

Reassembly of the nucleolus after mitosis is a sequential step-wise process. The

pre-rRNA processing components are initially associated with the perichromosomal

regions in prophase and metaphase, however during anaphase and telophase, these

components are also found in large cytoplasmic particles termed nucleolus-derived foci

(NDF) (Reviewed in (Olson et al. 2000)). NDF are composed of many pre-rRNA processing components, such as B23, fibrillarin, C23, U3 snoRNP, U8 snoRNP, and several nonribosomal nucleolar components (Dundr et al. 1997; Dundr and Olson 1998).

Further studies showed NDF also contain partially processed pre-rRNA, a mixture of 45S and 46S pre-rRNAs that lack the 5’ETS leader sequence (Dundr and Olson 1998). The purpose of the partially processed pre-rRNA found in NDF is unknown, however, it could be speculated that the partially processed pre-rRNA from the previous cell cycle serve as a scaffold for the processing proteins and RNA keeping them in close proximity for rapid utilization once ribosome production restarts at the end of mitosis (Reviewed in

(Olson and Dundr 2005)). In telophase, the number of NDF decreases until none remain in the cytoplasm. It appears that NDF are imported into the reforming nucleus and reassemble in the nucleoplasm as prenucleolar bodies (PNBs). Closer examination by

photobleaching showed that the nucleoplasmic PNBs have similar pre-rRNA processing

components as those found in the NDF, and that the PNBs do not contain any

transcriptional components (Gebranes-Younes et al. 1997; Dundr et al. 2000). This

suggests that during mitosis, processing components along with partially processed pre- 43 rRNA first aggregate to form cytoplasmic NDFs, and during early to late telophase, the

processing components and the pre-rRNA form the PNB particles when they are

imported back into the reforming nucleus. Once in the nucleoplasm, the PNBs are believed to migrate towards the NORs where they fuse and become the components of the newly built nucleolus (Figure 14) (Benavente et al. 1987; Scheer et al. 1993;

Fomproix et al. 1998).

The classic model of reformation of nucleolus after mitosis occurs in early

telophase when RNA Pol I transcription is reinitiated (Scheer et al. 1993; Fomproix et al.

1998). Once rDNA transcription is initiated, the processing proteins and partially

processed pre-rRNA will enter the nucleus and form PNBs. Scheer et al. suggested that

the fusion of PNBs at the NOR is dependent on RNA Pol I transcription (Scheer et al.

1993). Furthermore, inhibiting RNA Pol I function with either RNA Pol I antibodies or

with low dosage of Actinomycin D, which prevents RNA polymerases from functioning

by intercalating preferentially into G/C-rich regions, causes a disruption in the formation

of the nucleolus (Benavente et al. 1987; Dundr et al. 2000). The electron micrographs

revealed round structures containing dense fibrillar and granular elements (PNBs), which

shows that under these conditions, the typical compact nucleoli were not assembled as

cells progressed into G1. This implies that reformation of the nucleolus occurs only after

the reactivation of rDNA transcription.

However, more recent studies have shown that the reactivation of rDNA

transcription is not required for the initiation of the nucleolar assembly. In Xenopus

laevis embryogenesis, the processing proteins, such as fibrillarin and C23, are regrouped

44 around the rDNA/NOR before the activation of RNA Pol I transcription, suggesting that recruitment of PNBs to the NOR does not require active transcription (Verheggen et al.

1998; Verheggen et al. 2000). Rather, the recruitment of the processing proteins to the

NOR is the result of the maternal pre-rRNA being targeted to the NOR first. In the absence of the maternal pre-rRNAs, recruitment of the processing components to the

NOR is not observed (Verheggen et al. 2000). In studies carried out in mammalian cultured cells, Actinomycin D at 0.04ug/ml inhibits RNA Pol I transcription and fusion between PNBs at the NOR, but does not affect the recruitment of the processing factors to the NOR (Dousset et al. 2000). This suggests that recruitment of the PNBs to the NOR and fusion of the PNBs at the NOR are regulated by separate mechanisms.

In the presence of roscovitine, a Cdc2-Cyclin B inhibitor that releases cells from mitosis, rDNA transcription was restored in mitotic cells; however, pre-rRNA processing was not functional, thus 46S/47S pre-rRNA accumulated in these cells (Sirri et al. 2000;

Sirri et al. 2002). The accumulation of 46S pre-rRNA indicates that the processing events are inhibited by roscovitine. Furthermore, both mitotic and interphase cells treated with roscovitine exhibited a disruption in the nucleolus that was demonstrated by the different localization of the two nucleolar proteins, Nop52 and fibrillarin (Sirri et al.

2002). Fibrillarin was only partly localized to the NOR, whereas Nop52 appeared in

PNBs and was absent from the NOR. This inhibition of pre-rRNA processing disrupted nucleolar structure and nucleologenesis. These results further support the theory that transcription alone is not sufficient to initiate nucleolar assembly. Thus, reformation of

45 the nucleoli after mitosis requires (1) recruitment of the processing components to the

NOR, independent of active rDNA transcription, (2) active transcription, and (3) active

pre-rRNA processing.

There are many unanswered questions regarding the formation and migration of the PNBs, and their involvement in nucleologenesis. It has been proposed that there are

several distinct types of PNBs dedicated to early or late rRNA processing events (Savino

et al. 1999; Verheggen et al. 2000). For example, Verheggen et al. classified the PNBs

into two types; PNB I contain fibrillarin and C23, whereas PNB II contain B23

(Verheggen et al. 2000). Each processing component, such as fibrillarin, B23 and C23,

have different functions in pre-rRNA processing. Fibrillarin, for instance, is associated

with several snoRNAs that are required for pre-rRNA cleavage (U3, U8, U14, and U22

snoRNA) and methylation of rRNA (U14, U18, U24-63 snoRNA) (Tollervey et al. 1993).

In addition, the yeast homologue of fibrillarin, Nop1, is believed to play a role during the

first steps of rRNA processing, pre-rRNA modification and ribosome assembly

(Tollervey et al. 1993). C23 has been found to play a multifunctional role, such as early

cleavage processing and packaging nascent rRNAs (Ginisty et al. 1998). B23, on the

other hand, is involved in the late stages of rRNA processing (removing ITS2) as well as

in the late assembly steps (Savkur and Olson 1998). Nop52, which has a similar function as B23, is involved in removing ITS2 from the pre-rRNA. Recent studies have shown that fibrillarin appeared at the NOR prior to Nop52 (Savino et al. 1999). Since fibrillarin is involved in the early processing steps and Nop52 is involved in the later steps, this

suggests that the timing of pre-rRNA processing events could explain the differences in

the movement of PNBs towards the NOR (Figure 14 and 15). Experiments using time- 46 lapse video microscopy that showed fibrillarin-containing PNBs signal lasting only about

15 minutes ( + 5 minutes), whereas Nop52-containing PNBs were very stable (80

minutes + 5 minutes) before the processing proteins localize to the NOR (Savino et al.

2001). Quantative analysis in a more recent study showed that GC forms ~18 minutes

after the DFC and ~27 minutes after FC, which corresponds to the temporal recruitment

order of the processing proteins to the NOR (Leung et al. 2004).

The mechanism underlying the movement of PNB towards the NOR has been

uncertain for years. Studies based on the fluorescence recovery after photobleaching

experiments (FRAP), suggested that the transfer of PNB components to the newly formed

nucleoli occurs by a diffusion mechanism, whereas other data support the hypothesis of a

specific pathway (Sleeman and Lamond 1999; Dundr et al. 2000; Leung and Lamond

2003; Leung et al. 2004; Angelier et al. 2005). Using four-dimensional imaging, Savino et al. (2001) observed oscillating movement of PNBs towards the reforming nucleoli

(Savino et al. 2001). Furthermore, they observed bridges linking one PNB to another,

where the GFP signal diminished in one interconnected PNB and increased in the other in a unidirectional flow of nucleolar material via the connecting bridges. Thus, the

movement of the PNBs seems to be a combination of both the diffusion mechanism,

where the processing proteins diffuse out of the PNB and into adjacent PNBs via the

connecting bridges in a specific pathway. The PNB material continuously move from one PNB to another until it reaches a more stable and expanding NOR. The NOR continues to expand until the formation of a functional nucleolus.

47 Cell division is a step-wise process that first involves replicating its DNA in S phase. This is followed by a break down of nuclear envelope in order for the spindle microtubules to attach to the condensed chromosomes. Condensed chromosomes are then separated and pulled to the opposite poles. Finally, nuclear envelope begins to reform, and cytokinesis will pinch the maternal cell to generate two new daughter cells.

After completion of nuclear envelope formation, nuclear transport is required to recompartmentalized proteins to its proper location. When transport is inhibited by using antibodies to the nuclear pore complex or using wheat germ agglutinin in cell exiting mitosis, the cells are trapped in a telophase-like configuration and the absence of nucleolus. From these results, we wanted to determine the consequences of only inhibiting nuclear export, specifically the CRM1-mediated export via LMB, in cells exiting mitosis.

Chapter 1 describes the effects of LMB on mitotic exit cells. LMB is a known

CRM1-mediated nuclear export inhibitor, originally discovered in Streptomyces sp.

Strain (Nishi et al. 1994; Wolff et al. 1997; Kudo et al. 1998; Kudo et al. 1999).

Treatment of LMB in mitotic exit cells showed no effect on its release from mitosis.

However, mitotic exit cells treated with LMB exhibited an increase in apoptotic cells as early as 8 hours of treatment. In contrast, cells released from mitosis for 2 hours prior to the addition of the drug and interphase cells treated with LMB showed minimal apoptotic cells compared to mitotic release cells. This suggests that in mitotic release samples, cells were not able to have a functional nuclear export activity, thus, failing the recompartmentalization process. In the samples where cells were released from mitosis for 2 hours prior to the addition of LMB as well as interphase-LMB treated samples, 48 recompartmentalization is completed, therefore, the number of apoptotic cells was minimal. From these data, it suggests that recompartmentalization process is critical for cells to continue from M phase into G1 phase.

Inhibition of nuclear transport after completion of mitosis resulted in cells lacking nucleoli (Benavente et al. 1989; Benavente et al. 1989; Benavente 1991). Furthermore, the nucleolus has recently been suggested to be a sensor organelle (Rubbi and Milner

2003). Damage-inducing agents and chemicals cause the nucleoli to be disrupted and become non-functional. This resulted in the p53 stabilization and the induction of apoptosis. Since we observed apoptotic cells in mitotic exit cells and not in interphase or in cells released from mitosis for 2 hours prior to treatment with LMB, we speculate that

LMB-treatment during the mitosis to G1 transition resulted in the failure of nucleologenesis, or reformation of the nucleolus. In Chapter 2, we examined the effects of LMB on the nucleolus of mitotic exit cells using immunofluorescent images. In mitotic exit-LMB treated samples we observed that cells contained numerous small, rounded phase dense structures throughout the nucleus and not the intact nucleolus observed in the control cells. Immunolocalization of nucleolar proteins that are known to localize to the interphase nucleolus, such as B23, C23 and fibrillarin, were observed to accumulate to different phase dense structures, while B23 was observed to localize throughout the nucleoplasm. The different localization of the nucleolar proteins indicated that cells treated with LMB during mitotic release failed to reform the nucleolus. In contrast, LMB treatment in interphase cells or cells that had transitioned out of mitosis for 2 hours appeared to have no effect on the nucleolar structure: nucleolus appeared to have an irregular shape with nucleolar proteins markers accumulated to the nucleolus. 49 Thus, inhibition of recompartmentalization of proteins during M to G1 transition by LMB results in the failure of nucleologenesis, and eventually the induction of apoptosis.

In chapter 3, we examine the effects of ratjadone, actinomycin D, and roscovitine, agents that are known to disrupt the nucleolar structures, and compare them to the effects observed in LMB treated cells. Cells treated with ratjadone during M to G1 transition appeared to contain similar phase dense structures and nucleolar protein localization as cells treated with LMB during M to G1 transition. In addition, both interphase cells and cells transitioned from M to G1 phase for 2 hours prior to LMB treatment had no effect on the nucleolar structures, similar to the observations of LMB treated cells. In contrast, both roscovitine, and actinomycin D caused nucleolar disruptions in cells transitioning from M to G1 phase, in cells released from mitosis 2 hours prior to treatment, and in interphase cells (Ochs et al. 1985; Benavente et al. 1987; Dousset et al. 2000; Sirri et al.

2000; Sirri et al. 2002). These results suggest that the effect of LMB is specific and unique that is not observed in other nucleolar inhibitors.

The results presented in this thesis indicate that active nuclear transport is required for recompartmentalization of proteins immediately after mitosis. Inhibition of

CRM1-mediated nuclear export immediately after mitosis resulted in the failure of recompartmentalization and nucleologenesis. Accumulation of cytoplamsic proteins within the nucleus prevented the reassociation of the nucleolar proteins to form the nucleolus. The absence of an intact nucleolus induces downstream apoptotic events.

Therefore, functional nuclear transport is vital for cells to complete mitosis and enter G1 phase successfully.

50 Transport Receptor Function (Alternative Name) Importin β Import of ribosomal proteins, HIV Rev, HIV (p97, PTAC97, Tat, HTLV Rex Karyopherin β1) Importin β/ α Complex Import proteins containing a “classical” NLS

Importin β/Importin 7 Import of histone H1 heterodimer

Transportin 1 Import of hnRNP protein A1, import of (Improtin β2, ribosomal proteins karyopherins β2) Importin 7 (RanBP7) Import of ribosomal proteins

CRM1 Export of proteins containing leucine-rich NESs

CAS Export of importin α

Exportin t Export of tRNA

Table 1: Examples of Transport Receptors of the Importin β Superfamily

51

DFC: Dense Fibrillar Component PNB: PreNucleolar Bodies

FC: Fibrillar Center rDNA: Ribosomal DNA

GC: Granular Component snoRNP: Small Nucleolar

Ribonucleoprotein

NOR: Nucleolar Organizer Region r-proteins: ribosomal proteins

NDF: Nucleolar Derived Foci

Table 2: List of Acronyms Used in the Thesis

52 Cytoplasm Central Channel

Internal Filamen

NE

Spoke Spoke NE Lumen Complex Complex

Lamina Lamina

Internal Filament

Nucleus

Key

Transporter Nucleoplasmic Coaxial Ring

Cytoplasmic Filaments Nuclear Basket

Cytoplasmic Coaxial Ring Radial Arms

Diagram 1: Diagram of Nuclear Pore Complex Diagram adapted from (Allen et al. 2000)

53

Cytoplasm Ran GDP

GTP Pi

Ran Ran BP1 GAP

RanGEF/RCC1

Ran GDP GTP

Nucleus

Diagram 2: The Ran Cycle Diagram adapted from (Yoshiaki and Dasso 2000)

54 Cytoplasm

Cargo NLS Ran β GDP

Cargo Ran Ran NLS β BP1 GAP

Cargo NLS β

Ran GTP

Ran GTP β

Ran Cargo NLS GTP β Nucleus

Diagram 3: Model of Nuclear Import with Importin β Receptor Diagram adapted from (Izaurralde and Adam 1998; Gorlich and Kutay 1999)

55 Cytoplasm β β CAS Cargo NLS

Ran α α GDP

Cargo NLS α Ran Ran β BP1 GAP

CAS Cargo NLS α α β

α Ran Ran Ran Ran GTP GTP GTP β GTP CAS

Ran α Cargo NLS Nucleus GTP β

Diagram 4: Nuclear Import Model with Importin α/β Receptors Diagram adapted from (Izaurralde and Adam 1998; Gorlich and Kutay 1999)

56 Cytoplasm

Ran Ran GDP GDP NTF2 Pi

NTF2

Ran Ran BP1 GAP

Ran GTP β

NTF2 Ran GDP NTF2

GTP

Ran GTP RanGEF/RCC1

β GDP Nucleus

Diagram 5: The Ran Transport Model Diagram adapted from (Izaurralde and Adam 1998; Gorlich and Kutay 1999)

57 Cytoplasm Cargo NES

Ran Ran GDP GDP NTF2

NTF2

Pi

CRM1

Ran Ran BP1 GAP

Ran CRM1 NES Cargo GTP

Ran GDP NTF2

CRM1 GTP

Ran GTP RanGEF/RCC1

Nucleus Cargo NES GDP

Diagram 6: Model of CRM1-Mediated Nuclear Export

58

Diagram 7: Chemical Structure of LMB Structure obtained from Sigma.com

59

Nucleus

Nucleolus

Cytoplasm

Fibrillar Center Nucleus Dense Fibrillar Component Cytoplasm

Granular Component

Diagram 8: Ultrastructure of Nucleoli Diagram adapted from (Hernandez-Verdun and Junera 1995)

60 Reticulated Nucleolus A Compact Nucleolus B

Ring-Shaped Nucleolus C Segregated Nucleolus D

Granular Fibrillar Component (GC) Center (FC)

Dense Fibrillar Component Interstices (DFC) in GC

Diagram 9: Models of the 4 Types of Nucleolar Organization Diagram adapted from (Hernandez-Verdun and Junera 1995)

61 5’ETS ITS1 ITS2 3’ETS

Tandem rRNA Gene Repeats On Chromsome 5’ 3’

rRNA Gene 3’ 5’ 18S 5.8S 28S Methylation/ RNA Pol I Psuedouridylation Transcription by snoRNP Ψ Ψ Ψ Ψ Ψ Ψ Pre-rRNA Transcript 5’ 3’ CH3 CH3 CH3 CH3

Endonucleolytic Ψ Ψ Ψ Ψ and Exonucleolytic Cleavages

CH3 CH3 CH3

Ψ Ψ Ψ Ψ

CH3 CH3 CH3

Ψ Ψ Ψ Ψ

CH3 CH3 CH3

Ψ Ψ Ψ Ψ Ψ Ψ

rRNA CH3 CH3 CH3 CH3 18S 5.8S rRNA 28S rRNA

Ψ Pseudouridine rRNA Coding Sequence CH3 Methylation External non-transcirbed spacer Internal transcribed spacers Promoter External transcribed spacers

Diagram 10: General Scheme of rRNA Processing and Modification Diagram adapted from (Fatica and Tollervey 2002)

62 SL1

TAF TIF- IA/C TAF TAF RNA Transcription UBF TBP Pol I

5’ 3’ 18S 5.8S 28S rRNA Gene

rRNA Gene

External non-transcribed spacer rRNA coding sequence

Promoter Transcription factors

Internal transcribed spacers 47S pre-rRNA transcript

External transcribed spacers RNP Particles

Diagram 11: Schematic Model of the Electron Micrograph view of rDNA Transcription Diagram adapted from (Scheer et al. 1997)

63

rDNA

Transcription 47S pre-rRNA Pre-40S 18S 5.8S 28S Factors

5S rRNA Nucleolus Ribosomal proteins made in cytoplasm 90S Particle

Processing and Modification of pre-rRNA Nonribosomal Pre-60S proteins Factors

pre-60S subunit

pre-40S Nucleoplasm Subunit

Cytoplasm 28S 18S 5S

5.8S 40S subunit 60S subunit mRNA

Diagram 12: Ribosome Biogenesis Model Diagram adapted from (Alberts et al. 2002)

64

5’ 3’

Fibrillarin GAR RNP-2 α-helical

1aa 320aa

5’ 3’ C23/ RBD1 RBD2 RBD3 RBD4 GAR Nucleolin 1aa 707aa

B23/ 5’ 3’ Nummatrin/ Nucleophosmin 1aa 275aa

Nucleolar Localizng Signal Bipartite NLS

Glycine/Arginine Nonpolar Region Rich Domain (GAR)

RNA Binding Moderately Domain (RBD) Basic Residues

Acidic Domain Aromatic Domain

Basic Domain

Diagram 13: Nucleolar Protein Gene Structure

65

NOR

Late Anaphase

NOR

Early Telophase

NOR

Late Telophase

PNBs Nucleolus Containing C23

NDF Containing PNBs Nucleolar Proteins Containing B23

PNBs Contain Other Nucleolar Fibrillarin Proteins

Diagram 14: Nucleologenesis Model

66 67

CHAPTER 1

INHIBITION OF NUCLEAR EXPORT DURING M TO G1 TRANSITION

INDUCES APOPTOSIS

INTRODUCTION

One of the hallmark characteristics that separate bacterial cells from eukaryotic cells is the presence of a nuclear membrane in the higher organisms. Without a nuclear membrane, there is no separation of cytoplamsic and nuclear components. Since the proteins that are required for the DNA transcription and translation are located within the

same compartment in bacterial cells, transcription and translation occur simultaneously.

In contrast, eukaryotic genomes are contained within the nuclear membrane, where DNA

transcription occurs. Once transcription is completed, transcripts are processed and

matured to remove introns (non-codoning mRNA sequences) before being exported out

to the cytoplasm for translation into proteins. The spatial separation of transcription and translation allows eukaryotic cells to check and recheck any mistakes that could have been made during the process; one mechanism for minimizing the occurrence of mutations. The nuclear envelope must break down during mitosis, however, to allow

68 attachment of the mitotic spindle to the chromosomes. The absence of the barrier that normally separates the cytoplasmic and nucleoplasmic compartment allows protein components of each compartments to become mixed. After mitosis, the nuclear membrane is reformed around the chromosomes, and reestablishment of functional nuclear transport is required for cells to recompartmentalized and completely enter G1 phase. Inhibition of nuclear transport by WGA or by antibodies to the nuclear pore complex at the end of mitosis results in a block in mitotic exit in which cells retain telophase like condensed chromosomes and fail to form nucleoli (Benavente et al. 1987;

Benavente et al. 1989; Benavente et al. 1989). This indicates that nuclear transport after mitosis, and subsequent recompartmentalization, are critical steps to ensure that cells successfully complete mitosis and progress completely into G1 phase.

LMB, an antifungal isolated from Streptomyces strain, specifically inhibits the nuclear export of proteins that require CRM1 as the export receptor (Hamamoto et al.

1985; Komiyama et al. 1985; Yoshida et al. 1990; Kudo et al. 1998; Kudo et al. 1999).

Many studies have shown that proteins with classical NES export signals bind to the nuclear export receptor CRM1 (Nishi et al. 1994; Fornerod et al. 1997; Fukuda et al.

1997; Ossareh-Nazari et al. 1997; Wolff et al. 1997). The association of CRM1 with

NES is disrupted in the presence of LMB, which has been shown to act as a competitor for NES binding (Fornerod et al. 1997; Wolff et al. 1997; Kudo et al. 1998; Kudo et al.

1999). LMB binds to cysteine residue (Cys-529) in the central domain region of CRM1, and blocks the formation of ternary complexes in the nucleus consisting of CRM1,

RanGTP, and proteins that contain NES sequences (Fornerod et al. 1997; Fukuda et al.

69 1997; Ossareh-Nazari et al. 1997; Kudo et al. 1999). As a result, NES-containing proteins are trapped inside the nucleus and cannot be exported to the cytoplasm.

Constant accumulation of proteins within the nucleus has an aberrant effect on the cell cycle. For instance, inhibiting CRM1-mediated nuclear export has been shown to affect the cell cycle by arresting cells in G1 and G2 phases when applied to a logarithmically growing population of cells (Yoshida et al. 1990). In addition, it has been shown that LMB stabilizes the tumor suppressor protein, p53, by inhibiting nuclear export of the p53-Mdm-2 protein complex (Freedman and Levine 1998). Under normal conditions, p53 levels are kept low by the actions of Mdm2 proteins; targeting p53 for protein degradation and blocking the transctivation activity of p53 (Momand et al. 1992;

Oliner et al. 1993; Ljungman 2000). The intrinsic ubiquitin ligase activity of Mdm2 allows it to mark p53 for degradation, and shuttles the ubiquitinated p53 to the cytoplasm, via its leucine-rich nuclear export signal (NES), for degradation (Honda and Tanaka

1997; Tao and Levine 1999).

p53 stabilization in LMB treated cells could contribute to cell cycle arrest in G1 and G2 phases. For example, the interaction between Mdm2 and p53 could be disrupted, thus, stabilizing p53, and allowing it to function as a transcription factor that turns on downstream genes, such as p21, thus arresting the cells in G1 phase. Typically, the G1 arrest allows the cell to repair the DNA damage and proceed into S phase after the damage is repaired. In the event that the DNA is not repaired, p53 triggers apoptosis.

