THE KINETICS OF G2 AND M TRANSITIONS REGULATED BY B CYCLINS
by
YEHONG HUANG
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Advisor: Dr. James W. Jacobberger
Department of Molecular Biology and Microbiology
CASE WESTERN RESERVE UNIVERSITY
January 2014
CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of Yehong Huang, Candidate for the Doctor of Philosophy degree*.
Dr. James W Jacobberger, Ph.D. Thesis Advisor and Committee Member
Dr. Jonathan Karn, Ph.D. Committee Chair
Dr. Mark W Jackson, Ph.D. Committee Member
Dr. You-Wei, Ph.D. Committee Member
Date of Defense: November 7th, 2013
*We also certify that written approval has been obtained for any proprietary material contained therein.
2 TABLE OF CONTENTS
LIST OF TABLES 3
LIST OF FIGURES 9
LIST OF TABLES 12
LIST OF ABBREVIATIONS 13
ACKNOWLEGEMENTS 16
ABSTRACT 18
CHAPTER1: INTRODUCTION & BACKGROUND 20
1.1 INTRODUCTION TO CELL CYCLE 20
1.1.1 Concept of cell cycle 20
1.1.2 Events of cell cycle 21
1.1.3 The duration of cell cycle 22
1.1.4 Role of cell cycle in tumor formation 22
1.2 CONTROL OF CELL CYCLE 23
1.2.1 The cell-cycle control system 23
1.2.2 Cell cycle Checkpoints 24
1.2.3 Key regulators of cell cycle 25
1.2.4 Two alternative models of regulation of cell cycle by Cdk 28
1.3 REGULATION OF MITOSIS 29
1.3.1 History of discovery of protein kinase complex regulating mitosis 29
1.3.2 Components of MPF 30
1.3.2.1 P34 Cdc2 (Cdk1) 30
3 1.3.2.2 Mitotic cyclins 32
1.3.2.3 B- type cyclins 34
1.3.3 Regulation of M phase entry and exit 38
1.3.3.1 Regulation of M phase entry 38
1.3.3.2 Nuclear translocalization of cyclin B1-Cdk1 of mitotic
entry 41
1.3.3.3 Regulation of M phase exit 41
1.3.3.4 Mitotic Checkpoints 43
1.4 STAGES OF MITOSIS 44
1.4.1 Morphologic stages of mitosis 44
1.4.2 Re-staging mitosis 47
1.5 FUNCTIONS OF CYCLIN B 50
1.5.1 Function of cyclin B-Cdk1 during mitosis 50
1.5.2 Rate-limiting function of cyclin B 51
1.5.3 Functional redundancy of B cyclins 52
1.6 HYPOTHESIS AND SPECIFIC AIMS OF THESIS WORK 53
CHAPTER 2: STABLE CELL LINES THAT EXPRESS
CYCLIN B1- OR B2-EGFP FUSION PROTEINS 56
2.1 ABSTRACT 56
2.2 INTRODUCTION 57
4 2.3 MATERIALS AND METHODS 59
2.3.1 Plasmids and construction of Tet-Off response plasmids 59
2.3.2 Oligonucleotides of siRNA specific to cyclin B1, B2 and A2, and
construction of siRNA-resistant constructs 63
2.3.3 Cell culture, transfection and generation of stable cell lines 64
2.3.4 Cell fixation, intracellular staining 64
2.3.5 Fluorescence activated cell sorting and flow cytometry 65
2.3.6 Immunoblotting analysis 66
2.3.7 Microscopy imaging 66
2.4 RESULTS 67
2.4.1 Construction of Tet-Off response plamsids containing human
cyclin B1-, B2- or A2 –EGFP gene that is resistant to siRNA and
specific for cyclin B1, B2 or A2 67
2.4.2 Establishment stable cell lines 68
2.4.3 Expression of cyclin B1-EGFP and cyclin B2-EGFP is
reduced and tightly regulated under the control of doxycycline 69
2.4.4 Effect of fixation on the intensity of B cyclin EGFP fluorescence 70
2.4.5 Induction of cyclin B1- and B2-EGFP is rapid and regulated
by doxycycline in a dose dependent manner 71
2.4.6 Cell line resistance of ectopic cyclin B1-EGFP and cyclin
B2-EGFP with synonymous base changes to siRNA 72
2.4.7 Ectopic B cyclin -EGFPs activate Cdk1-mediated Bcl-2
phosphorylation 73
5 2.5 DISCUSSION 74
CHAPTER 3: THE KINETICS OF G2 AND M TRANSITIONS
REGULATED BY B CYCLINS 77
3.1 ABSTRACT 77
3.2 INTRODUCTION 78
3.3 MATERIALS AND METHODS 81
3.3.1 List of stable cell lines used for experiments 81
3.3.2 Cell culture and transfection 81
3.3.3 Cell fixation, intracellular staining and flow cytometry 82
3.3.4 BrdU labeling, G2 and M phase transit time analysis 83
3.3.5 Time lapse experiments 83
3.3.6 Immunoblotting 84
3.3.7 Software 84
3.4 RESULTS 85
3.4.1 Characterization of stable cell lines in this study 85
3.4.2 Cyclin B2 is rate controlling for G2 and M transitions 85
3.4.3 Over-expression of B cyclins does not affect the rates of
G2 and M transition 87
3.4.4 Over-expression of single B cyclins can restore cell cycling
when endogenous B cyclins are depleted 88
3.4.5 Expression of single B cyclins can completely rescue the
G2 arrest phenotype caused by cyclin B1 and B2 co-depletion 88
6 3.5 DISCUSSION 91
CHAPTER 4: LOCALIZATION OF CYCLIN B1- OR B2-EGFP
WHEN LACK OF CYCLIN B2 OR B1 101
4.1 ABSTRACT 102
4.2 INTRODUCTION 105
4.3 MATERIALS AND METHODS 105
4.3.1 Cell culture 105
4.3.2 Plasmids, small interfering RNA (siRNA) constructs and
transfection 105
4.3.3 CellLight Golgi-RFP living cell transduction 106
4.3.4 Cell fixation and intracellular staining 107
4.3.5 Time lapse microscopy 108
4.3.6 Confocal microscopy 108
4.3.7 Laser scanning cytometry 108
4.3.8 Immunoblotting analysis 109
4.4 RESULTS 109
4.4.1 Cytoplasmic cyclin B1-EGFP concentrates on the
centrosomes during interphase and translocates to the
nucleus at beginning of mitosis 109
4.4.2 Cyclin B2-EGFP localizes to Golgi apparatus and centrosomes
in interphase and translocalizes to the nucleus at the beginning of
mitosis 112
7 4.4.3 Study the localization of cyclin B1 to the Golgi when cyclin B2
knockdown 115
4.5 DISCUSSION 117
CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS 121
5.1. SUMMARY 121
5.2. FUTURE DIRECTIONS 127
5.2.1 To study the effect of single cyclin B1 or B2 on Golgi disassembly 127
5.2.2 To explore the residues that regulate human cyclin B2 nuclear
localization 127
5.2.3 To identify distinct sequences in cyclin B2 that bind to the
chromosome, centrosomes, and spindle during mitosis 128
5.2.4 To determine when and where cyclin B2-Cdk1 is first activated 129
5.2.5 To study the role of B cyclin –Cdk1 in regulation of chromosome
segregation 129
5.2.6 To explore the co-operation and redundancies of cyclin A2
and B cyclins on G2 and M transition 130
8 LIST OF FIGURES
Figure 1-1: The phase of cell cycle and the checkpoint of cell cycle 132
Figure 1-2: Two models for the Cdk control of cell cycle 133
Figure 1-3: positive feedback loop that regulates cyclinB-Cdk1 activity 134
Figure 2-1: Schematic of gene regulation in the Tet-Off Systems 135
Figure 2-2: Schematic representation of Tet-response constructs containing
EGFP, cyclinB1-EGFP and cyclin B2-EGFP genes 136
Figure 2-3: Transient expression of the tet-response constructs containing
cyclin B1-or B2- and EGFP genes in HeLa Tet-Off cells in the
presence of siRNA 137
Figure 2-4: Detection of doxycycline regulated cyclinB1- or B2-EGFP
expression in stable cells under immunofluorescence microscopy 138
Figure2-5: Induction of cyclin B-EGFP in selected HeLa Tet-Off clones
in the absence of Doxycycline 139
Figure2-6: Effect of fixation and permeabilization on intensity of
cyclin B-EGFP expression in the stable cell lines 140
Figure 3-1: Expression of cyclin B1- and cyclin B2-EGFP is reduced and
tightly regulated by Dox 141
Figure 3-2: Time and concentration dependence of doxycycline action on
a Tet-Off stable cell line 142
Figure 3-3: Effect of doxycycline and siRNA on the ectopic cyclin B-EGFP
expressed by stable cells 143
Figure 3-4: Bcl2 is phosphorylated as a function of cyclin B1- and
9 B2-EGFP expression 144
Figure 3-5. G2 and M phase transitions after cyclin B2 knockdown 145
Figure 3- 6: Over-expression of B cyclins does not alter G2 and M
phase transition times 146
Figure 3-7: B cyclin-EGFP expression rescues the G2 arrest phenotype
of B cyclin co-depletion 147
Figure 3-8: B cyclin co-depletion induces G2 arrest 148
Figure 3-9: Single B cyclin-EGFP can rescue the G2 arrest of
endogenous cyclin B1 and B2 co-depletion 149
Figure 3-10: Reanalysis of the primary data of the experiments
presented in Figure 9 150
Figure 3-11: Single B cyclin EGFP completely rescues the G2 arrest
of cyclin co-depletion as a function of dose 151
Figure3-12: Two possible models of cyclin B1 and B2 co-operation to regulate
G2 and M transition and progression 152
Figure 4-1: localization of ectopic cyclinB1-EGFP expressed in
p015 through the cell cycle 153
Figure 4-2: Titrations to determine optional concentration of
doxycycline to obtain ectopic cyclinB1/B2-EGFP level equivalent
to endogenous cyclin B1/B2 in p015 and p017 stable cells 154
Figure 4-3: Localization of ectopic cyclin B1-EGFP when expressed
in a level equivalent the endogenous cyclin B1 in the absence of
endogenous cyclin B1 155
10 Figure 4-4: Localization of transient transfected EGFP protein in
HeLa Tet-Off cells during cell cycle 156
Figure 4-5: Localization of ectopic cyclin B2-EGFP to the Golgi
determined by confocal microscopy 157
Figure 4-6: Localization of ectopic cyclinB2-EGFP expressed in
p017 through the cell cycle 158
Figure 4-7: Localization of wild type of cyclin B2 to the tubulin
(centrosomes) and chromosome determined by indirect
immunofluorescence 159
Figure 4-8: Localization of wild type cyclin B1 to the Golgi
determined by indirect immunofluorescence 160
Figure 4-9: Determination of the intensity of cyclin on the Golgi and in the
whole cell by laser scanning cytometry 161
11
LIST OF TABLES
Table 3-1. Synonymous base changes 162
Table 3-2. G2 and M phase times; effect of cyclin B2 knockdown 162
12 LIST OF ABBREVIATIONS
A488: Alexa Fluor 488
A555: Alexa Fluor 555
A647: Alexa Fluor 647
Amp: ampicillin
Ampr: ampicillin resistance genes
APC/C: anaphase promoting complex / cyclostomes
ATM: ataxia telangiectasia mutated
ATR: ATM and Rad3-related protein kinase
BSA: bovine serum albumin
CAK: cyclin activating kinase
Cdc: cell division cycle
Cdk: cyclin-dependent protein kinase
CKIs: Cdk inhibitor proteins
Cis: cisternae
CRS: cytoplasmic retention sequence
CycB1: cyclin B1
CycB2: cyclin B2
DAPI: 6-diamidino-2-phenylindole
DIC: differential interference contrast
Dox: doxycycline
EGFP: enhanced green fluorescence protein
FACS: fluorescence activated cell sorting
13 FBS: fetal bovine serum (FBS),
GVBD: germinal vesicle breakdown
Hyg: hygromycin
r Hyg : hygromycin resistance genes
KD: knockdown
LSM: Laser Scanning Microscope
MCS: multiple cloning sites
NEBD: nuclear envelope breakdown
NES: nuclear export signal
MPF: maturation promoting factor
MTOC: major microtubule-organizing center
ORF: the cDNA open reading frames pBcl2: phospho Bcl2
PCR: polymerase chain reaction
PE: phycoerythrin pHH3: phospho histone H3
Plk: Polo like kinase
Pmin CMV: minimal promoter of cytomegalovirus
RNAi: RNA interference
PP2A: protein phosphates 2A
PVDF: polyvinylidene difluoride
SAC: spindle assemble checkpoint
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis
14 Tet: tetracycline tetO: the tetracycline operator sequence
TetR: the tetracycline repressor protein
TRE: the tetracycline response element tTA: tetracycline-controlled transactivator
7:AAD: 7-amino-actinomycin D
15 ACKNOWLEDGEMENTS
I would like to express my sincere appreciation to my advisor, Dr. Dr James W.
Jacobberger, for his guidance and support throughout my graduate study, and also for his constructive criticism on my paper and dissertation. I especially appreciate his encouragement whenever I encountered challenges in my study.
I would like to express my gratitude to my graduate committee members, Drs. Jonathan
Karn, Mark W. Jackson, You-Wei Zhang and James W. Jacobberger for their support, advice and valuable suggestions during my thesis study.
I would like to express my thanks to all my co-workers in Jacobberger’s lab, Dr. Phyllis
S. Frisa, Tammy Stefan, Dr. Philip Woost, Blain Tesfaye Fente, Nataliya Pyatka for their support and friendship and for making the lab a pleasant place to study and work.
Especially, I would like to thank Phyllis for her helpful suggestion and discussion to my experiments and critical reading of my thesis. I would like to thank Tammy for training me in laser scanning cytometry and for her first-hand data that I can always use for antibody staining
I would like to express my thanks to all members in the Case Comprehensive Cancer
Center's Cytometry and Imaging Microscopy Core Facility. I wish to thank Mike
Sramkoski for his support, advice and suggestion on my flow cytometry experiments and image experiments. I also wish to thank Jonida Toska for her support and help in my
16 experiments. Also, thanks to Dr. Judy Drazba and Dr. John W. Peterson of the Lerner
Institute Imaging Core Facility for their help with confocal microscopy.
I would like to express my thanks to the faculty, staff, post-doct, and graduate students in the Department of Molecular Biology and Microbiology, and the Case Comprehensive
Cancer Center for their kind help. I would like to thank Brinn Omabegho, Tracy Rehl and
William Jacobberger for their support and help in administration.
At last, I am deeply grateful to my parents, my sister and my brother for their continuous support and encouragement and for believing in me throughout my studies. In particular,
I would like appreciate my husband Chi. His love, support and encouragement have made it possible for me to pursue this degree.
17 The Kinetics of G2 and M Transitions Regulated by B Cyclins
Abstract
by
YEHONG HUANG
B cyclin / cyclin dependent kinase 1 (Cdk1) complexes are essential for mitotic entry and progression. Cyclin B1 and B2 are the mitotic B cyclins in mammalian somatic cells.
Cyclin B1 is an essential gene for mouse embryogeneisis, but cyclin B2 is dispensable.
However, RNA interference (RNAi) studies in human somatic cells show that either cyclin B1 or B2 can promote mitosis when 95-99% of cyclin B1 or B2 are depleted. In this setting, cyclin B1 and B2 compensate each other for the gross ability to transit M phase. In this study, cell lines with inducible expression of RNAi resistant cyclin B1- or
B2-EGFP fusion proteins were established using the BD Tet-Off gene expression system and site-directed mutagenesis. These fusion proteins were shown by fluorescence imaging and confocal laser scanning microscopy to localize intracellularly to the same locations as endogenous, wild-type cyclin B1 and B2. With these lines, I analyzed the
G2 and M transition times in the absence of endogenous cyclin B2 and provided substantial evidence that cyclin B1 and B2 are rate limiting for mitotic entry and progression, but that they act in opposition for mitotic timing. Cyclin B2 depletion decreases mitotic residence time, but cyclin B1 depletion results in an increased mitotic time. In co-depletion studies, either B cyclin, could rescue the 4C DNA content phenotype (G2 arrest). This ability was dose-dependent and with sufficient expression the phenotype could be reversed beyond that of wild type cyclins. Therefore, I provided
18 quantitative evidence to demonstrate that cyclin B1 and cyclin B2 are interchangeable for ability to promote G2 and M transition in this experimental setting. Based on is this study,
I propose two possible models for cyclin B1 and B2 co-operation to regulate G2 and M transition. The first model posits that cyclin B1 is not necessary and both cyclins can promote mitosis. The second model provides a mechanism whereby cyclin B1 is necessary. Overall, the second model explains most data.
19 CHAPTER1: INTRODUCTION & BACKGROUND
1.1 INTRODUCTION TO CELL CYCLE
1.1.1 Concept of cell cycle
“Omnis cellula e cellula” (“every cell stems from another cell”), this famous aphorism, proposed by the German pathologist Rudolf Virchow (1821-1902) as a third tenet of the cell theory, carried with an important message that every cell originates from another existing cell like it and to generate more cells by division of those that already exist.
Cell reproduction is the series of events that take place in a cell leading to duplication of its contents and division into two, known as cell cycle. By cell division, unicellular species produces a complete new organism, and multicellular species produce a functional organism. After an organism is fully grown, cell division continues to function in renewal and repair by replacing cells that die from normal wear/tear or accidents. In cells without a nucleus (prokaryotic), the cell cycle occurs via a process termed binary fission. In cells with a nucleus (eukaryotes), the cell cycle can be divided into two periods: interphase during which the cell grows, doubling its mass and accumulating proteins needed for mitosis and duplicating its DNA, and the mitotic (M) phase, during which the nucleus divides then the cell splits itself into two distinct cells, often called "daughter cells" in the final M sub-phase, cytokinesis, where the new cell is completely divided.
The fundamental task of cell division is to pass its genetic information to the next generation of cells: the DNA in each chromosome must first be faithfully replicated, and the replicated chromosomes must then be accurately segregated to the two daughter cells,
20 so that each receives an identical copy of the entire parental genome. Cell cycle underlies growth and development in all living organisms and is central to their heredity and evolution. Therefore, it is an important problem in biology to understand how the cell cycle operates and is controlled.
1.1.2 Events of cell cycle
Cell cycle consists of (Figure 1-1)
G1 = growth and preparation of the chromosomes for replication; S = synthesis of DNA and duplication of the centrosome; G2 = preparation for Mitosis M = mitosis.
The eukaryotic cell cycle is divided into several different phases [1,2], In somatic cells, the first gap phase “G1” is the interval between M phase and S phase. During G1, cell grows in preparation for DNA replication, and production of structural and functional protein. The gap provides time for the cell to monitor its internal and external environment to ensure the condition is suitable before entering the next stage. If extracellular conditions are unfavorable, cells delay progression through G1 and enter a resting state, marked by G0 phase. If extracellular conditions are favorable and signals are committed to divide further, cells progress through a commitment point near the end of G1 known as the restriction point in mammalian cells. After passing this point, cells enter the “Synthesis phase” (S phase) during which the DNA replication occurs. The G2 phase is the second gap phase occurring between the end of DNA replication in
S phase and the beginning of mitosis, during which mitochondria and chloroplasts divide, mitotic spindle begins to form, and the cell can check to make sure that the entirety of its DNA and other intracellular components have been properly duplicated. M
21 phase (M= mitosis + cytokinesis) contains the most visually dramatic events in the cell cycle. This includes nuclear division and distribution of organelles and cytoplasm equally to each daughter cell.
1.1.3 The duration of cell cycle
The duration of the cell cycle varies from organism to organism and from cell ty pe to cell type within organisms. Most of the differences in cell cycle duration between species and cells are found in the duration of specific cell cycle phases, such as G1 phase.
Generally, the simpler the organisms, the faster DNA will replicate. An example is prokaryotes which have smaller genomes and not as much DNA for replication. In order to rapidly multiply for the development of the embryo, the early embryonic cell cycles often skip G1 and G2 phase, and quickly proceed through between S phase and mitosis.
G1 is the first gap for new cells to grow after cell division. It is usually the longest phase of the cell cycle. In the typical dividing mammalian cell in tissue culture, cells remain in
G1 for about 10 to 12 hr, S phase lasts 5-6 hr. G2 is shorter, lasting only 3 to 4 hr, and mitosis about 1 to 2 hr [3,4].