Thus, when cells are continuously arrested in G1/G2 phases by LMB in the absence of

DNA damage, it could eventually result in the induction of apoptosis by p53-dependent or p53-independent pathways. Previous studies on inhibiting nucleocytoplasmic

70 transport at the end of mitosis have not specifically examined the inhibition of import or

export processes selectively. Therefore, in this section, we will examine the effects on

cells if only nuclear export were inhibited via LMB during the M to G1 transition. Based

on previous results, we speculate that specifically inhibiting CRM1-mediated nuclear

export by LMB would prevent normal recompartmentalization of proteins, and may have

an adverse effect on cells transitioning from M to G1 phase.

We have investigated the effects of LMB, an inhibitor of CRM1-mediated nuclear

export, on M to G1 transition. CHO cells were arrested in mitosis with nocodazole, isolated by mitotic shake-off and cultured in fresh media or media containing LMB.

Mitotic cells treated with LMB exited mitosis rapidly and similarly to non-treated cells,

thus LMB had no effect on completion of mitosis. However, apoptosis was induced in

LMB-treated samples as early as 8 hours after treatment. This is in contrast to interphase

cells treated with LMB, where some apoptotic cells were observed in samples treated

with LMB, but only after 18 or more hours. Furthermore, a delayed treatment of CHO

cells with LMB after nocodazole release (2 hours) also showed minimal amount of

apoptotic cells but only after 18 hours. Hence, inhibition of CRM1 mediated nuclear

export during M to G1 phase results in a rapid induction of apoptosis. It is likely that the

accumulation of factor(s) in the nucleus after mitosis resulted in a disruption of the

functions that are associated with G1 phase.

71

MATERIALS AND METHODS

Cell Culture and Drug Treatment. CHO cells were maintained in F-10 medium

(Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco). Cells were grown in a

humidified incubator at 37˚C with 5.5% CO2 level. Cells were first blocked with

thymidine (Sigma) at 5mM for 15-16hrs. The cells were then released from the

thymidine block by removing the thymidine containing media and incubating with fresh

media for 3-4 hours. Cells were subsequently treated with 0.04µg/ml of nocodoazole for

8 hrs prior to collecting the blocked mitotic cells. Mitotic cells were isolated by mitotic shake-off, rinsed free of nocodazole, and then plated into fresh tissue culture flasks. The mitotic cells were incubated without further drug treatment (control), treated immediately with LMB, or grown for 2 hours prior to the addition of LMB at various concentrations

(see below). The mitotic cells released and incubated in the presence or absence of LMB were collected at selected times up to 36 hours.

To determine the mitotic index and apoptotic index, harvested mitotic cells were plated and treated with LMB at 0ng/ml, 2.5ng/ml, 5ng/ml, or 10ng/ml for 0 hours, 2, 8,

12, and 24 hours. In one set of the samples, DAPI (0.02µg/ml) was used to determine if

LMB had an effect on mitotic release. LMB-treated cells were collected by trypsinization, fixed with 4% paraformaldehyde in phosphate buffer saline (PBS:

140.0mM NaCl, 1.50mM KH2PO4, 2.70mM KCl, 6.50mM Na2HPO4 ) and lysed with

0.2% triton in PBS for each time point prior to staining with DAPI. Six counts of 100

72 DAPI stained cells were collected for each time point using a Zeiss Axioskop and epifluorescence illumination.

In another set of samples, the vital stain trypan blue (Sigma) was used to analyze

LMB-treated samples. Trypan blue does not stain cells with an intact cell membrane, but will stain apoptotic cells. Trypan blue was diluted to 0.8mM in PBS. Each of the LMB treated samples were collected by trypsinization, and mixed with trypan blue solution at

1:1 ratio for 5 minutes prior to collecting cell counts in a hemacytometer. Four separate counts were taken at each time point.

Flow Cytometry. LMB (7.5ng/ml) treated CHO cells, and control cells, were collected at 8, 12, 18, and 24 hours by trypsinization from the culture dishes. The collected cells were washed with PBS, and pelleted at 900rpm or 49.8 x g (IEC Clinical Centrifuge).

The pelted cells were resuspended and fixed with cold 70% ethanol (ETOH; Fisher

Bioscience) in dH2O for 12 -24 hours at 4˚C. The samples could also be stored in -20˚C for months. To examine cells by flow cytometry, samples were washed free of ETOH with PBS, and treated with DNA extraction buffer (0.2M phosphate citrate buffer, pH

7.8) as described in (Darzynkiewicz and Juan 1997) for 30 minutes at 37˚C prior to staining with propidium idodide (50µg). Ten thousand cells were analyzed for each data set using a Beckman-Coulter Elite flow cytometer.

Immunoblot Analysis. CHO cells treated with or without LMB as described were collected by trypsinization and pelleted to remove the trypsin. Cell pellets, collected from one 10 cm plate for each time point, were lysed in 100µl of RIPA buffer (50mM

73 Tris pH 7.5, 150mM NaCl, 1% NP-40, 0.1% SDS, 0.5% Deoxycholate, and protease

inhibitor cocktail (Sigma)) for 5 minutes on ice. Protein concentrations of the lysed samples were determined using the binchinchonic acid (BCA) assay (Pierce). Forty

micrograms of protein was loaded per lane in 10% SDS-polyacrylamide gels. Following

the separation of proteins by electrophoresis (200 volts), proteins were transferred to

either PVDF or nitrocellulose membrane (Millipore) for 2 hours at 100 volts in 4˚C.

Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline (TBS) plus

0.05% Tween-20 (TBST) for 1-2 hours and then probed with rabbit polyclonal anti- cleaved-PARP (poly (ADP-ribose) polymerase) antibodies (1:500) (Cell Signaling) over night at 4˚C. After 3 washes in TBST, membranes were incubated with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibodies (Molecular Probe;

1:10,000) for 1 hour. The bands were visualized by enhanced chemiluminescence (KPL) on X-ray films (Fuji).

74

RESULTS

To determine whether cells were exiting mitosis following LMB treatment, DAPI

stained cells were examined by epifluorescence microscopy. In the presence of LMB, the

mitotic cells were shown to exit mitosis and enter G1 phase independent of the LMB

concentration used (Figure 1.1A). LMB-treated mitotic cells completed mitosis within 2 hours after release from nocodazole, which was no different than entry into G1 phase by mitotic cells that were not treated with LMB (Figure 1.1A). This indicated that exposure

to LMB during mitosis had no effect upon completion of mitosis.

Although LMB has no effect on cells exiting mitosis, a dose dependent increase in

the number of apoptotic cells was observed at later time points in cells treated with LMB

(Figure 1.1B). Cells treated with 2.5ng/ml of LMB showed little or no formation of

apoptotic cells, similar to what was observed in cells that were not treated with LMB

(Figure 1.1B). However, cells treated with 5ng/ml of LMB showed significant apoptosis

within 8 hours. Furthermore, at 12 and 24 hours, cells treated with 5ng/ml of LMB

showed that 12 and 15%, respectively, of the cells were apoptotic. The levels of

apoptosis were even more pronounced in cells treated with 10ng/ml of LMB. By 8 hours,

approximately 10% of the cells were apoptotic, and at 12 and 24 hours, apoptosis had

increased to about 22% and 33%, respectively (Figure 1.1B). It should be noted that the

number of apoptotic cells counted in this experiment is likely an underestimate of the

total number of dead cells due to the detachment of apoptotic cells from the surface of the

culture dish prior to fixation. Nevertheless, this experiment showed that while LMB has

75 no effect on cells exiting mitosis, it does induce apoptosis in a time and concentration dependent manner following entry into G1 phase. Since LMB inhibits CRM1-mediated nuclear export, we believe that functional nuclear export after the reformation of the nuclear envelope at the end of mitosis is necessary for cells to progress through G1 phase.

Flow cytometry was used to confirm the LMB effects that were observed by examining the morphology of DAPI stained cells. Mitotic cells were accumulated and collected as described above. As a control, mitotic cells were released into normal media for 0 hour, 12 hours, 24 hours, and 32 hours (Figure 1.2). At the 0 time point, the vast majority of the cells were in mitosis as indicated by the 4N DNA peak (Figure 1.2A).

This indicates that our preparation of mitotic cells was minimally contaminated with cells in other phases of the cell cycle. As expected, a normal cell cycle distribution was observed for nocodazole-released cells at 12 hours, 24 hours, and 36 hours, respectively indicating that cells released from a nocodazole block exit mitosis, reenter mitosis, and exit mitosis again (Figures 1.2B-D).

Similar to the results obtained from DAPI staining, mitotic cells released from nocodazole and into LMB (MT-LMB) showed no inhibition of mitotic exit or entry into

G1 phase 6 hours after drug treatment (Figure 1.3A). However, the presence of apoptotic cells was observed in cells treated with LMB 12 hours after mitotic release (Figure 1.3B).

At 18 and 24 hours after LMB exposure, the apoptotic peak continued to increase while the G1 and G2/M peak decreases to almost indistinguishable levels (Figures 1.3C and

1.3D). These data support our previous observation based upon DAPI staining that LMB does not inhibit mitotic exit. In addition, LMB was also shown to induce more apoptosis in cells with increased incubation time in LMB. Again, the observed induction of

76 apoptosis following treatment of cells during the mitosis to interphase transition suggests

that functional nuclear export is required for cells to successfully reenter interphase after mitosis.

To determine whether the LMB effects were specific to a critical time range during the process of nuclear envelope reformation, we collected mitotic cells as described above, but first released the cells from nocodazole into normal media for 2 hours before the addition of LMB (2hrD-LMB). This allowed the cells to complete mitosis as well as the reformation of the nuclear membrane prior to exposure to the LMB.

After the 2 hour mitotic release, the cells were then treated with LMB for 6, 12, 18, and

24 hours. Similar to the mitotic-treated LMB cells, a normal cell cycle distribution was observed after 6 hours of LMB exposure in the 2-hour delayed samples (Figure 1.4A).

The similarities between the mitotic-treated LMB samples and the 2-hour delay LMB treated samples diverges after this time point. Unlike the mitotic-treated LMB samples, the 2-hour delay LMB samples showed only a minimal accumulation of apoptotic cells

(Figure 1.3 vs. 1.4B-D). Rather, starting with the 12 hour time point, the cells showed a broadening of the G1 phase peak indicating an accumulation of cells in S phase. Further accumulation of cells in S phase, with an additional accumulation of G2 cells, was observed at the 18 and 24 hour time points. Thus, LMB does not induce apoptosis in cells that have completed nuclear envelope reformation at the end of mitosis, but does arrest cells in G1 and G2 phase as a result of nuclear export inhibition. These data confirm that re-establishment of functional nuclear transport, specifically nuclear export, after mitotic exit is necessary for cell survival. These results further suggest that shortly after the formation of the nuclear envelope a critical component or factor must be

77 exported from the early G1 nucleus to maintain cell viability. Continual export of this factor does not appear to be required, since LMB does not induce apoptosis once the cells had an opportunity to recompartmentalize.

Since active nuclear transport just after the completion of mitosis appears to be required for cell survival, we wanted to determine that inhibition of nuclear export during interphase cells would not induce apoptosis. Cells were plated on culture flasks 24 hours prior to the addition of LMB (INT-LMB) for 0, 8, 12, 18, and 24 hours before analysis.

We observed a normal cell cycle distribution in interphase cells treated with LMB for 8 hours that is similar to the control interphase population, untreated mitotic cells released from nocodazole, and the 2 hour-delayed samples treated with LMB for 6 hours (Figures

1.4A-B vs. 1.5A-B). Similar to the 2 hour-delayed samples, apoptotic cells were absent in interphase cells treated with LMB for 8 and12 hours (Figures 1.5B and 1.5C). At 18 and 24 hours, there was a small amount of apoptotic cells, which is significantly below the levels of apoptotic cells detected in LMB treated cells during mitotic exit after the same amount of time exposed to LMB (Figures 1.3D-E vs. 1.5D-E). When interphase cells were treated with LMB for 12 hours, we noticed that the there are two distinct peaks for G1 and G2 phase cells, but as the LMB incubation time progresses from 12 hours to

24 hours, there was an accumulation of cells in G1 and S phase (Figures 1.5C-E). This suggests that although LMB does not induce apoptosis as observed in the mitotic release cells, it does arrest cell cycle progression in the interphase cells, consistent to what was observed in the 2 hour-delayed LMB treated samples.

To further confirm the results from DAPI staining and flow cytometry experiments, which showed an inhibition of nuclear export by LMB during the mitosis to

78 interphase transition induces subsequent apoptosis in interphase cells, we used Western immunoblot to examine for the presence of an early apoptotic marker. One of the downstream proteins cleaved by activated caspases is PARP, poly (ADP-ribose) polymerase, which is involved in repairing single-stranded DNA damage. PARP normally exists as a 116 kDa protein, but during apoptosis, caspases cleave PARP into two fragments: 89kDa and 24kDa. CHO cells were arrested in mitosis as described in the materials and methods section. After isolation of the mitotic cells and washing them free of nocodazole, the cells were cultured in the presence of LMB for 0, 8, 12, 18 and 24 hours. In parallel, interphase cells were treated with LMB for the same time and concentration as those in the mitotic release experiments. Mitotic cells treated with LMB exhibited observable, but weak cleaved PARP signal (89kDa) at 8 hours, and this signal increased from 8 to 18 hours and remained constant at 24 hours (Figure 1.6A). This result is consistent with the results from the flow cytometry experiments, where few if any apoptotic cells were present in mitotic cells released into LMB for 8 hours, but apoptosis was readily observed in cells treated with LMB for 12 or more hours (Figures

1.3 vs. Figure 1.6A). In contrast, PARP cleavage was not observed in interphase cells until 18 hours of LMB treatment (Figure 1.6B). Again, this is consistent with the flow cytometry results, where a small apoptotic peak was observed in the interphase population at 18 and 24 hours (Figures 1.5 vs. 1.6B). These results confirm the observation showing that active nuclear export is required for cells to fully exit mitosis and progress into G1 phase, and that inhibition of nuclear export immediately after mitosis results in the induction of apoptosis.

79

DISCUSSION

Functional nuclear transport after mitosis is essential for cell survival. In this chapter, we have shown that inhibiting CRM1-mediated nuclear export after mitosis results in the induction of apoptosis using morphology of cells stained with DAPI, flow cytometry, and Western immunoblot analysis. The induction of apoptosis was rapid (8-

12 hours) for mitotic cells treated with LMB, whereas after a 2 hour-delayed, or in interphase cells treated with LMB, little or no apoptotic response was apparent after 8 or

12 hours. Even after 18 and 24 hours of incubation, the induction of apoptosis in 2 hour- delayed LMB treated samples was minimal, whereas interphase cells showed a slight increase in apoptosis after 24 hours. These data indicate that after mitosis, NES- containing proteins needs to be exported out of the nucleus, and that inhibiting nuclear export after mitosis by LMB leads to cell death.

Prior to mitosis, the chromosomes are condensed, and the nuclear envelope breaks down in preparation for cell division. As a consequence of nuclear envelope breakdown the cytoplasmic and nuclear compartments become mixed. Upon reformation of the nuclear envelope during late anaphase or early telophase, many proteins could be displaced from their intended or normal cellular compartments. These proteins need to be recompartmentalized, which requires the reformation of a functional nuclear pore complex as well as transport receptors to allow for protein movement in and out of the nucleus. Earlier studies have shown that nuclear transport after mitosis is critical for cells to expand the nucleus and to fully enter the G1 phase (Benavente 1991). Following

80 microinjection into mitotic cells of wheat germ agglutinin (WGA) or antibodies such as

PI1, directed against the WGA-binding nuclear pore complex glycoproteins, these

investigations followed the nuclear organization of the daughter cells by

immunofluorescence and electron microscopy. They observed that WGA or PI1

antibodies prevented the incorporation of pore components into a functional nuclear pore

complex. Furthermore, they showed that even in the absence of a functional nuclear pore

complex, the resulting daughter cells still formed a double nuclear membrane, but

chromatin remained in a condensed telophase-like configuration. In addition, they also observed that the nuclei failed to increase in volume and there was an absence of functional nucleoli. This indicates that both nuclear import and nuclear export are required for decondensation of chromosomes and formation of a functional nucleus and nucleoli. In our studies, we extended these observations specifically inhibiting CRM1- mediated nuclear export with LMB, without affecting nuclear pore formation or nuclear import. Functional nuclear import is illustrated in our studies by several criteria including decondensation of chromosomes, swelling of the nucleus, and flow cytometry histograms that showed a normal cell cycle distribution containing a sharp G1 peak and a low G2/M peak indicating that treated cells had entered G1 phase completely. However, the inhibition of export of NES-containing proteins requiring CRM1 receptors results in the induction of apoptosis. Thus, the reestablishment of functional nuclear import after mitosis is not sufficient for cell cycle progression or survival after mitosis, but requires active nuclear export as well to reestablish functional cellular compartmentalization necessary for cell survival. In addition, LMB treatment in cells that had completed recompartmentalization had no induction of apoptosis.

81 A possible mechanism underlying the induction of apoptosis in LMB-treated cells

transitioning from M to G1 phase could involve the tumor suppressor protein, p53. p53

maintains genomic integrity in response to cellular stresses at the G1-S transition, a DNA damage checkpoint (Ko and Prives 1996; Levine 1997). Sequence analysis of p53 in

CHO cells has shown that p53 contains a mutation in codon 211, exon 6, in the DNA binding domain (Lee et al. 1997; Hu et al. 1999; Tzang et al. 1999). The presence of the mutation suggests that p53 is not functional and may not be inducible after irradiation in

these cells (Hu et al. 1999). Since the mutation in p53 occurs on the DNA binding

domain in CHO cells, such a mutation could prevent p53 from binding to DNA and activating down stream genes, such as p21. However, p53 would still retain its ability to

bind to other proteins. In addition to activating pro-apoptotic genes, p53 is also involved

in repression of RNA polymerase I transcription through its binding to SL1. Binding to

SL1 prevents SL1 interaction with TBP, which prevents the formation of the transcription

initiation complex (Zhai and Comai 2000). Studies have shown that inhibition of rDNA

transcription using actinomycin D results in segregation of nucleolar structures and

inhibition of nucleolar functions (Vagner-Capodano and Stahl 1980; Ochs et al. 1985).

Stabilization of p53 in response to LMB treatment during mitotic exit could lead to

suppression of RNA polymerase I transcription that could also lead to disruption of the

structure and function of nucleoli.

However, a study suggests that the mutation in the DNA binding domain of p53

in CHO cells had no effect on its functional properties (Tzang et al. 1999). This study

showed that p53 was able to induce its target proteins, such as Gadd45 after UV

irradiation and that p53 expression was elevated (Tzang et al. 1999). If p53 in CHO has

82 wild type functions, then the presence of LMB could lead to an accumulation of p53 in

the nucleus, since both p53 and Mdm-2 are dependent upon CRM1-mediated export.

Earlier studies have shown that LMB stabilizes p53 by preventing the export of the Mdm-

2-p53 protein complex into the cytoplasm, which is necessary for p53 proteasomal degradation (Freedman and Levine 1998). The resulting stabilization of p53 could lead to repression of RNA polymerase I transcription as described above, or allow it to turn on downstream genes, such as p21. Activation of p21 by p53, would result in cells being arrested in G1 phase and could trigger p53 mediated apoptosis.

Nucleolar function has also recently been associated with having a role as a stress sensor organelle (Rubbi and Milner 2003). In this role, functional nucleoli are required to maintain low levels of p53. However, when stress or other damage impairs the functionality of the nucleoli, levels of p53 are elevated (Rubbi and Milner 2003). Thus, in the presence of LMB, p53 cannot be exported out of the nucleus and into the cytoplasm for subsequent degradation. This could also allow p53 to inhibit rDNA transcription, which would further contribute to the disruption of the nucleolar structure.

In our study we observed apoptosis only in those cells transitioning from mitosis to G1 when exposed to LMB, but not in the 2 hour delayed or the interphase samples.

Whether p53 in CHO is functional or not, cells exiting mitosis when treated with LMB leads to the inhibition of CRM1-mediated nuclear export of a protein factor(s) that must be transported out of the newly formed nucleus and into the cytoplasm. The accumulation of such a cytoplasmic protein factor(s) inside the nucleus may allow for this protein to interfere directly or indirectly with p53 function; either causing G1 arrest or nucleolar disruption by suppressing rDNA transcription, both of which could induce

83 apoptosis. Thus, accumulation of cytoplasmic protein factor(s) inside the nucleus

immediately after nuclear envelope reformation would result in the induction of apoptosis

within several hours. Although p53 has been discussed here, it is certainly not the only

protein that could induce apoptosis in cells transitioning from M to G1 phase in the

presence of LMB.

We have demonstrated that inhibiting nuclear export in cells transitioning from M

to G1 phase results in the induction of apoptosis. In contrast, the 2 hour-delayed and

interphase samples treated with LMB showed very little or no accumulation of apoptotic

cells. Since nuclear import is functional in these cells, as observed by the decondensed

chromatin in DAPI stained cells, the inhibition of CRM1-mediated nuclear export alone

resulted in triggering downstream apoptosis, indicating that recompartmentalization of proteins after mitosis is a critical process. Addition of LMB to cells that had already completed the initial recompartmentalization process after mitosis did not trigger apoptosis. Cytoplamsic proteins trapped within the reforming interphase nucleus may hinder the functions of nuclear proteins, such as p53, or nucleolar proteins through direct or indirect mechanisms. Since nucleoli are now considered a stress sensor in cells, apoptosis triggered by inhibition of CRM1-mediated export using LMB may be mediated through nucleolar structure or function.

84

Figure 1.1: Mitotic Release Index and Apoptotic Index in Cells Treated with LMB.

Mitotic Cells were collected after 8 hours of nocodazole arrest, and released into LMB at

0ng/ml (-♦-), 2.5ng/ml (-■-), 5ng/ml (-▲-), or 10ng/ml (-x-) for 2, 8, 12 and 24 hours.

The cells were stained with DAPI, and examined microscopically for DNA staining to identify and quantify mitotic cells (Panel A) and apoptotic cells (Panel B).

85

86

Figure 1.2: Flow Cytometry Analysis of CHO Cells Released from Mitosis.

Mitotic cells were collected after 8 hours of nocodazole treatment, and were released into fresh media for 0 hour (Panel A), 12 hours (Panel B), 24 hours (Panel C), and 36 hours

(Panel D). At 0 hour, CHO cells were predominantly in G2/M phase (Panel A). 12 hours after mitotic release into fresh media, CHO cells displayed a typical cell cycle profile of DNA distribution, showing prominent G1 and G2 peaks (Panel B). 24 hours after mitotic release into fresh media, CHO cells displayed a decrease in the G1 peak and an increase in G2/M peak, indicating that cells were able to continue with the cell cycle progression (Panel C). After 36 hours released from mitosis, a typical cell cycle distribution was observed to indicate that CHO cells had completed one round cell cycle division, and that nocodazole had no effect on cells exiting mitosis (Panel D).

87

88

Figure 1.3: Flow Cytometry Analysis of CHO Cells Released from Mitosis and into

LMB for 6, 12, 18, and 24 hours.

Nocodazole arrested mitotic cells were collected and replaced with media containing

LMB (7.5ng/ml) for 6 hours (Panel A), 12 hours (Panel B), 18 hours (Panel C) and 24

hours (Panel D). After CHO mitotic cells were released from mitosis and into LMB for

6 hours, a typical cell cycle DNA distribution was observed (Panel A). After 12 hours of

LMB incubation, an increase in G2/M peak was observed (Panel B). In addition, a sharp peak was observed below 200 on the FL2-A axis to indicate apoptosis (Panel B). At 18

hours, the apoptosis peak increased further, while the G1 and G2 peak decreased (Panel

C). After 24 hours of LMB incubation, CHO cells showed only a wide peak to represent

apoptosis while the G1 and G2/M peaks were not distinguishable (Panel C).

89

90

Figure 1.4: Flow Cytometry Analysis of CHO cells Released from Mitosis for 2 hours Prior to the Addition of LMB for 6, 12, 18, and 24 hours.

Mitotic cells were collected as described above and released from mitosis for 2 hours prior to the addition of LMB for 6 hours (Panel A),12 hours (Panel B), 18 hours (Panel

C), and 24 hours (Panel D). At 6 hours of LMB incubation, CHO cells were displayed a typical cell cycle profile of DNA distribution, giving a prominent G1 and G2 peaks

(Panel A). After 12 hours of LMB incubation, CHO cells appeared to initiate an S phase arrest (Panel B). At 18 hours of LMB incubation, the S phase arrest continued as well as the initiation of the G2/M phase arrest (Panel C). After 24 hours of LMB treatment,

CHO continued to display an S phase and G2/M phase arrest while showing a prominent

G1 peak (Panel D).