1.1.4 Role of cell cycle in tumor formation
Cell cycle has implications for medicine, particularly cancer and immune function disorders where the controls of cell growth and division are defective. The highly organized and regulated cell cycle process is responsible for duplication of the cell. Tight regulation and timing ensure that DNA is replicated once during the S phase (without
22 errors), and that identical chromosomes are equally delivered to daughter cells during the
M phase [5,6]. The cell cycling process is carefully regulated and responds to the specific needs of a certain tissue or cell type. Normally, in adult tissue, there is a delicate balance between cell death (programmed cell death or apoptosis) and proliferation (cell division) producing a steady state. Disruption of this equilibrium by loss of cell cycle control may contribute to tumor development [5]. Uncontrolled cell proliferation is one of the main hallmarks of cancer, and tumor cells have acquired damage to genes that are directly involved in regulating the cell cycle [5]. Damage is caused by mutations producing an oncogene with a dominant gain of function, and/or by mutations in tumor suppressor genes causing a recessive loss of function [7,8]. Regardless of the genetic damage or type of cancer, the common feature is a disrupted cell cycle.
1.2 CONTROL OF CELL CYCLE
1.2.1 The cell-cycle control system
The cell cycle is a series of discrete events in eukaryotic cells. The most important events of the cell cycle are faithful replication of the genome and precise segregation of the daughter genomes into the daughter cells formed at division [9]. The cell-cycle control system triggers the major processes of the cell cycle to ensure: 1) all cell cycle events occur in the correct ordered and sequential progression from G1 to M phase, 2) each event is triggered only once per cycle and in a complete, irreversible fashion, and 3) the cell cycle works properly even if parts of the system malfunction [2]. Checkpoints or surveillance mechanisms and biochemically oscillating activity of cyclin-dependent protein kinases (Cdks) are two major principles of control of cell cycle progression.
23 1.2.2 Cell cycle Checkpoints
Cell cycle checkpoints are cellular surveillance pathways respond to damage or in conditions unfavorable for cell division by delaying the cell cycle progression to provide time for repair [10,11,12]. They must have a sensor to detect a mistake or damage and generate a signal through a transduction system and a response mechanism to achieve the correction[13].
There are several checkpoints during cell cycle (Figure 1-1). The G1 checkpoint is the first checkpoint and decides whether the cell should divide, delay division, or enter a resting stage (G0 phase). Contact inhibition, growth factor withdrawal, or DNA damage activates this checkpoint [12]. Upon DNA damage, the G1 checkpoint delays the cell cycle in the G1 phase by inhibiting the initiation of replication [14,15] and maintaining
G1 arrest [16,17,18,19]. The S phase checkpoint is activated by DNA damage, nucleotide depletion, or unrepaired damage that escapes the G1/S checkpoint and leads to block in
DNA synthesis [20,21,22]. The S-phase checkpoint response co-ordinates DNA replication, DNA repair and cell-cycle progression[23]. The G2 checkpoint is an important cell cycle checkpoint in eukaryotic organisms ranging from yeast to mammals.
It is triggered by unreplicated DNA or DNA damage and prevents the damaged cell from undergoing mitosis before damaged DNA is repaired by suppressing Mitosis Promoting
Factor (MPF) through inhibition of Cdc25 and maintenance of Wee1 activity [19]. The spindle assembly checkpoint is activated and prevents the onset of the anaphase if chromosomes are not properly attached to the mitotic spindle [24,25].
24 1.2.3 Key regulators of cell cycle
The Nobel Prize in Physiology or Medicine 2001 was awarded jointly to Leland H.
Hartwell, Tim Hunt and Sir Paul M. Nurse “ for their discoveries for key regulators of the cell cycle” [26,27,28]. Using Saccharomyces cerevisiae (budding yeast) as the model organism to study the cell cycle, Leland H. Hartwell identified more than 100 genes, named cell division cycle (Cdc) genes that involved in cell cycle control. One gene,
Cdc28 was proved to be essential for initiation of each cell cycle [29]. He also noticed that the cell cycle arrested after DNA damage [30]. Based on his observation, he introduced the concept of cell cycle checkpoint[11]. Paul Nurse studied the cell cycle using Schizosaccharomyces Pombe (fission yeast). He identified the Cdc gene in fission yeast [31] which is the same gene of Cdc28 in budding yeast [32] and Cdc2 in human
[33]. He discovered that Cdc2 had the kinase activity during cell cycle [34], which was confirmed earlier in Cdc28 [35]. It was demonstrated that the kinase activity of Cdc2 varied during the cell cycle [36] and was required for onset of both S phase and M phase
[31,37]. Tim Hunt worked with sea urchin eggs as a model organism. He discovered another protein that was periodically synthesized during the cell cycle [38]. He named this protein cyclin. Further, he discovered that this protein formed a complex with the proteins discovered by Drs. Hartwell and Nurse [39,40]. These fundamentally important discoveries indicated the some of the central molecules that determine the cell’s progress through the cell cycle are the Cdc2 kinases and their activating cyclins [41]. This level of cell cycle control was conserved in yeast and all higher organisms.
Cyclin-dependent kinases (Cdks)
25 The Cdks are essential regulators of cell cycle control. The Cdks are a family of serine/threonine protein kinases with small molecular weight (~30-40 KD). In yeast, a single essential Cdk, called Cdk1 (Cdc2 in budding yeast and Cdc28 in fission yeast) control all cell cycle events. In higher eukaryotes, of the twenty Cdks that have been identified in humans, only several Cdks are involved in the cell cycle [42,43]. Cdk1 and
Cdk2 are functionally homologous to yeast Cdc2/Cdc28 operating in S phase and M phase. Cdk4 and Cdk6 are required in response to the extracellular growth signal in regulating cells entry into the cell cycle and G1 phase progression [42,44]. Cdk function has been conserved during evolution. One early key experiments that showed this was that the human Cdc2 gene could fully replace the fission yeast Cdc2 gene for yeast cells to proliferate [33]. The cellular level of Cdk1 is in constant excess during the cell cycle, however, the activity varies depending primarily on corresponding oscillation of cyclin subunits levels [36,44]. Cdks have a two-lobed structure like other protein kinases. The crystal structure study of the human Cdk2 shows that there are two structure modifications that make the Cdk inactive in the absence of cyclins [45]. The one is the protein substrate binding site, which is blocked by an extended loop termed the T-loop, and the other is that ATP is poorly positioned for kinase reaction because of the incorrectly orientation of the side chain in the active site [42]. Cyclin binding initiates
Cdk activity by changing the conformation of the Cdk active site. Full Cdk activation requires phosphorylation of a threonine residue adjacent to the kinase active site catalyzed by Cdk-activating kinases (CAKs). There are three mechanisms to suppress
Cdk activity: Cdk inhibitor proteins (CKIs) bind to Cdks stoichiometrically and inhibit its activity by phosphorylation of a conserved threonine-tyrosine pair that alter the T-loop to
26 block the binding of the protein substrates, and control over cyclin destruction and synthesis [46].
Cyclins
Cyclins are activators of Cdks with the size range from 35 kDa to 90 kDa. Unlike the
Cdks, the concentration of most cyclins is low and oscillates during the cell cyclin [38].
The cyclins are expressed at undetectable levels to single peak levels during the cell cycle, generating a single oscillation of Cdk activity per cell cycle [27]. The regulation of concentration of cyclins is primary on the process of gene expression by transcription and proteolysis-mediated protein destruction [47]. The cyclin family can be divided into three main classes based on the timing of their expression and their function in the cell cycle [48,49,50]. The “G1 cyclins” include Cln1-3 in budding yeast [51,52,53], puc1 in fission yeast [54], and D and E cyclins in higher eukaryotes [41,42]. These proteins accumulate in G1 to regulate the G1 checkpoint, control origin complex loading, and trigger G1 to S phase progression. “S phase cyclins” are Clb5-6 in budding yeast, cig1 and cig2 in fission yeast, and E and A cyclins in higher eukaryotes. They are responsible for stimulating monitoring DNA replication. The “mitotic cyclins” are Clb1-4 in budding yeast, Cdc13 in fission yeast, and the A and B cyclins in higher eukaryotes. These cyclins control the G2 to M transition and mitotic progression. Cyclins are quite different in amino acid sequences, however, they possess a conserved 100-residue region known as the cyclin box, which is necessary for Cdk binding and activation [55,56]. Despite variation in their amino acid sequences, all cyclins have similar tertiary structure known as the cyclin fold containing two domains, one of which corresponds to cyclin box [42].
Outside of cyclin fold are N-terminal and C-terminal region. The length of N-terminus is
27 varied in each cyclin class. The N-terminus contains regulatory and targeting domains, such as the destruction box motif for proteolysis, that are specific to each cyclin
[44,57,58] .
1.2.4 Two alternative models of regulation of cell cycle by Cdk
Cell cycle progression is directed by cyclin-Cdk complexes [59]. The expression level of cyclin is regulated by oscillatory synthesis and degradation during the cell cycle. The concentration of the kinase is constant and at levels that are approximately 10 fold over cyclin expression [60]. Activated cyclin-Cdk complexes phosphorylate protein substrates to regulate subcellular events and drive the cell cycle progression [60]. Members of Cdk and cyclin families can form many different cyclin-CDK combinations. Numerous experiments to delete or replace individual cyclins have shown that different cyclin-Cdk complexes are responsible for state-specific cell cycle events [61,62,63,64]. For example, cyclin E-Cdk2 is important in S phase initiation and centrosome duplication, and B cyclin
/ Cdk1 complexes are master regulators, required for mitosis in mammalian cells. One of the cell cycle regulation models is termed the qualitative model (Figure 1-2A). According to this model, regulatory subunits of Cdks, the cyclins determines the biological specificity of cyclin-Cdk complex by controlling the substrate selection through altering the affinity of the Cdk for particular substrate, localizing to the subcellular position, or by directly binding to preferred substrates [65,66,67,68]. Thus, different cyclins expressing in the different stage of cell cycle help to promote the correct timing of CDK substrate phosphorylation that to assist to make ordered cell cycle progression occur [69].
However, a natural single cyclin B Cdk kinase complex in fission yeast as well as an experimental single B cyclin in budding yeast can promote largely normal cell cycles of
28 growth and division [70,71]. Based on this observation, a quantitative model for cell cycle was proposed [72]. In this model, different level of Cdk activity triggers sequential events of cell cycle: low to moderate level of kinase activity initiates S phase, and a further increase of activity to a high level initiates mitosis [72,73] (Figure 1-2B). In this model, it is proposed that the phosphorylation status of Cdk substrates is determined by the competition of Cdks and Cdk-counteracting phosphates, and that each substrate has different affinities for the kinases and phosphatases, therefore, a single cyclin-Cdk complex can regulate cell cycle progression with correct time [74,75]. These two models are not mutually exclusive and hybrid models have been proposed to explain key aspects for how cell division cycle includes an ordered series of events [74]
1.3 REGULATION OF MITOSIS
1.3.1 History of discovery of protein kinase complex regulating mitosis
In mitosis the genetic materials are faithfully transmitted to two daughter cells. In addition, each daughter cell receives a copy of an equal amount of cytoplasm and cytoplasmic organelles. In cell fusion experiments, fusion of M phase cells with G1, S or
G2 cells, induced inappropriate mitosis, suggesting that there is a diffusible factor that can promote M phase, and this factor could promote every stage of cell cycle [76]. This factor was first identified in extracts from frog eggs and was named maturation promoting factor (MPF). MPF was the cytoplasmic content in unfertilized frog eggs that could induce germinal vesicle breakdown (GVBD) and cause the oocyes to undertake meiosis (mature) when injected into immature oocytes [77,78]. A similar activity was also found in starfish extracts and proved to be active in the starfish oocye’s maturation
29 [79,80]. In the 1980s, cell-free systems established from frog eggs and Xenopus embryos were used to examine the oscillatory behavior of MPF activity in vitro [81,82]. MPF has been found both in cells spanning all eukaryotic kingdoms [83,84]. The function of MPF was demonstrated to be evolutionarily conserved based on the experiments that cytoplasm from egg of mouse was able to induce meiotic maturation of Xenopus laevis
[85] and starfish oocytes [86], and cytoplasm from mitotically active embryonic cells [87] and G2 phase mammalian tissue culture cells can induce meiotic maturation of Xenopus oocytes [88]. Eventually, MPF purified from Xenopus unfertilized eggs was found to be a protein kinase complex consisting of two subunits: a 45-kDa and a 32-kDa protein that acted as a histone H1 kinase [89]. Thus, MPF also named as “M phase promoting factor”.
1.3.2 Components of MPF
From starfish and Xenopus oocytes, with biochemical studies and gene sequencing, the two essential subunits of MPF complex were revealed as a B type cyclin and the Cdc2 kinase [90,91,92,93].
1.3.2.1 P34 Cdc2 (Cdk1)
In fission yeast, DNA sequence analysis and antisera identification indicated that MPF consisted of a 34 kDa protein kinase, a product of Cdc2 gene [34,94], the function and structure of which was equivalent to Cdc28 in budding yeast. In fission yeast, p34cdc2 was believed to be rate limiting for G2 progression by controlling the initiation of mitosis.
Gain-of–function mutants of this gene accelerated the onset of mitosis at a small cell size, thus shortening interphase time [95]. Loss-of-function mutants delayed mitotic onset and led either to cell cycle arrest or division at an increased cell size [31,95]. Thus, the timing
30 of mitosis was controlled by this gene. The protein sequence of the Cdc28 gene in budding yeast has 62% identity with Cdc2 in fission yeast [94]. The budding yeast
Cdc28 kinase was thought to play a role in G1 to S phase transition, but it also played a role in mitotic entry as its mutants were blocked at mitosis [96]. Furthermore, genetic and biochemical studies had demonstrated that Cdc28 protein kinase activity accumulated to high levels during the G2-M interval and functioned on mitotic entry, analogous to that of the Cdc2 protein kinase of fission yeast [97]. The human homologue of the Cdc2 gene was selected by complementation using a fission yeast Cdc2 mutant strain from at least
500 protein kinases in the human genome. The human Cdc2 gene encoded a 34K protein, and displayed 63% identity in sequence with the yeast gene [33]. Microinjection of anti- human P34cdc2 antibody into rat fibroblasts prevented cells from entering mitosis but had no effect on DNA replication. This implied that human P34cdc2 was important for regulating mitosis [98]. The homologue of Cdc2 gene also was identified in other species, such as clam, Drosophila, plants, and protists. This gene was functionally interchangeable between different species. The Cdc28 of budding yeast is able to rescue Cdc2 mutants and promote cell division [32]. The human Cdc2 gene can completely substitute for the yeast Cdc2 gene [33]. Antibodies raised against the fission yeast P34cdc2 can recognize the homologues in budding yeast and human cells [99], and also can detect a 32 kDa protein kinase from Xenopus eggs [90] and starfish oocytes [100,101]. Evidence from these experiments and other work strongly suggested that the Cdc2 gene was remarkably conserved in both structure and function to regulate mitosis from yeast to human, and in all other eukaryotes [102]. In 1990 and 1991, a number of different close relatives of
Cdc2 were discovered [103,104,105,106]. These kinases played different roles from Cdc2.
31 They were required for the initiation of S phase [107,108], and also bound cyclins. A new convention for naming the Cdc2 kinases was established: “kinases that are associated with cyclins would be called: cyclin-dependent kinases, or Cdks. And so
Cdc2 became Cdk1” [40]. In mammals, Cdk2 was a close relative that bound the E and
A cyclins in vivo; Cdk4 and Cdk6 bound the D cyclins, etc. See Table X for a list of
Cdks and their partner cyclins.
1.3.2.2 Mitotic cyclins
B cyclins are an essential component of MPF. Cyclins were initially identified in marine invertebrates as a class of proteins that accumulate at interphase of each cell cycle and destroyed at every mitosis [38]. Cyclins are involved in regulation of M-phase. The initial proof was that injection of clam cyclin A mRNA released Xenopus oocytes from
G2 arrest and induced meiotic M phase [109]. Later, Murray and Kirschner showed in cell-free systems that addition of exogenous Xenopus cyclin mRNA was able to induce multiple cell cycles in the absence of any other endogenous mRNA [110]. In the same system, entry into M phase was blocked if antisense oligonucleotides were used to reduce cyclin mRNA levels [111]. If all mRNAs were depleted with RNAase, M phase was blocked, but this block was reversed after addition of RNAase inhibitor and cyclin message [110]. These findings indicated that cyclin synthesis was required for M phase.
Co-immunoprecipitation experiments showed that cyclins could be associated with p34cdc2 in clam extracts, suggesting cyclins are a component of MPF [112]. The direct evidence came from an experiment sequencing the 47 kDs component of purified starfish
MPF which was found to be a B cyclin [93].
32
Cyclins are activators of p34cdc2. In fission yeast, Cdc13, whose sequence was identified as a B-type cyclin [113,114] was associated with p34cdc2 and required for entry into mitosis [114]. Cdc13 depletion caused low level of p34cdc2 kinase activity and cells were blocked prior to M phase [36]. In Xenopus, destruction of B cyclin mRNA prevented
MPF activity [110,111], and mutant Xenopus cyclin induced M entry in oocyte extracts, but meiosis was incomplete [47]. In humans, p34cdc2 became very active as a histone H1 kinase when associated with a 62 kDa protein [115], which was later identified as cyclin
B1 [116]. All these experiments suggested that B cyclin is an essential component of
MPF to activate p34cdc2.
There are two classes of mitotic cyclins: A- type and B-type, based on the difference of sequences, kinetics accumulation, intracellular distribution, and association with different forms of Cdc2 [117] [118,119,120]. Most organisms contain both types except for yeast.
A- type and B- type cyclins share about 30% identity in sequence in the central portion, which has been named “cyclin box” [56]. Cyclin box contains a conserved motif that can be used to classify A- type or B- type cyclin. Both the A- and B-type cyclins can induce
M phase. In vitro, A- and B-type cyclins are able to replace each other to activate p34cdc2
[121], but they are not functionally complement each other, since null mutation in the cyclin A gene resulted embryonic-lethal of Drosophila [122,123] and cyclin A2 depletion in mice was lethal [124] despite the expression of B cyclins. A type cyclins are able to form kinase complexes with two different kinase subunits: p33cdc2 (Cdk1) and p34cdc2 in somatic animal cells [125]. S phase progression [126] [127] and entry into mitosis [128] was blocked with anti-cyclin A antibody injections suggested that cyclin A played a role
33 at the initiation of both S phase and M phase. A and B type cyclins have different localizations in the cell. Cyclin A is localized in the nucleus but cyclin B remains in the cytoplasm until mitosis [129]. Cyclin A plays a role earlier in cell cycle and Cyclin B is the major mitotic cyclin that associates with p34cdc2. This conclusion came from evidence that histone H1 kinase activity associated with cyclin A appeared earlier and with less activity than that associated with B cyclin in the Xenopus [92,119], clams [112] and in human somatic cells [130].
1.3.2.3 B- type cyclins
Unlike A-type cyclin, which was discovered as a single functional gene in invertebrates or two genes in vertebrates, B-type cyclins were identified in multiple numbers in diverse organisms such as yeast, Xenopus, clams, mouse and human. The number of B-type cyclins varies in different organisms. Cyclin B1 and B2 are well characterized in vertebrates, while cyclin B3 is less well understood. In mammalians, cyclin B3 is distant from cyclin B1 and B2. Cyclin B3 is expressed in germ cells and localizes to the nucleus through the cell cycle which is able to is associated with both Cdc2 and Cdk2[131,132].
The RNA is expressed in testes and skeletal muscle [133]. While the chemistry and functional roles of most mitotic cyclins are well investigated, substrate specificity, and precise models are less well understood. In general, B-type cyclins co-express during S,
G2, and M in most proliferating cells, and all investigated higher organisms contain at least one mammalian cyclin B1-like gene and a cyclin B3 gene [134]. There are different numbers of B-type cyclins in different organisms probably as an adaption to their distinct developmental strategies. The phylogenetic tree for selected B-type cyclins is shown in[134]. The different B-type cyclins identified in organisms are briefly described below.
34
B-type cyclins in yeast
There are six B-type cyclins (Clb1 to Clb6) in the budding yeast that are functionally redundant [135]. These six B-type cyclins were grouped in three classes according to the similarity of the sequences and the time of expression. Clb1 and Clb2 encode very similar proteins which appear around nuclear division [136], whereas Clb3 and Clb4 encode a different pair of related proteins whose level rise much earlier, about the beginning of S phase in cell cycle [137]. Clb5 and Clb6 is another class of B-type cyclin in budding yeast that encode related proteins whose transcripts appear in late G1 phase having a specific role in S phase entry [138]. Disruption of any single Clb gene, the mutants were viable, whereas various multiple disruptions resulted in lethality, suggesting no single Clb was essential for cell cycle, and Clbs were redundant. Clb2 and
Clb5 seems to be more important genes, as Clb2 and Clb5 disruption caused mitotic delay or extend S phase, and all the lethal combinations of disruptions included the Clb2 gene. Mitotic spindle formation may require Cdc28 kinase activity. Multiple disruptions of Clb genes that resulted in defective mitosis produced cells that were arrested at G2 phase without a mitotic spindle. This finding suggested that Clb/Cdc28 kinase activity was necessary for spindle formation in yeast. Thus, the three classes of B-type cyclins in budding yeast are expressed at different stages of the cell cycle and have different and redundant function.