91

92

Figure 1.5: Flow Cytometry Analysis of CHO Interphase Cells Incubated with

LMB for 8, 12, 18, and 24 hours.

Asynchronize popoluation of CHO cells were not treated (Panel A) or treated with LMB

for 8 hours (Panel B), 12 hours (Panel C), 18 hours (Panel D), and 24 hours (Panel E).

The untreated (control) CHO cells displayed a normal cell cycle profile of DNA

distribution, showing a prominent G1 and G2 peak (Panel A). After 8 hours of LMB

treatment, CHO cells displayed a cell cycle distribution as that of the control cells (Panel

B). Cells treated for 12 hours with LMB incubation showed an increase in the G2/M

peak to suggest that LMB arrests cells in G2/M phase in interphase cells (Panel C).

After 18 hours of incubation, CHO cells showed an increase in S and G2/M phase arrest

(Panel D). The S and G2/M phase arrest was even more prominent in cells that were

treated with LMB for 24 hours (Panel E). Furthermore, apoptotic cells were observed in

cells treated for 24 hours in LMB.

93

94

Figure 1.6: Western Analysis of CHO Mitotic Cells Released into LMB and

Interphase Cells Incubated with LMB.

(Panel A) Mitotic arrested cells were collected, washed, and released into media

containing LMB (7.5ng/ml) for 8, 12, 18 and 24 hours. Cell were lysed in RIPA buffer

and prepared as described in the materials and methods section. 40µg of proteins were

loaded into each lane to examine the cleaved PARP product via anti cleaved PARP

antibody (1:500). In cells released from mitosis and into LMB, an increase in the

intensity of the cleaved PARP product was observed.

(Panel B) Exponentially growing cells were treated with LMB (7.5ng/ml) for 6, 8, 12,

18, and 24 hours. Cells were prepared as described above. 40µg of proteins were also used to examine the cleaved PARP product. Cleaved PARP product was not observed in

LMB treated cells until after 18 hours. This is opposite of what was observed in mitotic

cells released into LMB.

95

96 CHAPTER 2

THE EFFECTS OF LMB ARE SPECIFIC FOR M TO G1 TRANSITION, WHICH

CORRELATES TO NUCLEOLOGENSIS, A SPECIFIC PROCESS TO THE M

TO G1 TRANSITION

INTRODUCTION

The nucleolus is a distinct and highly organized organelle within the nucleus. It

assembles around clusters of tandemly repeated rDNA genes, where it performs its major

function, ribosome biogenesis. Assembly of the ribosomes involves rDNA transcription,

pre-rRNA processing and assembly of the mature rRNAs along with ribosomal proteins

(Olson et al. 2000). Recently, the nucleolus was also reported to be a plurifunctional

nuclear structure that is involved in cell cycle control, nuclear protein export, and serve as

a stress sensor for cell survival (Olson et al. 2000; Visintin and Amon 2000; Rubbi and

Milner 2003). Therefore, the full functionality of the nucleolus not only determines

ribosome production, but also the ability of the cell to survive and proliferate.

The function of the nucleolus as a stress sensor was first suggested by Rubbi and

Milner (Rubbi and Milner 2003). They observed that a disruption of the nucleolus causes

97 the translocation of a nucleolar protein, B23 to the nucleoplasm, as well as stabilization

of p53. Furthermore, the level of p53 increased during nucleologenesis, the process of

nucleolar assembly, which occurs after mitosis (David-Pfeuty 1999). Thus, a functional

nucleolus appears to promote p53 degradation, and in response to cellular stresses or

processes that cause nucleolar disruption, p53 is stabilized.

There are several lines of evidence that suggest a relationship between the

nucleolus and p53. First, ARF, a suppressor protein for Mdm2 activity, is predominately

localized to the nucleolus. Normally Mdm2 forms a complex with p53 and these

complexes have been postulated to travel to the nucleolus before CRM1-mediated export

of the proteins out of the nucleus and into the cytoplasm where p53 is degraded (Tao and

Levine 1999). In response to cellular stress, ARF disrupts the interaction between p53

and Mdm2, and sequesters the latter to the nucleolus, thus stabilizing p53 (Tao and

Levine 1999). Since Mdm2-p53 complexes are not formed, the complex therefore is not

exported to the cytoplasm. Second, the nucleolar protein B23 interacts directly with p53

and possibly regulates its activity (Colombo et al. 2002). Third, under conditions of cell

stress, another nucleolar protein, C23, translocates from the nucleolus to the nucleoplasm

and interacts with p53 (Daniely et al. 2002). These results suggest that disruption of the

nucleolus stabilizes p53 by either promoting ARF sequestration of Mdm2 to the

nucleolus, and/or by the interactions of both B23 and C23 with p53.

During mitosis, transcriptional activity and other nucleolar activities are abolished and the nucleolar structure is no longer maintained. This results in the disassembly of the nucleolus, which can be shown by the staining of the nucleolar processing proteins (B23, C23, and fibrillarin) throughout the cytoplasm in the mitotic

98 cells localized to structures termed nucleolar derived foci or NDFs, and also staining for

these proteins that surrounds the chromosome periphery (Ochs et al. 1983; Spector et al.

1984; Chou and Yung 1995; Dundr et al. 1997; Dundr and Olson 1998).

Reformation of the nucleolus after mitosis, or nucleologenesis, is a complicated

process that is not fully understood. Sequential temporal recruitment of rRNA processing

proteins to the nucleolar organizing region (NOR) is correlated with pre-rRNA

maturation steps. These processing proteins are imported into the newly formed nucleus,

from the pools of NDFs in the cytoplasm, which aggregate to form prenucleolar bodies

(PNBs) within the nucleoplasm along with the processing proteins that were located near

the chromosomal periphery (Savino et al. 1999; Dundr et al. 2000). Different types of

PNBs have been proposed based on the temporal sequence in which the different processing proteins migrate to the NOR during reassembly of the nucleolus. (Savino et al. 1999; Savino et al. 2001). For instance, processing proteins involved in the early maturation steps of pre-rRNA, such as fibrillarin and C23, were observed in the PNBs earlier than B23, which is involved in the late pre-rRNA processing steps. Therefore, inhibition of the sequential recruitment of these processing components to the NOR would result in the inability of the cell to reform the nucleolus

Previously, we have shown that inhibition of CRM1-mediated nuclear export by

LMB in cells transitioning from M to G1 phase resulted in a rapid induction of apoptosis.

This could be due to the stabilization and accumulation of p53 in LMB treated cells.

Similarly, accumulation, and stabilization of p53 was observed in cells with a disrupted nucleolus (Freedman and Levine 1998; Rubbi and Milner 2003). Thus, we wanted to determine whether LMB has an effect on nucleolar structure. Based on the role of the

99 nucleolus as a stress sensor, and the presence of apoptotic cells after LMB treatment during the transition from M to G1 phase, we speculated that inhibition of nuclear export during mitotic release causes a cellular stress that would similarly result in the disruption of nucleolar structures and function.

We analyzed the effect of LMB on nucleolar structure and nucleologenesis using immunofluorescence staining and electron microscopy. Mitotic HeLa and CHO cells were collected and either cultured in fresh media for 2 hours before adding LMB or placed in LMB containing media immediately after mitotic release. Mitotic cells released into fresh media without LMB treatment were used as a control. In control cells, the nucleolar proteins B23, C23 and fibrillarin, were observed to colocalize to the nucleolus in an irregular shaped region of staining that overlapped with the position of the nucleolus determined by phase contrast microscopy. However, in the mitotic cells treated with LMB during the transition to interphase, an irregular shaped nucleolus was not observed, rather multiple small rounded phase dense structures were present within the nucleus. Both C23 and fibrillarin were observed to localize to these phase dense structures, but were not always colocalized to the same structure. In contrast, B23 was distributed evenly throughout the nucleoplasm, and was not observed in the phase dense structures. Ultrastructural morphology confirmed that the normal nucleolar structures were disrupted. Nucleolar proteins in both interphase cells and the 2 hour delayed LMB treated cells localized to the nucleolus. From these studies, we conclude that LMB disrupts nucleologenesis in cells transitioning from M to G1 phase, but not in interphase cells or cells allowed to complete mitotic exit and reform a nuclear envelope prior to

LMB treatment. Thus, we hypothesize that inhibition of CRM1 mediated nuclear export

100 during the M to G1 transition inhibits nucleologenesis through the accumulation of a postulated nucleolar assembly licensing factor(s) in the nucleus, which prevents the completion of nucleologenesis.

101

MATERIALS AND METHODS

Cell Culture. HeLa cells were cultured in DMEM media (Gibco) and CHO cells were

cultured in F-10 media (Gibco). Both were supplemented with 10% fetal bovine serum

(Gibco) and 10µg/ml of gentamycin (Gibco). Cells were grown in a humidified

incubator at 37°C with 5.5% carbon dioxide. Populations of cells were grown to 50%

confluence and then treated with 5mM of thymidine for 16 hours. The thymidine

blocked cells were then released from the thymidine and into fresh media for 6 hours

prior to the addition of 0.04µg/ml nocodazole for 8 hours to arrest the cells in M phase.

Mitotic cells were isolated by mitotic shake-off, rinsed free of nocodazole, and plated on glass cover slips in the presence or absence of 10ng/ml LMB (Sigma) for 12 hours. In some cases, cells were incubated in nocodazole-free media for 2 hours prior to the addition of LMB for 12 hours (2hr delayed LMB).

Immunofluorescence Labeling. LMB-treated or non-treated HeLa cells were either fixed and lysed with cold methanol at -20˚C for 6 minutes, or fixed in 4% paraformaldehyde for 10 minutes followed by 0.1% triton in PBS (140.00mM NaCl,

1.50mM KH2PO4, 2.70mM KCl, 6.50mM Na2HPO4) for another 10 minutes. Cells were

washed with PBS three times (5 minutes/wash) between the fixation and the lysing steps.

HeLa cells then were incubated with either 4% normal goat serum or 4% normal donkey serum for 30 minutes at 37˚C to block non-specific antibody binding. Samples fixed in methanol were incubated with mouse monoclonal-anti-C23 (Santa Cruz; 1:100) followed

102 by Alexa 488 goat-anti-mouse (Jackson Immuno Research; 1:250). HeLa cells fixed in paraformaldehyde were incubated with goat polyclonal-anti-B23 (Santa Cruz; 1:100) and subsequently incubated with Alexa 488 donkey-anti-goat (Jackson Immuno Research;

1:250). The primary antibodies were incubated at 37˚C for 1 hour, whereas the secondary antibodies were incubated at 37˚C for 30 minutes.

Similar procedures for CHO immunofluorescence labeling were preformed as that described for HeLa cells with the following modifications. LMB-treated or non-treated

CHO cells were fixed in 4% paraformaldehyde in PBS for 10 minutes and subsequently lysed in 0.1% SDS in PBS. CHO cells were then incubated in 4% normal donkey serum

(Gibco) for 30 minutes at 37˚C. For the double labeling of CHO cells, cells were first incubated with a mixture of goat polyclonal-anti B23 (1:100) and mouse monoclonal-anti

C23 (1:100), goat polyclonal-anti B23 (1:100) and rabbit polyclonal-anti fibrillarin

(Abcam; 1:100), or mouse monoclonal-anti C23 (1:100) and rabbit polyclonal-anti fibrillarin (1:100) for 1 hour at 37˚C. Samples were subsequently labeled with Cyanine

Cy3 donkey-anti-goat antibody (Jackson Immuno Research) and Alexa 488 donkey-anti- mouse antibody (Molecular Probe), Cyanine Cy3 donkey-anti-goat antibody and Alexa

488 donkey-anti-rabbit, or Alexa 488 donkey-anti-mouse antibody and Cyanine Cy3 donkey-anti-rabbit antibody for 30 minutes at 37˚C. The dilution of the secondary antibodies used was 1:250. Cells were washed with PBS three times between the primary antibody incubation and the secondary antibody incubation.

Both HeLa and CHO samples were stained with 0.02µg/ml DAPI (Sigma) during the final rinse steps. Cover slips were mounted onto slides with Prolong anti-fade kit

(Molecular Probe) and examined with a Zeiss Axioskop by epifluorescence.

103

Preparation for EM. After release from nocodazole, mitotic cells were collected and

plated on to 25mm coverslips in the presence or absence of 10ng/ml LMB for 12 hours.

Cells were fixed in 0.1M PS buffer (0.2M PS stock solution pH 7.4: 0.2M NaH2PO4-

H2O, 0.2M Na2HPO4) with 2% glutaraldehyde for 1 hour followed by 3 washes (10

minutes/wash) with PS + 1.8% sucrose. The fixed cells were then incubated with 1% of

OsO4 in 0.1M PS for 1 hour, followed by 3 washes with PS + 1.8% sucrose. Cells were dehydrated for 10 minutes each in 30, 50, 60, 70, 90, 95, and 100% ETOH followed by two washes in proplyene oxide (PO) for 5 minutes. A mixture of PO and EPON (2:1)

was added to the cells and incubated for 1 hour, and then replaced by PO and EPON (1:1)

mixture and incubated over night at room temperature. After removal of the PO and

EPON (1:1), 100% of EPON was added to the cells for an hour, and fresh EPON was

added for addition changes. Finally, coverslips with the cells side down were placed on

beam capsules filled with EPON, and incubated at 60˚C for 24-48 hours. After removal of the coverslips, EPON embedded samples were cut into thin sections using a Richert

Ultracut E ultramicrotome with a diamond knife. Sections were stained in 2% uranyl acetate for 15 minutes followed by Reynolds lead citrate staining for 4 minutes. Sections were examined in Philips CM-12 at 60KV.

104

RESULTS

To ensure that LMB was inhibiting CRM1-mediated nuclear export, we examined the distribution of cyclin B in the presence of the drug. Cyclin B has been shown to be a

substrate of CRM1-mediated nuclear export during interphase (Yang et al. 1998). HeLa

cells were first blocked in thymidine for 16 hours to arrest the cells in S phase, followed

by the addition of LMB for another 4 hours. In the cells treated with only thymidine,

cyclin B was observed to accumulate in the cytoplasm and not in the nucleus (Figure

2.1A-C). This staining pattern was due to the continuous shuttling of inactive cyclin B

from the cytoplasm into the nucleus, and rapid CRM1-mediated export from the nucleus

back to the cytoplasm during S and G2 phase (Hagting et al. 1998). As a result, in S

phase arrested cells, cyclin B appears to be localized in the cytoplasm. In the presence of

thymidine and LMB, however, we observed an accumulation of cyclin B within the

nucleus (Figure 2.1D- F). This shows LMB is inhibiting CRM1-mediated export, which

prevents the nuclear export of cyclin B, but has no affect nuclear import.

Having established that LMB was inhibiting CRM1-mediated export, we

proceeded to determine whether LMB has an effect on nucleolar structure when applied

to cells released from mitosis, hereafter referred to as MT-LMB. Nocodazole

synchronized mitotic cells were collected and washed free of nocodazole and

subsequently treated with or without LMB for 12 hours. Cells were examined by

immunofluoresence microscopy to determine the localization of the nucleolar proteins

B23, C23, and fibrillarin. Morphological examination of HeLa MT-LMB cells revealed

105 that while chromosome decondensation and nuclear swelling appeared normal, the nucleoli of the treated cells showed differences from those observed in cells without

LMB (Figure 2.2). In contrast to the large, irregular shaped nucleoli in control cells,

HeLa MT-LMB cells exhibited multiple small round phase dense structures within the nuclei (Figure 2.2A-B vs. 2.2G-H). To investigate whether these altered structures also contained protein components of normal nucleoli, we examined the localization of antibodies to C23 and B23, proteins which are known morphological markers that localize to the nucleolus during interphase (Chan et al. 1989; Ginisty et al. 1999). The immunostaining pattern of B23 and C23 in HeLa control cells showed both proteins localized to a large and irregular shaped region, which corresponded to the shape of the nucleolus in the phase contrast micrograph (Figure 2.2A-D). In the presence of LMB,

C23 staining was localized to the small round phase dense structures within the nucleus

(Figure 2.2J). B23 staining was observed to be distributed throughout the nucleoplasm, however, staining intensity was decreased or absent in the phase dense structures (Figure

2.2I). These results indicate that when cells exiting mitosis are treated with LMB, the formation of the nucleolus is disrupted upon return to interphase.

In addition to C23 and B23, another nucleolar protein, fibrillarin, was also used as a morphological marker for nucleolar structures in CHO cells (Ochs et al. 1985). Similar to HeLa control cells, B23, C23 and fibrillarin staining in CHO control cells was also localized to the nucleolus, as staining was shown to correspond to the same irregular shape as the nucleoli observed in the phase contrast images (Figure 2.3A-c, 2.4A-c, and

2.5A-c). Furthermore, an overlay of the fluorescent images of C23 and fibrillarin staining, B23 and C23 staining, and B23 and fibrillarin staining in the CHO cells showed

106 that these nucleolar markers colocalized to the nucleolus (Figures 2.3D-d, 2.4D-d, and

2.5D-d). Ultrastructural studies of the nucleolus have shown that C23 is localized to the

fibrillar center (FC), dense fibrillarin component (DFC), and granular component (GC).

This is the staining pattern we observed in both HeLa and CHO cells (Figures 2.2D,

2.3B-b, and 2.5B-b). B23, on the other hand, is only found in the DFC and GC of the

nucleolus. Since the FC is typically smaller than the other two nucleolar regions, B23

also appears to stain the whole nucleolus (Figures 2.2C, 2.3C-c, and 2.4C-c). Fibrillarin

is only found in the DFC regions of the nucleolus. As a result, the staining of fibrillarin

appears to be punctate throughout the nucleolus where some areas are brightly stained

and others are not (Figures 2.4B, 2.4D, 2.5C, and 2.5D). This is most readily observed in

the enlarged micrographs (Figures 2.4b, 2.4d, 2.5c, and 2.5d)

In contrast to the control cells, CHO MT-LMB cells were observed to have many

small round phase dense structures in the nucleus, similar to those observed in HeLa MT-

LMB cells (Figures 2.3E-e, 2.4E-e, and 2.5E-e). In the presence of LMB, the B23 and

C23 staining exhibited different localization patterns compared to the non-treated cells.

C23 staining formed small rounded structures similar to the phase dense structures

observed by phase contrast microscopy (Figures 2.3F-f). Unlike C23, B23 stained

uniformly throughout the nucleoplasm, and staining was decreased at the position

corresponding to the phase dense structures (Figures 2.3G-g). Overlaying the two images

showed that C23 staining was present at the position where B23 staining was decreased

or absent (Figures 2.2H- h). Fibrillarin and B23 exhibited similar staining patterns as

those for B23 and C23 in the presence of LMB (Figures 2.4E-h). Like C23, fibrillarin

stained the small spherical phase dense structures observed in the phase contrast (Figures

107 2.4F-f), whereas B23 stained throughout the nucleoplasm, but not the phase dense

structures (Figures 2.4G-g). Merging the two fluorescence staining patterns showed that

fibrillarin and B23 do not overlap (Figures 2.4H-h).

While both C23 and fibrillarin appeared to localize to the small round phase dense

structures present in the nucleus of LMB treated cells released from mitosis, there were

some phase dense structures that only labeled with fibrillarin antibodies (Figures 2.5F-f,

and 2.5G-g: arrow indicating fibrillarin staining only). When the two immunofluorescent

images were merged, it is clear that not all of the stained structures overlapped with C23

staining (Figures 2.5H, arrows in 2.5h). These results indicate that the phase dense

structures formed as a result of LMB treatment were not equivalent. Rather, some of these structures are simpler in composition and may contain as few as one type of nucleolar protein (i.e. fibrillarin), while others are more complex and contain mixtures of nucleolar proteins (i.e. fibrillarin and C23). The immunolocalization results suggest that inhibition of CRM1 nuclear export by LMB during exit from mitosis disrupts nucleologenesis in early stages of interphase. While LMB treatment had no affect on the ability of nucleolar proteins to enter the nucleus after reformation of the nuclear envelope

at the end of mitosis, the inhibition of nucleologenesis appears to be the result of a defect

in reassembly of the nucleolar components around the NOR.

Ultrastructural analysis has shown that the nucleolus is composed of three

components, the FC, DFC, and GC. These components are organized such that the FC

(Figures 2.6B and 2.6F; red arrows) is surrounded by the DFC (Figures 2.6B and 2.6F;

white arrows), which in turn is surrounded by the GC (Figures 2.6B and 2.6F; yellow

arrows). Each of these components has specific functions in ribosome biogenesis. rDNA

108 is located in the FC, and as a result, the transition zone between the FC and DFC is where

rDNA transcription occurs. The newly synthesized transcripts are then processed and

matured in the DFC, while the assembly of the ribosomes occurs in the GC. Under light

microscopy, MT-LMB treated cells showed multiple small round phase dense structures in the nucleus containing nucleolar proteins C23 and fibrillarin, but not B23. The presence of these small phase dense structures, and the different localizations of the

nucleolar proteins, suggested that nucleolar structural organization was also disrupted in

LMB treated cells. Therefore, electron microscopy was used to examine if the ultrastructure of the nucleolus in MT-LMB treated cells (Figure 2.6).

The nucleoli of the control HeLa and CHO cells appeared to be large and irregular in shape with distinct central lighter FC regions surrounded by the more electron dense

DFC, which was surrounded by the GC (Figures 2.6A, B, E and F). In contrast, typical nucleolar organization was not observed in either HeLa or CHO MT-LMB treated samples (Figures 2.6C, D, G, H, I, and J). In HeLa cells, less electron dense and larger irregular shaped components were present that appeared in contact with numerous small electron dense structures (Figure 2.6C). Additional small round electron dense structures were also spread across the nucleus. Upon examination under higher magnification, it was observed that many electron dense structures were associated with the periphery of the larger less electron dense nucleolar-like structures (Figure 2.6D).

In the nucleus of CHO MT-LMB treated cells, we observed two different types of cells each with a distinct morphology of electron dense structures. In the first type, the electron dense structures were small, and circular in structure and were prominently distributed throughout the nucleus (Figure 2.6G). Under higher magnification, the

109 electron dense structures exhibited some variability in structure and density of their

matrix (Figure 2.6H). This type of the electron dense nucleolar-like structures may

correspond to the phase dense structures observed by phase contrast microscopy. In the second type of cells, which were not as common as the first type, there appeared to be

more aggregation of the electron dense structures (Figure 2.6I). After examining these

structures at higher magnification, the less electron dense material often seemed to form a doughnut-like shaped structure that appeared to interact with round electron dense structures (Figure 2.6J). Thus, the EM analysis confirmed that in mitotic cells treated with LMB, the reformation of the nucleolus was disrupted. Nucleolar components appeared to be segregated into at least two aggregated structures that differed in electron density, however, we have not determined the protein composition of these structures.

Since LMB has an effect on nucleologenesis when applied to cells during mitotic exit cells, we wanted to determine if LMB has the same effects on cells that have reformed their nuclear envelope prior to the addition of the drug, hereafter referred to as

2hr-delayed LMB. Isolated mitotic CHO cells were collected as described above and seeded on to plates for two hours prior to the addition of LMB. Cells were fixed and stained as described above. In the phase contrast micrographs, we observed an irregular shaped nucleolus, similar to those present in the control cells (Figures 2.7A-a, 2.8A-a, and 2.9A-a). B23, C23, and fibrillarin all localized to the nucleolus, corresponding to the same irregular shaped structure present in the phase contrast micrographs (Figures 2.7B- c, 2.8B-c, and 2.9B-c). Furthermore, when overlaying the fluorescent images, a yellow color was observed that indicated an overlap of the individual red and green fluorescence staining of the nucleolar proteins (Figures 2.7D-d, 2.8D-d, and 2.9D-d). These results

110 showed that LMB had no effect on nucleologenesis when cells were allowed to exit

mitosis for 2 hours prior to the addition of LMB. In the 2 hour time frame after mitosis, cells were able to reform their nuclear envelope and initiate, if not complete, recompartmentalization of proteins prior to the addition of LMB. Therefore, we observed intact nucleoli in these cells, which support our conclusion, that inhibition of recompartmentalization after mitosis results in a failure of nucleologenesis.