In fission yeast S. pombe, three B-type cyclins are discovered: Cdc13, Cig1 and Cig2.
Cdc13 is a mitotic B-type cyclin required for mitostic entry by activation of Cdc2
35 [113,139], rearrangement microtubules into a mitotic spindle in G2 [140], and promoting
Cdc2 nuclear translocation [139,141], and spindle pole bodies formation [142]. Cdc13 may be the only determinant to control the cell cycle by forming a complex with Cdc2 in
G2 to promote mitosis and prevent S phase [143]. Cig1 and Cig2 are closely related to
Clb3 and Clb4 of budding yeast and Cdc13. The role of Cig1 in the cell cycle is not clear.
Cig2 is expressed at the G1/S boundary of cell cycle and plays a role in S phase initiation
[144].
B-type cyclins in amphibians
Xenopus oocytes express five B-type cyclins genes (Cyclin B1-B5) were identified,
Cyclin genes B1, B2 and B3 were readily identifiable within human genome sequence, and cyclin B4 and B5 were unique to amphibians [134]. The larger number of B-type cyclins existent in Xenopus may be to adapt to the large size of eggs [145]. Ablation of cyclin B1 or B2 mRNA alone with antisense oligonucleotides in Xenopus oocytes did not block entry into mitosis, but, inhibition of synthesis of both cyclin B1 and B2 with antisense oligonucleotides prevented entry into mitosis, suggesting cyclin B1 and B2 were functional redundant for driving cells into mitosis [111]. Cyclin B4 and B5 are expressed during oocyte maturation and the early embryonic cell cycle [145]. Cyclin B4 is similar to cyclin B1, and cyclin B5 is similar to cyclin B2. Ablation of each B-type cyclin using specific antisense oligonucleotides did not cause obvious defects in oocytes maturation further indicating that there is significant functional overlap between these four B-type cyclins [146].
B-type cyclins in Caenorhabditis elegans
36 Four B-type cyclin genes were identified in C. elegans: Cyclin B (ZC168.4), cyclin B3
(To6E6.2), and other two additional genes: H31G24.4 and Y43E12A.1. The latter two genes have 87% identity as polypeptides. Both have same amino-acid exon and intron structure as the cyclin B gene, and H31G24.4 has 60% identity to cyclin B and
Y43E12A.1 has 65% identity at amino acid level. The sequence alignment shows the high degree conservation between the four B-type cyclins, particularly over the cyclin box. However, all four B-type cyclins differ in the size and sequence of their N-termini, which may determine cellular function. RNA interference of nematode cyclin B resulted in embryonic lethality. Because RNAi may not have distinguished the four B-type cyclins which are highly identical to each other and consequently the function of all four B-type cyclins were lost, the best interpretation of the RNAi phenotype possible is that at least one of these B-type cyclins is required [147].
B-type cyclins in Drosophila melanogaster
Genetic analysis illustrated there were only three cyclins genes in Drosophila: one A-type cyclin and two B-type cyclins, cyclin B and B3 [148,149,150]. These three cyclins are expressed in all mitotically proliferating cells; however, most work has been done on drosophila embryos. Mutational analysis suggested that cyclin A is the only required cyclin for mitosis [123]. Neither cyclin B nor cyclin B3 are essential for viability. In the absence of cyclin B, embryonic cells division were slower than wild type, and cyclin B3 mutant embryonic cells showed normal mitoses, however, flies lacking both cyclin B and cyclin B3 displayed embryonic lethality, suggesting cyclin B and cyclin B3 were at least partly functionally redundant. Double mutant and triple mutant analysis indicated that these three mitotic cyclin were to some extent functionally overlapping and co-operated
37 during mitosis in Drosophila [150]. Recent work shows that each of these cyclins is degraded at different times during mitosis and is hypothesized to time mitosis [151].
Further, these cyclins may time S phase[152].
B-type cyclins in mammalians
Three B-type cyclins were identified in mammals, cyclin B1, B2 and B3. Cyclin B1 and
B2 are mitotic cyclins. Cyclin B1 and B2 mutant mice displayed different phenotypes.
Cyclin B1 null mice die in utero, whereas cyclin B2 null mice develop normally, suggesting cyclin B2 was not essential, or cyclin B1 can compensate its function [153].
Cyclin B3 was in the nucleus and form a complex with Cdk2 and Cdk1 having a modest histone H1 kinase activity [154]. Endogenous cyclin B3 is hard to detect at the protein level in most cell types. It may be involved in the both male and female meiosis [132].
1.3.3 Regulation of M phase entry and exit
1.3.3.1 Regulation of M phase entry
Mitotic entry and exit is mediated by cyclin B-Cdk1 complex [155]. Activity of cyclin
B-Cdk1 complex is regulated by cyclin B, inhibitory kinases Wee1 and Myt1, and an activator Cdc25 phosphatase [156].
Cyclin B-Cdk1 gains activity when cyclin B binds Cdk1. At least in some systems, it times the cell cycle as it reaches threshold levels, however, the abrupt stimulation of high levels of activity that corresponds to mitotic entry require additional biochemistry [157].
Cyclin B levels oscillate during cell cycle by specific transcription and proteolysis mechanism. In mammals, cyclins B1 and B2 begin to accumulate during late G1 phase
38 and rise dramatically in late S and G2 phases [158,159], Cells enter mitosis at or near peak levels; levels remain high until metaphase when degradation begins [129,159,160].
Regulation of cyclin B transcription is facilitated by several promoter elements, co- activators and transcription factors [161]. During mitosis, the promoter of cyclin B1 maintains an open chromatin configuration and is occupied by the NF-Y transcriptional activator, thus cyclin B1 gene remains at a high rate of transcription during mitosis [162].
Binding of cyclin B to Cdk1 makes Thr161, a residue in the T-loop of Cdk1 more accessible to be phosphorylated by an activating kinase. Phosphorylation on Thr161 by cyclin activating kinase (CAK), which is composed of CDK7, cyclin H, and MAT1, is required for Cdk1 activation [163]. However, binding of cyclin B to Cdk1 does not sufficient to fully activate cyclin B-Cdk1 complex. Phosphorylation of Cdk1 at
Thr14/Tyr15 residues by Myt1 and Wee1 kinase keeps it inactivated [155]. Wee1 is soluble and predominantly nuclear and phosphorylates Cdk1 only at Tyr15 [164], while
Myt1 is bound to membrane structures of endoplasmic reticulum and Golgi apparatus and phosphorylates both Thr14 and Tyr15 of Cdk1, but prefers Thr14 [165]. In late G2 phase, Cdk1 is abruptly activated by dephosphorylation of Tyr15 and Thr14 by dual- action phosphatases of the Cdc25 family. The abruptly activation of the cyclin B-Cdk1 complex by Cdc25 drives cell entry into mitosis. Once active, cyclin B-Cdk1 complex can phosphorylate Myt1 and Wee1, thereby further amplifying Cdk1 activity. Cdk1 phosphorylates 25 in an activating reaction, which induces a positive feedback loop that is responsible for very rapid accumulation of B cyclin/Cdk1 activity [155] (Figure 1-3), but how this autocatalytic system is initiated remains to be elucidated. Cyclin A may play a role in cyclin B1-Cdk1 activation [166]. Vertebrate cells contain three Cdc25 isoforms:
39 Cdc25A, Cdc25B and Cdc25C. These isoforms shuttle between the nucleus and the cytoplasm and play significant roles in controlling G2/M phase transition [167].
Several kinases are involved in the regulation of CyclinB-Cdk1 auto-amplification loop
[168]. Polo like kinase 1 (Plk1) is believed to directly initiate the Cyclin B-Cdk1 autocatalytic reaction. Plk1 phosphorylates Cdc25C as well as Cdc25B and promotes accumulation of Cdc25C and Cdc25B in the nucleus. In addition, Plk1-dependent phosphorylation of Wee1 and Myt1 results in inhibition of Wee1 and Myt1 activity.
Thus, Plk1 strengthens and modulates cyclin B-Cdk1 autocatalysis through phosphorylation of Cdc25, Wee1 and Myt1. Aurora A is another kinase that activates
Cdc25 phosphatases at the centrosome, and also phosphorylates T210 residue of Plk1 which is required for Plk1 activation, and therefore, may initiate cyclin B –Cdk1 activity.
A kinase called PIM1 (proto-oncogene serine/Threonine-protein kinase 1) also can activate Cdc25 by direct phosphorylation or indirectly through C-TAK1 kinase (CDC twenty-five C-associated protein kinase 1). Cyclin A-Cdk1/2 may participate in the activation of cyclin B-Cdk1 through inactivation of Wee1. Other kinases that influence
Cyclin B-Cdk1 the auto-amplification loop including DNA damage checkpoint protein
CHK1, the NIMA-related kinase NEK2, and kinase-related protein Eg5, all of which inhibit Cdc25 activity. Recently, Greatwall kinase was found to be involved in regulation of mitotic entry and stabilization [169]. Greatwall promotes mitotic entry by negatively regulating a crucial phosphatase that inhibits Cdc25 activity. Greatwall itself is activated by Cyclin B-Cdk1 phosphorylation. Activated Greatwall inhibits protein phosphates 2A
(PP2A) activity by phosphorylating its substrate Endosulphine. PP2A works as
40 antagonist of Polo-like kinases by dephosphorylating phosphorylated Cdc25 and Wee1.
So, Greatwall maintains the mitotic state by inhibiting PP2A. In conclusion, Cyclin B-
Cdk1 activity is regulated by multiple feedback loops. Cdk1 substrates regulated Cdk1 and this creates positive-feedback loops that enhance cyclin B-Cdk1 activity at different levels to promotes mitotic entry and stabilize the mitotic states.
1.3.3.2 Nuclear translocalization of cyclin B1-Cdk1 of mitotic entry
Of the three mammalian B cyclins, only cyclin B1 and B2 are mitotic cyclins. During interphase, cyclin B1-Cdk1 complex is primarily co-localized with microtubules and centrosomes and is translocated rapidly from the cytoplasm to the nucleus in prophase
[170,171,172,173]. The N-terminus of cyclin B1 contains a cytoplasmic retention sequence (CRS) and a leucine-rich nuclear export signal (NES) that binds to the export receptor Crm1. During interphase, cyclin B1 shuttles between cytoplasm and nucleus.
The rate of export exceeds the rate of import, and thus the instant location is primarily in the cytoplasm [174,175]. At the onset of mitosis, cyclin B1 is phosphorylated at
CRS/NES region resulting inhibition of cyclin B1 binding to Crm1, the export of cyclin
B1 from the nucleus is blocked, and cyclin B1 accumulates in the nucleus [176,177].
Cyclin B1 might be phosphorylated by Cyclin B1-Cdk1 itself with Plk1 during mitosis, suggesting that cyclin B1-Cdk1 may collaborate with Plk1 to promote its own translocation to the nucleus [178,179]. Cyclin B2 is colocalized with Golgi apparatus in the interphase and dispersed into cytoplasm in mitosis [180].
1.3.3.3 Regulation of M phase exit
41 Mitosis exit is regulated by APC/C (anaphase promoting complex / cyclostome) mediated ubiquitination and degradation of mitotic proteins [181]. APC/C is a multisubunit ubiquitin ligase, the activity of which is tightly regulated by the activators, inhibitors, and phosphorylation so as to ensure proper and timely degradation of cell cycle regulators. Cdc20 and Cdh1/Hct are two activators of APC/C identified in human cells and yeast. They bind transiently to APC/C to determine APC/C activity [182,183].
Emi1 and SAC are the inhibitors of APC/C. Cdc20 and Cdh1/Hct work at different times during the cell cycle and confer different substrate specificity on APC. Cdc20-dependent
APC/C is activated during the early stages of mitosis, while Cdh1-dependent APC/C is activated from late mitosis to G1 phase, perhaps to early S phase, and then the activity of
APC/C is in check by Emi1 [184]. During mitosis, APC/C is first activated at prophase by
SCF-mediated degradation of Emi1 to target cyclin A for degradation [185,186], and later the
APC/C activity is restrained by the inhibitor signal generated by SAC. APC/CCdc20 is activated at the beginning of metaphase when spindle assemble checkpoint (SAC) is silenced. Activated APC/CCdc20 catalyzes the degradation of securin to initiate anaphase onset by releasing the Separase which cleaves Cohesin to trigger sister chromatid disjunction [187]. APC/CCdc20 also recognizes the destruction box containing consensus amino acid sequence RXXLXXXN at the N-terminal of mitotic cyclins [188], and targets cyclin B for destruction to inactivate Cdk1, which is required for mitosis exit
[189]. The precise timing of cyclin B1-GFP fusion protein destruction was measured when injected into somatic vertebrate cells. The initial of destruction occurs at the beginning of metaphase and nearly completes during anaphase [190]. During mitosis,
Cdh1 is phosphorylated by cyclin B1-Cdk1, which prevents binding of Cdh1 to APC.
APC/CCdh1 is activated when cyclin B1 is destroyed. Activation of APC/CCdh1 leads to
42 the degradation of Cdc20 and any remaining of cyclin B1, and also destruction of several important mitotic regulators including Plk1, Cdc25A, and Aurora A [168]. APC/CCdh1 is turned off to allow to the next mitosis by APC/C inhibitor Emi1, which rises at late G1 by transcription [191], as well as by cyclin A2-Cdk1 and cyclin A2-Cdk2 mediated Cdh1 phosphorylation to dissociate Cdh1 from APC [192].
1.3.3.4 Mitotic Checkpoints
Cells entering and transitioning M are guarded by at least two checkpoint control pathways including a G2 phase checkpoint and the spindle assembly checkpoint pathway.
G2 phase checkpoint
The purpose of cell division is to distribute accurate copies of each replicated genome to the daughter cells. The G2 checkpoint is a surveillance mechanism that prevents mitotic entry when DNA is damaged or chromosomes are only partially replicated [193]. The
G2 phase checkpoint is controlled by the ATM (ataxia telangiectasia mutated) and ATR
(ATM and Rad3-related) protein kinases. These two kinases are conserved from yeast to humans and regulate DNA synthesis, transcription of the genes involved in DNA synthesis and repair, and proteins that regulate cell cycle progression in response to DNA damage and unreplicated DNA. When the G2 checkpoint is activated, ATM and ATR phosphorylate effector kinases Chk1 and Chk2 and stimulate their kinase activities. In turn, activated Chk1 and Chk2 phosphorylate mitotic entry kinase Cdc25 and Wee1 to inhibit Cdc25 and activate Wee1, which maintains Cdk1 in an inactivation state. The G2 checkpoint is reversible. When DNA damage is repaired or DNA replication is complete, the checkpoint is inactivated and cells can proceed to M phase.
43
Spindle-assembly checkpoint
The spindle assembly checkpoint (SAC) is another cell cycle surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis. By inhibiting the activity of APC/C, the SAC determines anaphase onset until all of the chromosomes have achieved bipolar attachment to the mitotic spindle. Bipolar attachment is necessary for segregating each daughter chromatid to the correct daughter cell. The mechanisms that activate the spindle checkpoint are not completely understood, but a single unattached kinetochore can provoke and maintain SAC activity [194]. The basic components of spindle checkpoint include Mad1, Mad2 and Mad3 (BubR1 in human), Bub1, Bub3, and
Mps1 [195]. These genes were first identified in budding yeast, and are conserved in all eukaryotes. Activation of the SAC involves kinetochore localization of these spindle checkpoint proteins. The localization of different checkpoint proteins follows a defined order, and is interdependent on each other. Assembly of checkpoint proteins at kinetochores generates a signal to inhibit APC/CCdc20 activity by sequestration of Cdc20.
In this state, progress to anaphase and proteolytic destruction of securin and B cyclins is prevented. The SAC is inactivated upon kinetochore-microtubule attachment and chromosome bi-orientation. Inactivation allows degradation of securin, which induces anaphase.
1.4 STAGES OF MITOSIS
1.4.1 Morphologic stages of mitosis
In mammalian cells as well as many others, the central, microscopically observable events of mitosis are chromatin condensation, centrosome separation and movement to
44 180 degree separation, bi-polar spindle assembly, nuclear membrane breakdown, chromosome reorganization on the spindle, sister-chromatid separation and segregation, and reformation of the nuclear envelope. M phase also includes cytokinesis with distinctly observable microscopic events as well. These include contractile ring formation, cleavage furrow appearance, and formation of a midbody/bridge. The early events occur simultaneously with the late stages of mitosis, but the midbody/bridge is the last observable event in cell division. Centrosome duplication occurs during G1 and S phase. Chromosomes are replicated (called sister-chromatids) in S and are tightly associated by proteins (cohesions) that form bridges between the two chromatids. The centrosome is the major microtubule-organizing center (MTOC), on which initiates the formation of the two poles of mitotic spindle. The mitotic spindle is composed of microtubules and associated proteins. It plays an essential function for segregation of chromosomes and formation of two daughter cells. Traditionally, mitosis is divided into five distinct stages: prophase, prometaphase, metaphase, anaphase and telophase [2].
These five stages occur in sequential order, involving the coordinated movement of the centrosomes, reorganization of the cytoskeleton, and movement of the chromosomes. At prophase, the replicated chromosomes undergo condensation in the nucleus. Outside the nucleus, the centrosomes move from a single peri-nuclear location to perinuclear positions that are 180 degrees apart and initiate spindle assembly. These events constitute prophase. Prometaphase begins with nuclear envelope breakdown and continues till chromosomes have completely attached to the spindle microtubules via kinetochores and moved to the central region, termed the metaphase plate. During metaphase, all chromosomes are aligned at the metaphase plate, midway between the
45 spindle poles, and the sister chromatids are attached to kinetochore microtubules emanating from the opposite poles of the mitotic spindle. The most dramatic events in the cell cycle occur at the anaphase. With the abrupt release of the cohesion links between sister chromatids, the sisters immediately separate and move to the opposite spindle poles. This process includes two independent and overlapping movements of chromosomes know as anaphase A and anaphase B. In anaphase A, the kinetochore microtubules that attach to the chromatids get shorter, and in anaphase B, the spindle poles move farther apart from each other. Both contribute to segregation of the sister chromatids. Mitosis is completed in telophase, when daughter chromosomes arrive at the poles of spindle, decondense, and repackage into identical daughter nuclei. The nuclear envelope reassembles around each daughter genome and the spindle is disassembled, leaving a single centrosome. A contractile ring starts to form near the original metaphase plate to initiate the division of the cytoplasm, or cytokinesis. At cytokinesis, contractile ring creates a cleavage furrow on the cell surface, and the furrow rapidly pinches and spreads until it completely divides the cell into two. The cleavage furrow can begin earlier, and cytokinesis can overlap mitosis.
The traditional stages of mitosis were originally defined as a period in which the changes of spindle and chromosomes in the structure and behavior are observed under microscope.
However, this terminology is limited for some reasons. In some cells, the condensation of chromosomes is not visible under microscope, or even never happens. Furthermore, in some organisms, such as yeast and fungi, the nuclear envelope never breaks down, while in Caenorhabditis elegans it continues until anaphase. Moreover, formation of the
46 metaphase plate is not a universal feature of mitosis, nor is it a requirement for anaphase onset even in higher organisms. As a result, the stages of mitosis are approximated in some organisms because of indistinguishable criteria. A new terminology was proposed to more accurately re-define the stages of mitosis by J. Pine and C.L. Rieder[196]. Based on the activity of cell cycle regulators Cdk1 and APC, mitosis was divided into five transitional phases, independent of chromosome structure and the state of the nuclear envelope [196].
1.4.2 Re-staging mitosis
The five transition stages of redefined mitosis are briefly described as follows [196].
Transition 1: Antephase and the end of G2
Antephase is defined as the period at late G2 phase just before the chromosome condensation becomes visible. Antephase is unique in that the process of chromosome condensation is reversible at this stage. Normal cells arrest in antephase upon the
ATM/ATR mediated DNA-damage or drug treatment to disassemble microtubules. At this stage, cyclin A-Cdk activity is maximal and regulates events that occur in preparation for mitosis. Cyclin B-Cdk1 is inactive and remains in the cytoplasm. Other kinase such as Plk and aurora B play an important role during this period. Plk is required for formation of the spindle pole and activation of Cdc25 phosphatase, and aurora B kinase phosphorylates histone H3 and induces chromosome condensation. Once cells pass antephase, cells are committed to mitosis independent of environmental conditions except death can prevent the progression. In many higher animals, the point of no return is in
47 traditionally termed late prophase. Since this description, there are papers that suggest that cells can return from mitosis to antephase [197].
Transition 2: The commitment to mitosis
This transition is the commitment of cells entry to mitosis. At this transition, cyclin B-
Cdk1 is fully activated and accumulated in the nucleus. In animal cells, cyclin B1-Cdk1 shuttles between the nucleus and the cytoplasm throughout G2 phase. At the end of G2 phase, cyclin B1-Cdk1 is phosphorylated at the amino terminus and activated, first on the centrosome [198], resulting in rapid accumulation of cyclin B1-Cdk1 within the nucleus.
The exact mechanism that triggers the sudden translocation of cyclin B1-Cdk1 to the nucleus remains unknown, but the sudden accumulation of cyclin B1-Cdk1 within the nucleus induces changes that commit cells to mitosis past the point of no return. Once past this point cells have lost their ability to inactivate cyclin B-Cdk1 until cell exit from mitosis or undergo endoreduplication.
Transition 3: Satisfying the criteria to exit mitosis
This transition is characterized by the fully activated cyclin B1-Cdk1 and activation of
APC/C. Once cell commit to mitosis, APC/CCdc20 is activated and targets cyclin A and B for ubiquitin-mediated degradation. However, destruction of securin and cyclin B by
APC/CCdc20 is inhibited by spindle assembly checkpoint induced by unattached kinetochores, so that chromosome cohesion is maintained until sister chromatids have congressed. A current view is that SAC proteins are recruited to the unattached kinetochore to sequester Cdc20 by forming Mad-Cdc20 complexes, so that Cdc20 cannot bind to the APC. It is not clear how APC/CCdc20 can target and degrade cyclin A but not
48 cyclin B for destruction during prometaphase. It is possible that cyclin A compete with
SAC protein Mad to bind to Cdc20 directly for degradation [199]. Transition 3 is equivalent to the traditional stage of prometaphase.
Transition 4: Escaping from mitosis
This transition is marked by the full activation of APC/CCdc20 and rapidly degradation of cyclin B. Once all the kinetochores are attached to microtubules, the SAC is silenced and inhibition of Cdc20 is relieved inducing full activation of APC/CCdc20 which is essential for mitotic exit. Activated APC/CCdc20 regulates mitosis exit by initiation of two pathways. First, activated APC/CCdc20 induces destruction of securin releasing the separatase protease which then cleaves the cohesion complex and separates sister chromatids to start anaphase, and second, activated APC/CCdc20 mediates cyclin B degradation which turns results in rapid loss of Cdk1 activity resulting in activation of
APC/CCdh1 and the hallmark changes of telophase. Securin and cyclin B destruction are prerequisites for exit from mitosis and production of two daughter cells.
Transition 5: Returning to interphase
At transition 5, a new nuclear envelope is assembled around the two separated genomes, and a midbody forms between the two new daughter nuclei to induce cytokinesis and separate the cell in two. Then each cell enters into interphase and persists in this state until it committed to another round of DNA replication. APC/C activator Cdc20 is degraded and replaced by Cdh1. APC/CCdh1 can recognize both the Ken- and D-box motifs. Cdc20 itself and any remaining of cyclin B are destroyed by APC/CCdh1.
49 APC/CCdh1 keeps cells in an anti-mitotic state throughout interphase until the restriction point in G1.
1.5 FUNCTIONS OF CYCLIN B
1.5.1 Function of cyclin B-Cdk1 during mitosis
Activation of cyclin B-Cdk1 triggers mitosis in most eukaryotes. Cyclin B-Cdk1 regulates a large number of mitotic events by phosphorylation of numerous substrates both in the nucleus and cytoplasm at various mitotic stages. These mitotic events include early mitotic events, such as centrosome separation [200], nuclear envelope breakdown
[198], and chromosome condensation [201]. Cyclin B-Cdk1 is also involved in the regulation of spindle assembly and chromosome movements [202], as well as spindle checkpoint to control anaphase onset and mitotic exit [203].
There are three B type cyclins in human somatic cells, cyclin B1, B2 and B3. The genes of cyclin B1 and B2 are very similar. They are coexpressed in most tissues, but high cyclin B3 RNA expression is restricted to the testis [131]. Evidence is quite good that different B cyclins in yeast and drosophila play specific roles during mitosis. For the human genes, it is not as clear. Different localization suggests different function[170].
Cyclin B1 is translocated to nucleus during mitosis promoting chromosome condensation, nuclear lamina resolution and mitotic spindle assembly. Cyclin B2 is associated with
Golgi and is expressed at a low level [170], and may be involved in Golgi disassembly
[180]. Additionally, if the levels of either cyclin B1 or B2 are reduced by RNA
50 interference, the phenotype is mild cell cycle perturbation. But if both are knocked-down,
G2 arrest occurs [197,204]. This could suggest different function, but could also be explained by cyclin levels. Human cyclin B3 is expressed in G1 or resting cells and appears different in its properties from cyclinB1 and B2 [132,133]. It is unlike a mitotic cyclin.
1.5.2 Rate-limiting function of cyclin B
Rate-limiting function of cyclins was first discovered when adding of exogenous cyclin mRNA was shown to be sufficient to induce multiple cell cycle in frog egg extraction in which endogenous mRNA was destroyed [110]. Subsequently, it was demonstrated that cyclin D is a rate limiting for cell cycle commitment and progression from G1 to S phase since microinjected antibody or antisense directed to cyclin D blocked G1 progression
[205,206] while constitutive expression of cyclin D shortened the G1 phase [206,207].
Cyclin E and cyclin A had also been proved to be rate limiting, since inducible expression of cyclin E accelerates G1 progression [208] and overexpression of cyclin A accelerate the entry of G1 phase cells into S phase [209]
Ectopic expression of cyclin B1 and cyclin B2 mRNA in early Xenopus embryos was able to accelerate mitotic entry, suggesting that cyclin B level controls cell cycle timing
[210]. Microinjection of active cyclin B1-Cdk1 can drive G2 phase into mitosis, indicating that cyclin B1 is rate-limiting in the mammalian cell cycle [211]. Inhibition of
Cdk1 activity by chemicals lengthened mid-G2 to prophase time in mammalian cells, suggesting that Cdk1 activity is rate-limiting for cell cycle progression during a mid-G2 through prophase [212]. Furthermore, RNA interference studies show that cyclin B1
51 depletion produces both G2 phase and M phase delay at prometaphase and metaphase, while cyclin B2 knockdown produces increased numbers of G2 cells and reduced numbers of M cells [204]. These data suggest that both B cyclins are rate-limiting and perhaps with different roles on mitotic time regulation. Several groups have studied the effect of cyclin B1 on progression through mitosis by overexpression of nondegradable cyclin B1 in mammalian cells, however, their work was focused on effect of cyclin B1 on metaphase to anaphase transition or anaphase onset [213,214]. It is little known about the effect of overexpressing B cyclins on mitotic timing in mammalian cells.
1.5.3 Functional redundancy of B cyclins
In mammalian cells, four regulatory cyclins (D, E, A and B) associate specifically with four Cdk types (Cdk4/6, Cdk2 and Cdk1) to control the cell cycle process. Some of these cyclins and Cdks have shown functional redundancy for cell cycle progression [215,216].
The first evidence came from the experiments by Sicinski et al that showed survival of cyclin D1 null mice with several abnormal phenotypes, and rescue of all phenotypes by replacement with cyclin E [217]. In mammals, it was believed that cyclin B1 was essential and un-compensated for mitosis entry. Cyclin B1 null mice were embryonic lethal, while cyclin B2 deletion mice were viable implying that cyclin B1 is necessary for development and may compensate for any cyclin B2 function [153]; Microinjection of active cyclin B1-Cdk1 complexes into CHO cells could drive G0/G1 cells into mitosis, but cyclin B2-Cdk1 complexes could not [180]. All these data lead to the idea that cyclin
B1 could compensate for the lack of B2, but cyclin B2 could not in replace cyclin B1.
However, several studies with RNA interference-mediated cyclin B1 and cyclin B2 knockdown in mammalian cell lines carried out by different research groups have shown
52 mild consequences phenotype with cell cycle perturbation [197,204,218,219]. These are difficult to explain if cyclin B1 is necessary. First, the levels of cyclin B1 after knock down were less than 3-5% of peak cyclin B1 levels. Second, if cyclin B2 was co- depleted, a profound G2 arrest developed, which is reminiscent of Cdk1 inhibition. If B1 is necessary, then the critical substrate must be taken care of by very low levels of B1, and critically, adequate levels of B2 must be present for that low level to be functional.
A less complex model is that a threshold of B cyclin necessary, and that threshold is equal to the level of B2 + the residual B1. This model states that cyclin B1 and B2 can fully compensate each other. Currently, this model is most likely and cyclin B2 can compensate cyclin B1 for mitosis progression. Re-expression of ectopic expressing cyclin
B2 can rescue all phenotypes caused by cyclin B1 and cyclin B2 co-depletion [197], however, this rescue was performed by over-expression of cyclin B2, and just restores the single cyclin knock-down state. Therefore, it remains unknown whether cyclin B1 and cyclin B2 are redundant for mitotic entry and progression.
1.6 HYPOTHESIS AND SPECIFIC AIMS OF THESIS WORK
In summary, activation of mitosis-promoting factor triggers transition into M and regulates transition through M by phosphorylation of multiple proteins. This has been documented in starfish, sea urchin, and Xenopus oocytes, three different yeasts, plants, and mammalians among others. In somatic mammalian cells, cyclin B1- and B2- Cdk1 are the catalytic activities at the heart of MPF. Different localization of cyclin B1 and B2 is in favor of different roles of cyclins B1 and B2 in mitosis. From the evidence obtained from siRNA studies, cyclin B1 and B2 depletion showed that a B cyclin is necessary for
G2 to M transition but could not distinguish whether the two cyclins are completely
53 redundant or whether cyclin B1, expressed at less than 5% of peak values, has a critical role. Therefore, there is evidence that these two cyclins overlap in function and there is evidence that they have specific roles. The work presented herein quantitatively explores the effect of cyclin B2 knock down on cell cycle kinetics. This aspect has not previously been explored in any depth. Additionally, the work firms up the reported effect of cyclin
B1 knock down by quantitatively restoring the kinetic phenotype of wild-type cyclin B1.
And finally, points to a distinct difference between the two cyclins for the ability to regulate mitotic time. We hypothesize that cyclin B1 and B2 are functional redundant for mitotic entry and are rate limiting for mitotic progression, but have opposite timing roles.
Cyclin B2 retards mitotic progression and cyclin B1 promotes mitotic progression in to cell cycle.
The purpose of this thesis work is to study the role of cyclin B1 and B2 on mitotic timing and to discover the possible underling mechanism of redundant function of cyclin B1 and
B2 for mitotic entry and progress in somatic mammalian cells. For this purpose, we have established tetracycline-dependent inducible Tet-off HeLa cells that express siRNA resistant cyclin B1-EGFP or cyclin B2-EGFP. In this system, ectopic cyclin B1-or B2-
EGFP cannot be recognized by siRNA specific to cyclin B1 or B2, but is inducible and expressed in the absence of Doxycycline (Dox). The specific aims of this work was: 1)
To quantitatively study the ability of cyclin B1 or cyclin B2 to regulate mitotic entry and progression; 2) To quantitatively test the ability of recombinant cyclin B1-EGFP or cyclin B2-EGFP to restore wild-type cell cycle kinetics in double depletion background - that is, to investigate the relationship between quantity of single cyclin B expression and
54 the ability to enter and go through mitosis, and 3) To explore the localization of cyclin B1 and B2 when the other has been depleted.
55 CHAPTER 2
STABLE CELL LINES THAT EXPRESS CYCLIN B1- OR B2-EGFP
FUSION PROTEINS
2.1 ABSTRACT
To study the effect of B cyclins on the mitotic entry and progression, we produced stable cell lines with inducible expression of ectopic, RNAi (RNA interference) resistant cyclin
B1or B2 fused with Enhanced Green Fluorescent Protein (CyclinB1-EGFP, CyclinB2-
EGFP). Full-length, wild-type Cyclin B1 or CyclinB2 and EGFP cDNAs were cloned into the tetracycline (Tet) response vector, which is an inducible system with tight quantitative and temporal control of gene expression. Synonymous mutations were generated within the CyclinB1- or B2-specific siRNA recognition sites in the constructs.
Constructs were tested that contained 4, 6, or 7 mutations. Tet response vectors encoding
CyclinB1- or cyclinB2-EGFP genes were transfected into a commercial HeLa Tet-Off cell line. Cells were selected for hygromycin-resistance and subjected to fluorescence activated cell sorting (FACS) to produce stable clones. The expression of ectopic B cyclin-EGFP could be turned on or turned off and regulated in a dose- and time- dependent manner by doxycycline. The ectopic CyclinB1- or CyclinB2-EGFP encoded by constructs with synonymous mutations were expressed in RNAi-treated cells in which endogenous cyclins were specifically depleted. Constructs with 5 or 7 mutations worked best. The EGFP fusion proteins displayed Cdk1 activity, measured by phosphorylation of
Bcl2 in mitotic cells.
56 2.2 INTRODUCTION
The cell cycle is regulated by specific cyclin-dependent kinases (Cdks) and their activator cyclins at each cell cycle stage (reviewed in [59,220,221,222]). Cyclins A2, B1 and B2 are the principle activators that bind and activate Cdk1 to regulate mitotic entry and transition in mammalian cells [116]. Cyclin B1 is essential for embryonic viability while cyclin B2 is dispensable [153]. However, siRNA interference studies from several groups show that depletion of cyclin B1 in human somatic cell lines has a mild phenotype, inducing some cell cycle perturbations, enhancing chromosomal abnormalities, and increasing propensity for apoptosis [197,204,223]. In our studies, cyclin B1 depletion increased the G2 and M phase fractions, and kinetic experiments showed that these increases were the result of increased G2 and M phase times. Cyclin B2 depletion induced a slight increase in the G2 fraction but a decreased M fraction. Cyclin B1 and
B2 co-depleted cells arrested in G2 and later entered a 4C cell cycle state. Additionally, mitotic cells were very rare in co-depleted populations. This is consistent with Bellanger et al. [197], who observed entry into mitosis microscopically, but cells failed to remain in a mitotic state and re-entered interphase. These data suggest that cyclin B1 and B2 can compensate for each other for regulation of mitotic entry and progression but the frequencies of M phase fractions suggest that there could be subtle differences as well.
To explore quantitatively any dose dependent gain of function of cyclin B1 and B2 on G2 and M phase time and the ability of cyclin B1 and B2 to compensate for each other, and to fully regulating mitotic entry and progression, we created a system in which cyclin B1 or cyclin B2 levels can be induced and precisely regulated and measured in cell in which endogenous cyclins have been depleted.
57
The Tet-regulated gene express system is a tightly regulated, inducible, high-level gene expression system which is well studied and widely. This system, first described by
Gossen & Bujard [224], consists of two critical components: a regulatory protein and a response plasmid containing cDNAs under control of the tetracycline-response element
(TRE). In the Tet-Off system [225], the regulatory protein is known as the tetracycline- controlled transactivator (tTA) and is encoded by the pTet-Off regulator plasmid. This plasmid contains the neomycin-resistance gene to permit selection of stable transformants. tTA is a fusion of amino acids 1-207 of the tet repressor protein (TetR) and the C- terminal activation domain of the Herpes simplex virus, VP16. The TRE is located just upstream of a minimal CMV promoter (Pmin CMV) that lacks the strong enhancer elements of the wild type CMV promoter. In the absence of doxycycline (Dox), tTA binds to TRE to activate transcription of the genes of interest, while in the presence of Dox, Dox binds tTA and prevents the binding to the TRE and gene expression is shut off (Figure 2-1).
We decided to apply the Tet-Off system with conditional expression of cyclin B1-EGFP or B2-EGFP fusion proteins containing synonymous mutations at the cyclin B1 or B2 siRNA recognition site. In combination with small RNAi technology, the endogenous cyclin B1 or B2 can be knocked down, while the ectopic cyclin B1- or B2-EGFP will be expressed and regulated by Dox. At the same time, EGFP is a marker and reporter of the ectopic cyclin B1 or B2 expression. The d4EGFP has a half-life of approximately four hours. This may or may not have been needed, as the B cyclins are specifically degraded by the Anaphase Promoting Complex / Cyclosome (APC/C). The intensity of EGFP fluorescence is proportional to the expression level of ectopic cyclin B1 or B2, which provides the opportunity to study the quantitative relationship between cyclin B1 and B2
58 during mitotic entry and progression by comparing the intensity of EGFP fluorescence of ectopic cyclin B1- or B2-EGFP.
In this study, we have constructed Tet-Off response plasmids that encode wild type human cyclin B1, B2 and A2, and cyclin B1, B2 and A2 with synonymous mutations at their siRNA recognition site, and established and validated HeLa Tet-Off stable cell lines that express ectopic cyclin B1- or B2-EGFP under control of Dox. The reduction in cyclin B1- or B2- EGFP in the stable cell lines is rapid in response to Dox and tightly regulated by Dox. The ectopic cyclin B1- or B2- EGFP is resistant to the siRNA specific to cyclin B1 or B2. The ectopic cyclin B1- or B2-EGFP can activate Cdk1-mediated Bcl2 phosphorylation, indicating that the ectopic cyclin B1- or B2-EGFP are active.
2.3 MATERIALS AND METHODS
2.3.1 Plasmids and construction of Tet-Off response plasmids
The pd4EGFP vector and Tet-Off response vector pTRE2hyg were purchased from
Clontech (Mountain View, CA). Plasmids pcDNA3-cyclin B1 and pcDNA3-cyclin B2 containing full length cDNAs for human cyclin B1 and B2 were a gifts from Dr. Denis
Templeton (University of Virginia) and Dr. Joelle Sobczak-Thepot (University P & M
Curie), respectively. Plasmid pCMV-cyclin A2 contains full length human cyclin A2.
Construction of pTRE2-EGFP(NheI/NotI) (pTRE2-001) and pTRE2-EGFP (BamHI/NotI)
(pTRE2-002): An EGFP cDNA fragments with NheI /NotI or BamH1/NotI restriction sites, obtained by performing double digestion with NheI/NotI or BamH1/NotI enzymes, were cloned into the multiple cloning sites (MCS) of pTRE2hyg vector to generate pTRE2-001 (pTRE2-EGFP(NheI/NotI)) and pTRE2-002 (pTRE2-EGFP(BamH1/NotI))
59 constructs. The integrity of the cDNA open reading frames (ORF) was confirmed by both double digestion and DNA sequencing (Genomic Core Facility of Case Western Reserve
University, Cleveland, OH). Figure 2-2 shows a schematic describing the process, starting and resulting plasmids.
Construction of pTRE2-cyclin B1-EGFP (pTRE2-006) and pTRE2-m4cyclin B1-EGFP
(pTRE2-015), pTRE2-m7cyclin B1-EGFP (pTRE2-016) with 4 or 7 synonymous mutations at cyclin B1 siRNA recognition site: Human cyclin B1 cDNA ORF was amplified by polymerase chain reaction (PCR) with pcDNA3-cyclin B1 plasmid using
Taq polymerase and a set of primers containing NheI and AgeI restriction sites as follows:
CCNB1NheI-5’: -GGGGCTAGCGCCGCCACCATGGCGCTCCGAG
CCNB1AgeI-3’: -CCCACCGGTGCCACCTTTGCCACAGC
The EGFP cDNA fragment was obtained from the pd4EGFP vector with AgeI/NotI digestion. To construct the Tet-Off response plasmid pTRE2-cyclin B1-EGFP (pTRE2-
006), which encodes cyclin B1 gene with an EGFP tag, cyclin B1 (NheI/AgeI) cDNA and
EGFP (AgeI/NotI) cDNA were inserted into the MCS of pTRE2hyg with NheI/NotI by triple ligation. In order to allow proper expression of the cyclin B1-EGFP fusion protein at the carboxyl (C) terminus, the primers were designed to add a NheI restriction site and a Kozak consensus sequence at the 5’ end; eliminate the stop code, and add an AgeI restriction site at 3’ end. There are two extra nucleotides added just between the AgeI restriction site and the coding sequence of protein cyclin B1 at the 3’ end primer to make the DNA sequence of cyclin B1 protein in the same reading frame as EGFP tag at the C terminus. The integrity of the cDNA (pTRE2-006) open reading frame was confirmed by both double enzyme digestion and DNA sequencing. To produce cyclin B1 mutants that
60 cannot be recognized by cyclin B1 specific siRNA, 4 and 7 nucleotide mutations were introduced at cyclin B1 siRNA recognition sites by PCR-mediated site-directed mutagenesis with pTRE2-006 plasmids using the Quick-change kit (Stratagene, La Jolla,
CA) according to the manufacturer’s instructions. The mutants conserve the same amino acid sequence as wild type cyclin B1. The sequence of the mutants was confirmed by
DNA sequencing. The mutagenesis products were named pTRE2-m4cyclin B1-EGFP
(pTRE2-015) and PRE2-m7cyclin B1-EGFP (pTRE2-017).