While we established that LMB treatment has an effect on the process of

nucleologenesis when applied to cells exiting mitosis but not in cells released from

mitosis for 2 hours prior to the addition of the drug, we wanted to determine whether

LMB has any effect on nucleolar structure in cells that have fully completed

recompartmentalization and nucleologenesis. Therefore, we examined a logarithmically

growing population of CHO cells to determine if LMB treatment had any effect on

nucleolar structure. In control cell populations, we observed co-localized staining of

B23, C23 and fibrillarin to the irregular shaped nucleolar structure (Figures 2.10A-d,

2.11A-d, and 2.12A-d). Interphase cells incubated with LMB also had an irregular

shaped nucleolus that corresponded to the localization of the three nucleolar markers

(Figures 2.10E-h, 2.11E-h, and 2.12E-h). These results indicate that LMB has no effect

on the structure of the nucleolus in interphase cells. Thus, nuclear export is not required for the maintenance of nucleolar structure in interphase cells. As a result, the nucleolus remains intact in the presence of LMB, which further supports our claim that inhibition of nuclear export after mitosis results in a failure of recompartmentalization and

111 nucleologenesis. Therefore, the effects of LMB on nucleolar structure are specific to the process of nucleologenesis, and restricted temporally to the period of time in which cells are reconstructing the nuclear envelope at the end of mitosis.

112

DISCUSSION

In this chapter, we have shown that the presence of LMB during mitotic exit and entry into interphase inhibits the reformation of the nucleolus, or nucleologenesis. We noticed two prominent morphological characteristics in mitotic cells treated with LMB.

First, there are numerous small, round phase dense structures in the nucleus in place of the typical irregular shaped nucleoli observed in the control cells. At the ultrastructural level, the typical organization of the three nucleolar components was not observed in

LMB treated cells. Rather, in the MT-LMB treated cells, different nucleolar components appeared segregated from one another. Secondly, immunolocalization studies showed that the nucleolar proteins no longer co-localized with each other within the nucleolus.

Although both C23 and fibrillarin localized to the phase dense structures in MT-LMB treated cells, they did not always appear in the same phase dense structures. This suggests that different types of nucleolar protein aggregates were present that contained different processing proteins. We are uncertain of the identity of the phase dense structures, however, since both fibrillarin and C23 are localized to phase dense structures, we believe that these structures could possibly represent different types of PNBs.

Unlike MT-LMB cells, the nucleolus remained intact in interphase cells treated with LMB or the 2hr-delayed LMB treated cells. In both of these experiments, the nucleolus appeared to be irregular in shape, which corresponded with the localization of each of the three nucleolar markers used in our studies. Since nucleologenesis has been completed in interphase cells and intact nucleoli are present, inhibition of CRM1-

113 mediated nuclear export has no effect on nucleolar structure. In the 2hr-delayed LMB

samples, recompartmentalization had initiated in the 2 hours after mitotic release and

prior to the addition of LMB. As a result, inhibiting CRM1-mediated nuclear export after

the 2 hour time frame did not have an effect on nucleologenesis. Thus,

recompartmentalization of proteins immediately after mitosis appears to be required for nucleologenesis to occur.

During mitosis, nuclear and cytoplamsic proteins are mixed due to the breakdown of the nuclear envelope. Once the nuclear envelope reforms, these proteins need to be recompartmentalized to allow cells to enter G1 phase. In previous studies, both nuclear import and export were inhibited after mitosis, resulting in cells retaining condensed telophase-like chromosomes and nuclei that failed to enlarge (Benavente et al. 1989;

Benavente et al. 1989; Benavente 1991). However, we showed that by inhibiting only

CRM1-mediated nuclear export during and immediately after mitosis, chromosomes decondensed, the nuclei enlarged, but the formation of nucleolus was inhibited, eventually inducing apoptosis. The induction of apoptosis could be explained through stress sensor function of the nucleolus.

Under stress-free conditions, the nucleolus helps maintain a low level of p53 through mechanisms that promote its degradation. In the absence of a functional

nucleolus, p53 is stabilized and would induce apoptosis if the damage, in this case nucleolar assembly, is not repairable. In our studies (described in the previous chapter),

we observed apoptosis in mitotic exit cells treated with LMB as early as 8 hours after

treatment. In this chapter, immunofluorescence localization of nucleolar markers showed that there was a disruption in nucleolar structure. In the absence of intact nucleoli several

114 studies have shown that p53 is stabilized (Freedman and Levine 1998; Rubbi and Milner

2003). Therefore, the observed increase in apoptotic cells may result from a stabilization of p53 due to the concentration and time dependent affects of LMB when applied during mitosis. In the 2hr delayed LMB treated cells, as well as the interphase cells treated with

LMB, nucleolar proteins were shown to remain localized to the nucleolus, indicating that the nucleolus remained intact. Furthermore, in these samples, the amount of apoptotic cells observed were minimal. Thus, in interphase and 2 hour-delayed LMB treated samples, the intact and functional nucleoli help maintain the p53 levels at a low basal level. Thus, in those samples, we observed a minimal level of apoptotic cells.

How then does LMB inhibit the formation of the nucleolus after mitosis? One possibility is that inhibition of CRM1-mediated nuclear export by LMB traps certain cytoplasmic protein factor(s) within the nucleus, thus preventing the nucleolar proteins from associating with one another. We would define these potential proteins as nucleologenesis licensing factor(s). Earlier studies have shown that B23, C23, and fibrillarin interact with each other during interphase (Ochs et al. 1983; Li et al. 1996; Liu and Yung 1999; Pinol-Roma 1999). However, during mitosis as the nucleolus disassembles, B23, C23, and fibrillarin are no longer associated with each other. Instead, they are located throughout the cell, and at the chromosome periphery (Ochs et al. 1983;

Gautier et al. 1992; Liu and Yung 1999). We believe that it is possible that during

mitosis nucleolar proteins become associated with the nucleologenesis licensing factor(s),

which prevents the nucleolar proteins from interacting with each other. These licensing

factor(s) are either associating with the nucleolar proteins in the cytoplasm, and are then

imported into the nucleus along with the import of the nucleolar processing proteins, or

115 they are trapped in the newly reformed nucleus at the end of mitosis and become

associated with the nucleolar proteins upon their import into the nucleus. In either

scenario, the nucleologenesis licensing factor(s) must be exported out of the nucleus via

the CRM1-mediated nuclear export pathway to allow the nucleolar proteins to reassociate

with one another, and participate in the formation of the nucleolus.

Reassembly of the nucleolus is a temporal sequential event that is correlated to the processing of pre-rRNA. Nucleolar proteins that are involved in the early pre-rRNA processing are recruited to the NOR prior to proteins that are involved in the later processing steps (Savino et al. 1999; Savino et al. 2001; Angelier et al. 2005). Both fibrillarin and C23 are involved in the early pre-rRNA processing, however, several studies have shown that fibrillarin is one of the earliest nucleolar proteins to be recruited to the NOR (Savino et al. 1999; Savino et al. 2001; Angelier et al. 2005). Therefore, we used fibrillarin as a makers for the NOR. B23, on the other hand is known for its involvement during late pre-rRNA processing, thus, it is recruited to the NOR later than

fibrillarin and C23 (Savino et al. 1999).

LMB binds to the same binding site on CRM1 as NES substrates. Thus, LMB out

competes the NES sequence on the licensing factor(s) for the binding site on CRM1

(Kudo et al. 1998; Kudo et al. 1999). As a result, the trapped nucleologenesis licensing

factor(s) continued to prevent the proper reassociation and assembly of the nucleolar

proteins. In MT-LMB treated cells, both fibrillarin and C23 were able to form PNB

aggregates, however, not all C23 was able to fuse with fibrillarin. B23, on the other

hand, was unable to form PNB aggregates. This suggests that inhibition of CRM1-

mediated nuclear export by LMB immediately after mitosis prevented the reassociation

116 of the nucleolar proteins. Therefore, the temporal sequence of recruitment of PNBs to the

NOR, and the formation of PNBs containing nucleolar proteins downstream of the PNB

containing C23 are inhibited (Figure 11).

In this chapter, we have further demonstrated the importance of

recompartmentalization after mitosis. Furthermore, we have illustrated that only

inhibition of CRM1-mediated nuclear export immediately after mitotic exit resulted in

the failure of nucleologenesis. This effect was not seen in interphase cells or in cells that

have completed mitosis 2 hours prior to the addition of the inhibitor. Inhibiting CRM1-

mediated nuclear export via LMB during mitotic exit prevents critical aspects of the recompartmentalization process. We propose that the accumulation of nucleologenesis licensing factor(s) within the nucleus binds to nucleolar proteins and prevents them from

properly reassociating. As a result, the nucleolus fails to reform and induces downstream

apoptotic events. Inhibition of nucleologenesis by LMB is a specific and unique effect of

the drug that has not been observed by others. There are several other compounds that

have been shown to disrupt the nucleolus, which also eventually lead to the induction of

apoptosis. However, other inhibitors that disrupt nucleologenesis or nucleolar function

and structure have known mechanisms of action that are different from that of LMB. In the next chapter we will examine the similarities and differences between these inhibitors of the nucleolus.

117

Figure 2.1: The Effects of LMB on the Localization of Cyclin B

HeLa cells that were blocked in 500nM thymidine for 16 hours were either not treated

(Panels A, B, and C) or treated with LMB (Panels D, E, and F) for 4 hours. Cyclin B

was observed to localize in the cytoplasm in cells treated with only thymidine (Panel A), whereas in the presence of LMB, cyclin B appeared to accumulate in the nucleus (Panel

D). 0.02µg/ml of DAPI was used to stain the DNA of HeLa cells treated with only thymidine (Panel B) or HeLa cells treated with thymidine and subsequently with LMB

(Panel E). Images were examined with a Zeiss Axioskop at 60X magnification. The size bar in panels C and F represent 20µm.

118

119

Figure 2.2: Localization of B23 and C23 in HeLa Control Cells vs. Mitotic Cells

Released into LMB

Mitotic HeLa cells were collected after 8 hours of nocodazole arrest (0.04µg/ml). Mitotic cells were then washed, and either plated onto coverslips in the absence (Panels A-F) or presence of LMB (Panels G-L) for 12 hours. The nucleoli of control cells appeared large with an irregular shape (Panels A and B). In the control cells, both nucleolar proteins

B23 (Panel C) and C23 (Panel D) were localized to the nucleolus. The DAPI stain

(Panels E and F) indicated that the observation of the nucleolar protein stain in the control cells were located in the nucleus. Cells released from mitosis and into LMB were observed to contain small round phase dense structures (Panels G and H). Unlike the control cells, B23 was observed to localize throughout the nucleoplasm and not in the phase dense structures (Panel I), while, C23 accumulated in the phase dense structures

(Panel J). DAPI stains of the LMB treated cells showed a decondensed DNA (Panels K and L). The cells were examined at 100X magnification. The size bar in panel A, B, G, and H equals 10µm.

120

121

Figure 2.3: Localization of C23 and B23 in CHO Control Cells vs. Mitotic Cells

Released into LMB

CHO mitotic cells were collected as described in the materials and methods section. The collected mitotic cells were washed and plated onto coverslips in the absence of LMB

(Panels A-d) or in the presence of LMB (Panels E-h) for 12 hours. In the control cells, the nucleoli were observed to be large and irregular in shape (Panel A-a). Both the C23 and B23 were observed to localize to the nucleolus (Panels B-c). B23 and C23 colocalization was observed in the overlap image (Panel D-d). The red square in panel

A-D is the expanded view of a selected nucleus (Panels a-d).

In CHO mitotic cells released into LMB, the nucleus contained small round structures

(Panel E-e) which is the localization of C23 (Panel F-f). B23 was observed to be distributed throughout the nucleus and not in the phase dense structures (Panel G-g).

Overlay the two nucleolar staining showed that the localization of B23 and C23 do not

overlap (Panel H-h). The red square in panels E-H represents expanded view of a

nucleus (Panels e-h). The cells were examined in 100X magnification. The size bar in

panel A and E is 10 µm.

122

123

Figure 2.4: Localization of Fibrillarin and B23 in CHO Control Cells vs. Mitotic

Cells Released into LMB

CHO mitotic cells were collected as described in the materials and methods section. The collected mitotic cells were washed, and plated onto coverslips in the absence of LMB

(Panels A-d) or in the presence of LMB (Panels E-h) for 12 hours. In the phase contrast of the control cells, the nucleoli were observed to be large and irregular in shape (Panel

A-a). Fibrillarin and B23 were observed to localize to the nucleolus (Panels B-c), and the overlay of the two staining showed colocalization of fibrillarin with B23 (Panel D-d).

The red square in panel A-D is the expanded view of a selected nucleus (Panels a-d).

In CHO mitotic cells released into LMB, the nucleus contained small round structures

(Panel E-e). Fibrillarin was observed to localize to the phase dense structures (Panel F- f), while B23 was distributed throughout the nucleus and not in the phase dense structures

(Panel G-g). Overlay the two nucleolar staining showed that the localization of B23 and fibrillarin do not overlap (Panel H-h). The red square in panels E-H represents expanded view of a selected nucleus (Panels e-h). The cells were examined in 100X magnification. The size bar in panel A and E equals 10 µm.

124

125

Figure 2.5: Localization of C23 and Fibrillarin in CHO Control Cells vs. Mitotic

Cells Released into LMB

CHO mitotic cells were collected as described in the materials and methods section. The collected mitotic cells were washed and plated onto coverslips in the absence of LMB

(Panels A-d) or in the presence of LMB (Panels E-h) for 12 hours. The nucleoli appeared to be large and irregular in shape in the phase contrast of the control cells

(Panel A-a). Both the C23 and fibrillarin were observed to localize to the nucleolus

(Panels B-c). Overlay of the C23 and fibrillarin staining showed that the two nucleolar proteins colocalized to the nucleolus (Panel D-d). The red square in panel A-D is the expanded view of a selected nucleus (Panels a-d).

In CHO mitotic cells released into LMB, the nucleus contained small round structures

(Panel E-e). Both C23 and fibrillarin appeared to localize to the phase dense structures

(Panels F-g). However, closer examination showed that there are different types of phase dense structures (Panels f-g). Overlay the two nucleolar staining showed that the some fibrillarin and C23 colocalize to the same phase dense structures (Panels H- h),

while some phase dense structures only contain fibrillarin (Panel h, arrow). The red

square in panels E-H represents expanded view of a nucleus (Panels e-h). The cells were

examined in 100X magnification. The size bar in panel A and E equals 10 µm.

126

127

Figure 2.6: Ultrastructure of the Nucleolus in Control Cells vs. Mitotic Cells

Released into LMB

EM micrographs of HeLa control cells (Panels A and B). Typical nucleoli with distinct

FC (red arrows), DFC (white arrows), and GC (yellow arrows) domains are observed.

HeLa cell nuclei after LMB treatment showed dissociation of nucleolar structures

(Panels C and D).

In the EM micrographs of CHO control cells, the FC (red arrows) are found within the electron dense semi-circle shaped structures of DFC (white arrows), and the lighter gray area that surrounds the DFC is the GC (yellow arrows) (Panels E and F). In the majority of CHO cells treated with LMB, the EM micrographs showed numerous small phase dense circular structures (Panels G and H). Occasionally CHO cells treated with LMB exhibited numerous small phase dense particles scattered in the nucleoplasm adjacent to the less electron dense structures that seemed to form a doughnut-like shaped structure

(Panels I and J).

The red square in panels A, C, E, G, and I represent the magnified image of the section in panels B, D, F, H, and J. The size bar in panel A and E equals 1µm, and the size bar in panel B and F equals 0.5µm.

128

129

Figure 2.7: Localization of C23 and B23 in CHO Mitotic Arrested Cells Released

from Mitosis for 2 Hours Prior to the Addition of LMB

CHO mitotic cells were collected as described in the materials and methods section. The collected mitotic cells were washed and plated onto coverslips in fresh media without drug for 2 hours prior to the addition of LMB for 12 hours. In the 2 hour delayed LMB treatment, the nucleoli appeared to be large and irregular in shape (Panel A-a). Both the

C23 and B23 were observed to localize to the nucleolus (Panels B-c). Overlay of the

C23 and B23 staining showed that the two nucleolar proteins colocalized to the nucleolus

(Panel D-d). These images suggest that LMB has no effect on the nucleolar structure in cells treated with LMB after 2 hours of mitotic release. The red square in panel A-D is the expanded view of a selected nucleus (Panels a-d). The cells were examined in 100X magnification. The size bar in panel A equals 10 µm.

130

131

Figure 2.8: Localization of Fibrillarin and B23 in CHO Mitotic Arrested Cells

Released from Mitosis for 2 Hours Prior to the Addition of LMB

CHO mitotic cells were collected as described in the materials and methods section. The collected mitotic cells were washed and plated onto coverslips in fresh media without drug for 2 hours prior to the addition of LMB for 12 hours. In the delayed LMB treated cells, the nucleoli appeared to be large and irregular in shape (Panel A-a). Similar to the

C23 and B23, the fibrillarin and B23 were observed to localize to the nucleolus that formed a staining pattern similar to the nucleolus observed in the phase contrast (Panels

B-c). Overlay of the fibrillarin and B23 staining showed that the two nucleolar proteins

colocalized to the nucleolus (Panel D-d). The red square in panel A-D is the expanded

view of a selected nucleus (Panels a-d). The cells were examined in 100X magnification. The size bar in panel A equals 10 µm.

132

133

Figure 2.9: Localization of C23 and Fibrillarin in Mitotic Arrested Cells Released

from Mitosis for 2 Hours Prior to the Addition of LMB

CHO mitotic cells were collected as described in the materials and methods section. The collected mitotic cells were washed and plated onto coverslips in fresh media without drug for 2 hours prior to the addition of LMB for 12 hours. In the 2 hour delayed LMB treatment, the nucleoli appeared to be large and irregular in shape (Panel A-a). Both the

C23 and fibrillarin were observed to localize to the nucleolus (Panels B-c). Overlay of the C23 and fibrillarin staining showed that the two nucleolar proteins colocalized to the nucleolus (Panel D-d). LMB does not appear to have an effect on cells released from mitosis for 2 hours prior to the addition of LMB. The red square in panel A-D is the expanded view of a selected nucleus (Panels a-d). The cells were examined in 100X magnification. The size bar in panel A equals 10 µm.

134

135

Figure 2.10: Localization of C23 and B23 in CHO Control Cells vs. Interphase Cells

Treated with LMB

Asynchronize population of cells were plated for 24 hours onto coverslips, and were

either not treated (Panels A-d: control cells) or treated with LMB (Panels E-h: LMB

treated) for 12 hours. The nucleoli appeared to be large and irregular in shape in the

phase contrast of the control cells (Panel A-a). Both the C23 and B23 were observed to

localize to the nucleolus (Panels B-c). Overlay of the C23 and fibrillarin staining

showed that the two nucleolar proteins colocalized to the nucleolus in the control cells

(Panel D-d). The red square in panel A-D is the expanded view of a selected nucleus

(Panels a-d).

In interphase cells treated with LMB, the nucleus also contained large irregular shaped

nucleoli, similar to the control cells (Panel E-e). Both C23 and B23 appeared to localize

to the nucleolus (Panels F-g), and the overlay image showed that both C23 and B23

colocalized to the nucleolus (Panels H-h).

The red square in panels E-H represents expanded view of a nucleus (Panels e-h). The

cells were examined in 100X magnification. The size bar in panel A and E equals 10 µm.

136

137

Figure 2.11: Localization of Fibrillarin and B23 in CHO Interphase Cells Treated with LMB

Asynchronize population of cells were plated for 24 hours onto coverslips, and were either not treated (Panels A-d: control cells) or treated with LMB (Panels E-h: LMB treated) for 12 hours. The nucleoli appeared to be large and irregular in shape in the phase contrast of the control cells (Panel A-a). Both the Fibrillarin and B23 were observed to localize to the nucleolus (Panels B-c). Overlay of the C23 and fibrillarin staining showed that the two nucleolar proteins colocalized to the nucleolus in the control cells (Panel D-d). Furthermore, the fibrillarin staining appeared as punctate compared to the uniform staining of B23. The red square in panel A-D is the expanded view of a selected nucleus (Panels a-d).

In interphase cells treated with LMB, the nucleus also contained large irregular shaped nucleoli, similar to the control cells (Panel E-e). Both fibrillarin and B23 appeared to localize to the nucleolus (Panels F-g), and the overlay image showed that both fibrillarin and B23 colocalized to the nucleolus (Panels H-h). In addition, fibrillarin showed a punctate staining pattern similar to the fibrillarin staining observed in the control cells.

The red square in panels E-H represents expanded view of a nucleus (Panels e-h). The cells were examined in 100X magnification. The size bar in panel A and E equals 10 µm.

138

139

Figure 2.12: Localization of C23 and Fibrillarin in CHO Interphase Cells Treated

with LMB

Asynchronize population of cells were plated for 24 hours onto coverslips, and were

either not treated (Panels A-d: control cells) or treated with LMB (Panels E-h: LMB

treated) for 12 hours. The nucleoli appeared to be large and irregular in shape in the

phase contrast of the control cells (Panel A-a). Both the C23 and fibrillarin were

observed to localize to the nucleolus that formed a staining pattern corresponded to the

shape of the nucleolus observed in the phase contrast (Panels B-c). Overlay of the C23

and fibrillarin staining showed that the two nucleolar proteins colocalized to the nucleolus in the control cells (Panel D-d). The red square in panel A-D is the expanded view of a selected nucleus (Panels a-d).

In interphase cells treated with LMB, the nucleus also contained large irregular shaped nucleoli, similar to the control cells (Panel E-e). Both C23 and fibrillarin appeared to localize to the nucleolus (Panels F-g), and the overlay image showed that both C23 and

B23 colocalized to the nucleolus (Panels H-h). These results suggest that LMB has no effect on the nucleolar structure in interphase cells.

The red square in panels E-H represents expanded view of a nucleus (Panels e-h). The cells were examined in 100X magnification. The size bar in panel A and E equals 10 µm.

140

141

Figure 2.13: A Model of the Effects of LMB on Temporal Sequence of PNBs to the

NOR

The recruitment of PNBs to the NOR in the presence of LMB is inhibited in our proposed model. Our immunofluorescence studies indicated that both fibrillarin and C23 were able to form PNBs, while B23 was observed to be distributed throughout the nucleoplasm.

The recruitment of the PNBs to the NOR is related to the timing of the proteins involved in the pre-rRNA processing. Fibrillarin is known to be one of the earliest nucleolar proteins involved in the pre-rRNA processing steps, therefore it is recruited to the NOR before C23 and B23. Thus, in our studies, fibrillarin acts as a marker for the NOR. In the presence of LMB, C23 was observed to locate in PNBs that were different than those containing fibrillarin (NOR). This suggested that LMB does not have an effect on the formation of PNBs containing C23, but our model suggests that LMB inhibits the recruitment of the PNB containing C23 to the NOR and all events downstream of C23 recruitment, including the formation of PNBs containing B23.

142 22 143 CHAPTER 3

THE EFFECTS OF LMB ON NUCLEOLAR STRUCTURES ARE DISTINCT

FROM OTHER NUCLEOLAR INHIBITORS

INTRODUCTION

Nucleoli are highly dynamic structures showing great structural variability as a function of activity. The number of nucleoli, their size, and their activity increase in exponentially growing cells, therefore these parameters reflect the proliferating activity of the cells. Fully active nucleoli are large with extensive intermingling of subdomains, which includes the fibrillar center, dense fibrillar component, and granular component.

Under phase-contrast microscopy, active nucleoli appear as large irregular shaped structures within the nucleus that are more phase dense than other cellular organelles

(Hernandez-Verdun and Junera 1995; DiMaro 2004). In contrast, quiescent cells, or those with nucleolar inactivation following induced transcriptional arrest have small and compact nucleoli. The distinct morphological subcomponents segregate from one another in inactivated nucleoli to form separate but juxtaposed structures. The disruption of the nucleoli often results in cell cycle arrest and eventually ends in cell death. Thus, intact nucleoli are critical for cell survival.

144 Nucleolar disruption can be caused by various agents that target different cellular processes. DNA damage by UV-irradiation, and transcriptional inhibition, for example, are two of the most common events that cause nucleolar defragmentation

(Vagner-Capodano and Stahl 1980; Dousset et al. 2000; Rubbi and Milner 2003).