Construction of pTRE2-cyclin B2-EGFP (pTRE2-010) and pTRE2-m4cyclin B2-EGFP
(pTRE2-017), pTRE2-m6cyclin B2-EGFP (p023) with 4 or 6 synonymous mutations at cyclin B2 siRNA recognition site: Human cyclin B2 cDNA was amplified by PCR using pcDNA3-cyclin B2 plasmid as a template with the primers containing NheI and AgeI restriction sites as following:
CCNB2NheI-5’: -GGGGCTAGCGCCGCCACCATGGCGCTGCTCCG
CCNB2AgeI-3’: -GGGACCGGTGCGGACCTTCCTATCAGTG
The EGFP cDNA fragment was obtained from the pd4EGFP vector with AgeI/NotI digestion. To construct Tet-Off response plasmid pTRE2-cyclin B2-EGFP (pTRE2-010) encoding cyclin B2 gene with the EGFP tag, cyclin B2 (NheI/AgeI) cDNA and EGFP
(AgeI/NotI) cDNA were cloned into the MCS of pTRE2hyg with NheI/NotI by triple ligation. The primers were designed to add a NheI restriction site and a Kozak consensus sequence at the 5’ end, and eliminate the stop codon and add an AgeI restriction site at the 3’ end. There are two extra nucleotides added just between the AgeI restriction site and the coding sequence of cyclin B2 at the 3’ end primer so that the DNA sequence of the cyclin B2 protein is in the same reading frame as the EGFP tag at the C terminus.
61 Four and six nucleotide mutations were introduced into the cyclin B2 siRNA recognition sites by PCR-mediated site-directed mutagenesis with p006 plasmids using the Quick- change kit producing HeLa-m4cyclin B2-EGFP (pTRE2-017) and HeLa-m6cyclin B2-
EGFP (pTRE2-023) constructs. The mutants conserve the same amino acid sequence as wild type cyclin B2 but cannot be recognized by siRNA specific to cyclin B2. The DNA sequence of all constructs was confirmed by DNA sequencing.
Construction of pTRE2-cyclin A2-EGFP (pTRE2-008) and pTRE2-m4cyclin A2-EGFP
(pTRE2-025), pTRE2-m6cyclin A2-EGFP (pTRE2-026) with 4 or 6 synonymous mutations at cyclin A2 siRNA recognition site: Human cyclin A2 cDNA with BamHI and an AgeI restriction sites, amplified by PCR using pCMV-cyclin A2 plasmid as a template, and the EGFP cDNA fragment obtained from the pd4EGFP vector by an AgeI/NotI enzyme digestion were cloned into the MCS of pTRE2hyg with BamH1/NotI to construct pTRE2-cyclin A2-EGFP (pTRE2-008). The primers (see below) were designed to add a
BamHI restriction site and a Kozak consensus sequence at the 5’ end, eliminate the stop codon, and add an AgeI restriction site at the 3’ end. Two extra nucleotides were added just between the AgeI restriction site and the coding sequence of cyclin A2 at the 3’ end primer to put the DNA sequence of cyclin A2 protein in the same reading frame as EGFP tag at the C terminus. The primers were:
CCNA2BamHI-5’: -GGGGGATCCGCCGCCACCATGTTGGGCAACTC
CCNA2AgeI-3’: -GGGACCGGTGCCAGATTTAGTGTCTCTG
Four and six nucleotide mutations were introduced at cyclin A2 siRNA recognition sites by PCR-mediated site-directed mutagenesis with pTRE2-008 plasmids to produce HeLa- m4cyclin A2-EGFP (pTRE2-025) and HeLa-m6cyclin A2-EGFP (pTRE2-026)
62 constructs. The mutants conserve the same amino acid sequence as wild type cyclin A2.
The DNA sequence of all constructs was confirmed by DNA sequencing.
2.3.2 Oligonucleotides of siRNA specific to cyclin B1, B2 and A2, and construction of siRNA-resistant constructs siRNA sequences against human cyclin B1 and B2 were designed as previously described
[204], and synthesized by Themo Scientific. Oligonucleotides targeting cyclin B1 correspond to the cDNA sequences of human cyclin B1 coding region 340-360, as follows: 5’-AAACTTTCGCCTGAGCCTATT-3’. pTRE2-m4cyclin B1-EGFP and pTRE2-m7cyclin B1-EGFP were created by directed mutagenesis. The mutations are as follows: 5’-AAACTCTCCCCTGAACCGATT -3’and 5’-
AAGCTCTCCCCGGAACCGATC-3’ respectively. Oligonucleotides targeting cyclin
B2 correspond to the cDNA sequences of human cyclin B2 coding region 847-866 as follows: 5’-CAGCACACTTTAGCCAAGT-3’. The sequence of mutations in pTRE2- m4cyclin B2-EGFP and pTRE2-m6cyclin B2-EGFP is as follows: 5’-
CAGCATACCTTAGCGAAAT -3’and 5’-CAACATACCTTGGCGAAAT-3’ respectively. Oligonucleotides of siRNA specific to cyclin A2 correspond to the cDNA sequences of human cyclin A2 coding region 711-730 as follows: 5’-
CTACATTGATAGGTTCCTG-3’. The sequence of mutations in pTRE2-m4cyclin A2-
EGFP and pTRE2-m6cyclin A2-EGFP is as follows 5’-CTATATCGATAGATTTCTG-3’ and 5’-CTATATCGACAGATTTCTA-3’ respectively. Scrambled oligonucleotides with approximately 50% G + C content was purchased from Dharmacon and used as a negative control.
63 2.3.3 Cell culture, transfection and generation of stable cell lines
The HeLa cell line was purchased from ATCC (Manassas, VA). The HeLa Tet-Off line expressing tetracycline transactivator (tTA) was purchased from Clontech. HeLa or HeLa
Tet-Off cells were cultured in RPMI (Invitrogen, Grand Island, NY) medium supplemented with 10% fetal bovine serum (FBS), 50ug/ml gentamicin sulphate, and
100ug/ml G418 (HeLa Tet-Off only) and grown at 37oC in a humidified incubator with 5%
CO2 atmosphere.
For transfection, cells were plated in 10cm or 6-well tissue culture plates with growth medium and grown to 70% confluence at the time of transfection. Transfection was performed using lipofectamine T2000 reagent (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. For transient transfection, cells were collected 24 hours after transfection and subjected to various assays. For generation of stable cell lines, 48 hours after transfection, EGFP expressing cells were sorted by FACS. Single sorted cells were deposited into p96-well plates and selected with 200ug/ml hygromycin in 100ug/ml G418 medium. After 6 weeks of serial culture, the surviving clones were analyzed by flow cytometry or Western blotting to determine expression of exogenous and endogenous cyclins. Established stable cell lines were maintained in RPMI medium supplemented with 10% tetracycline-free FBS, 50ug/ml gentamicin, 100ug/ml G418 and
5 ng/ml doxycycline.
2.3.4 Cell fixation, intracellular staining
Cells dissociated with trypsin were fixed with 0.125% formaldehyde in culture media at
37oC for 10 min, washed, then suspended in methanol (90% final) at -20oC as previously described [226]. Before antibody staining, fixed cells were washed twice with cold PBS
64 to remove methanol and once with PBS/BSA (20mg/ml bovine serum albumin, 10 mM
NaPO4, 150 mM NaCl) to reduce non-specific antibody binding. For indirect staining, 1-
2 million cells were incubated with primary antibody for 1h at 4oC, washed twice with
PBS/BSA, followed by the secondary antibody for another 1h at 4oC, and a final three washes. Finally, the cells were stained with 1 ug 4', 6-diamidino-2-phenylindole (DAPI) for DNA staining and analyzed by flow cytometry. For conjugated primary antibodies, the second antibody step was omitted. The following antibodies and amounts were used for cell staining: anti-cyclin B1, 0.125 ug GNS1 conjugated with Alexa Fluor 647 (A647) or 0.125 ug GNS1 un-conjugated (BD Biosciences, San Jose, CA); anti-cyclin B2, 0.34 ug antibody (#sc22776, Santa Cruz Biotechnologies, Dallas, Texas ), anti-phospho-S10- histone H3 (PHH3), 0.125 ug conjugated with Alexa Fluor 488 (A488) or A647
(Millipore-Upstate, Billerica, MA); anti-phospho-T56-Bcl2, 0.25ug (Cell Signaling
Technology, Beverly, MA); Secondary antibodies with labels Alexa Fluor 488 (A488),
A555 and A647 were obtained from Invitrogen and used at 2:1 ratio w/w and 1ug DAPI
(Invitrogen, Grand Island, NY). Staining was performed in 50 ul phosphate buffered saline with 2% bovine serum albumin (BSA, Sigma, St. Louis, MO).
2.3.5 Fluorescence activated cell sorting and flow cytometry
Sorting of EGFP positive cells was performed with a BD Biosystems FACS Aria. Two color fluorescence measurements of EGFP and DNA were made on Beckman Coulter XL.
Multi-color fluorescences were measured on a BD Biosystems LSR II or Beckman
Coulter (Miami, FL) Gallios. On all instruments, doublets were excluded from the analysis based on the peak height or width versus integrated DNA content signals. The
65 stop count was set at 15,000 to 30,000 events. The fluorescence data was analyzed with
WinList 3D 7.0 software (Verity Software House, Topsham, ME).
2.3.6 Immunoblotting analysis
Cells pellets were lysed in whole cell lysis buffer (0.137 M saline, 2% NP-40, 20mM Tris,
1% SDS, 1% deoxycholate). 10-15 ug total protein were separated using 8 or 10% acrylamide gels in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (EDM,
Millipore, Billerica, MA). The following primary antibodies were used according to the manufacturer’s instructions. Mouse anti-cyclin B1 (#554177, BD Pharmingen, San Jose,
CA), rabbit anti-cyclin B2 (sc-22776, Santa Cruz, Dallas, TX), goat anti-cyclin B2 (#sc-
5335Santa Cruz, Dallas, TX), polyclonal Human anti-cyclin A (clone BF683,
Pharmingen, San Jose, CA) mouse anti-Cdk1/Cdc2 (#610038BD Biosciences, San Jose,
CA), mouse anti-GFP (#sc9996, Santa Cruz, Dallas, TX) and rabbit anti--tubulin
#ab6046-200, Abcam, Cambridge, MA). Alkaline phosphatase conjugated species specific secondary antibodies were purchased from Promega (Madison, WI). Secondary antibodies were diluted at 1:10,000 in PBS with 0.2% Iblock (Invitrogen, Grand Island,
NY). Membranes were developed by CDP-star chemiluminescent substrate (Sigma, St.
Louis, MO). Kodak X-OMAT AR films (Eastman Kodak Company, Rochester, NY) were used to detect chemiluminescence.
2.3.7 Microscopy imaging p015 and p017 cells were grown in optical plates (Ibidi, Martin Reid, Germany) in RPMI plus 10% FBS with or without Dox for 24hr. Fluorescence and differential imaging
66 contrast (DIC) images were collected with a Leica AF6000 fluorescence microscope using 10X objective. Images were processed using Leica AF imaging software.
2.4 RESULTS
2.4.1 Construction of Tet-Off response plamsids containing human cyclin B1-, B2- or A2 –EGFP gene that is resistant to siRNA and specific for cyclin B1, B2 or A2
Details of the construction of pTRE2-cyclin A2-EGFP, pTRE2-cyclin B1-EGFP and pTRE2-cyclin B2-EGFP are given in the materials and methods. Human cyclin and
EGFP cDNAs were amplified by PCR and inserted in the pTRE2hyg Tet-Off response vector to construct pTRE2-cyclin-EGFP plasmids encoding cyclin-C-terminal-EGFP fusion proteins. Synonymous mutations of 4, 6, or 7 nucleotides at the cyclin siRNA recognition sites were introduced into a subset of these plasmids to generate Tet-Off response plasmids that express C-terminal EGFP-tagged cyclins and resistance to cyclin siRNA. All constructs and their encoding genes are listed in Table 3-1.
To examine the resistance of B cyclin-EGFP genes to siRNA knockdown, HeLa Tet-Off cells were transfected with wild-type (pTRE2-006, pTRE2-010) or (pTRE2-016, pTRE2-
017) B cyclin or negative control (pTRE2-p001) plasmids and treated with cyclin B1 or
B2 siRNA for 24 hr, followed by immunoblotting analysis (Figure 2-3). Ectopic cyclin
B1- or B2-EGFP was expressed from the constructs 24 hr of transfection. Endogenous cyclin B1 or B2 was undetectable after siRNA treatment. Ectopic cyclin B1- or B2-EGFP with wild type sequences (pTRE2-006 and pTRE2-010) was efficiently reduced by siRNA treatment while the levels of ectopic cyclin B1- or B2-EGFP with either 7 or 4 synonymous mutations (pTRE2-p016, pTRE2-p017, respectively) were unaffected. By
67 specific siRNAs, RNAi treatment did not change the level of the non-targeted endogenous cyclins A2, B1, or B2. Therefore, the designed plasmids encoded the expected ectopic B cyclin–EGFP constructs and the specific siRNAs were efficient and specificity was validated.
2.4.2 Establishment stable cell lines
HeLa Tet-Off cells were transfected with plasmids pTRE2-001, pTRE2-006, pTRE2-015, pTRE2-016, pTRE2-010, pTRE2-017 and pTRE2-023 to generate stable cell lines: P001,
P006, P015, P016, P010, P017, and P023, respectively. Single EGFP expressing green cells were sorted into 96-well plates and selected with both hygromycin and G418 for 6 weeks. One antibiotic resistant clone from both pTRE2-015 and pTRE2-p017 transfected cells was examined by fluorescence microscopy in the presence or absence of Dox. The images (Figure 2-4) show that, in the absence of Dox, the cyclin B-EGFP fluorescence was expressed and detectable. In the cyclin B1-EGFP clone (P015), green fluorescence is distinctly cytoplasmic with prominent centriole-like features (arrows). In the cyclin
B2-EGFP clone (P017) less extensive cytoplasmic fluorescence is evident with peri- nuclear, Golgi-like features (arrows). Both clones display complete staining in late mitotic cells (metaphase, thick arrows). These patterns are the expected distributions for the two B cyclins. Treatment with Dox abolished fluorescence detection in both clones.
Several antibiotic resistant clones from each stable cell line were selected to perform flow cytometry to measure the EGFP fluorescence intensity quantitatively in the presence or absence of doxycycline 24 hr post-transfection. Measurements were made after fixation and staining with DAPI for DNA content. Figure 2-5A shows typical flow cytometry fluorescence patterns for clones of either B cyclin. There are several features to note.
68 First, without Dox, there is distinct, bimodal EGFP expression in G1. The low peak of fluorescence likely represents cells in the APC/CCdh1 dominated (pre-committed) part of
G1. Second, the high expression in G1 is expected but distinctly abnormal – wild-type cyclins are expressed at very low levels in G1 that exponentially increase at two distinct rates in S and G2[227]. Presumably, this is not injurious to the cell because B cyclin/Cdk1 is inactive until the G2 to M transition. Finally, treatment with Dox reduces fluorescence below the presumed APCCdh1 population. This means that some B cyclin-
EGFP is expressed in all cells when Dox is absent. The quantified expression of induced ectopic B cyclin-EGFP for each clone is show in Figure 2-5B. These data were generated by subtracting the suppressed (+Dox) levels from the same cells cultured without Dox.
Individual clones hr varied by as much as 10 fold in maximum expression. We selected clones P001-1 (EGFP), p006-14, p015-4 and p016-4 (cyclin B1-EGFP), p010-5, p017-4 and p023-1 (cyclin B2-EGFP) for further study. For convenience, the clone names will be abbreviated leaving off the clone number in the text and figures, but will be included in Figure legends.
2.4.3 Expression of cyclin B1-EGFP and cyclin B2-EGFP is reduced and tightly regulated under the control of doxycycline
To assess expression of cyclin B1- and B2-EGFP further in the Tet-Off stable cell lines,
HeLa- p006, p015, p016, p010, p017 and p023 were incubated in the presence or absence of 10ng/ml doxycycline for 24 hr and EGFP expression was analyzed by flow cytometry or SDS-PAGE and immunoblotting (Figure 3-1). Immunoblotting analysis showed that ectopic cyclin B-EGFP protein in HeLa-p006, p015, p016, p010, p017 or p023 stable cells or EGFP protein in HeLa-p001 control cells were detected by anti-GFP antibody in
69 the cell lysates in the absence of doxycycline, while EGFP expression declined in the cell lysates after addition of doxycycline (Figure 3-1A). Flow cytometry data showed a strong upshift in cyclin B-EGFP fluorescence in each of these stable clones in the absence of doxycycline compared to that in the presence of doxycycline (Figure 3-1B).
These results show that expression of B cyclin-EGFP expression in the stable cell lines is tightly regulated under the control of doxycycline.
2.4.4 Effect of fixation on the intensity of B cyclin EGFP fluorescence
To perform flow cytometry or microscopy analysis, cells were fixed and permeabilized before antibody staining. There are a few of examples of the GFP signal becoming faded and undetectable after fixation and permeabilization. Since we need to perform multi- parameter cell cycle analysis on these cells lines, it is important to determine whether fixation affects the expressed levels of B cyclin-EGFP. To compare the intensity of the
EGFP signal before and after fixation and permeabilization, HeLa p006 cells were plated in 6-well plates and incubated in the medium with different concentrations of Dox for 24 hr. The EGFP fluorescence was measured by flow cytometry with living cells, or after fixation with formaldehyde and methanol as described in Materials and Methods. Figure
2-6 shows the correlation between the intensity of EGFP fluorescence in living or fixed cells. The data show that the EGFP signal is lowered after fixation and permeabilization, however, the slopes of both curves are similar, suggesting that the effect of fixation and permeablilization on the intensity of EGFP fluorescence is uniform and acceptable.
70 2.4.5 Induction of cyclin B1- and B2-EGFP is rapid and regulated by doxycycline in a dose dependent manner
To evaluate the kinetics of ectopic B cyclin-EGFP expression changes upon removal or addition of doxycycline, time course experiments were performed with stable cell lines.
Cells were incubated with medium in the presence or absence of doxycycline for 24 hr, then fixed, and EGFP fluorescence was measured at indicated time points by flow cytometry after removal or addition of doxycycline from the medium. Figure 3-2A shows the change in cyclin B1-EGFP in the HeLa-p006 cell line in response to removal or addition of doxycycline. In P006, removal of doxycycline led to an induction of cyclin
B1-EGFP expression that reached half maximum by 6 hr and an asymptotic maximum by
14 hr after removing doxycycline. Reduction of cyclin B1-EGFP was observed with addition of doxycycline, but with a half-life of almost 10 hr. The expression of cyclin
B1-EGFP was almost completely absent 17 hr after adding doxycycline. Similar results were obtained from other stable cell lines (data not shown). All subsequent experiments were performed at least 17 hr after removal or addition of Dox so that the expression of ectopic B cyclin-EGFP was at a stable level. These data show that expression of B cyclin-
EGFP levels can be changed relatively quickly in response to doxycycline in the B- cyclin-EGFT expressing cell lines.
To measure the responsiveness of stable cell lines to different doxycycline concentrations.
Stable cells were incubated with medium containing different doses of doxycycline, from
0 to 10ng/ml. After 24 hr incubation, EGFP fluorescence was analyzed by flow cytometry. Figure 3-2B shows the dose-response curve for HeLa-p016. Doxycycline
71 concentrations as little as 0.002ng/ml reduced ectopic cyclin B1-EGFP expression, while concentrations from 0.25ng/ml and higher led to a relatively complete reduction in B cyclin B-EGFP levels. Half maximal expression occurred at ~0.1ng/ml. Other cell lines behaved similarly.
2.4.6 Cell line resistance of ectopic cyclin B1-EGFP and cyclin B2-EGFP with synonymous base changes to siRNA
To investigate the effect of siRNA specific to cyclin B1 or B2 on ectopic cyclin B1- and
B2-EGFP expression in the cell lines, RNA interference and immunoblotting experiments were performed. HeLa-p015, p016, p017 and p023, and control cells, HeLa-p006, and p010 were transfected with siRNA against cyclin B1 or B2 in the presence or absence of doxycycline. After 18 hr, cells were lysed and blotted with anti-cyclin B1 or B2 antibody.
As shown in Figure 2-8, and in agreement with the previous data, ectopic cyclin B1- or
B2-EGFP was expressed in the stable cells in the absence of doxycycline, while ectopic cyclin B1 or B2-EGFP was repressed in the presence of doxycycline. Endogenous cyclin
B1 or cyclin B2 in the stable cells was efficiently depleted by cyclin B1 or cyclin B2 siRNA. Expression of ectopic cyclin B1- or B2-EGFP in HeLa-p006 and HeLa-p010 was greatly decreased or almost undetectable after cyclin B1 or cyclin B2 siRNA treatment (lane 4 in Figure 3-3A and 3-3B). However, the levels of ectopic cyclin B1- or
B2-EGFP in HeLa-p015, p016, p017 and p023 stable cell lines treated with siRNA were comparable to cells treated with control siRNA (lane 8 and 10 in Figure 3-3A and 3-3B).