Inhibition of pre-rRNA processing has also been shown to result in the disruption of the nucleolus (Sirri et al. 2000; David-Pfeuty et al. 2001; Sirri et al. 2002). These results suggest that both active rDNA transcription and pre-rRNA processing events are required to maintain intact nucleoli. Since various nucleolar inhibitors function through different mechanisms, it would be of interest to compare the effects of some other nucleolar inhibitors, such as ratjadone, actinomycin D, and roscovitine to those that we have demonstrated for LMB.

Ratjadone, originally isolated as a from the myxobacterium

Sorangium cellulosum, is a polyketide compound that has a chemical structure distinct from that of LMB (Figures 3.1 Panel A vs. B). Several recent reports suggest that the mechanism of action of ratjadone is similar to that of LMB. First, cell cycle analysis showed an increase in the percentage of cells in G1 cell cycle arrest when treated with ratjadone at a concentration of 10nM to 100nM (Burzlaff et al. 2003). The cell cycle arrest in G1 observed is similar to that of cells treated with LMB. The maximal percentage of G1 phase arrested cells was observed after treatment with 10nM ratjadone.

Furthermore, ratjadone caused a dose-dependent decrease in cell proliferation activity, which corresponds to the observation that cells are in G1 arrest. Secondly, ratjadone was shown to inhibit CRM1-mediated nuclear export. In addition, both ratjadone and LMB

145 have the same binding site, Cys 529, on CRM1 (Koster et al. 2003; Meissner et al. 2004).

These results indicate that the biochemical mechanism of action of ratjadone is similar, if

not identical, to that of LMB.

The second inhibitor that we examined is actinomycin D (Act D), a polypeptide

antibiotic isolated from the bacteria strain Streptomyces (Figure 3.1, Panel C). It

intercalates into DNA with a preference for GC-rich sequences, such as those common in

rDNA, and prevents RNA Polymerase I from gliding on the DNA strand for continuous

transcriptional activities. Several earlier studies used Act D in concentrations that ranged

from 0.04µg/ml to 0.2µg/ml to block RNA Pol I transcription, but not Pol II or Pol III transcription (Ochs et al. 1985; Benavente et al. 1987; Dousset et al. 2000). This selective inhibition of RNA Pol I transcription by Act D provided a useful tool for studies in nucleologenesis. The transcriptional arrest caused by low concentrations of Act D resulted in the segregation of the nucleolar components (Vagner-Capodano and Stahl

1980; Thiry and Goessens 1996). Furthermore, during nucleologenesis, PNBs were

observed in cells treated with 0.04µg/ml of Act D, however, it prevented the fusion of

PNBs at the NORs (Ochs et al. 1985; Dousset et al. 2000). This indicates that active

transcription is required for both maintaining the intact structure of mature nucleoli and fusion of PNBs at the NOR after mitosis.

Roscovitine (Figure 3.1, Panel D) is a Cdk inhibitor that is also known to disrupt nucleolar structure. It is a purine analog that competes with ATP for the ATP-binding site on the CDKs, such as Cdk1 (cdc2), Cdk2, and Cdk5 (Meijer et al. 1997). Entry into mitosis requires the activation of Cdk1. This is a multi-step process that initiates with the association of cyclin B to Cdk1 during late S phase when cyclin B levels are increased,

146 and the dephosphorylation of Cdk1 by Cdc25C just prior to entry into mitosis. During

mitosis, RNA Pol I transcription factors, SL1 and upstream binding factor (UBF) are

phosphorylated and inactivated by Cdk1-cyclin B, which causes a repression of rDNA

transcription (Heix et al. 1998; Kuhn et al. 1998; Klein and Grummt 1999; Sirri et al.

2000). Thus, phosphorylation of UBF and SL1 prevents the formation of the transcription initiation complex. Reactivation of transcription after mitosis requires dephosphorylation of SL1 and UBF, and phosphorylation of UBF at another site (Voit et al. 1999). Other research has shown that inhibition of rDNA transcription caused nucleolar defragmentation. However, even after restoration of rDNA transcription in mitotic cells following treatment with roscovitine, PNBs were unable to fuse at the NOR, or process the newly synthesized 47S pre-rRNA into 28S or 18S rRNA species (Sirri et al. 2000). Thus, the activities of rRNA processing machinery were not restored when

Cdk1-cyclin B activity was represessed by roscovitine. This suggests that rDNA transcription and processing of pre-rRNA are regulated by separate mechanisms, and that

activation of both processes are required for nucleologenesis to occur.

Actinomycin D and roscovitine disrupt the nucleolar components at the

transcriptional level or at the pre-rRNA processing level, respectively. A comparison

between these nucleolar inhibitors and LMB has not been made. In the previous chapter,

we have shown that inhibition of nuclear export immediately after mitotic release resulted in the failure of nucleologenesis. However, this effect was not observed in cells that have either initiated the recompartmentalization process after mitotic release or in interphase cells where the recompartmentalization has been completed. Therefore, we wanted to examine whether other nucleolar inhibitors have the same effects as those observed for

147 LMB in interphase cells and the delayed treated cells. Since ratjadone has also been shown to inhibit CRM1-mediated nuclear export, we wanted to confirm that the failure of

nucleologenesis after mitotic release is the result of the inhibition of nuclear export

immediately after mitotic release using this compound as well.

Immunostaining of the nucleolar proteins, C23, B23, and fibrillarin were used to

compare the effects of the different nucleolar inhibitors, such as ratjadone, actinomycin

D, and roscovitine with that of LMB. CHO cells were arrested in mitosis with

nocodazole, isolated by mitotic shake-off, and cultured in fresh media or media

containing drugs. Mitotic cells treated with ratjadone had a similar nucleolar staining

pattern as that of LMB treated cells; both C23 and fibrillarin appeared to localize to

round phase dense structures within the nucleus, while B23 was localized throughout the

nucleoplasm. While, nucleolar disruption caused by actinomycin D in cells exiting

mitosis also produced many phase dense structures, in contrast to ratjadone or LMB

treated cells, these structures exhibited a cap-like structure associated with them that was

more phase dense. Immunostaining showed that C23 and fibrillarin localized to the

round phase dense structures, however, only C23 appeared to localize at the cap like

structure. In addition to its distribution throughout the nucleoplasm, B23 also

accumulated to the cap like structures in actinomycin D treated mitotic cells. Treatment

of cells exiting mitosis with roscovitine also produced a different pattern of nucleolar

protein staining than that observed for either LMB treated cells or actinomycin D treated

cells. All three nucleolar proteins appeared to be distributed throughout the nucleoplasm

with a slight accumulation in the phase dense structures present in the nuclei of

148 roscovitine treated cells. Thus, treatment with actinomcyin D or roscovitine during

mitotic release produced a different nucleolar protein staining pattern than that of LMB

and ratjadone.

In cells released from mitosis for 2 hours prior to the addition of the inhibitors, or

in interphase cells treated with the inhibitors, only ratjadone treated cells showed a

similar nucleolar staining pattern to that observed in LMB treated cells. In these ratjadone treated cells, the three nucleolar protein markers were colocalized to the nucleolus. This indicated that ratjadone, like LMB, had no effect on nucleolar structure when applied to cells that were allowed to release from mitosis 2 hours prior to the addition of ratjadone or in interphase cells treated with ratjadone. In the cells treated with actinomycin D or roscovitine after a 2 hour delay, and interphase cells treated with

actinomycin D or roscovitine, a similar distribution of nucleolar proteins was observed as

the staining pattern obtained in mitotic cells released into actinomycin D or roscovitine.

The different nucleolar protein localization patterns resulting from roscovitine and

actinomycin D treatment further suggest that the nucleolar disruption caused by LMB is a

specific and unique effect due to inhibition of CRM1-mediated nuclear export during the

mitosis to interphase transition.

The effects of LMB appeared to be reversible. After 8 hours of LMB incubation,

cells were washed free of LMB and replaced with fresh media without drugs for 12 hours

prior to fixation. We observed that all three nucleolar proteins were localized to the

nucleolus similar to that of nontreated control cells. This suggested that incubation with

LMB in cells transitioning from M to G1 phase for 8 hours is reversible.

149 From these studies, we conclude that nucleolar inhibitors, such as actinomycin D

and roscovitine, have a different effect on nucleolar structure and function than LMB and ratjadone. Furthermore, the similarity in nucleolar protein localization between mitotic cells released into ratjadone and LMB, the 2 hour delay treatment, and interphase cells treated with these two drugs, support our hypothesis that inhibition of CRM1-mediated nuclear export immediately after mitosis inhibits nucleologenesis due to a failure to export a nucleologenesis licensing factor. Thus, inhibition of nucleologenesis by LMB is a specific and unique effect on nucleolar structure and function that is not observed using nucleolar inhibitors that act through mechanism other than inhibition of CRM1-mediated

nuclear export.

150

MATERIALS AND METHODS

Cell Culture and Drug Treatments. CHO cells were maintained in F-10 medium

(Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco). Cells were kept in

humidified incubator at 37˚C with 5.5% CO2 level. Cells were first blocked with

thymidine (Sigma) at 5mM for 15-16hrs. The cells were then released from the

thymidine block by removing the thymidine containing media and incubating with fresh

media for 3-4 hours. Cells were subsequently treated with 0.04µg/ml of nocodoazole for

8 hrs prior to collecting the blocked mitotic cells. Mitotic cells were isolated by mitotic shake-off, rinsed free of nocodazole, and then plated into fresh tissue culture flasks. The mitotic cells were incubated without further drug treatment (control), treated immediately with nucleolar inhibitors (see below) or grown for 2 hours prior to the addition of nucleolar inhibitors (see below) (2hr-Delayed). The inhibitors used in this chapter are:

Actinomycin D (Sigma, 0.04µg/ml), Roscovitine (Alexis Biochemicals, 75µM),

Ratjadone (Alexis Biochemicals, 60mM). For Interphase cells treated with inhibitors, the cells were plated for 24 hours before the addition of inhibitors for 12 hours. All cells were fixed, lysed and stained after 12 hours of incubation (see below).

LMB Reversibility Experiment: mitotic cells were collected as described above and plated onto coverslips in the presence of LMB (10ng/ml) for 8 hours. After incubation in

LMB, it was removed with fresh media for 12 hours. Cells were finally fixed, lysed, and stained with nucleolar antibodies (see below).

151 Immunofluorescence Labeling. LMB-treated or non-treated CHO cells were fixed in

4% paraformaldehyde in PBS for 10 minutes and subsequently lysed in 0.1% SDS in

PBS. CHO cells were then incubated in 4% normal donkey serum (Gibco) for 30 minutes at 37˚C. The double labeling of CHO cells required incubation with goat polyclonal-anti B23 (1:100) and mouse monoclonal-anti C23 (1:100), goat polyclonal- anti B23 (1:100) and rabbit polyclonal-anti fibrillarin (Abcam; 1:100), or mouse monoclonal-anti C23 (1:100) and rabbit polyclonal-anti fibrillarin (1:100) for 1 hour at

37˚C. Samples were subsequently labeled with Cyanine Cy3 donkey-anti-goat antibody

(Jackson Immuno Research) and Alexa 488 donkey-anti-mouse antibody (Molecular

Probe), Cyanine Cy3 donkey-anti-goat antibody and Alexa 488 donkey-anti-rabbit, or

Alexa 488 donkey-anti-mouse antibody and Cyanine Cy3 donkey-anti-rabbit antibody for

30 minutes at 37˚C. The dilution of the secondary antibodies used was at 1:250. Cells were washed with PBS three times between the primary antibody incubation and the secondary antibody incubation. CHO samples were stained with 0.02µg/ml DAPI

(Sigma) during the final rinse steps. Cover slips were mounted onto slides with Prolong anti-fade kit (Molecular Probe) and examined with a Zeiss Axioskop by epifluorescence.

152

RESULTS

Since ratjadone has been reported to inhibit CRM1-mediated nuclear export, we wanted to determine whether the effects of ratjadone on the localization of nucleolar

proteins was similar to that of LMB. Nocodazole arrested CHO cells were collected and

released into media with or without inhibitors for 12 hours. Similar to cells treated with

LMB (MT-LMB), cells treated with ratjadone during M to G1 transition (MT-Ratjadone) also exhibited several small round phase dense structures in the nucleus (Figures 3.2A-a vs. 3.3A-a). Immunostaining of C23 appeared to localize to these same phase dense structures (Figures 3.2B-b vs. 3.3B-b), whereas B23 was distributed throughout the nucleus (Figure 3.2C-c vs. 3.3C-c). In addition, C23 and B23 staining did not appear to colocalize when images of the staining patterns were merged. This result is also similar to the localization of C23 and B23 observed in MT-LMB cells (Figures 3.2D-d vs. 3.3D- d). In contrast, both C23 and B23 were localized to the nucleolus in control cells (Figure

3.4). The results from the immunolocalization studies indicated that the distribution of the nucleolar proteins B23 and C23 was the same in ratjadone and LMB treated cells.

Immunostaining of fibrillarin and B23 also showed similarities between the effects of ratjadone and LMB. In CHO MT-ratjadone treated samples, fibrillarin was observed to localize to the phase dense nuclear structures (Figures 3.5A-b). Furthermore, overlay of the two immunostaining pattern indicated that B23 was located throughout the nucleus and not in these phase dense structures (Figures 3.5B-d). Again, this was similar

153 to the staining pattern of fibrillarin and B23 observed in MT-LMB treated cells and not

with the staining pattern observed in the control cells (Figures 3.5, 3.6, and 3.7).

Double immunofluoresence staining for C23 and fibrillarin in MT-Ratjadone cells

showed localization of both proteins to the phase dense nucleolar-like structures (Figures

3.8A-c). However, similar to MT-LMB treated samples, C23 and fibrillarin were not always observed colocalized to the same phase dense structure (Figures 3.8D-d; arrows and 3.9D-d; arrows). While some C23 and fibrillarin staining was colocalized to the same phase dense structures, other phase dense structures contained only C23 or fibrillarin. These results are different than the control cells where both fibrillarin and

C23 were observed to colocalize to the nucleolus (Figure 3.10). The localizations of

C23, B23, and fibrillarin in MT-Ratjadone treated cells was indistinguishable from the staining patterns of these nucleolar proteins in MT-LMB cells. Therefore, we conclude that ratjadone, another CRM1-mediated inhibitor, has a similar effect on nucleologenesis and nucleolar structure as that of LMB.

Next, we wanted to compare the nucleolar protein localization following the exposure of mitotic cells to nucleolar inhibitors with known mechanisms of action other than inhibition of CRM1-mediated transport, such as actinomycin D (MT-ActD), with

LMB and ratjadone treated cells. Similar to MT-LMB and MT-Ratjadone, nuclei of MT-

ActD cells contained numerous small round phase dense structures. However, the phase dense structures in the nucleus of MT-ActD treated cells appeared to have two distinct morphologies. The first type were the small round phase dense structures similar to those observed in the MT-LMB and MT-Ratjadone samples (Figures 3.11A-a, 3.12A-a, and

3.13A-a). The second type of cells contained small round phase dense structures that

154 were adjacent to a more phase dense cap-like structure (Figures 3.11E-e, 3.12E-e, and

3.13E-e). The presence of similar cap-like structures were never observed in MT-LMB or MT-Ratjadone treated cells.

Double immunolocalization of C23 and B23 in MT-ActD treated cells showed that C23 localized to the phase dense structures as well as being distributed throughout the nucleoplasm, while B23 was observed to locate mainly to the nucleoplasm (Figures

3.11B-c and 3.11F-g). In addition, B23 was also observed to accumulate in the cap-like

structures unique to MT-ActD treated cells, but not in the other phase dense structures

(Figures 3.11G-g; arrow indicate cap-like structure). Overlay of B23 and C23

immunostaining patterns showed a colocalization of B23 and C23 at the cap-like

structures, while only C23 was present in the phase dense structures (Figures 3.11D-d

and 3.11H-h; arrow indicate cap-like structure).

Immunostaining of fibrillarin and B23 in MT-ActD treated cells showed a similar

localization pattern to that of C23 and B23. Fibrillarin also appeared to accumulate to the

phase dense structures with some localization to the nucleoplasm, similar to that observed

for C23 (Figures 3.12B-b and 3.12F-f). However, a difference between C23 and

fibrillarin staining was apparent; fibrillarin did not appear to accumulate to the cap-like

structure (Figure 3.12F-f). Overlay of the two protein localization patterns showed that

fibrillarin only localized to the phase dense structures, while B23 staining was present in

both the cap-like structures and the nucleoplasm (Figures 3.12D-d and 3.12H-h; arrow

indicate cap-like structure).

Both fibrillarin and C23 localized to the intranuclear phase dense structures in

MT-ActD treated cells, but overlay of the two protein staining patterns showed some

155 differences in the distribution of the two proteins within the phase dense structures

(Figures 3.13D-d and 3.13H-h). Only C23 was shown to be present in the cap-like structures, while the remaining C23 and all of the fibrillarin staining was localized to the rest of the phase dense structures. Closer examination of the phase dense structures showed that fibrillarin staining appeared to accumulate at the opposite end from the cap- like structure when compared to C23 staining (Figures 3.13H and 3.13h; arrow indicate cap-like structure and pink arrow indicate fibrillarin staining ). Although LMB, ratjadone, and actinomycin D all have the ability to disrupt nucleologenesis, the nucleolar disruption caused by actinomycin D resulted in a different distribution of the nucleolar proteins than that observed in LMB and ratjadone treated cells.

CHO mitotic cells released into roscovitine also exhibited different nucleolar protein localization than cells treated with LMB or ratjadone. First, while the nucleus appeared to be swollen and chromosomes decondensed in the interphase cells, no observable nucleolus or intranuclear phase dense structures were present in mitotic cells after being released into roscovitine (Figures 3.14A-a, 3.15A-a, and 3.16A-a). Secondly, neither C23 nor fibrillarin appeared to have accumulated within the nucleus into distinct aggregates as that in the MT-LMB and MT-Ratjadone treated samples. Rather, both C23 and fibrillarin were distributed throughout the nucleus with only a slight increase in staining intensity in selected spots (Figures 3.14B-b, 3.15B-b, 3.16B-c). In MT-

Roscovitine treated cells, B23 appeared to also have some nuclear accumulation that was not observed in MT-LMB or MT-Ratjadone cells (Figures 3.14C-c and 3.15C-c). These observations suggested that cells treated with roscovitine during M to G1 transition also have different nucleolar protein distribution than LMB or ratjadone treated cells.

156 In the previous chapter, we demonstrated that CHO cells released from mitosis for

2 hours prior to the addition of LMB (2hr-Delay LMB) showed no disruption in nucleolar structure. All three nucleolar markers were colocalized to the nucleolus, as confirmed by their localization with the position of the nucleolus observed by phase contrast (Figures

3.17, 3.18, and 3.19). Since ratjadone has the same biological target as LMB, and showed similarities to the immunostaining patterns of nucleolar proteins in cells transitioning from M to G1 phase when treated, we expected that ratjadone would have the same effects as LMB on the 2 hour delayed treated samples. This was confirmed when observing mitotic cells released from mitosis for 2 hours prior to the addition of ratjadone (2hr-Delay Ratjadone) (Figures 3.20, 3.21 and 3.22). Each case, the double immunolocalization of C23 with B23, fibrillarin with B23, and C23 with fibrillarin in the

2hr-Delay ratjadone samples showed that all the nucleolar proteins localized to the nucleolus (Figures 3.20, 3.21, and 3.22). Thus the presence of an intact nucleolus in both the 2hr-Delayed Ratjadone samples and the 2hr-Delayed LMB samples indicated that inhibition of CRM1-mediated nuclear export after 2 hours of mitotic release has no effect on nucleolar structure.

In contrast, cells released from mitosis for 2 hours prior to treatment with actinomycin D (2hr-Delay ActD) showed a different pattern of nucleolar protein localization than either the 2hr-Delay LMB or 2hr-Delay Ratjadone. Instead, 2hr-Delay

ActD had a similar nucleolar protein localization as that observed in the MT-ActD samples (Figures 3.23, 3.24, 3.25 vs. 3.11, 3.12, and 3.13). In 2hr-Delay ActD cells, cells also appeared to have two types of the nucleolar like structures: round phase dense structures and round phase dense structures with a cap-like structure (Figures 3.23A-a,

157 3.24A-a, 3.25A-a). Both fibrillarin and C23 were accumulated to the round phase dense

structures (Figures 3.23B-b, 3.24B-b, 3.25B-c). B23 appeared to accumulate to the cap

like structures, as well as being distributed throughout the nucleus. B23 staining was not

observed in the rounded phase dense structures (Figure 3.23C-c and 3.24C-c; arrow point

to cap-like structure). Overlay of the C23 and B23 immunostaining patterns showed a

colocalization of B23 and C23 to the cap-like structures, however, only C23 appeared to localize to the round phase dense structures (Figures 3.23D-d; arrow point to cap-like structure). Double immunolocalization of fibrillarin and B23 showed that only B23 was found at the cap-like structures, while fibrillarin accumulated to the rest of the phase dense structures (Figures 3.24D-d; arrow point to cap-like structure). These two nucleolar proteins did not appear to overlap in localization. The double localizations of

C23 and fibrillarin in the 2 hr-Delay ActD cells also showed a similar staining pattern as that of MT-ActD cells. Both C23 and fibrillarin appeared to localize to the phase dense structure, but only C23 appeared to accumulate to the cap-like structures (Figure 3.25D; white arrow). Again, closer examination of the phase dense structures showed that fibrillarin accumulated more at the opposite side of the phase dense structure to that of the cap-like structure (Figure 3.25d; pink arrow point to fibrillarin localization). The observed disruption of the nucleolus in the 2hr-Delayed ActD cells is opposite to that of the 2hr-Delayed LMB and the 2hr-Delayed Ratjadone cells. Thus the nucleolar inhibitory effects of LMB and ratjadone are clearly different from the nucleolar inhibitory effects of actinomycin D.

Nucleolar protein localization in 2hr-Delay Roscovitine treated cells also did not resemble the 2hr-Delay LMB and 2hr-Delay Ratjadone cells. Both C23 and fibrillarin

158 appeared to accumulate to the phase dense structures, and also throughout the

nucleoplasm (Figures 3.26B-b, 3.27B-b, 3.28B-c). The majority of the B23 signal was

distributed throughout the nucleus, however, some B23 appeared to accumulate near the

phase dense structures (Figures 3.26C-c and 3.27C-c). Overlay of the nucleolar staining

showed some colocalization between C23 and B23, fibrillarin and B23, and C23 and

fibrillarin, however a significant amount of staining was localized to the nucleoplasm

(Figures 3.26D-d, 3.27D-d, and 3.28D-d). Thus, roscovitine treatment also has an effect on nucleolar structure that is different than LMB or ratjadone treated cells.

We have also shown that interphase cells treated with LMB showed no effects on nucleolar structure (See chapter 2 and Figures 3.29, 3.30, and 3.31). Interphase cells treated with ratjadone (INT-Ratjadone) showed a similar nucleolar protein staining pattern as the interphase cells treated with LMB and control cells. All three nucleolar proteins appeared to colocalize to the nucleolus, and they colocalized to the same

irregular shaped nucleoli observed by phase contrast microscopy (Figures 3.32, 3.33, and

3.34). Furthermore, fibrillarin also appeared to have a punctate staining pattern when it

overlaps with C23 or B23 (Figures 3.33D-d and 3.34D-d). Thus, the control-like

nucleolar staining pattern in interphase cells treated with ratjadone indicated that there

was no nucleolar disruption by this drug, which is similar to the interphase cells treated

with LMB.

Interphase cells treated with actinomycin D (INT-ActD) however, showed a

disruption in the localization of nucleolar components. Unlike INT-LMB and INT-

Ratjadone, INT-ActD treatment resulted in cells that contained numerous round phase

dense structures within the nucleus, both with and without the cap like structures (Figures

159 3.35, 3.36, and 3.37). In the double immunolocalization studies of C23 and B23, both

nucleolar proteins were observed to distribute evenly throughout the nucleus (Figures

3.35B-c, and 3.35F-g). However, B23 also accumulated to the cap like structures, but not

in the rounded phase dense structures (Figures 3.35G-g and 3.35H-h; white arrow point

to the cap-like structure; blue arrow point to the round phase dense structure). C23

appeared to localize to both the round phase dense structures and the cap like structures

(Figures 3.35C-c and 3.35F-f). Although the double immunolocalization studies of fibrillarin and B23 showed a similar staining pattern as that of C23 and B23, the only difference is that fibrillarin did not accumulate in the cap like structure, similar to MT-

ActD and 2hr-Delayed ActD cells (Figure 3.36B-b and 3.36F-f). When the immunostaining patterns were merged, the cap like structure was clearly shown to contain only B23 and not fibrillarin (Figure 3.36D-d and 3.36H-h; white arrow point to cap-like structure). Examination of C23 and fibrillarin localization in INT-ActD cells revealed a staining pattern that was similar to MT-ActD and 2hr-Delay ActD cells

(Figures 3.37 vs. 3.13 and 3.25). Both C23 and fibrillarin were colocalized to the round phase dense structures and throughout the nucleus, but only C23 was observed in the cap like structures (Figures 3.37H-h; arrow point to cap-like structure). In addition, fibrillarin appeared to accumulate more at the opposite end of the phase dense structure that contains the cap-like structure (Figures 3.37D-d, and 3.37H-h; white arrow point to cap- like structure). Unlike LMB and ratjadone, which have no observable effect on nucleolar structure in interphase cells, actinomycin D disrupts the nucleolar structure when applied to the interphase cells.

160 Addition of roscovitine to interphase cells (INT-Roscovitine) also did not resemble the INT-LMB and INT-Ratjadone cells (Figures 3.38, 3.39, and 3.40). We observed that all three nucleolar proteins were localized throughout the nucleus in INT-

Roscovitine cells (Figures 3.38, 3.39 and 3.40). B23, C23, and fibrillarin all appeared to have some localized accumulation in the nucleus, but the accumulation was not defined or distinct (Figures 3.38B-c, 3.39B-c, and 3.40B-c). Overlapping the immunolocalization images indicated that there was some colocalization of proteins between fibrillarin and

B23, and C23 and fibrillarin (Figures 3.38D-d, 3.39D-d, and 3.40D-d). However, the colocalization of these nucleolar proteins was not the same as the INT-LMB and INT-

Ratjadone cells. This also demonstrates the nucleolar inhibitory effects of LMB and ratjadone are different from the inhibitory effects of roscovitine.

Finally, we wanted to determine if the effects of LMB on nucleologenesis were reversible. Mitotic cells were collected as described in the materials and methods section and plated on the coverslips in the presence of LMB for 8 hours. Cells were washed free of LMB, and cultured with fresh media for 12 hours. We observed that the nucleolar proteins all localized to the nucleolus similar to the control cells (Figures 3.41, 3.42, 3.43 vs. 3.4, 3.7, and 3.10). This indicated that treatment of cells released from mitosis into

LMB is reversible.

161

DISCUSSION

In this Chapter, we have presented several lines of evidence observing that among the three nucleolar inhibitors we have compared to LMB, only ratjadone had a similar effect on the nucleolar structure. First, the four inhibitors all disrupted the nucleologenesis in cells transitioning from M to G1 phase, however, the localization of the three nucleolar markers in actinomycin D and roscovitine treated cells were markedly different than that in ratjadone and LMB treated cells. Ratjadone and LMB treated cells both exhibited localization of C23 and fibrillarin to phase dense structures, whereas B23 was distributed throughout the nucleus. In actinomycin D treated cells, while both B23 and C23 were distributed throughout the nucleoplasm there was also an accumulation of these proteins in cap-like structures, which were only observed in actinomycin D treated cells, in association with intranuclear phase dense bodies. Both C23 and fibrillarin were also localized to the intranuclear phase dense structures. Localization of the three nucleolar protein makers in roscovitine treated cells showed a distribution throughout the nucleoplasm with some accumulation or aggregation of staining. Secondly, treatment with LMB or ratjadone in cells that had first transitioned out of M phase for 2 hours had no apparent effect on nucleolar structure. In contrast, both actinomycin D and roscovitine treatment of cells pre-released from mitosis exhibited a disruption of the nucleolus similar to that present in cells transitioning from M to G1 phase when exposed to the drugs. Finally, LMB and ratjadone treatment of interphase cells also had no effect

162 on nucleolar structure, whereas both actinomycin D and roscovitine disrupted nucleolar

structure similar to that observed after the treatment of cells during the M to G1 transition.

Although all four inhibitors disrupted nucleolar structure, each of the inhibitors target different cellular functions, which we believe, is directly responsible for the

different morphological effects on the nucleolus that were observed. Similar to LMB, ratjadone disrupted the nucleolar structures in mitotic samples by inhibiting CRM1- mediated nuclear export (Burzlaff et al. 2003; Koster et al. 2003; Meissner et al. 2004).

The fact that ratjadone, a different compound with the same molecular target as LMB,

was shown to have similar effects on nucleologenesis and nucleolar structure as those of

LMB support our hypothesis that recompartmentalization immediately after mitosis is

necessary for nucleologenesis and cell survival. As a result of inhibiting nuclear export

immediately after mitotic release, our results suggest that a nucleologenesis licensing

factor(s) is trapped inside the nucleus. This licensing factor prevents the proper

reassembly of nucleolar proteins around the NOR and a failure of nucleologenesis.

Interphase cells, and cells treated with ratjadone 2 hours after mitotic release do not

require additional recompartmentalization, which indicates that nuclear export of the

nucleologenesis licensing factor occurs immediately after reformation of the nuclear

envelope. Once completed, the factor does not need to be continually removed from the

nucleus, since it is not imported back into the nucleus once nuclear envelope reformation

is completed. This indicates that the nucleologenesis licensing factor can only gain

access to the nucleus upon subsequent entry of the cell into mitosis and the associated

breakdown of the nuclear envelope.

163 Actinomycin D is known to inhibit rDNA transcription at low concentrations

(0.04µg/ml to 0.2µg/ml) (Ochs et al. 1985; Benavente et al. 1987; Dousset et al. 2000). It intercalates into DNA with a preference for GC-rich sequences, such as those in rDNA.

Several studies have shown that reformation of intact functional nucleoli after mitosis

appears to be dependent on rDNA transcription (Ochs et al. 1985; Benavente et al. 1987;

Dousset et al. 2000). Injection of RNA Polymerase I antibodies to prometaphase and

metaphase cells resulted in daughter cells without intact nucleoli (Benavente et al. 1987).

Furthermore, in another study, the Topoisomerase I inhibitor, camptothecin, was used to

block the reinitiation of NOR transcription in telophase, which resulted in the lack of

nucleoli in the newly formed daughter cells (Weisenberger et al. 1993). These results

indicate that inhibition rDNA transcription prevents the reformation of the nucleolus after

mitosis. As a result, actinomycin D was used in several studies to decipher the

mechanisms of nucleologenesis.

When cells are treated with actinomycin D in either mitosis or interphase it has

been shown that two types of nucleolus-related structures are formed: small round phase

dense structures that resemble typical PNBs, and phase dense structures with a cap-like

structure (Ochs et al. 1985; Benavente et al. 1987; Dousset et al. 2000). Ultrastructural

examination of actinomycin D treated cells has shown that nucleolar structural disruption,

is characterized by the segregation of the nucleolar components, GC, DFC, and FC, forming separate but juxtaposed regions (Vagner-Capodano and Stahl 1980; Hernandez-

Verdun et al. 1984; Puvion-Dutilleul et al. 1992; Thiry and Goessens 1996; Dousset et al.

2000; Hernandez-Verdun 2006). Since the DFC is more electron dense than either the

GC or FC, the DFC appears as a concave cap-like structure next to the GC. A more

164 recent study showed that the cap-like structures (or dense nuclear cap; DNC) contain pre- rRNA transcripts, nucleolar proteins, PML or Cajal body proteins and several proteins related to the RNA Pol I transcription machineries (Shav-Tal et al. 2005). In addition, segregation of the nucleolar components is accompanied by the release of several GC proteins into the nucleoplasm. These results are similar to our observations using actinomycin D and support our conclusion that nucleolar disruption by inactivation of rDNA transcription, like that caused by actinomycin D treatment, has a different nucleolar disruption pattern than the nucleolar morphologies caused by inhibition of

CRM1-mediated nuclear export.

Roscovitine has a distinct effect on nucleolar structure that differs from actinomycin D, ratjadone, or LMB due to its mode of action as an inhibitor of Cdks.

Normally, both rDNA transcription and pre-rRNA processing are arrested as cells enter mitosis, and are restored when cells exit from mitosis. Silencing of the rDNA transcription prior to mitosis is due to the phosphorylation of SL1, a transcription initiation factor, by cdk1-cyclin B (Heix et al. 1998; Kuhn et al. 1998). Phosphorylation of SL1 impaired the interaction with UBF (upstream binding factor) to form the pre- initiation complex that is required for the activation of rDNA transcription. Treatment of roscovitine in mitotic cells resulted in a premature reinitiation of rDNA transcription

(Sirri et al. 2000). Interestingly, activation of rDNA transcription, however, did not result in the reinitiation of 47S pre-rRNA processing into mature transcripts of 5.8S, 18S or 28S rRNAs (Sirri et al. 2000). This suggested that inhibition of a Cdk or Cdk-like activity prevented the reactivation of pre-rRNA processing. Furthermore, fibrillarin immunofluorescent staining studies of roscovitine treated cells showed some intranuclear

165 accumulation of fibrillarin in cells transitioning from M to G1 phase as well as in

interphase cells (Sirri et al. 2002). Again, we obtained similar immunolocalization

results in cells treated with roscovitine. These results showed that the restoration of

rDNA transcription, but not the pre-rRNA processing also has a different effect on

nucleolar morphology than that observed in LMB or ratjadone treated cells.

In this chapter, we have compared the effects of different nucleolar inhibitors and demonstrated that the disruption of nucleolar structure observed after LMB treatment was similar to that caused by ratjadone, but were unique effects that were not seen using other nucleolar inhibitors. One of the main differences among the nucleolar inhibitors examined is that only LMB and ratjadone had no effect on nucleolar structure in interphase cells or cells that had completed mitosis for 2 hours prior to treatment. On the contrary, the nucleolus was disrupted in both actinomycin D and roscovitine treated interphase cells. This indicated that both active transcription and pre-rRNA processing activities are required to maintain interphase nucleoli, whereas continued export of proteins from the nucleus into the cytoplasm is not. The only known function of LMB is as an inhibitor of CRM1-mediated nuclear export (Yoshida et al. 1990; Nishi et al. 1994;

Kudo et al. 1998; Kudo et al. 1999; Meissner et al. 2004). However, our observations show that LMB is also an inhibitor of nucleologenesis. We believe that the inhibition of nucleologenesis caused by LMB is also a direct result of its inhibition of CRM1-mediated export. The presence of a nucleologenesis licensing factor(s) within the newly reformed nucleus disrupts the reassociation of the nucleolar proteins and reformation of the nucleolus around the NOR. In the absence of a nucleolus downstream apoptotic events are subsequently activated. This specific chain of events was only observed immediately

166 after mitotic release. This supports our conclusion that the nucleolar disruption caused by

LMB is specific to the M to G1 transition, and is a unique effect that has not been observed with other nucleolar inhibitors.

167

Figure 3.1: Chemical Structures of the Nucleolar Disrupting Agents

Chemical structures of ratjadone (Panel A), Leptomycin B (Panel B), actinomycin D

(Panel C), and roscovitine (Panel D).

168 Ratjadone Mol. Wt. 456.6g/mol A

Leptomycin B B Mol. Wt. 540.7g/mol

Actinomycin D Roscovitine Mol. Wt. 1255.5g/mol C Mol. Wt. 354.5g/mol D

Figure 3.1: Chemical Structures of the Nucleolar Disrupting Agents

169

Figure 3.2: Localization of C23 and B23 in CHO Mitotic Cells Released into

Ratjadone.

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in media containing ratjadone for 12 hours prior to fixing and staining of the cells. Phase contrast views of mitotic cells released

into ratjadone showed numerous small round phase dense structures in the nucleus

(Panels A and a). Immunostaining of C23 showed localization to the phase dense structures (Panels B and b). B23 immunostaining illustrated a distributed localization

throughout the nucleus in mitotic cells released into ratjadone (Panels C and c). A

distinct green color stain of C23 showed that the overlay of the two nucleolar proteins

C23 and B23 do not overlap (Panels D and d). The red squares in panels A-D illustrate

the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals

10µm.

170 A a

Phase

B b

C23

C c

B23

D d

Merge

Figure 3.2: Localization of C23 and B23 in Mitotic Cells Released into

Ratjadone

171

Figure 3.3: Localization of C23 and B23 in CHO Mitotic Cells Released into LMB

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed free of nocodazole and plated onto coverslips in media containing LMB for 12 hours prior to fixing and staining of the cells. Phase contrast views of mitotic cells released into LMB showed numerous small round phase dense structures in the nucleus (Panels A and a). Immunostaining of C23 showed an accumulation to the phase dense structures similar to the C23 localization in ratjadone treated cells (Panels B and b). B23 immunostaining illustrated a distributed localization throughout the nucleus and not in the location of the phase dense structures (Panels C and c). Overlay of the two immunostaining showed that the localization of C23 and B23 do not overlap (Panels D and d). The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

172 A a

Phase

B b

C23

C c

B23

D d

Merge

Figure 3.3: Localization of C23 and B23 in CHO Mitotic Cells

Released into LMB

173

Figure 3.4: Localization of C23 and B23 in CHO Control Cells

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without drugs for 12 hours prior to fixing and staining of the cells. Phase contrast views of mitotic cells released

into fresh media exhibited large irregular shaped nucleolus, opposite of the LMB treated and ratjadone treated cells (Panels A and a). C23 immunostaining accumulated to the nucleolus forming a shape similar to the nucleolus observed in the phase contrast (Panels

B and b). Similar to C23, B23 also accumulated to the nucleolus (Panels C and c).

Overlay of the two immunostaining showed a yellow color staining to indicate the

colocalization of C23 and B23 (Panels D and d). The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

174

175

Figure 3.5: Localization of Fibrillarin and B23 in CHO Mitotic Cells Released into

Ratjadone

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed free of nocodazole and plated onto coverslips in media containing ratjadone for 12 hours prior to fixing and staining of the cells. Phase contrast views of mitotic cells released

into ratjadone showed numerous small round phase dense structures in the nucleus

(Panels A and a). Immunostaining showed that C23 accumulated to the phase dense structures (Panels B and b). B23 immunostaining illustrated a distributed localization

throughout the nucleus and not in the location of the phase dense structures (Panels C

and c). Overlay of the two immunostaining showed that the localization of fibrillarin and

B23 do not overlap (Panels D and d). The red squares in panels A-D illustrate the

expanded nucleus shown in panels a-d. The size marker in the phase contrast equals

10µm.

176

177

Figure 3.6: Localization of Fibrillarin and B32 in CHO Mitotic Cells Released into

LMB

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed free of nocodazole and plated onto coverslips in media containing LMB for 12 hours prior to fixing and staining of the cells. Phase contrast views of mitotic cells released into LMB showed numerous small round phase dense structures in the nucleus (Panels A and a). Similar to ratjadone treated cells, fibrillarin was observed to accumulate to the phase dense structures and not in the nucleoplasm (Panels B and b). B23 immunostaining illustrated a distributed localization throughout the nucleus and not in the location of the phase dense structures (Panels C and c). Overlay of the two immunostaining showed that the localization of fibrillarin and B23 do not overlap in

LMB treated cells (Panels D and d). The similar immunolocalizations of fibrillarin and

B23 between LMB treated and ratjadone treated cells indicate that both drugs have the same effects on cells transitioning from M to G1 phase. The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

178

179

Figure 3.7: Localization of Fibrillarin and B23 in CHO Control Cells

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without any drugs for 12 hours prior to fixing and staining of the cells. Phase contrast views of mitotic cells released into fresh media showed many irregular shaped nucleolus (Panels A and a).

Opposite of LMB treated and ratjadone treated cells, immunostaining of fibrillarin was observed to accumulate to the irregular shaped nucleolus in the control cells (Panels B and b). Fibrillarin appeared as punctuate, where some areas of the nucleolus were stained brightly with fibrillarin and other areas were not. In addition, B23 immunostaining was also localized to the nucleolus forming the same nucleolar shape as the phase contrast (Panels C and c). Overlay of the two immunostaining showed that the localization of C23 and B23 overlap one another (Panels D and d). The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

180

181

Figure 3.8: Localization of C23 and Fibrillarin in CHO Mitotic Cells Released into

Ratjadone

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in media containing ratjadone for 12 hours prior to fixing and staining of the cells. Phase contrast views of mitotic cells released

into ratjadone showed numerous small round phase dense structures in the nucleus

(Panels A and a). Immunostaining of C23 showed an accumulation to the phase dense structures (Panels B and b). Fibrillarin staining exhibited localization to the phase dense structures as well (Panels C and c). Overlay of the two immunostaining showed that

some C23 and fibrillarin localized to the same phase dense structures, while other phase

dense structures contain either C23 or fibrillarin proteins (Panels D and d). This

indicates that there are different types of phase dense structures with different nucleolar

proteins in ratjadone treated cells. The red squares in panels A-D illustrate the expanded

nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

182 A a

Phase

B b

C23

C c

Fibrillarin

D d

Merge

Figure 3.8: Localization of C23 and Fibrillarin in Mitotic Cells Released

into Ratjadone

183

Figure 3.9: Localization of C23 and Fibrillarin in CHO Mitotic Cells Released into

LMB

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed free of nocodazole and plated onto coverslips in media containing LMB for 12 hours prior to fixing and staining of the cells. Phase contrast views of mitotic cells released into LMB showed numerous small round phase dense structures in the nucleus, similar to the ratjadone treated cells (Panels A and a). Immunostaining of C23 appeared to accumulate to the phase dense structures (Panels B and b). Fibrillarin also localized to the phase dense structures (Panels C and c). However, overlay of the two immunostaining showed that similar to ratjadone treated cells, some C23 and fibrillarin accumulated to the same phase dense structures while other phase dense structures contain only fibrillarin or C23 (Panels D and d). Similar to ratjadone treated cells, LMB treated cells transitioned from M to G1 phase also contained different types of phase dense structures. The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

184 A a

Phase

B b

C23

C c

Fibrillarin

D d

Merge

Figure 3.9: Localization of C23 and Fibrillarin in CHO Mitotic Cells

Released into LMB

185

Figure 3.10: Localization of C23 and Fibrillarin in CHO Control Cells

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without drugs for 12 hours prior to fixing and staining of the cells. Phase contrast views of mitotic cells released

into fresh media showed many irregular shaped nucleolus (Panels A and a). C23 appeared to accumulate to the irregular shaped nucleolus forming the same shape as the nucleolus observed in the phase contrast (Panels B and b). Fibrillarin also localized to the nucleolus and exhibited punctuate staining pattern, where some areas of the nucleolus were stained brightly with fibrillarin (Panels C and c). Overlay of the two immunostaining showed that both C23 and fibrillarin colocalized to the nucleolus

(Panels D and d). The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

186 A a

Phase

B b

C23

C c

Fibrillarin

d

Merge

Figure 3.10: Localization of C23 and Fibrillarin in Control Cells

187 Figure 3.11: Localization of C23 and B23 in CHO Mitotic Cells Released into

Actinomycin D

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in media containing Actinomycin D for 12 hours. Treatment with actinomcyin D produced two types of phase dense structures and nucleolar staining. In type one, round phase dense structures was observed in the phase

contrast (Panels A and a). C23 immunostaining accumulated to the phase dense

structures and throughout the nucleoplasm (Panels B and b). B23 was distributed

throughout the nucleus (Panels C and c). Overlay of the two immunostaining showed

that the localization of C23 and B23 in the nucleoplasm overlap while C23 accumulated

to the phase dense structures more pronounced than B23 in the same area (Panels D and

d). The second type of cells contained phase dense structures with a more phase dense

cap-like structures adjacent to it (Panels E and e). C23 appeared to localize to the round

phase dense structures, the cap-like structures, and throughout the nucleoplasm (Panels F

and f). B23 was distributed throughout the nucleoplasm, but not in the round phase

dense structures (Panels G and g). Furthermore, B23 appeared to accumulate to the cap-

like structures (Panel G; arrow in the expanded view). Overlay of the two staining

showed that both B23 and C23 colocalized to the cap-like structures while, only C23

appeared in the round phase dense structures (Panels H and h; arrow point to the cap-

like structures in the expanded nucleolus view). The red squares in panels A-H

illustrate the expanded nucleus shown in panels a-h. The red square in panels e-h

represent the expanded view of the nucleolus. The size marker in the phase contrast

equals 10µm.

188 f e g h F E G H Mitotic Cells Released into Actinomycin D B23 C23 Phase Merge c a d b B D C A Figure 3.11: Localization of C23 and B23 in CHO B23 C23 Phase Merge

189 Figure 3.12: Localization of Fibrillarin and B23 in CHO Mitotic Cells Released into

Actinomycin D

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in media containing actinomycin D for 12 hours. Treatment with actinomycin D produced two types of phase dense structures and nucleolar staining. In type one, round phase dense structures was observed in the phase

contrast (Panels A and a). Fibrillarin immunostaining accumulated to the phase dense

structures and throughout the nucleoplasm (Panels B and b). B23 was distributed

throughout the nucleus (Panels C and c). Overlay of the two immunostaining showed

that the localization of fibrillarin and B23 in the nucleoplasm overlap while fibrillarin

accumulated to the phase dense structures more pronounced than B23 in the same area

(Panels D and d). The second type of cells contained phase dense structures with a more phase dense cap-like structures adjacent to it (Panels E and e). Fibrillarin appeared to localize to the round phase dense structures, throughout the nucleoplasm, and not at the cap-like structures (Panels F and f; arrow in the expanded nucleolus view). B23 was distributed throughout the nucleoplasm, and to the cap-like structures, but not in the round phase dense structures (Panels G and g; arrow in the expanded nucleolus view).

Overlay of the two staining showed only fibrillarin appeared in the round phase dense structures, while B23 localized to the cap-like structures (Panels H and h; arrow point to the cap-like structures in the expanded nucleolus view). The red squares in panels

A-H illustrate the expanded nucleus shown in panels a-h. The red square in panels e-h represent the expanded view of the nucleolus. The size marker in the phase contrast equals 10µm.

190

191 Figure 3.13: Localization of C23 and Fibrillarin in CHO Mitotic Cells Released into

Actinomycin D

Mitotic cells were collected by mitotic shake off and washed free of nocodazole and plated onto coverslips in media containing actinomycin D for 12 hours. Treatment with actinomcyin D produced two types of phase dense structures and nucleolar staining. In the first type, round phase dense structures was observed in the phase contrast (Panels A and a). C23 immunostaining accumulated to numerous phase dense structures and throughout the nucleoplasm (Panels B and b). Fibrillarin also localized to phase dense structures (Panels C and c). Overlay image showed that C23 and fibrillarin localized to the same phase dense structures or to different phase dense structures (Panels D and d).

The second type of cells contained phase dense structures with a more phase dense cap- like structures adjacent to it (Panels E and e). C23 appeared to localize to the round phase dense structures, the cap-like structures, and throughout the nucleoplasm (Panels F and f; arrow point to cap-like structure). Fibrillarin was accumulated to the phase dense structures, but not in the cap-like structures (Panels G and g; arrow point to the cap-like structure). Overlay of the two staining showed that both C23 and fibrillarin colocalized to the round phase dense structures while, only C23 appeared in the cap-like structures (Panels H and h; arrow point to the cap-like structures). Closer examination of the nucleolus showed that fibrillarin localized more completely than C23 to the round phase dense structures, thus creating red color ring opposite end of the cap- like structures (Panel h: pink arrow). The red squares in panels A-H is the expanded nucleus shown in panels a-h. The red square in panels e-h is the expanded view of the nucleolus. The size marker in the phase contrast equals 10µm.