Cdk1 levels were unaffected by either doxycycline or siRNA treatment. These data suggest that expression of ectopic cyclin B1- or B2-EGFP was tightly regulated by Dox,
72 that Cdk1 and endogenous B cyclins were unaffected by Dox, and that exogenous cyclin with synonymous mutations were resistant to specific siRNA. There is an unknown band in some preparations of the cell line that is recognized by the highly specific GNS1 antibody (cyclin B1 cell lines) and the cyclin B2 antibody (cyclin B2 lines). It appears to be most prominent in cell lines that express exogenous B cyclin-EGFPs at the highest level. This is likely to be a degraded intermediate.
2.4.7 Ectopic B cyclin -EGFPs activate Cdk1-mediated Bcl-2 phosphorylation
B cyclins are the principle activators of the master mitotic kinase Cdk1. To examine the activity of the ectopic cyclin B1- or B2-EGFP in the stable cell lines, we examined the ability of cyclin B1- or B2-EGFP to activate Cdk1-mediated Bcl2 phosphorylation. Bcl-2 is a member of Bcl-2 family which has anti-apoptotic properties[228] It is transiently phosphorylated by the cyclin B/Cdk1 kinase complex during normal mitosis [229,230].
The ability of cyclin B1- or B2-EGFP to activate Cdk1 can be determined by the phosphorylation level of Bcl2 in the presence of ectopic cyclin B1- or B2-EGFP during mitosis. To measure the intensity of phospho-Bcl2 induced by ectopic cyclin B1, stable cells as well as the parent cell line (HeLa Tet-Off) were transfected with specific or control siRNA, in the presence or absence of doxycycline. The expectation is that the cells express: 1) both endogenous B cyclins and ectopic cyclin B1- or B2-EGFP (control siRNA without Dox); 2) endogenous B cyclins (control siRNA with Dox); 3) ectopic cyclin B1- or B2-EGFP (specific B cyclin siRNA without Dox); 4) neither endogenous nor ectopic B cyclins (specific B cyclin siRNA with Dox). After 24 hr, cells were fixed and stained for the mitotic marker phospho-S10-histone H3 (pHH3), phospho-T56-Bcl2
73 (pBcl2) and DNA content, and analyzed by flow cytometry. Figure 3-4A shows the signal generated for G2 cells (R5) or mitotic cells (R2) from which the pBcl2 levels were calculated. The results show in figure 3-4B. As expected, the level of p-Bcl2 was unaffected by Dox treatment to the parent cell, but was markedly reduced by Dox treatment in cell lines expressing cyclin B1-EGFP (p006, p015 and p016) or cyclin B2-
EGFP (p010, p017). Specific siRNA treatment reduced pBcl2 levels by variable amounts, and siRNA+Dox treatment reduced levels maximally, except in the parent HeLa Tet-Off cells. Therefore, the EGFP fusion protein catalyzes Cdk1 activity equivalent to or greater than endogenous B cyclin proteins.
2.5 DISCUSSION
Tet-regulated gene expression system is a valuable tool that facilitates quantitative and temporal control of a gene of interest. To study the effect of cyclin B1 and B2 on mitotic entry and progression, we chose the tet-regulated gene expression system to conditionally express cyclin B1- or B2-EGFP. In this Chapter, I have documented the creation of unique cell lines and validated that with these, the levels of an active B cyclin-EGFP protein can be modulated after the endogenous B cyclins have been depleted. Following the transfection of tet-response plasmids into HeLa Tet-Off cells and antibiotic selection, the stable cell lines that express wild type cyclin B1- or B2-EGFP (p006 and p010), and siRNA-unresponsive cyclin B1- and B2-EGFP (p015, p016, and p017, p023) were successfully established with the tet-regulated gene expression system.
Several clones of each stable line were analyzed with flow cytometry, immunofluorescence microscopy, and western blotting to examine the expression level of
74 ectopic cyclin B1- or B2-EGFP. In the absence of Dox, variations of expression level in clones of some stable cell lines, as shown in Figure 2-5, might result from different vector integration sites when cyclin B1- or B2-EGFP cDNA was stably transfected into the genome of HeLa Tet-Off. This make sense for a minimal promoter that would be more sensitive to integration site effects than a strong promoter [231]. However, the minimal CMV promoter in the response element also minimizes background expression
[232]. In the presence of Dox, the basal level of ectopic cyclin B1 or B2 EGFP in most clones was reduced to an extremely low level, comparable to that of the control cell line.
The time course of Dox action (Figure 2-7A) has shown that induction of cyclin B-EGFP reached the maximum steady-state level within 13 hr after removal of Dox. When Dox was added, the expression of cyclin B-EGFP was reduced 50% within 9 hr and to the basal level within 17 hr. These data suggest that the expression of ectopic cyclin B-EGFP is regulated by Dox rapidly and efficiently. To assess the proper function of cyclin B1- or cyclin B2-EGFP in the stable cell lines, ectopic cyclin B-EGFP mediating Bcl-2 phosphorylation level was measured by flow cytometry. The data demonstrated that ectopic cyclin B1- or B2 –EGFP was as active as the endogenous B cyclins.
Furthermore, dose response data (Figure 2-7B) indicated that the expression of ectopic cyclin B-EGFP was quantitatively regulated by Dox when the concentration of Dox was between 0.002 to 0.25 ng/ml. siRNA depletion experiments demonstrated that ectopic cyclin B-EGFP with synonymous mutations at siRNA recognition sites specific to cyclin
B is resistant to siRNA knockdown (Figure 2-8). Combining the siRNA technology and
Tet-Off gene expression system allowed control of the cyclin B level in a set of stable-
75 expression cell lines. We tried to create stable cell line that inducible expression of cyclin
A2-EGFP. Unfortunately, clones failed to survive, suggesting overexpression of cyclin
A2 may be toxic to the cells.
In summary, using the Tet-Off system, I created stable cell lines with inducible expression of cyclin B1- or B2-EGFP recombinant genes. These recombinant genes could be made resistant to siRNA depletion by creating 4 or more synonymous mutations within the siRNA recognition sites in the expression plasmid. The levels of exogenous gene expression could be precisely monitored by flow cytometry and precisely modulated by doxycycline. RNAi could be employed to specifically deplete the endogenous B cyclins to background levels, and specific siRNA could be combined to facilitate experiments in which the effect of the quantity of cyclins B1 or B2 determine with respect to cell cycle progression (Chapter 3).
76 CHAPTER 3
Adapt from manuscript:
THE KINETICS OF G2 AND M TRANSITIONS REGULATED BY B CYCLINS
Yehong Huang, R. Michael Sramkoski, and James W. Jacobberger
Accepted by PLOS ONE, Decision# [PONE-D-13-32592R1]
3.1 ABSTRACT
B cyclins regulate mitotic entry and transition. Because human somatic cells continue to cycle after RNA interference (RNAi) mediated reduction of either cyclin B1 (cycB1) or cyclin B2 (cycB2) to <5% of peak levels, and because cycB2 knockout mice are viable, the existence of two genes should be an optimization. To explore this idea, we generated
HeLa BD Tet-Off cell lines with inducible cyclin B1- or B2-EGFP that were resistant to RNAi. Exponential cultures were treated with RNAi and/or doxycycline (Dox) and bromodeoxyuridine. We measured G2 and M transit times and 4C cell accumulation. In the absence of ectopic B cyclin expression, knockdown (kd) of either cyclin increased G2 transit. M transit was increased by cycB1 kd but decreased by cycB2 depletion. This novel difference was further supported by time-lapse microscopy experiments. Mitotic time reduction after cycB2 knockdown suggests that cycB2 tunes mitotic timing, and we speculate that this is through regulation of a Golgi checkpoint. In the presence of endogenous cyclins, expression of active B cyclin-EGFP did not affect G2 or M phase times. As previously shown, B cyclin co-depletion induced G2 arrest. Expression of either B cyclin-EGFP completely rescued knockdown of the respective endogenous
77 cyclin in single kd experiments, and either exogenous B cyclin-EGFP completely rescued endogenous cyclin co-depletion. Most of the rescue occurred at relatively low levels of exogenous cyclin expression. Therefore, cycB1 and cycB2 are interchangeable for ability to promote G2 and M transition in this experimental setting. Cyclin B1 is thought to be required for the mammalian somatic cell cycle and cyclin B2 is thought to be dispensable. However, residual levels of cyclin B1 or cyclin B2 in double knockdown experiments are not sufficient to promote successful mitosis, yet presumably lower residual levels are sufficient to promote mitosis in the presence of cyclin B2. We discuss the simplest model that would explain most data if cyclin B1 is necessary.
3.2 INTRODUCTION
Eukaryotic cell cycle progression is regulated by cyclin-dependent kinases (Cdks) and their regulatory, cyclin subunits [54,59,220,221]. Cdk cell cycle expression is proportional to cell mass in excess of cyclins, which are limiting and expressed periodically. This periodicity, in part, creates periods of activity for specific cyclin complexes that correlate roughly with cell cycle phases and/or major cell cycle events[222]. Assignment of cyclin/Cdk activity to major cell cycle events has been studied in most model organisms, and cyclin/Cdk complexes activate transcription
[233,234], enable DNA replication[235,236], and catalyze mitosis[222].
Cdc2 or cyclin-dependent kinase 1 (Cdk1) regulates mitotic entry and progression [237].
Expression of a kinase-dead mutant or immunodepletion causes G2 arrest in human cells
[61,98]. Conditional, down-regulation of Cdk1 stops HT2-19 human cell division and promotes endoreduplication[238]. Chemical inhibition of Cdk1 arrests interphase cells in
78 G2, but in mitotic cells, results in premature origin licensing and mitotic exit [239]. In mitosis, A and B type cyclins activate Cdk1. Cyclin A is required for G2 to M transition and nuclear envelope breakdown [128,223], however B cyclin is the principal activator of
Cdk1. Cyclin B-Cdk1 complexes are activated by a cdc25 phosphatase [167]. The activated complex then phosphorylates a large number of substrates to regulate sub- cellular events, including mitotic entry, chromosome condensation, nuclear envelope breakdown, spindle assembly, Golgi fragmentation, and the spindle checkpoint (reviewed in[237]. The complex is inactivated at the metaphase to anaphase transition when B cyclins are degraded by the anaphase promoting complex/cyclostome (APC/C)[240]. In mammals, there are three B cyclins: B1, B2 and B3. Cyclin B3 is expressed in the human testis and in developing germ cells in the mouse [131,132]. Cyclin B1 and B2 differ in the first 100 residues, and are 57% identical in the remaining sequences [153,241].
Mammalian cyclins B1 and B2 are co-expressed. They are detectable beginning in G1, rise slowly through S phase then rapidly in G2, peaking in late G2 or early M, and degraded approximately after metaphase [119,130,159,170]. Cyclin B1 shuttles between the cytoplasm and nucleus but is mostly cytoplasmic during interphase and mostly nuclear in prophase with initial activation on the centrosome [129,170,173,242]. Cyclin
B2 localizes to the Golgi apparatus and evidence supports a role in regulating Golgi fragmentation [170,180,243,244,245,246,247].
Different localization suggests different roles for cyclin B1 and cyclin B2, and exogenous expression in G1 cells coupled with amino termini swapping demonstrated that cyclin B1 regulated mitotic events like cell rounding, chromatin condensation, aster formation, and nuclear membrane breakdown while cyclin B2 regulated Golgi fragmentation. However,
79 cyclin B1 with a B2 amino terminus was capable of Golgi fragmentation while cyclin B2 with amino terminal B1 residues was not capable of nuclear mitotic functions despite apparently correct cytoplasmic localization [180]. Since these exogenous proteins were all expressed at about the same levels, the experiments suggested that localization may have a significant effect on substrate specificity, but the termini swapping also suggested substrate differences between the two cyclins that are not dependent on localization.
However, experiments with B cyclin Null mice have shown that cyclin B1 is required for the viability of embryos, but cyclin B2 is not [153]. Thus, mammalian B cyclins appear to have distinctly different functions; different substrates are implied, but cyclin B2 function is dispensable.
RNA interference (siRNA) experiments for both cyclins B1 and B2 in human somatic cell lines results in G2 arrest while knockdown of either cyclin B1 or cyclin B2 alone results in mild perturbations of G2 and M time[197,204]. Cyclin B1 depletion produces a delay in mitotic entry and transit, and we reported some evidence that cyclin B2 depletion might accelerate mitosis [204]. Thus, cyclin B2 appears to redundantly permit mitosis in cells that express less than 5% of peak Cyclin B1 levels in G2 [204]. These data fit models whereby cyclin B2 can either compensate for cyclin B1 loss or cyclin B2 can catalyze effective use of low, normally insufficient levels of cyclin B1. To explore the differential effects of these B cyclins on mitotic time, we examined quantitatively the effect of cyclin B1 or cyclin B2 expression on mitotic time and the ability of either cyclin to overcome the G2 arrest imposed by depletion of both B cyclins. To do this, we generated stable HeLa cell lines with inducible expression of cyclin B1-EGFP or cyclin
B2–EGFP recombinant genes that are not recognized by cyclin B1 and B2 siRNAs.
80 3.3 MATERIALS AND METHODS
3.3.1 List of stable cell lines used for experiments
Cells were single-cell sorted by FACS for B cyclin-EGFP or EGFP expression into 96 well plates. The following clones were used in this study: p001-1, (expresses EGFP); p006-14, p015-1, p015-4, p015-14 and p016-4 (express cyclin B1-EGFP); p010-5, p017-
4, and p023-1 (express cyclin B2-EGFP). p015-1, p015-4, p015-14 and p017-4 expressed fusion RNA encoding 4 synonymous mutations in the siRNA recognition site, and p016-4 and p023-1 expressed fusion RNA encoding 7 and 6 synonymous mutations, respectively. p006-14 and p010-5 expressed fusion RNA with wild-type sequences.
Since the same clones are used repeatedly throughout the study, the clone names have been abbreviated leaving off the clone number in the text and figures. Clone numbers are in Figure legends.
3.3.2 Cell culture and transfection
HeLa Tet-Off cells and stable cell lines established in chapter 2 were plated in 6 or 10cm tissue culture plates, maintained in RPMI (Invitrogen, catalog# 11835-055) supplemented with 10% fetal bovine serum (FBS, Gibco/Life Technologies, catalog #631101), 50 ug/ml gentamicin (Sigma, catalog# G1264-5G), 100 ug/ml G418 (not for HeLa Tet-Off cells), and 5ng/ml Doxycycline (Sigma, catalog# D9891) at 37oC in a humidified atmosphere containing 5% CO2.
The target sequences against cyclin B1 and cyclin B2 have been previously described
[204]. Briefly, oligonucleotides targeting cyclin B1 and cyclin B2 corresponding to the cDNA sequences of human cyclin B1 coding region 340-360 and human cyclin B2
81 coding region 845-855 were designed as recommended [248] and synthesized by Thermo
Scientific. For siRNA transfection, cells were plated in 6cm plates, and Lipofectamine
2000 (Invitrogen, catalog #11668-019) was used according to the manufacturers’ protocols.
3.3.3 Cell fixation, intracellular staining and flow cytometry
Cells were fixed with 0.125% formaldehyde in culture media at 37oC for 10 min, washed, then followed by ice-cold methanol (90% final) for at least 20 min at -20oC as previously described[226]. For indirect staining, 1-2 million cells were incubated with antibody for
o 1h at 4 C, washed twice with PBS/BSA (20mg/ml bovine serum albumin, 10 mM NaPO4,
150 mM NaCl), followed by the secondary antibody for another 1h at 4oC, and a final three washes. Finally, the cells were stained with 1 ug 4', 6-diamidino-2-phenylindole
(DAPI) for DNA staining and analyzed by flow cytometry. For direct staining, the second antibody step was omitted. The following antibodies and amounts were used for cell staining: cyclin B1, 0.125 ug GNS1 conjugated with Alexa Fluor 647 (A647) or
0.125 ug GNS1 unconjugated (clone GNS1, BD Biosciences, catalog #554177); cyclin
B2, 0.34 ug antibody (clone H105, catalog #sc-22776, Santa Cruz Biotechnologies), phospho-S10-histone H3 (PHH3), 0.125 ug conjugated with Alexa Fluor 488 (A488)
(catalog #9708) or A647 (catalog #9716, Millipore-Upstate); phospho-T56-Bcl2, 0.25ug
(catalog #2875, Cell Signaling Technology); 5-bromo-2'-deoxyuridine (BrdU), 0.5 ug conjugated with A647 (clone PRB-1, Phoenix Flow Systems); Secondary antibodies with labels A488, Alexa Fluor 555 (A555), phycoerythrin (PE), and A647 were obtained from
Invitrogen and used at 2:1 ratio w/w and 1ug DAPI (Invitrogen). Stainings were
82 performed in 50 ul phosphate buffered saline with 2% bovine serum albumin (BSA,
Sigma, catalog #A7030-100G).
Multi-color fluorescence emissions were measured using a BD Biosystems LSR II or
Beckman Coulter (Miami, FL) Gallios. Two color measurements of EGFP and DNA were made on a Beckman Coulter XL. Cell sorting was performed with a BD
Biosystems FACS Aria.
3.3.4 BrdU labeling, G2 and M phase transit time analysis
Cells were continuously labeled with 20 uM BrdU (Sigma, catalog B9285) for various periods of time. At specific times, cells were harvested and fixed for Brdu staining as previously described [204]. Unlabeled G2 and M phase cells were quantified as a percentage of the population by Boolean classification and histogram analysis. G2 and M phase transit times were calculated as described previously [204]except that data were normalized to a top of 1 and bottom of 0 and analyzed by non-linear regression using
1 decay equations following the form fx() 1 10m() t x where x = the fraction of unlabeled G2 or M cells and t = time in hours.
3.3.5 Time lapse experiments
Live cell images were obtained with a Leica AF6000 fluorescence microscope. Cells were cultured in glass-bottom dishes (ibidi, #81156) were placed into a covered sample chamber supplied with 5% CO2 and 37C for 30 min before acquiring images. Images were collected with a Hamamatsu C 10600-10B (ORCA-R2) camera through an HCX PL
83 FLUOTAR L 20x0.40 DRY objective lens. Images were captured with 100-800ms exposure times in 3 or 5 minute intervals for 18-36 hours.
3.3.6 Immunoblotting
Cells were lysed in whole cell lysis buffer (0.137 M saline, 2% NP-40, 20mM Tris, and 1% sodium dodecyl sulfate (SDS), 1% deoxycholate). 10-15 ug total protein was subjected to
8 or 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (EDM, Millipore, catalog #IPVH09120). The following primary antibodies were used according to the manufacturer’s instructions.
Mouse anti-cyclin B1; Rabbit anti-cyclin B2; Goat anti-cyclin B2 (N-20, Santa Cruz, catalog #sc 5235); Rabbit anti-human cyclin A (upstate biotechnology, catalog #06-138);
Mouse anti-Cdk1/Cdc2 (BD Transduction Laboratories, catalog #610038), Mouse anti-
GFP (Santa Cruz, catalog #SC 9996) and Rabbit anti--tubulin (Abcam, catalog
#ab6046-200). Alkaline phosphatase conjugated secondary antibodies were purchased from Promega. Membranes were developed by CDP-star chemiluminescent substrates
(Sigma, catalog #C0712-100ML). Kodak X-OMAT AR films (Eastman Kodak company, catalog #894 1114) were used to detect chemiluminescence.
3.3.7 Software
Multi-parametric cytometry fluorescence data were analyzed using WinList 3D 7.0 software (Verity Software House). Cell cycle analysis was performed with ModFit LT
3.1 (Verity Software House) using single trapezoid S phase components and floating G1 and G2+M means and standard deviations. Resulting statistics and some secondary calculations were compiled and performed in Excel 2007 (Microsoft Corporation).
84 Kinetic data were transformed and analyzed by non-linear regression in GraphPad Prism
5.04 (GraphPad Software). Prism was also used to construct x-y plots for Figures.
Bivariate histograms were configured with WinList 3D and complex Figures composed with Adobe Photoshop 11.0.2 (Adobe Systems, Inc). Time lapse images were processed using Leica AF6000 imaging software.
3.4 RESULTS
3.4.1 Characterization of stable cell lines in this study
In order to study the effect of cyclin B1 and cyclin B2 on mitotic time and G2 arrest, in chapter 2, we have created stable HeLa cells conditionally express human cyclins B1- and B2-EGFP with anonymous mutation at cylcin B1 or B2 specific siRNA recognition sites. Immunoblotting and flow cytometry showed that ectopic EGFP or B cyclin-EGFP proteins of the stable cell lines were expressed or repressed in the absence or presence of
Doxycycline (Dox), respectively (Figurer3-1). Time course experiments showed that expression of ectopic B cyclin-EGFP was maximal and stable by 13 hours after removal of Dox and minimal and stable by 17 hours after addition of 10 ng/ml of Dox (Figure 3-
2). Figure 3-2B showed that expression of cyclin B1-EGFP is in dose dependent manner.