192

f e g h F E G H Released into Actinomycin D Released C23 Phase Merge Fibrillarin a b d c Fibrillarin in CHO Mitotic Cells B C A D Figure 3.13: Localization of C23 and C23 Phase Merge Fibrillarin

193

Figure 3.14: Localization of C23 and B23 in CHO Mitotic Cells Released into

Roscovitine

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed free of nocodazole and plated onto coverslips in media containing roscovitine for 2 hours prior to fixing and staining of the cells. Phase contrast views of mitotic cells released into roscovitine showed that nucleus appeared swollen with unexpanded cytoplasm

(Panels A and a). Immunostaining of C23 appeared to have some accumulation to structures that were not observable in the phase contrast, as well as localization to the nucleoplasm (Panels B and b). B23 appeared to be distributed throughout the nucleoplasm (Panels C and c). Overlay of the two immunostaining showed that both

C23 and B23 colocalized to the nucleoplasm, while some C23 accumulated more pronounced than B23 in the nucleus (Panels D and d). The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

194 A a

Phase

B b

C23

C c

B23

D d

Merge

Figure 3.14: Localization of C23 and B23 in CHO Mitotic Cells Released into

Roscovitine

195

Figure 3.15: Localization of Fibrillarin and B23 in CHO Mitotic Cells Released into

Roscovitine

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed free of nocodazole and plated onto coverslips in media containing roscovitine for 2 hours prior to fixing and staining of the cells. Phase contrast views of mitotic cells released into roscovitine showed that nucleus appeared swollen with unexpanded cytoplasm

(Panels A and a). Fibrillarin immunostaining appeared to have some accumulation to structures that were not observable in the phase contrast, as well as localization to the nucleoplasm (Panels B and b). B23 appeared to be distributed throughout the nucleoplasm with some accumulation (Panels C and c). Overlay of the two immunostaining showed that both fibrillarin and B23 colocalized to the nucleoplasm

(Panels D and d). The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

196 A a

Phase

B b

Fibrillarin

C c

B23

D d

Merge

Figure 3.15: Localization of Fibrillarin and B23 in CHO Mitotic Cells Released

into Roscovitine

197

Figure 3.16: Localization of C23 and Fibrillarin in CHO Mitotic Cells Released into

Roscovitine

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in media containing roscovitine for 2 hours

prior to fixing and staining of the cells. Phase contrast views of mitotic cells released

into roscovitine showed that nucleus appeared swollen with unexpanded cytoplasm

(Panels A and a). Immunostaining of C23 appeared to have some accumulation to

structures that were not observable in the phase contrast, as well as localization to the

nucleoplasm (Panels B and b). Fibrillarin appeared to be distributed throughout the

nucleoplasm and some accumulation was also observed (Panels C and c). Overlay of

the two immunostaining showed that the accumulation observed in C23 and fibrillarin

individual staining were colocalized to each other (Panels D and d). The red squares in

panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the

phase contrast equals 10µm.

198 A a

Phase

B b

C23

C c

Fibrillarin

D d

Merge

Figure 3.16: Localization of C23 and Fibrillarin in CHO Mitotic Cells Released

into Roscovitine

199

Figure 3.17: Localization of C23 and B23 in CHO Cells Released from Mitosis 2

Hours Prior to the Addition of LMB

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without drugs for 2 hours prior to the addition of LMB. Phase contrast views showed that the nucleus contained 4-

5 irregular shaped nucleolus (Panels A and a). C23 immunostaining appeared to

accumulate to the irregular shaped nucleolus (Panels B and b). B23 also localized to the

nucleolus forming the same nucleolar shape (Panels C and c). Overlay of the two immunostaining showed that both C23 and B23 colocalized to the nucleolus (Panels D and d). These data indicate that the 2 hour delayed LMB treatment in cells exiting mitosis had no effect on the nucleolus. The red squares in panels A-D illustrate the

expanded nucleus shown in panels a-d. The size marker in the phase contrast equals

10µm.

200 A a

Phase

B b

C23

C c

B23

D d

Merge

Figure 3.17: Localization of C23 and B23 in CHO Released from

Mitosis 2 Hours Prior to the addition of LMB

201

Figure 3.18: Localization of Fibrillarin and B23 in CHO Mitotic Cells Released from Mitosis 2 Hours Prior to the Addition of LMB

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated on to coverslips in fresh media without drugs for 2 hours prior to the addition of LMB. Phase contrast views showed that the nucleus contained various numbers of irregular shaped nucleolus (Panels A and a). Fibrillarin

immunostaining appeared to accumulate to the irregular shaped nucleolus with punctuate

localization, where some areas of the nucleolus was brightly stained with fibrillarin and

other areas were not (Panels B and b). B23 was observed to localize to the nucleolus

forming the same nucleolar shape (Panels C and c). Overlay of the two immunostaining

showed that both fibrillarin and B23 colocalized to the nucleolus, with some areas of the

nucleolus brightly stained with fibrillarin (Panels D and d). The 2 hour delayed

treatment of LMB in cells released from mitosis appeared to have no effects on the

localization of fibrillarin and B23. The red squares in panels A-D illustrate the expanded

nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

202 A A

Phase

B b

Fibrillarin

C c

B23

D d

Merge

Figure 3.18: Localization of Fibrillarin and B23 in CHO Released

from Mitosis 2 Hours Prior to the addition of LMB

203

Figure 3.19: Localization of C23 and Fibrillarin in CHO Mitotic Cells Released

from Mitosis 2 Hours Prior to the Addition of LMB

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without drugs for 2 hours prior to the addition of LMB. Phase contrast views showed that the nucleus contained various number of irregular shaped nucleolus (Panels A and a). C23 immunostaining appeared to accumulate to the irregular shaped nucleolus (Panels B and b). Fibrillarin was also localized to the nucleolus exhibiting similar staining pattern as that of C23

(Panels C and c). Overlay of the two immunostaining showed that both C23 and fibrillarin colocalized to the nucleolus (Panels D and d). These results indicate that

LMB had no effect on the localization of C23 and fibrillarin. The red squares in panels

A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

204

205

Figure 3.20: Localization of C23 and B23 in CHO Mitotic Cells Released From

Mitosis for 2 Hours Prior to the Addition of Ratjadone

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without drugs for 2 hours prior to the addition of ratjadone. Phase contrast views showed that the nucleus

contained various number of irregular shaped nucleolus similar to the delayed ratjadone

treated cells (Panels A and a). C23 immunostaining appeared to accumulate to the irregular shaped nucleolus (Panels B and b). B23 also localized to the nucleolus

forming the same nucleolar shape (Panels C and c). Overlay of the two immunostaining

showed that both C23 and B23 colocalized to the nucleolus (Panels D and d). These

results suggest that similar to delayed LMB treated cells, ratjadone has no effects on the

localization of C23 and B23 nucleolar proteins, thus no effect on the nucleolar structures.

The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The

size marker in the phase contrast equals 10µm.

206 A a

Phase

B b

C23

C c

B23

D d

Merge

Figure 3.20: Localization of B23 and C23 in CHO Mitotic Cells Released

from Mitosis for 2 Hours Prior to the Addition of Ratjadone

207

Figure 3.21: Localization of Fibrillarin and B23 in CHO Mitotic Cells Released from Mitosis for 2 Hours Prior to the Addition of Ratjadone

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without drugs for 2 hours prior to the addition of ratjadone. Phase contrast views showed that the nucleus

contained various number of irregular shaped nucleolus similar to the delayed ratjadone

treated cells (Panels A and a). Fibrillarin immunostaining accumulated to the irregular shaped nucleolus (Panels B and b). Similar to the 2 hour delayed LMB treated cells, fibrillarin appeared to have a punctate staining. B23 was also observed to localize to the nucleolus, forming the same nucleolar shape (Panels C and c). Overlay of the two immunostaining showed that both fibrillarin and B23 colocalized to the nucleolus, with some areas of the nucleolus stained more with fibrillarin (Panels D and d). The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

208 A a

Phase

B b

Fibrillarin

C c

B23

D d

Merge

Figure 3.21: Localization of Fibrillarin and B23 in CHO Mitotic Cells

Released from Mitosis for 2 Hours Prior to the Addition of Ratjadone

209

Figure 3.22: Localization of C23 and Fibrillarin in CHO Mitotic Cells Released

from Mitosis for 2 Hours Prior to the Addition of Ratjadone

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without drugs for 2 hours prior to the addition of ratjadone. Phase contrast views showed that the nucleus

contained various number of irregular shaped nucleolus similar to the delayed LMB

treated cells (Panels A and a). Similar to the delayed LMB treated cells and the control cells, C23 immunostaining appeared to accumulate to the irregular shaped nucleolus

(Panels B and b). In addition, fibrillarin also localized to the nucleolus (Panels C and

c). Fibrillarin immunostaining appeared to be punctate, where some areas of the

nucleolus were brightly stained with fibrillarin and other areas were not. Overlay of the

two immunostaining showed that both C23 and fibrillarin colocalized to the nucleolus

with areas of bright fibrillarin staining (Panels D and d). zxThe red squares in panels A-

D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase

contrast equals 10µm.

210 A a

Phase

B b

C23

C c

Fibrillarin

D d

Merge

Figure 3.22: Localization of C23 and Fibrillarin in CHO Mitotic Cells

Released from Mitosis for 2 Hours Prior to the Addition of Ratjadone

211

Figure 3.23: Localization of C23 and B23 in CHO Mitotic Cells Released from

Mitosis for 2 Hours Prior to the Addition of Actinomycin D

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without drugs for 2 hours prior to the addition of actinomycin D. Phase contrast views showed that the nucleus contained numerous small round phase dense structures with and without the more phase dense cap-like structures, similar to the mitotic cells released into actinomycin D (Panels

A and a; arrow indicate cap-like structures). C23 immunostaining appeared to accumulate to the round phase dense structures and the cap-like structures (Panels B and b; arrow indicate cap-like structures). B23 was also observed to localize to the more phase dense cap-like structures as well as distributed evenly throughout the nucleoplasm

(Panels C and c; arrow indicate cap-like structures). Overlay of the two immunostaining showed that both C23 and B23 colocalized to the cap-like structures but only C23 was found in the round phase dense strucutres (Panels D and d; arrow indicate cap-like structures). These results are different than the 2 hour delayed LMB treated or ratjadone treated cells. The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

212 A a

Phase

B b

C23

C c

B23

D d

Merge

Figure 3.23: Localization of C23 and B23 in CHO Mitotic Cells Released

from Mitosis for 2 Hours Prior to the Addition of Actinomycin D

213

Figure 3.24: Localization of Fibrillarin and B23 in CHO Mitotic Cells Released from Mitosis for 2 Hours Prior to the Addition of Actinomycin D

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed free of nocodazole and plated onto coverslips in fresh media without drugs for 2 hours prior to the addition of actinomycin D. Phase contrast views showed that the nucleus contained numerous small round phase dense structures with and without the more phase dense cap-like structures, similar to the mitotic cells released into actinomycin D (Panels

A and a; arrow indicate cap-like structures). Fibrillarin immunostaining appeared to accumulate to the round phase dense structures and throughout the nucleoplasm, but not to the cap-like structures (Panels B and b; arrow indicate cap-like structures). B23 was also observed to localize to the more phase dense cap-like structures as well as distributed evenly throughout the nucleoplasm (Panels C and c; arrow indicate cap-like structures). Overlay of the two immunostaining showed that only B23 localized to the cap-like structures while fibrillarin was found in the round phase dense structures; the two staining did not overlap (Panels D and d; arrow indicate cap-like structures). The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

214 A a

Phase

B b

Fibrillarin

C c

B23

D d

Merge

Figure 3.24: Localization of Fibrillarin and B23 in CHO Mitotic Cells

Released from Mitosis for 2 Hours Prior to the Addition of Actinomycin D

215

Figure 3.25: Localization of C23 and Fibrillarin in CHO Mitotic Cells Released

from Mitosis for 2 Hours Prior to the Addition of Actinomycin D

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without drugs for 2 hours prior to the addition of actinomycin D. Phase contrast views showed that the nucleus contained numerous small round phase dense structures with and without the more phase dense cap-like structures, similar to the mitotic cells released into actinomycin D (Panels

A and a; arrow indicate cap-like structures). C23 immunostaining appeared to accumulate to the round phase dense structures, the cap-like structures, and throughout the nucleoplasm (Panels B and b; arrow indicate cap-like structures). Fibrillarin localized to the round phase dense structures as well as in the nucleoplasm but not in the cap-like structures (Panels C and c; arrow indicate cap-like structures). Overlay of the two immunostaining showed that both C23 and fibrillarin localized to the round phase dense structures, however, only C23 localized to the cap-like structures (Panels D and d; arrow indicate cap-like structures). Furthermore, the overlay image showed that while C23 accumulated to the cap-like structures, fibrillarin appeared to localize more completely to the round phase dense structures than C23 at the opposite end of cap-like structures (Panel d; pink arrow). Thus, the round phase dense structures appeared to be composed of two structures; one with both C23 and fibrillarin and the other one with only fibrillarin. The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

216 A a

Phase

B b

C23

C c

Fibrillarin

D d

Merge

Figure 3.25: Localization of C23 and Fibrillarin in CHO Mitotic Cells

Released from Mitosis for 2 Hours Prior to the Addition of Actinomycin D

217

Figure 3.26: Localization of C23 and B23 in CHO Mitotic Cells Released from

Mitosis for 2 Hours Prior to the Addition of Roscovitine

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without drugs for 2 hours prior to the addition of roscovitine. Phase contrast views showed that the nucleus appeared to be swollen with some appearance of the phase dense structures (Panels A and a). Furthermore, the cytoplasm does not appear to be fully expanded. C23 immunostaining appeared to accumulate to the phase dense structures as well as to the nucleoplasm (Panels B and b). Immunostaining of B23 exhibited a uniform distribution to the nucleoplasm with some observable accumulation to the phase dense structures

(Panels C and c). Overlay of the two immunostaining showed that B23 did not localize completely to the phase dense structures as C23 (Panels D and d). These results do not resemble the delayed LMB and the delayed ratjadone treated cells. The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

218 A a

Phase

B b

C23

C c

B23

D d

Merge

Figure 3.26: Localization of C23 and B23 in CHO Mitotic Cells Released

from Mitosis for 2 Hours Prior to the Addition of Roscovitine

219

Figure 3.27: Localization of Fibrillarin and B23 in CHO Mitotic Cells Released from Mitosis 2 Hours Prior to the Addition of Roscovitine

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed free of nocodazole and plated onto coverslips in fresh media without drugs for 2 hours prior to the addition of roscovitine. Phase contrast views showed that the nucleus appeared to be swollen with the observance of the phase dense structures (Panels A and a). Furthermore, the cytoplasm does not appear to be fully expanded. Fibrillarin immunostaining appeared to accumulate to the phase dense structures as well as to the nucleoplasm (Panels B and b). Immunostaining of B23 exhibited a distributed localization to the nucleoplasm that contained some observable accumulation to the phase dense structures (Panels C and c). Overlay of the two immunostaining showed that B23 did not localize completely to the phase dense structures as C23 (Panels D and d).

These results do not resemble the delayed LMB and the delayed ratjadone treated cells.

The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

220 A a

Phase

B b

Fibrillarin

C c

B23

D d

Merge

Figure 3.27: Localization of Fibrillarin and B23 in CHO Mitotic Cells

Released from Mitosis for 2 Hours Prior to the Addition of Roscovitine

221

Figure 3.28: Localization of C23 and Fibrillarin in CHO Mitotic Cells Released

from Mitosis 2 Hours Prior to the Addition of Roscovitine

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in fresh media without drugs for 2 hours prior to the addition of roscovitine. Phase contrast views showed that the nucleus appeared to be swollen with some appearance of the phase dense structures (Panels A and a). Furthermore, the cytoplasm does not appear to be fully expanded. C23 immunostaining appeared to accumulate to the phase dense structures as well as to the nucleoplasm (Panels B and b). Immunostaining of fibrillarin exhibited an accumulation to the phase dense structures as well as localized to the nucleoplasm (Panels C and c).

Overlay of the two immunostaining showed that the localization of C23 and fibrillarin

overlapped (Panels D and d). In addition, both C23 and fibrillarin also appeared to

accumulate in different structures near the phase dense structures. These results do not

resemble the delayed LMB and the delayed ratjadone treated cells. The red squares in

panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the

phase contrast equals 10µm.

222 A a

Phase

B b

C23

C c

Fibrillarin

D d

Merge

Figure 3.28: Localization of C23 and Fibrillarin in CHO Mitotic Cells

Released from Mitosis for 2 Hours Prior to the Addition of Roscovitine

223

Figure 3.29: Localization of C23 and B23 in CHO Interphase Cells Treated with

LMB

Asynchronized population of the cells were treated with LMB for 12 hours prior to the fixation and the staining. In the phase contrast views, the cells contained various number of irregular shaped nucleolus (Panels A and a). Immunostaining of C23 appeared to accumulate to the irregular shaped nucleolus (Panels B and b). In addition, B23 was also observed to localize to the nucleolus (Panels C and c). Overlay of the two immunostaining showed that both C23 and B23 colocalized to the nucleolus (Panels D and d). These results suggested that LMB had no effect on the localization of the nucleolar proteins, thus, no effect on the nucleolar structure. The red squares in panels

A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

224 A a

Phase

B b

C23

C c

B23

D d

Merge

Figure 3.29: Localization of C23 and B23 in CHO Interphase Cells Treated

with LMB

225

Figure 3.30: Localization of Fibrillarin and B23 in CHO Interphase Cells Treated with LMB

Asynchronized population of the cells were treated with LMB for 12 hours prior to the fixation and the staining. In the phase contrast views, the cells contained various number

of irregular shaped nucleolus, similar to the nucleolus observed in the control cells

(Panels A and a). Immunostaining of fibrillarin appeared to accumulate to the irregular

shaped nucleolus (Panels B and b). Furthermore, fibrillarin staining appeared to have a

punctate pattern, where some areas of the nucleolus were brightly stained with fibrillarin

and other areas were not. B23 was also localized to the nucleolus (Panels C and c).

Overlay of the two immunostaining showed that both fibrillarin and B23 colocalized to

the nucleolus (Panels D and d). However, the overlay showed fibrillarin stained in a

punctate pattern to that of B23. These results suggested that LMB had no effect on the

localization of the nucleolar proteins, thus, no effect on the nucleolar structure. The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

226 A a

Phase

B b

Fibrillarin

C c

B23

D d

Merge

Figure 3.30: Localization of Fibrillarin and B23 in CHO Interphase Cells

Treated with LMB

227

Figure 3.31: Localization of C23 and Fibrillarin in CHO Interphase Cells Treated

with LMB

Asynchronized population of the cells were treated with LMB for 12 hours prior to the fixation and the staining. In the phase contrast views, the cells contained various number

of irregular shaped nucleolus, similar to the nucleolus observed in the control cells

(Panels A and a). Immunostaining of C23 exhibited localization to the nucleolus,

(Panels B and b). Immunostaining of fibrillarin also appeared to accumulate to the

irregular shaped nucleolus forming the same shape as the nucleolus observed in the phase

contrast (Panels C and c). However, fibrillarin staining appeared to have a punctate

pattern, where some areas of the nucleolus were brightly stained with fibrillarin and other

areas were not. Overlay of the two immunostaining showed that both C23 and fibrillarin

colocalized to the nucleolus (Panels D and d). However, the overlay showed fibrillarin

stained in a punctate pattern to that of C23. These observations are similar to the

immunostaining of the control cells, thus suggesting that LMB had no effect on the

localization of the nucleolar proteins; no effect on the nucleolar structure. The red

squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size

marker in the phase contrast equals 10µm.

228 A a

Phase

B b

C23

C c

Fibrillarin

D d

Merge

Figure 3.31: Localization of C23 and Fibrillarin in CHO Interphase Cells

Treated with LMB

229

Figure 3.32: Localization of C23 and B23 in CHO Interphase Cells Treated with

Ratjadone

Asynchronized population of the cells were treated with ratjadone for 12 hours prior to the fixation and the staining. In the phase contrast views, the cells contained various number of irregular shaped nucleolus, similar to the nucleolus observed in the control and in the LMB treated interphase cells (Panels A and a). Immunostaining of C23 appeared to localize to the nucleolus (Panels B and b). B23 was also localized to the nucleolus

(Panels C and c). Overlay of the two immunostaining showed that both C23 and B23 colocalized to the nucleolus (Panels D and d). The nucleolar protein staining in asynchronized population of cells treated with ratjadone appeared to be similar to the nucleolar protein localization in the control cells. This suggested that ratjadone had no effect on the localization of the nucleolar proteins, thus, no effect on the nucleolar structure. The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

230 A a

Phase

B b

C23

C c

B23

D d

Merge

Figure 3.32: Localization of C23 and B23 in CHO Interphase Cells Treated

with Ratjadone

231

Figure 3.33: Localization of B23 and Fibrillarin in CHO Interphase Cells Treated

with Ratjadone

Asynchronized population of the cells were treated with ratjadone for 12 hours prior to the fixation and the staining. In the phase contrast views, the cells contained various number of irregular shaped nucleolus, similar to the nucleolus observed in the control and in the LMB treated interphase cells (Panels A and a). Immunostaining of fibrillarin appeared to localize to the nucleolus (Panels B and b). Furthermore, fibrillarin appeared to stain in a punctate pattern. B23 was also localized to the nucleolus (Panels C and c).

Overlay of the two immunostaining showed that both fibrillarin and B23 colocalized to

the nucleolus (Panels D and d). In addition, fibrillarin localization appeared to be

punctate in pattern against B23 localization. The nucleolar protein staining in

asynchronized population of cells treated with ratjadone appeared to be similar to the

nucleolar protein localization in the control cells. This suggested that ratjadone had no

effect on the localization of the nucleolar proteins, thus, no effect on the nucleolar

structure. The red squares in panels A-D illustrate the expanded nucleus shown in panels

a-d. The size marker in the phase contrast equals 10µm.

232 A a

Phase

B b

Fibrillarin

C c

B23

D d

Merge

Figure 3.33: Localization of B23 and Fibrillarin in CHO Interphase Cells

Treated with Ratjadone

233

Figure 3.34: Localization of C23 and Fibrillarin in CHO Interphase Cells Treated

with Ratjadone

Asynchronized population of the cells were treated with ratjadone for 12 hours prior to the fixation and the staining. In the phase contrast views, the cells contained various number of irregular shaped nucleolus, similar to the nucleolus observed in the control and in the LMB treated interphase cells (Panels A and a). C23 protein was observed to localize to the nucleolus (Panels B and b). Immunostaining of fibrillarin appeared to localize to the nucleolus (Panels C and c). Furthermore, fibrillarin appeared to stain in a punctate pattern. Overlay of the two immunostaining showed that both C23 and fibrillarin colocalized to the nucleolus (Panels D and d). In addition, fibrillarin localization appeared to be punctate in pattern against C23 localization. The nucleolar protein staining in asynchronized population of cells treated with ratjadone appeared to

be similar to the nucleolar protein staining in the control cells. This suggested that

ratjadone had no effect on the localization of the nucleolar proteins, thus, no effect on the

nucleolar structure. The red squares in panels A-D illustrate the expanded nucleus shown

in panels a-d. The size marker in the phase contrast equals 10µm.

234 A a

Phase

B b

C23

C c

Fibrillarin

D d

Merge

Figure 3.34: Localization of C23 and Fibrillarin in CHO Interphase Cells

Treated with Ratjadone

235 Figure 3.35: Localization of C23 and B23 in CHO Interphase Cells Treated with

Actinomycin D

Asynchronized population of CHO cells were treated with actinomycin D for 12 hours prior to the fixation. Treatment with actinomcyin D produced two types of cells with different nucleolar staining, similar to the mitotic and the 2 hour delayed cells treated with actinomycin D. In the first type, round phase dense structures was observed in the phase contrast (Panels A and a). C23 immunostaining accumulated to the phase dense structures and throughout the nucleoplasm (Panels B and b). B23 was also distributed throughout the nucleus (Panels C and c). Overlay of the two immunostaining showed that the localization of C23 and B23 in the nucleoplasm overlap while C23 accumulated to the phase dense structures more pronounced than B23 in the same area (Panels D and d). The second type of cells contained phase dense structures with a more phase dense cap-like structures adjacent to it (Panels E and e). C23 appeared to localize to the round phase dense structures, the cap-like structures, and throughout the nucleoplasm (Panels F and f). B23 was distributed throughout the nucleoplasm, but not in the round phase dense structures (Panels G and g). Furthermore, B23 appeared to accumulate to the cap- like structures (Panel G; arrow in the expanded view). Overlay of the two staining showed that both B23 and C23 colocalized to the cap-like structures (forming a yellow color stain) while, only C23 appeared in the round phase dense structures (Panels H and h; arrow point to the cap-like structures in the expanded nucleolus view). The red squares in panels A-H illustrate the expanded nucleus shown in panels a-h. The red square in panels e-h represent the expanded view of the nucleolus. The size marker in the phase contrast equals 10µm.

236 f g e h cin D y E F G H hase Cells Treated with Actinom p B23 C23 Phase Merge c a b d B C D A ure 3.35: Localization of C23 and B23 in CHO Inter g Fi B23 C23 Phase Merge

237 Figure 3.36: Localization of Fibrillarin and B23 in CHO Interphase Cells Treated with Actinomycin D

Asynchronized population of CHO cells were treated with actinomycin D for 12 hours

prior to the fixation. Treatment with actinomcyin D also produced two types of cells with different nucleolar staining, similar to the mitotic and the 2 hour delayed cells treated with actinomycin D. In the first type, round phase dense structures was observed in the phase contrast (Panels A and a). Fibrillarin immunostaining accumulated to the phase dense structures and throughout the nucleoplasm (Panels B and b). B23 was distributed throughout the nucleus (Panels C and c). Overlay of the two immunostaining showed that the localization of fibrillarin and B23 in the nucleoplasm overlap while fibrillarin accumulated to the phase dense structures more pronounced than B23 in the same area

(Panels D and d). The second type of cells contained phase dense structures with a more phase dense cap-like structures adjacent to it (Panels E and e). Fibrillarin appeared to localize to the round phase dense structures, the cap-like structures, and throughout the nucleoplasm (Panels F and f). B23 was distributed throughout the nucleoplasm, but not in the round phase dense structures (Panels G and g). Furthermore, B23 appeared to accumulate to the cap-like structures (Panel G; arrow in the expanded view). Overlay of the two staining showed that both B23 and fibrillarin colocalized to the cap-like structures (forming a yellow color stain) while, only fibrillarin appeared in the round phase dense structures (Panels H and h; arrow point to the cap-like structures in the expanded nucleolus view). The red squares in panels A-H illustrate the expanded nucleus shown in panels a-h. The red square in panels e-h represent the expanded view of the nucleolus. The size marker in the phase contrast equals 10µm.

238 f e g h F E H G ed with Actinomycin D B23 C23 Phase Merge CHO Interphase Cells Treat c a b d A B C D Figure 3.36: Localization of Fibrillarin and B23 in B23 Phase Merge Fibrillarin

239 Figure 3.37: Localization of C23 and Fibrillarin in CHO Interphase Cells Treated

with Actinomycin D

Asynchronized population of CHO cells were treated with actinomycin D for 12 hours

prior to the fixation. Treatment with actinomcyin D also produced two types of cells with different nucleolar staining, similar to the mitotic and the 2 hour delayed cells treated with actinomycin D. In the first type, round phase dense structures was observed in the phase contrast (Panels A and a). Immunostaining of C23 exhibited localization to the round phase dense structures and throughout the nucleoplasm (Panels B and b).

Fibrillarin immunostaining also accumulated to the phase dense structures and throughout the nucleoplasm (Panels C and c). Overlay of the two immunostaining showed a yellow color staining to indicate colocalization of C23 and fibrillarin (Panels D and d). The second type of cells contained phase dense structures with a more phase dense cap-like structures adjacent to it (Panels E and e; arrow points to the cap-like structures). C23 appeared to localize to the round phase dense structures, the cap-like structures, and throughout the nucleoplasm (Panels F and f). Fibrillarin localized to the round phase dense structures and the nucleoplasm, but not to the cap-like structures (Panels G and g).

Overlay of the two staining showed that both C23 and fibrillarin colocalized to the round phase dense structures (yellow color stain), however, only C23 appeared in the cap-like structures, and fibrillarin accumulated more to the phase dense structures opposite of the cap-like structures (Panels H and h; arrow point to the cap-like structures). The red squares in panels A-H illustrate the expanded nucleus shown in panels a-h. The red square in panels e-h represent the expanded view of the nucleolus. The size marker in the phase contrast equals 10µm.

240 f e h g F E G H Cells Treated with Actinomycin D C23 Phase Merge Fibrillarin a c b d nd Fibrillarin in CHO Interphase A D B C Figure 3.37: Localization of C23 a C23 Phase Merge Fibrillarin

241

Figure 3.38: Localization of C23 and B23 in CHO Interphase Cells Treated with

Roscovitine

Asynchronized cells were treated with roscovitine for 2 hours. Cells were then fixed and stained with C23 and B23 antibodies. Phase contrast views showed that the nucleus appeared to be swollen with some appearance of the phase dense structures in the nucleus

(Panels A and a). C23 immunostaining was distributed throughout the nucleoplasm

(Panels B and b). Immunostaining of B23 exhibited a slight accumulation to the phase dense structures but majority of the protein localized throughout the nucleoplasm (Panels

C and c). Overlay of the two immunostaining showed that C23 and B23 colocalized to the nucleoplasm (Panels D and d). These results do not resemble the interphase cells treated with LMB or interphase cells treated with ratjadone. The red squares in panels A-

D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

242 A a

Phase

B b

C23

C c

B23

D d

Merge

Figure 3.38: Localization of C23 and B23 in CHO Interphase Cells Treated

with Roscovitine

243

Figure 3.39: Localization of Fibrillarin and B23 in CHO Interphase Cells Treated with Roscovitine

Asynchronized cells were treated with roscovitine for 2 hours. Cells were then fixed and

stained with fibrillarin and B23 antibodies. Phase contrast views showed that the nucleus

appeared to be swollen with some appearance of the phase dense structures in the nucleus

(Panels A and a). Fibrillarin immunostaining was distributed throughout the

nucleoplasm with distinct localization to the phase dense structures (Panels B and b).

Immunostaining of B23 exhibited a slight accumulation to the phase dense structures but

majority of the protein localized throughout the nucleoplasm (Panels C and c). Overlay

of the two immunostaining showed fibrillarin and B23 colocalized with each other, and

separately from each other (Panels D and d). These results do not resemble the

interphase cells treated with LMB or interphase cells treated with ratjadone. The red

squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size

marker in the phase contrast equals 10µm.

244 A a

Phase

B b

Fibrillarin

C c

B23

D d

Merge

Figure 3.39: Localization of Fibrillarin and B23 in CHO Interphase Cells

Treated with Roscovitine

245

Figure 3.40: Localization of C23 and Fibrillarin in CHO Interphase Cells Treated

with Roscovitine

Asynchronized cells were treated with roscovitine for 2 hours. Cells were then fixed and

stained with C23 and fibrillarin antibodies. Phase contrast views showed that the nucleus

appeared to be swollen with some appearance of the phase dense structures in the nucleus

(Panels A and a). C23 immunostaining was distributed throughout the nucleoplasm with

some accumulation observed (Panels B and b). Immunostaining of fibrillarin exhibited

numerous accumulations in the nucleus as well as localization to the nucleoplasm (Panels

C and c). Overlay of the two immunostaining showed that some of the C23

accumulations and fibrillarin accumulations colocalized in the nucleoplasm (Panels D

and d). These results do not resemble the interphase cells treated with LMB or

interphase cells treated with ratjadone. The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals

10µm.

246 A a

Phase

B b

C23

C c

Fibrillarin

D d

Merge

Figure 3.40: Localization of C23 and Fibrillarin in CHO Interphase Cells

Treated with Roscovitine

247

Figure 3.41: Localization of C23 and B23 in CHO LMB Reversibility Experiment

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated onto coverslips in media containing LMB for 8 hours prior to fixing and staining of the cells. Phase contrast showed various irregular shaped nucleolus in the nucleus, similar to control cells (Panels A and a). C23 was observed to localize to the nucleolus, forming the same shape as the nucleolus (Panels B and b).

B23 immunostaining also localized to the nucleolus (Panels C and c). A yellow color stain appeared in the overlay of C23 and B23 to indicate colocalization of the nucleolar proteins (Panels D and d). The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals 10µm.

248 A a

Phase

B b

C23

C c

B23

D d

Merge

Figure 3.41: Localization of C23 and B23 in CHO LMB Reversibility

Experiment

249

Figure 3.42: Localization of Fibrillarin and B23 in CHO LMB Reversibility

Experiment

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated on to coverslips in media containing LMB for 8 hours prior to fixing and staining of the cells. Phase contrast showed various irregular shaped nucleolus in the nucleus, similar to control cells (Panels A and a). Fibrillarin was observed to localize to the nucleolus, forming the same shape as the nucleolus (Panels B and b). B23 immunostaining also localized to the nucleolus (Panels C and c). A yellow color stain appeared in the overlay of fibrillarin and B23 to indicate colocalization of the nucleolar proteins (Panels D and d). The red squares in panels A-D illustrate the expanded nucleus shown in panels a-d. The size marker in the phase contrast equals

10µm.

250 A a

Phase

B b

Fibrillarin

C c

B23

D d

Merge

Figure 3.42: Localization of Fibrillarin and B23 in CHO LMB Reversibility

Experiment

251

Figure 3.43: Localization of C23 and Fibrillarin in CHO LMB Reversibility

Experiment

Nocodazole synchronized mitotic cells were collected by mitotic shake off and washed

free of nocodazole and plated on to coverslips in media containing LMB for 8 hours prior to fixing and staining of the cells. Phase contrast showed various irregular shaped nucleolus in the nucleus, similar to control cells (Panels A and a). C23 was observed to localize to the nucleolus, forming the same shape as the nucleolus (Panels B and b).

Fibrillarin immunostaining also localized to the nucleolus (Panels C and c). A yellow

color stain appeared in the overlay of C23 and fibrillarin to indicate colocalization of the

nucleolar proteins (Panels D and d). The red squares in panels A-D illustrate the

expanded nucleus shown in panels a-d. The size marker in the phase contrast equals

10µm.

252 A a

Phase

B b

C23

C c

Fibrillarin

D d

Merge

Figure 3.43: Localization of Fibrillarin and C23 in CHO LMB Reversibility

Experiment

253 SUMMARY

The results presented in this thesis have shown that recompartmentalization of proteins after mitosis is a vital cellular process that is required for cells to progress through G1 phase. During mitosis, the chromosomes condense, the nucleolus disassembles, and the nuclear envelope breaks down to allow cell division to occur. This result in the mixing of proteins normally segregated within the nuclear and cytoplasmic compartments. As cells exit mitosis, the nuclear envelope is reformed, and nuclear transport is reestablished, which allows for selective movement of proteins in and out of the nucleus. In the absence of nuclear transport immediately following mitosis, daughter cells failed to decondense chromosomes and remained in a telophase-like condition

(Benavente et al. 1987; Benavente et al. 1989; Benavente et al. 1989). This indicates that

active nuclear transport is critical for cells to completely exit mitosis and enter G1 phase.

However, in these studies, both nuclear import and export was inhibited in the cells

transitioning from M to G1 phase. In our studies, we solely focused on the effects of

inhibition of nuclear export. To carry out these studies, we used LMB, which is an agent

that inhibits CRM1-mediated nuclear export by competing with the NES of its cargo for

the binding site on CRM1 (Nishi et al. 1994; Kudo et al. 1998; Kudo et al. 1999; Kutay

and Guttinger 2005).

254 The results presented in chapter 1 illustrated that inhibition of CRM1-mediated

nuclear export immediately after mitosis resulted in the induction of apoptosis. Mitotic

cells were arrested in nocodazole and released into fresh media or LMB containing

media. Both control cells and cells treated with LMB exited mitosis at the same rate, thus

indicating that LMB had no effect on the ability of cells to exit mitosis. However, apoptosis was observed after the mitotic cells that were released into LMB had

transitioned into interphase. In contrast, cells that were released from mitosis for 2 hours prior to LMB treatment, or interphase cells within logarithmically growing cultures treated with LMB, had little or no apoptotic response after 8 hours of LMB treatment.

These data demonstrate that functional CRM1-mediated nuclear export is necessary to recompartmentalize proteins immediately after mitosis; thus preventing the induction of

apoptosis.

In the second chapter, immunostaining of three nucleolar protein markers showed

that LMB prevented the reassociation of the nucleolar proteins at the nucleolus,

indicating inhibition of nucleologenesis. Again, inhibition of nucleologenesis was only observed in cells treated during mitotic exit, and not in interphase cells or cells released from mitosis for 2 hours prior to the addition of the drug. I have used Venn diagrams to illustrate the relationship between the localization of these nucleolar proteins. This analysis shows that there is a close association between all three nucleolar proteins in the control cells, the 2 hour-delay LMB treated samples, and in the interphase LMB-treated samples, but only a limited association in the MT-LMB treated samples (Diagrams 16,

17, and 18). This correlated with the results from chapter one; induction of apoptosis occurred only in the absence of the nucleolus when LMB was applied to cells

255 transitioning from M to G1 phase, but not in interphase cells or 2 hour delayed LMB treated cells where the nucleolus remained intact. As represented in Diagram 16, the nucleolar immunostaining in mitotic cells treated with LMB exhibited a different pattern of protein localization than nontreated cells: B23 was distributed throughout the nucleoplasm, while both C23 and fibrillarin were found localized to small intranuclear phase dense structures. While a significant amount of C23 and fibrillarin staining colocalized to the same phase dense structures, separate staining also defined at least two different types of phase dense structures that stained exclusively for fibrillarin or C23

(Diagram 17 and 18). In contrast, interphase cells and cells treated with LMB 2 hours after the M to G1 transition showed no effect on the localization of nucleolar proteins.

All three nucleolar proteins in those samples were observed to colocalize to the nucleolus in these cells, however, there were distinct labeling of subregions within the nucleolus.

The results presented in chapter 2 illustrated that inhibition of CRM1-mediated nuclear export during the M to G1 transition resulted in the disruption of nucleologenesis.

Numerous agents, such as ratjadone, Actinomycin D, and roscovitine, are also known to cause disruption of nucleolar structure and function. The data presented in chapter 3 demonstrated that nucleolar disruption by LMB is a specific effect that occurred only during the M to G1 transition, and is a unique effect that was not observed with nucleolar inhibitors that have different mechanisms of action. Only ratjadone, an inhibitor of CRM1-mediated nuclear export, caused a similar effect on nucleolar structure as that observed in LMB treated cells. Immunolocalization of nucleolar proteins in ratjadone treated cells was similar to that of LMB treated cells transitioning from M to

G1 phase. In addition, ratjadone also had no effect in interphase cells or cells that had

256 transitioned from M to G1 phase for 2 hours prior to treatment with the drug. Thus, two different inhibitors of CRM1-mediated nuclear export exhibited similar effects on nucleolar structure and nucleologenesis in cells transitioning from M to G1 phase, but showed no effect on nucleolar structure in cells treated with these inhibitors after nucleologenesis was completed. Therefore, CRM1-mediated recompartmentalization of proteins is necessary immediately after mitosis, but is not required for maintaining interphase nucleoli.

The four nucleolar inhibitors, LMB, ratjadone, actinomycin D, and roscovitine all disrupted nucleologenesis in cells released from mitosis, however, the similarities end there. First, cells transitioning from M to G1 phase treated with actinomycin D and roscovitine had different patterns of nucleolar protein localization than those observed in

LMB and ratjadone treated cells (Summarized in Diagram 16). Next, both actinomycin

D and roscovitine also affected the nucleolar structure in interphase cells, including cells that had been released from mitosis for only 2 hours, while LMB and ratjadone had no effect on these same cells (Summarized in Diagram 17). In addition, actinomycin D and roscovitine treatment caused a similar reorganization of nucleolar proteins in interphase cells as that observed in mitotic cells released into the inhibitors (Summarized in

Diagrams 16 and 18). Therefore, both transcription and pre-rRNA processing, the activities that actinomycin D and roscovitine inhibit respectively, are required to maintain interphase structure and function of interphase nucleoli. CRM1-mediated nuclear export, however, is not required to maintain the nucleolar structure as demonstrated by the presence of intact nucleoli in interphase cells and the cells treated with either LMB or ratjadone after a 2 hour delay. Thus, the differences in nucleolar protein localization

257 observed in actinomycin D and roscovitine treated cells further demonstrate that the

effects of LMB are specific, and unlike those of other types of nucleolar inhibitors.

The data presented in this dissertation illustrates the importance of nuclear export,

specifically the CRM1-mediated nuclear export, immediately after mitotic release.

Export of proteins out of the nucleus after mitosis allows the cells to move cytoplasmic

proteins that may have become trapped inside the nucleus after the reformation of the

nuclear envelope back into the cytoplasm. In the presence of LMB or ratjadone, CRM1-

mediated nuclear export is inhibited, which led to a failure of nucleologenesis and the

subsequent induction of apoptosis. We speculate that after the reformation of the nuclear envelope, a nucleologenesis licensing factor(s) is trapped inside the nucleus. After exit from mitosis, there is a requirement to shuttle this factor out of the nucleus to allow nucleolar proteins to reassociate with each other to form the nucleolus (Diagram 19).

However, in the presence of LMB or ratjadone, export of the factor(s) is inhibited. The trapped licensing factor(s) may interact with nucleolar proteins, and prevent the proper association with one another, which interrupts the temporal sequence of nucleolar protein

recruitment around the NOR (Diagram 20). This would explain the observations we have

made showing that C23 and fibrillarin accumulate to different phase dense intranuclear

structures, while B23 is distributed throughout the nucleoplasm in cells treated with LMB

during M to G1 transition.

The nucleolus is a dynamic structure composed of different proteins many of

which exchange continuously and rapidly with the surrounding nucleoplasm. The three

subdomains (or tripartite nucleolus; FC, DFC, and GC) of the nucleolus observed in

mammalian cells are not typically found in all eukaryotic cells. In fact, the typical

258 tripartite nucleolus appeared in amniotes and is only found in organisms higher in the evolutionary tree (Diagram 21; green shade). This indicates that the acquisition of the nucleolus as a three compartment structure is a relatively recent evolutionary event. Most eukaryotic organisms, such as yeast Saccharomyces cerevisiae, contains only two nucleolar domains (or a bipartite nucleolus), the fibrillar component and the granular component (Thiry and Lafontaine 2005)(Diagram 21; pink shade). The fibrillar component observed in S. cerevisiae is a reminiscent of the mammalian fibrillar center

(FC), which contains rDNA, however, it lacks the transcriptional machineries and the pre-rRNAs that are typically found in mammalian FCs (Leger-Silvestre et al. 1999).

Furthermore, in their report, they also noted that the intergenic spacer of the rDNA gene in the species that contain tripartite nucleolus are longer than the rDNA transcription unit

(Leger-Silvestre et al. 1999). This is opposite of the species that contain a bipartite nucleolus where the transcription unit of the rDNA is longer or about the same in size as the intergenic spacer (Leger-Silvestre et al. 1999). These results led to the speculation that the increase in the rDNA intergenic region in species containing a tripartite nucleolus allowed for a physical separation of ribosome biosynthesis functions that were initially carried out in one compartment in the yeast. The acquisition of the third nucleolar compartments may indicate the complexity of the evolved cell regulation in higher eukaryotes. Regardless of the tripartite structural of the nucleolus, all eukaryotic organisms have a nucleolus. Thus like the nucleus, the nucleolus is an organelle that represents one of the fundamental differences between eukaryotic and prokaryotic organisms. All metazoans undergo an open mitosis that allows mixing of the nuclear and cytoplamsic components. These compartments must be restored at the end of mitosis as

259 we have shown that inhibitor of this process leads to apoptosis. As such,

recompartmentalization at the end of mitosis (at least in those organism with an open

mitosis), is a fundamental process that might be under cell cycle checkpoint regulation.

There are several studies that have shown a close relationship between p53 and

the nucleolus. In LMB treated cells it was shown that p53 is stabilized (Freedman and

Levine 1998). Since nuclear export of the p53-Mdm2 complex is dependent on the

CRM1-mediated export pathway, in the presence of LMB, accumulation of this complex in the nucleus is expected. This would lead to the stabilization of p53. Accumulation of

p53 within the nucleus arrests cells in G1 phase, and turns on DNA repair factors. If the

damage is too severe, p53 would induce apoptosis. Nucleolar proteins, such as B23 and

C23 have also been shown to translocate from the nucleolus to the nucleoplasm and interact with p53 in cells that were exposed to ionizing radiation, UV irradiation, or other

drugs (Colombo et al. 2002; Daniely et al. 2002). The loss of the nucleolus allowed for

the interaction between p53 with C23 and/or B23 in the nucleoplasm, thus stabilizing p53

and eventually leading to the induction of apoptosis. In addition, p53 is also involved in the repression of RNA Polymerase I transcription (Zhai and Comai 2000). p53 binds to

SL1 and prevents SL1 interaction with TBP, thus inhibiting the formation of the transcription initiation complex. Finally, a recent report suggested that an intact and functional nucleolus is required for maintaining p53 at a minimal level (Rubbi and Milner

2003). Nucleolar disruption by radiation or drugs would thus, induce higher p53 levels.

In the absence of intact nucleoli, p53 would then activate downstream genes leading to

p53-mediated cell cycle arrest or apoptosis. Therefore, stabilization of p53 could explain

260 the induction of apoptosis in cells that failed to reform the nucleolus after being treated

with LMB in cells transitioning from M to G1 phase.

The tumor suppressor protein p53 is involved in many biological functions,

including cell cycle control, genome stability, DNA repair, apoptosis, and senescence.

Furthermore, p53 is also involved in the DNA damage checkpoint during the G1/S transition. The induction of apoptosis in cells treated with LMB during the M to G1

transition, along with studies associating p53 with the nucleolus, led us to speculate that p53 may be involved in a mitotic exit checkpoint. Cell cycle checkpoints are regulatory

pathways that ensure the initiation of a later event is coupled to the completion of an

early cell cycle event. In addition, checkpoints respond to damage by arresting cells at a

certain point in the cell cycle to allow for repair and/or induce apoptosis if the damage is

unrepairable. Failure of the checkpoints may result in the loss of genomic stability and

contribute to uncontrollable cell division. Thus, the absence of checkpoints has been

implicated in the transformation of normal cells into cancer cells. The level of p53 protein may act as a checkpoint sensor that responds to the presence of an intact nucleolus. Failure of nucleologenesis would trigger the checkpoint, and if unrepaired, the lack of a functional nucleolus would trigger apoptosis. Further studies are required to elucidate the levels of p53 and proteins that mediate the p53 levels in cells after being treated with LMB during the M to G1 transition.

Cell cycle regulation is often maintained by exporting proteins out of the nucleus after each cell cycle for proteosomal degradation. The transition of proteins in and out of the nucleus after each event is similar to the shift changes in factories, where a group of people install car doors during shift 1 and another group adds color to the doors in shift 2.

261 These events are regulated specifically by the types of proteins (or people) found in each

of the cell cycle phases (or shift hours). For instance, cyclin B shuttles continuously into

and out of the nucleus until it is needed to initiate mitosis. The constant export of cyclin

B by CRM1-mediated nuclear export prevents the premature initiation of M phase.

Inhibition of CRM1-mediated nuclear export by LMB, however, results in the

accumulation of CRM1-mediated proteins, which includes the nucleologenesis licensing

factor(s). Identification of the nucleologenesis licensing factor(s) that results in the

inhibition of nucleologenesis immediately after mitosis is required to understand the

mechanisms of nucleologenesis regulation.

This dissertation has presented data that demonstrates the importance of cellular

recompartmentalization after mitosis. Inhibition of CRM1-mediated nuclear export

immediately after mitosis by LMB likely prevents trapped cytoplasmic proteins from

being recompartmentalized. As a result, cells failed to reform the nucleolus, which

eventually leads to the induction of apoptosis. This is a specific effect only observed by

inhibiting CRM1-mediated nuclear export by LMB, which is different than other

nucleolar inhibitors. In addition, our data suggests that the presence of a nucleologenesis licensing factor(s) within the nucleus prevented nucleolar proteins from associating with

one another to form an intact nucleolus. The identification of the nucleologenesis

licensing factor(s) could provide further insight into the molecular mechanism of cells

transitioning from M to G1 phase. The possibility of p53 involvement in the M to G1

checkpoint is very intriguing. Many anticancer drugs are now being designed to target

proteins that are involved in cell cycle regulation, such as Cdks and p53 pathways. Thus,

the identification of a new checkpoint could provide another target for cancer

262 therapeutics. Similarly, the identification of the nucleologenesis licensing factor(s) could also provide another target for cancer therapy. Either way, the findings presented in this dissertation may be useful not only in the understanding of cell cycle regulation, but also in the area of cancer treatment.

263 264 265 266 267 268

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