Cyclin B1- or B2-EGFP with anonymous mutations is resistant to the cyclinB1 and B2 specific siRNA (Figure3-3). Ectopic cyclinB1- or B2-EGFP had B cyclin activity to mediate Bcl2 phosphorylation (Figure3-4).
3.4.2 Cyclin B2 is rate controlling for G2 and M transitions
85 We have shown previously that depleting the levels of cyclin B1 produces an increased fraction of G2 and M phase cells, and that these increased frequencies result from delays in G2 and M transitions. Further, we reported that cyclin B2 knockdown increases the fraction of G2 phase but decreases the M phase fraction. This suggested rate limiting functions for cyclin B2, but the effects were small[204]. Further, the reduced M phase fraction could indicate that the M transit rate was either unchanged or increased by cyclin
B2. This might make sense if a function of cyclin B2 is to regulate Golgi fragmentation, provided that Golgi fragmentation and dispersal is necessary, and that there is a checkpoint function involved[229]. To explore the rate limiting nature of cyclin B2, we performed kinetic experiments to determine G2 and M phase times for cells with depleted cyclin B2. Figure 3-5 shows typical kinetic data and Table 3- 2 shows the results of several experiments with two B2-EGFP cell lines and with a HeLa cell lab strain that is distantly related to the parental Tet-Off cell line. In all experiments, cyclin B1 depletion always resulted in lengthened G2 and M phases (data not shown). Most of the time, cyclin B2 depletion resulted in a longer G2 but shorter M, supporting our original observations[204]. In some experiments where cells were growing slower (tG2 > 3h), cyclin B2 depletion resulted in longer M phase times, therefore, the shortening of M by cyclin B2 depletion didn’t always occur, or there were experimental problems that we could not define. To investigate this further, we did a series of time-lapse analyses.
Average data for 7 experiments showed an average mitotic time of 81 and 50 minutes for control and cyclin B2 knockdown cells, respectively (N = 34; N = 38). The results were significant (p < 0.0001) using a Mann Whitney test, since the values were not normally distributed for knockdown cells. These experiments were done without a nuclear marker,
86 and we did not track cell density. We performed four more experiments after transfection of histone H2b-GFP to mark the chromatin. Each experiment showed significant differences after cyclin B2 knockdown with an average reduction in mitotic time of 23%
(range = 19 – 28%; p = 0.025, 0.0009, <0.0001, <0.0001); N = 12 and 15; 8 and 10, 45 and 48, and 20 and 11 for controls and knockdown, respectively). This last series was done at three different cell densities. The forgoing provide two lines of evidence for rate limiting roles for both B cyclins and opposing effects on M phase time.
3.4.3 Over-expression of B cyclins does not affect the rates of G2 and M transition
Unless the endogenous B cyclins are expressed at saturating conditions for their ability to affect cell cycle time, we expected that increasing the B cyclin concentrations would result in increased transition rates through G2 for both cyclins and also through M for cyclin B1. Presumably, (under the simplest model) expressing cyclin B2 at higher levels would decrease the M phase transition relative to controls. To address this, we performed kinetic experiments to examine effects of over-expression of B cyclin-EGFP on G2 and
M times in the conditional expressing cell lines. We compared the rates of transition in two cell lines for each cyclin and two experiments for each line. The cells were cultured with and without Dox, providing either endogenous cyclin levels (Dox) or over- expression of B cyclin-EGFP in excess of endogenous B cyclins. Figure 3-6A, C shows typical results and Figure 3-6B and D show the summary of experiments. In both cases, the transition rates in the presence of exogenous B cyclin were not significantly different from the rates in the presence of Dox, although the majority of the samples registered a slight increase in transition rates (faster transit). This indicates that endogenous cyclins
87 are expressed at "saturating" levels for the ability to control G2 and M transition times under exponential growth conditions. This could be considered tight regulation of B cyclin activities under "wild-type" conditions.
3.4.4 Over-expression of single B cyclins can restore cell cycling when endogenous B cyclins are depleted
Double knockdown of both B cyclins results in a profound G2 arrest followed by 4C cell cycles [197,204]. This profound arrest led us to conclude that either B cyclin could promote mitotic transition, since the arrest argued that any residual level of cyclin B1 in single-cyclin B1 depletion experiments was insufficient to catalyze mitotic transit unless normal levels of cyclin B2 were present. Therefore, with these conditional expression cell lines, we could ask whether a single cyclin could rescue cells from the profound G2 arrest and promote mitotic transition. B1 and B2-EGFP cell lines were treated with either control siRNA and Dox (expressing endogenous, normal levels of B cyclins) or B cyclin specific siRNA without Dox (over-expressing specific B cyclin-EGFPs). Eighteen hours after transfection, cells were continuously labeled with BrdU over a period of 10 hours, sampled and subjected to kinetic analysis. The results, shown in Figure 3-7, demonstrate that either B cyclin-EGFP could rescue cells from the effects of double siRNA mediated
B cyclin depletion. For both cyclins, the rescue was almost to the transition rates of controls for both G2 and M.
3.4.5 Expression of single B cyclins can completely rescue the G2 arrest phenotype caused by cyclin B1 and B2 co-depletion
88 The experiments presented above and in Figure 3-6 require many samples and are demanding. To further examine the ability of a single B cyclin to overcome cell cycle arrest induced by knockdown of both B cyclins, we measured the fraction of 4C cells by
DNA content flow cytometry after siRNA knockdown and Dox treatment. Cell lines, p015 and p017, were transfected with B cyclin specific siRNAs in the presence or absence of Dox for 24-30 hours, and the levels of endogenous B cyclin co-depletion was measured by immunoblotting (Figure3- 8A). Compared to control siRNA, endogenous B cyclins were barely detectable and cyclin A2 and Cdk1 levels were invariant. The reduction of cyclin B1, measured by flow cytometry as previously reported [204]was 99% of peak levels, and 67% of 4C cells displayed cyclin B1 immunofluorescence levels equal to that of late mitotic cells. Cyclin B2 knockdown is more difficult to determine, but our calculations based on immunofluorescence suggest that it approaches 100% for 80% of the 4C cells. Figure 3-8E shows the G2 arrest induced by siRNA treatment + Dox.
Figure3- 8D shows the control DNA content distribution when cells are not treated with siRNA. Figure3- 8 (B, C) shows an example of EGFP expression for p017 cells cultured without (B) or with (C) Dox. Figure3- 9 presents data for the two clonal cell lines and two experiments each. The fractions of 4C cells were measured by DNA content modeling as shown in Figure3- 8 (D, E insets). When both endogenous cyclins were knocked down and cells were treated with high concentrations of Dox (Figure 3-9, None), the fractions of 4C cells were between 2 and 3 times the fractions in cells treated with
Dox alone and expressing only endogenous B cyclins (Figure 3-9, Endo). When cells express both endogenous and exogenous B cyclins (Figure3- 9, Endo+Exo), the fractions of 4C cells were equivalent to cells expressing only endogenous cyclins - emphasizing
89 the previous conclusion that exogenous cyclins do not further affect cell cycle timing in the presence of endogenous cyclins. This is the case for both B cyclins. Full expression of exogenous cyclin B1-EGFP did not fully rescue the G2 arrest phenotype in these experiments, whereas cyclin B2-EGFP did (Figure 3-9, Exo). However, we noticed that cells that rescued least well were cells that either expressed lower levels of exogenous B- cyclin EGFP or the cultures contained S and G2 cells that were still negative for EGFP after full induction (no Dox). An example of this type of clone is shown in Figure 3-10A.
We then re-analyzed the primary data from the experiments of Figure3- 9, performing cell cycle analysis on only the EGFP positive fraction (R2 gate in Figure3- 10A). This is presumably equivalent to cell cycle analysis on cycling cells after the loss of Cdh1 activity in G1. The comparable results are presented in Figure 3-10C, D. In Figure 3-
10E and F, the ratios of 4C cells in the rescued cells to those in the knockdown are plotted versus EGFP expression levels. A ratio of 1 is equivalent to full rescue. The analyses presented in Figure 3-10 indicate that the co-depletion phenotype can be fully rescued when B cyclin EGFP expression is high enough. All of the analysis in Figure 3-
10 takes advantage of the fact that expression of exogenous B cyclin EGFP in the presence of endogenous cyclins does not alter cell cycle time (Figures3- 6, 3-8)
Therefore, we used another p015 clone, selected for high expression (p015-1) and sorted the p017 clone by FACS to obtain a sub-line that fully expressed high levels of B2 cyclin
EGFP. We then performed an experiment, measuring the 4C fraction of cells as a function of B cyclin EGFP expression dose. The results are presented in Figure 3-11.
For cyclin B1, expression of B1-EGFP could fully compensate the phenotype, and for
90 cyclin B2-EGFP, the fraction of 4C cells could be lowered significantly below the levels produced by both endogenous B cyclins. This experiment was repeated with similar results. The simplest explanation, especially in view of the data presented in Figure 3-7 for reduction of 4C cells, is that cells do not arrest in G2 and continue through M to G1.
This experiment also shows that in the double knockdown cells, full expression of either
B cyclin-EGFP is less than full expression of B cyclin-EGFP in control cells with endogenous cyclins. We do not know the reason for this, but checking all related experiments, the finding was consistent with about 15% less expression when the endogenous cyclins were not present.
3.5 DISCUSSION
The effects of B cyclin knockdown in cell lines are mild. We were surprised when we first observed the results of siRNA knockdown of cyclin B1[204]. We expected that cells that had not entered M would arrest in G2, and that cells in mitosis would prematurely exit mitosis, since it was known that high levels of cyclin B1/Cdk1 activity prevented mitotic exit[214]. We expected that we would see a high fraction of G2 phase cells and reduced or approximately zero M cells. Instead, we showed that >95% knockdown of cyclin B1 resulted in ~20% increases in the G2 and M transit times. An increased rate of endoreduplication was an associated cyclin B1 knockdown phenotype. We associated cyclin B1 knockdown with induction of high levels of maloriented chromosomes during metaphase (present only in HeLa cells) and sporadically increased cell death (present in both HeLa and RPE cells). In the same study, we also demonstrated that knockdown of cyclin B2 resulted in a slightly elevated G2 fraction and a reduced fraction of M cells.
91 Finally, we and others [197] showed that double knockdown of cyclins B1 and B2 resulted in a profound G2 arrest that resolved eventually by transit to a 4C cell cycle.
Bellanger et al. showed that this 4C phenotype could be reversed by transfection of cyclin
B2 [197]. We have shown quantitatively that it could be completely reversed.
Here, we focused on similarities and differences between cyclin B1 and B2 with respect to G2 and M transition rates. First, we completed the kinetic studies on cyclin B2 knockdown to provide more clarity to any differing cell cycle roles for the two cyclins, and second, we asked whether single cyclins could quantitatively compensate for the major effect of B cyclin co-depletion (G2 arrest). We found that the elevated fractions of
G2 cells were due to longer G2 transit times, which is expected for a mitotic cyclin. We also found that the lower levels of M phase cells, previously noted upon cyclin B2 knockdown, resulted from shorter M transit times. On average, for a 2.2 hour M phase time, knockdown of cyclin B2 resulted in an M phase that was 22 minutes shorter, or a reduction of 17% (Table 3-2). We supported this with similar results from time lapse microscopy that showed an average 23% reduction. This is opposite of the effect of cyclin B1 knockdown. It was an expected outcome based on our previous frequency results, but in general somewhat surprising. The G2 lengthening was expected, but the magnitude is high. The average increase was 1.1 hour longer than the 2.1 hour average
G2 time in control cells. This is approximately a 50% increase, which is the strongest B cyclin knockdown phenotype noted. Second, we asked whether over-expression of a single B cyclin could decrease transit times in the presence of endogenous cyclins. We could not find any evidence that either exogenous B cyclin was able to reduce G2 and M
92 times in the presence of endogenous cyclins (Figure3- 6). Thus, although knockdown experiments indicate that the B cyclins are rate controlling, the expectation that these molecules could produce the opposite effects when over-expressed was not demonstrated.
On another tack, we asked whether a single B cyclin could rescue the profound G2 arrest induced by knockdown of both endogenous B cyclins. The evidence from kinetic experiments is that either cyclin can rescue this phenotype (Figure3- 7). Expression of exogenous cyclin B2-EGFP nearly restored G2 transit time, but cyclin B2-EGFP could completely restore M transit, and in fact, reduce M transit times (Figure 3-7). This latter result is perplexing in that given the knockdown results, the simplest model would suggest that restoration of phenotype by B2 would lengthen M phase. However, the cyclin B2 knockdown experiments differ from the rescue experiments in that endogenous cyclin B1 is present in the single knockdown experiments. At this stage, the best explanation is that the system is more complex with respect to time than any simple model suggests. Expression of cyclin B1-EGFP in the absence of endogenous B cyclins nearly restored G2 and M transit times to those of endogenous cyclins (Figure3- 7). We supported these kinetic studies with experiments that monitored the fraction of 4C cells
(G2+M) in cells that expressed only endogenous cyclins (treated with Dox), cells with siRNA knockdown of endogenous cyclins + Dox suppression of exogenous B cyclins
(treated with B cyclin siRNAs and Dox), cells that expressed both endogenous and a single exogenous B cyclin (treated with control siRNA), and cells that expressed only a single exogenous B cyclin (treated with B cyclin siRNAs). These experiments reinforced the ability of either B cyclin to rescue the cell cycle arrest phenotype of double B cyclin knockdown (Figures3- 8 and 3-9). Finally, since we noticed that cells with partial
93 expression or lower expression of exogenous cyclins appeared to rescue less well (Figure
3-10), we tested the ability of either B cyclin to restore the cell cycle arrest phenotype of
B cyclin co-depletion by measuring the fractions of 4C cells as a function of single B cyclin-EGFP expression (Dox concentration). These results showed that when the expression of the exogenous B cyclin was high enough, cyclin B1 could completely restore the phenotype and cyclin B2 could exceed the phenotype - presumably by reducing G2 and M phase times (Figure3- 11). Thus, aside from the special phenotype of M transit shortening in the presence of endogenous cyclin B1, the two B cyclins appear completely redundant for the ability to regulate G2 and M phase transit times.
Since the experiments of Figure 3-11 were analyzed by cytometry on the same day and same instrument settings, the levels of EGFP expression are comparable, molecule for molecule. Curiously, cyclin B2 appears to be more potent for restoring the G2 arrest phenotype than cyclin B1. Cyclin B2 restores the levels of 4C cells to control levels at
4000 EGFP units while cyclin B1 requires 7800 units. This could be trivial in that cyclin
B2-EGFP could be more active than cyclin B1-EGFP. Since cyclin B2-EGFP was able to reduce the 4C cell fraction below the level of cells expressing endogenous cyclins only or cells expressing endogenous plus exogenous cyclins at lower levels than the endogenous + exogenous expressing cells, the fact that timing is more complex than simple models suggest is emphasized.
Implications for cell cycle models. There are three conceptual models for the general ability of cyclins to promote movement through the cell cycle. The first is a threshold model in which high affinity substrates can be phosphorylated at low cyclin
94 concentrations and lower affinity substrates are phosphorylated at higher cyclin concentrations, which occur at a later point in time. There is significant evidence for this model[214]. The model works best to explain organisms like fission yeast with a single mitotic cyclin and a single Cdk, but evidence supports the model in more complex organisms[172,249]. The second model is that specific cyclins direct Cdks to specific substrates. This model explains the periodic control by different cyclins and Cdks and is suited to organisms that display periodic expression of several cyclins and several Cdks
(e.g., mammals) or one Cdk (budding yeast). There is also significant evidence for this model[249]. Finally, a third model considers that cyclin localization affects the ability to phosphorylate substrates. Thus, premature activation of nuclear targets by cyclin
B1/Cdk1 are prevented (or, ensured) by dominant nuclear export until cyclin B1/Cdk1 activation at the beginning of mitosis[180]. These three models are not mutually exclusive and the most sensible, current model for mammalian cells is that all three concepts are in play[250].
Evidence for the role of localization is best illustrated by the B cyclins of mammals because localization is organelle-based and readily observed. Cyclin B1 is mainly cytoplasmic during interphase and abruptly accumulates in the nucleus during prophase, prior to nuclear envelope breakdown (NEB). In the cytoplasm, cyclin B1 is localized to microtubules and the centrosomes, and in the nucleus it is localized to chromatin and kinetochores. As mitosis progresses, cyclin B1 disappears from kinetochores first, then chromatin, and finally the spindle and centrosomes - the latter occurring at the same time as cyclin B1 degradation[251]. Cyclin B2 co-localizes with Golgi markers during
95 interphase but is cytoplasmic after Golgi fragmentation, and disperses throughout the cells after NEB [180]. A small amount could be shown to localize with the spindle at metaphase when cells were fixed with acetone/methanol and stained immunofluorescently[170]. We have detected both the cyclin B2-EGFP and endogenous cyclin B2 in the nucleus during mitosis prior to nuclear membrane breakdown and co- localized with centrosomes and the spindle (data not shown).
Currently, the division of function for the two somatic B cyclins rests with Golgi fragmentation for cyclin B2 and nuclear mitotic functions for cyclin B1, including important roles in nuclear membrane breakdown, spindle assembly, and spindle checkpoint induction and maintenance[237]. Cyclin B1 shuttles between the cytoplasm and nucleus until activation of cyclin B1/Cdk1. Then, a dramatic change in nuclear import rate occurs, and cyclin B1 is retained in the nucleus [174,175,242,252]. Peptide swapping experiments showed that a cyclin B1 fragment could be targeted to the Golgi by a cyclin B2 fragment, but cyclin B2 could not be targeted to the nucleus with an equivalent cyclin B1 fragment[180]. Equally, the localization of cyclin B1 to chromatin, kinetochores, the centrosome, and spindle could largely be attributed to specific peptide fragments[250].
Alternative models
If cyclin B1 is unnecessary for mitosis in human somatic cells - Model (1) (Figure 3-
Taking all of the forgoing into consideration, the simplest explanation for the high level of redundancy for G2 and M timing in B cyclin knockdown experiments is that in the
96 absence of cyclin B2, cyclin B1 can bind to the Golgi and function well enough to regulate Golgi fragmentation. In the absence of cyclin B1, cyclin B2 functions well enough to compensate for cyclin B1, at least in short term tissue culture. This should mean that cyclin B2 binds to centrosomes, and either prior or after NEB, to chromatin, kinetochores, and the spindle. Jackman et al. first reported human cyclin B2 Golgi localization[170]. In that paper, centrosomal-like objects can be seen to be stained, but were not reported on. In addition, an image was presented that shows possible nuclear and cytoplasmic staining but was either interpreted to be diffuse cytoplasmic staining or was interpreted as evidence complete re-location (like cyclin B1) was not occurring.
Binding to the spindle was observed. Further studies from the same group focused on microinjected G1 cells to obtain localization and functional information [180]. The functional evidence presented is very convincing for different actions of the two cyclins and supports essential mitotic functions to cyclin B1 (aster formation and NRB). We have evidence for the increased localization of cyclin B1 to Golgi when cyclin B2 is knocked down (Huang, Jacobberger, unpublished), suggesting that this part of the model is consistent. Since cyclin B1/Cdk1 has been observed to induce Golgi fragmentation
"better" than cyclin B2[180], the decrease in M phase transit time that we observe when cyclin B2 is knocked down could result from an ability of cyclin B1 to fragment the
Golgi more efficiently (therefore faster) and satisfy a Golgi fragmentation checkpoint sooner [175]and thereby increase M transit rate (in absence of cyclin B2). This is speculative, since the putative Golgi checkpoint is only known to arrest cells in G2, and the rate limiting function we are concerned with should be in early mitosis[243]. We do not have evidence for increased localization of cyclin B2 to cyclin B1 structures;
97 however, we have observed cyclin B2 immunostaining localized to the Golgi, centrosomes, the mitotic spindle, and in the nucleus in control and single knockdown experiments. We have also observed cyclin B2-EGFP localized to these same structures and observed nuclear entry at the beginning of mitosis in time lapse experiments. Thus, there are few arguments that cyclin B2 would not be in the right place to perform cyclin
B1 functions (we have not detected it on kinetochores). However, the functional of study of Draviam [180] argues that cyclin B2 would be very inefficient regulating spindle assembly or NEB.
If cyclin B1 is necessary for mitosis in mammalian somatic cells - Model (2)
Recently, unpublished data was cited [172] to indicate that mouse cells genetically null for cyclin B1 arrest in G2. If this finding is supported, then a working model of full redundancy would be incorrect for mouse cells, and either a major difference between mouse and human somatic cells exists, or a different model is needed to explain all data.
Assuming similarity between mouse and human, critical high affinity substrates that are phosphorylated at low levels of cyclin B1 (less than 3-5% of prophase levels) need to be postulated. The residual levels of cyclin B1 in double knockdown experiments would have to fall below that threshold, and for the simplest model, in single cyclin B1 knockdown experiments, the achieved knockdown levels would necessarily be above that critical threshold level. This possibility is the opposite of expected since double knockdown experiments are less efficient than single knockdown experiments [204]. An attractive but more complex model is to postulate at least one critical substrate with primary and secondary B cyclin/Cdk1 phosphorylation sites. The primary site(s) would
98 be low affinity and phosphorylated by high levels of either B cyclin, and phosphorylation would be required prior to phosphorylation of the secondary site(s). The secondary site(s) would be cyclin B1/Cdk1-specific, high affinity site(s) that could be phosphorylated by the low, residual levels of cyclin B1/Cdk1 after knockdown. Such substrates would explain the single and double knockdown data equally well and any data supporting a necessary role for cyclin B1. Because the high affinity site(s) would require low levels of cyclin B1, single cyclin B1 knockdown would result in continued cycling. The low affinity site(s) would be phosphorylated by high levels of cyclin B2/Cdk1 and the high affinity site(s) would be phosphorylated by residual cyclin B1/Cdk1. In double knockdown, neither cyclinB1 nor B2 would reach a level needed to phosphorylate the low affinity site(s), and cell cycle arrest would occur despite the presence of enough residual cyclin B1/Cdk1 to phosphorylate the secondary high affinity site(s).
The idea that the B cyclins are 100% redundant does not seem very likely given the long evolutionary time in which fitness should select for the most efficient system. Currently, our kinetic results suggest that there is a cost to the loss of cyclin B2 - less efficient transition into M and a faster rate through M. Both of these features might reduce to an ability to modify and optimize the putative Golgi fragmentation checkpoint [229,243].
99
100 CHAPTER 4
LOCALIZATION OF CYCLIN B1- OR B2-EGFP WHEN LACK OF CYCLIN B2 OR B1
4.1 ABSTRACT
Cyclin B1 and B2 are the key mitotic cyclins in mammalian somatic cells. At least one B type cyclin is required to promote G2/M phase transition and M phase progression, and there is evidence that cyclin B1 is essential for mammalian embryogenesis. Most authors have assumed that cyclin B1 is also required for somatic cell division as well. Either cyclin B1 or B2 can promote mitotic entry with different role on regulating mitotic timing.
Cyclin B1 and B2 regulate mitotic events though activating Cdk1 kinase and phosphorylating their substrates. Subcellular localization of cyclin B1 and B2 could play an important role on the function of cyclin B1 and B2 during mitosis. It is known that the wild type cyclin B1 is primarily localized in the cytoplasm in the interphase, and localized to centrosomes, chromatin, the spindle and kinetochore during mitosis, while cyclin B2 is primarily localized with the Golgi during interphase. In this study, we confirmed that ectopic cyclin B1- and cyclin B2-Cdk1 are localized on the correct localization by time lapse microscopy and fluorescence confocal microscopy. In addition, we find that cyclin B2, like cyclin B1, is localized to chromosomes and associates with centrosomes, chromatin, and the spindle during mitosis. These data partially explain why cyclin B1 and B2 are able to complement each other for mitotic entry and progression in siRNA experiments.
101 4.2 INTRODUCTION
Mitotic entry and progression are regulated by two post-translational mechanisms: protein phosphorylation and protein degradation. The precise temporal activation of mitotic protein kinases results in phosphorylation of particular substrates at specific stages to properly execute distinct mitotic events. The mitotic protein kinases include the cyclin-dependent kinases, Cdk1, the polo-like kinases, aurora kinases, and the NIMA family. Cdk1 is the most prominent mitotic kinase, and cyclins A and B are the cyclins that bind and activate Cdk1. Different cyclin-Cdk complexes phosphorylate different proteins relying on different cyclins to select substrates containing different motifs.
Cyclin A directly interacts with substrates containing an Rxl/Cy motif [253], while cyclin
B phosphorylates substrates containing an S/TRxK/R motif [65]. Compared to A cyclins,
B cyclins have a higher intrinsic Cdk1 kinase activity ([44]. In addition to substrate selection, differential sub-cellular localizations of cyclins A and cyclin B could provide substrates selection despite substrate binding overlap [129]. This may be important for the sub-types of A or B cyclins. B-type cyclins, which include cyclins B1 and B2, are the principle mitotic cyclins in somatic mammalian cells. Cyclin B1 and B2 show some identical respects during the cell cycle. Although they are different in first N terminal 100 amino acid sequences, they are 57% identical in the C terminal 300 amino acid. Both cyclin B1 and B2 accumulate through S and G2 phase, peak at mitosis and become undetectable in late mitosis through most of the G1 phase [116,170]. They possess a conserved cyclin box for binding to the Cdk1 subunit, and a conserved D-box for APC/C- mediated destruction. Their associated Cdk1 protein kinase activity is abruptly raised as
102 cells enter mitosis and rapidly lost as the cyclin is degraded from at metaphase to anaphase transition [170].
Cyclin B1 and B2 are thought to have overlapping and distinct functions in mitosis, largely because they are localized differently within the cell [170]. In the interphase, cyclin B1 is primarily cytoplasmic. It co-localizes with microtubules [170] and is also associated with centrosomes throughout its expression range [171]. Phospho-specific immunoblotting and immunofluorescence microscopy indicated that the cyclin B1-Cdk1 complex was first activated on centrosomes in prophase [198]. Centrosomes may increase the local concentration of cyclin B1-Cdk1 so that accelerate mitotic entry. [198]. At the beginning of prophase, cyclin B1 is rapidly trans-located into nucleus just before nuclear envelope breakdown [129,254], then associates with the mitotic spindle, chromosomes, and unattached kinetochores during mitosis [129,251,255,256]. Several Cdk1 substrates are localized in the chromosome. These include a subunit of condensin I, the phosphorylation of which is required for chromosome condensation [257,258]; nuclear lamins, the phosphorylation of which causes nuclear envelope disassembly [259,260]; the chromatin-associated protein, RCC1, which is important for spindle assembly [261], and separase, which when phosphorylated initiates anaphase onset [262]. Cdc20 is a likely
Cdk1 substrate at unattached kinetochores before chromosomes attach to the mitotic spindle. Cdk1 may maintain transient interaction of Cdc20 and the spindle assembly checkpoint proteins Mad2 to restrain APC/C activation [263]. Correlated to the localization of cyclin B1, cyclin B1-Cdk1 regulates chromosome condensation, nuclear envelope breakdown (NEBD), mitotic spindle assembly, the spindle assembly checkpoint,
103 and organelle reorganization [129,237,255,264] . Cyclin B2 is thought to localize primarily with the Golgi apparatus in interphase and is dispersed into cytoplasm as small vesicles during mitosis [129,170]. Cyclin B2 induces Golgi apparatus disassembly [180].
Golgi complex fragmentation may constitute a novel checkpoint for G2/M transition
[265,266]. Replacing of N terminus of cyclin B1 with region of cyclin B2, which is required to target cyclin B2 to the Golgi, directed a cyclin B2-B1 chimera to the Golgi apparatus [180]. The chimera was able to induce Golgi disassembly but unable to cause chromosome condensation and NEBD [180]. This observation further supported the idea that subcellular localization cyclin B1 and B2 controls different function of cyclin B1- and cyclin B2-Cdk1 complex in mitosis.
The studies in Chapter III show that cyclin B1 and B2 have different role on mitotic timing and that single cyclin B1- or B2-EGFP can promote successful G2/M transition.
To further characterize the fidelity with which these fusion proteins function, here, localizations of cyclin B1 and B2-EGFP expressing in the stable cell lines were examined during the cell cycle. Cyclin B1-EGFP was localized to the cytoplasm and centrosomes until mitosis in which it was translocated to the nucleus and associated with chromosomes, centrosomes, kinetochores and spindle similar to the endogenous cyclin
B1. Cyclin B2-EGFP was localized to the cytoplasm and Golgi, similar to endogenous cyclin B2. In contrast to previous studies, we found that both endogenous cyclin B2 and cyclin B2-EGFP translocated to the nucleus at the beginning of mitosis. To determine whether any differences occurred upon loss of either cyclin B1 or B2, we developed a quantitative assay to determine the the amount of cyclin B1 on the Golgi in the presence
104 or absence of cyclin B2 by laser scanning cytometry., This method will be applied to study the localization of cyclin B1 to the Golgi when cyclin B2 knocked down.
4.3 MATERIALS AND METHODS
4.3.1 Cell culture
HeLa Tet-off cells (BD Biosciences Clontech, Mountain View, CA) were cultured in
RPMI 1640 medium (Invitrogen) supplemented with 10% tetracycline-free fetal bovine serum (FBS) (BD Biosciences Clontech), 50ug/ml gentamicin sulphate (Sigma-aldrich,
St. Louis, MO) and 100ug/ml G418 (Fisher Scientific). p015, p016, p017 and p023 cell lines were cultured in RPMI-1640 supplemented with 10% FBS, 50ug/ml gentamicin sulphate, 100ug/ml G418, 100ug/ml Hygromycin (BD Biosciences Clontech), and
10ng/ml Doxycycline (Sigma-aldrich). All cells were grown at 37oC in a humidified incubator under 5% CO2. For time lapse imaging, laser scanning cytometry and confocal microscopy, cells were culture on 35mm coated glass bottom dishes (Ibidi).
4.3.2 Plasmids, small interfering RNA (siRNA) constructs and transfection
Histone H2B-GFP and H2B-RFP were purchased from BD Biosciences. siRNA sequences targeted to human cyclin B1 and B2 were synthesized by Thermo Scientific.
Oligonucleotides targeting cyclin B1 correspond to the cDNA sequences of human cyclin
B1 coding region 340-360, as follows: 5’-AAACTTTCGCCTGAGCCTATT-3’.
Oligonucleotides targeting cyclin B2 correspond to the cDNA sequences of human cyclin
B2 coding region 847-866 as follows: 5’-CAGCACACTTTAGCCAAGT-3’. Scrambled oligonucleotides with approximately 50% G+C content was purchased from Dharmacon
105 RNA Technologies and used as negative control. For transfection, cells were plated in
35mm ibidi dishes (30, 000 per dish) in RPMI medium supplemented with 10% FBS only, and allowed to proliferate for 24 hr. Before transfection, the medium was replaced in each dish with 1.5ml RPMI medium plus 10% FBS. Transfection was performed using lipofectamine T2000 reagent (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. Opti-MEM (Invitrogen), plasmid or siRNA and lipofectamine were mixed and incubated at room temperature for 20 min and then added to the cells. After 6 hr of incubation, the transfection mixtures were removed and replaced with 2ml fresh medium. The cells were incubated in the incubator until performing assay. In some experiments, when siRNA and plasmid co-transfection were required, two steps of transfection was applied to reduce the risk of cell death caused by reagent toxicity. Briefly, siRNAs were transfected to cells alone. After 24 hr post- transfection, medium was replaced with fresh medium and then the plasmids were transfected to cells.
4.3.3 CellLight○R Golgi-RFP living cell transduction
CellLight○R Golgi-RFP BacMam 2.0 was purchased from Invitrogen. The day before labeling, cells were plated in 35 ibidi dishes (30,000 per dish) with 2ml culture media and allowed to grow overnight. To transduce CellLight○R in to cells, 18ul reagent was added
(the viral reagent worked best with cells about 30 particles per cell) directly to the cells in complete cell medium and mixed well. Cells were incubated in the culture medium with
CellLight○R reagent in the incubator overnight (≥16 hr) before performing assays. For live cell imaging, fresh culture medium was replaced before imaging. For indirect
106 fluorescence imaging, cells were fixed with 1% formaldehyde solution in PBS for 25 minutes at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 5 minutes at room temperature, followed by the antibody staining.
4.3.4 Cell fixation and intracellular staining
Cells in ibidi dishes were fixed with 0.25% formaldehyde solution in cell culture medium for 10min at 37oC, washed and then dehydrated and permeabilized with methanol for at least 20 min at -20oC. CellLight Golgi-RFP transduction cells were treated differently as described previously. For intracellular staining, fixed cells were washed twice with PBS and once with PBS/BSA. Cells were then incubated with antibody for 1hr at 4oC, washed twice with PBS/BSA, followed by the secondary antibody for another 1hr at 4oC, and a final three, 15 minutes wash. Finally, the cells were stained with 0.5ug/ml 4’, 6- diamidino-2-phenylindole (DAPI) (Invitrogen) or 7-amino-actinomycin D (7-AAD)
(Invitrogen) PBS for DNA staining. For conjugated primary antibodies, the second antibody step was omitted. The following antibodies and amounts were used for cell staining: cyclin B1, 0.125 ug GNS1 conjugated with Alexa Fluor 647 (A647) or 0.125 ug
GNS1 un-conjugated (BD Biosciences, San Jose, CA); cyclin B2, 0.34 ug antibody
(Santa Cruz Biotechnologies, Dallas, Texas ), phospho-S10-histone H3 (PHH3), 0.125 ug conjugated with Alexa Fluor 488 (A488) or A647 (Millipore-Upstate, Billerica, MA); y- tubulin, 0.25ug ( Sigma ). Secondary antibodies labeled with A488, Alexa Fluor A555
(A555), and A647 were obtained from Invitrogen and used at 2:1 ratio (w/w, secondary:primary). Staining was performed in 150 ul phosphate buffered saline with 2% bovine serum albumin (BSA, Sigma).
107
4.3.5 Time lapse microscopy
Live-cell images were obtained with a Leica AF6000 fluorescence microscope. Cells in
o ibidi dishes were placed into a covered sample chamber supplied with 5% CO2 and 37 C for 30min before acquiring images. Images were collected with a Hamamatsu C 10600-
10B (ORCA-R2) camera and using an HCXPLFLUOTARL 20.0x0.40 DRY objective lens. Images were captured with 100-800ms exposure times in 3 or 5 minute intervals for
18-36 hours. Images were processed using Leica AF6000 imaging software.
4.3.6 Confocal microscopy
Indirect immunofluorescence images were acquired with a Zeiss Confocal Laser
Scanning Microscope LSM 510 Meta equipped with a Plan-Apochroma 63x (1.4NA) oil objective lens. The images were captured as 12 bit signals with a resolution of
1024x1024 pixels with zoom 1.6. All instrumental parameters pertaining to fluorescence detection were held constant to allow sample comparison. Images were processed using the LSM image browser.
4.3.7 Laser scanning cytometry
The iCyte Automated Imaging Cytometer (Compucyte Corp, Boston, MA) with iNovator software and iBrowser Data Intergration Software were used for fluorescent image acquisition and data processing. The iCyte was equipped with 405nm, 488nm, and
633nm lasers. A 40X objective lens was selected throughout the experiments.
108 4.3.8 Immunoblotting analysis
Cells were lysed in whole cell lysis buffer (0.137 M saline, 2% NP-40, 20mM Tris, 1%
SDS, 1% deoxycholate). 10ug total protein were separated by using 10% SDS-PAGE and transferred to PVDF membrane (EDM, Millipore, Billerica, MA). The following primary antibodies were used according to the manufacturer’s instructions. Mouse anti-cyclin B1
(BD Biosciences, St. Louis, MO), rabbit anti-cyclin B2 (Santa Cruz, Dallas, TX) and rabbit anti--tubulin (Abcam, Cambridge, MA). Alkaline phosphatase conjugated secondary antibodies were purchased from Promega. Secondary antibodies were diluted at 1:10,000 in PBS with 0.2% Iblock (Invitrogen). Membranes were developed by CDP- star chemiluminescent substrate (Sigma). Kodak X-OMAT AR films (Eastman Kodak
Company, Rochester, NY)) were used to detect chemiluminescence. The images were analyzed and quantified by Image J software.
4.4 RESULTS
4.4.1 Cytoplasmic cyclin B1-EGFP concentrates on the centrosomes during interphase and translocates to the nucleus at beginning of mitosis
In somatic mammalian cells, expression of cyclin B1 begins in G1 but increases exponentially such that in late S and G2 phases the levels are observable microscopically[160] and it is degraded at metaphase to anaphase transition [116,267].
Cyclin B1 is primarily cytoplasmic, associated with microtubules and centrosomes in interphase, and translocates to the nucleus at beginning of mitosis [129,171]. In mitosis, cyclin B1 binds to mitotic structures such as condensed chromosomes, centrosomes ,the spindle, and kinetochores [129,171,180,190,251,255]. To examine the expression and
109 subcellular distribution of ectopic cyclin B1-EGFP, time lapse microscopy was performed with live p015 cells transfected with the histone H2B-mCherry plasmid in the absence of Dox. Images of a typical cells in each stage of cell cycle is shown in Figure 4-
1A. Cyclin B1-EGFP fluorescence was observed in the cytoplasm and associated with small centrosome-like dots in interphase cells (Figure 4-1A, top row arrows). It was nuclear-localized in late prophase cells; chromosome-associated in metaphase and absent in anaphase (Figure 4-1A), In prophase through metaphase, the single cyclin B1-EGFP centrosome-like structures separated into two structures, eventually resting at the apexes of the spindle, identifying these as centrosomes (Figure 4-1A, top row) and at metaphase,
B1-EGFP fluorescence was observed binding to the metaphase spindle. The data show that the subcellular localization of cyclin B1-EGFP was the same as what has been previously shown for wild-type cyclin B1.
The subcellular localization of ectopic cyclin B1-EGFP described above was examined in a state of overexpression (full expression) and in presence of endogenous cyclin B1. To exclude the possibility that the overexpression of B1-EGFP caused artifactual localization, we examined the subcellular distribution of cyclin B1-EGFP at a level comparable with endogenous cyclin B1 and in the absence of endogenous cyclin B1. The ectopic cyclin
B1-EGFP level is tightly regulated by Doxycycline in p015 (Chapter 2). To determine the concentration of Dox that apply to the p015 to induce cyclin B1-EGFP to approximately the same level of endogenous cyclin B1, p015 cells were treated with different concentrations of Dox for 24hr and immunoblotted with anti-cyclin B1 antibody to detect endogenous cyclin B1 and ectopic cyclin B1-EGFP. These experiments showed that
110 when 0.20ng/ml Dox was added, the level of ectopic cyclin B1-EGFP, expressed by p015, was equivalent to endogenous cyclin B1 (Figure 4-2A and 4-2C). Time lapse microscopy was performed with p015 cells treated with cyclin B1 specific siRNA in the present of
0.20ng/ml Dox. Cells in interphase, G2 phase, and mitosis can be distinguished by the number (one or two) and migration of centrosomes, and differential interference contrast
(DIC) of cell, as centrosome matures in the S phase, duplicates completely in the G2 phase, and begins to separate at beginning of mitosis [268]. In the absence of endogenous cyclin B1, ectopic cyclin B1-EGFP in an endogenous cyclin B1 level was observed to colocalize with centrosomes in interphase translocate to the nucleus in prophase, and association with the spindle and chromosomes at prometaphase and metaphase (Figure 4-3). Maybe cyclin B1 localizes with chromatin at metaphase for a very short time and at 5 minute intervals in time-lapse microscopy, it is hard to capture this moment, the cell shown in figure 4-3B was not observed until cyclin B1-EGFP was chromatin-associated at metaphase. However, some cells showed cyclin B1-EGFP colocalized with chromatin at metaphase as shown in Figure 4-1C.
Unlike cyclin B1-EGFP expressing p015 cells, the fluorescence of pTRE2-EGFP transfected Tet-Off cells in time lapse experiments showed that EGFP is expressed in both the cytoplasm and nucleus throughout the cell cycle (Figure 4-4), indicating that the unique behavior of the cyclin B1-EGFP fusion protein is regulated by the cyclin B1 sequences and is exact accord with wild type cyclin B1 but not EGFP. All these data confirmed that ectopic cyclin B1-EGFP expressing from p015 either in the presence or absence of endogenous cyclin B1 confers cyclin B1 characters as described in the
111 previous studies, that is: localization to the cytoplasm and centrosomes in interphase and translocation into nucleus and association with chromosomes, centrosomes, and the metaphase spindle [129,190,251,255,269,270]. We attempted co-localization experiments with a kinetochore marker (CENP-A), and those experiments were suggestive but inconclusive. Unlike wild type cyclin B1, ectopic cyclin B1-EGFP was expressed at high levels in G1 (Chapters 2, 3), which is consistent with the promoter of the plasmid used to express the fusion protein. This is not thought to confer any phenotype on the cell, because cyclin B1-EGFP-Cdk1, like endogenous cyclin B1-Cdk1, is activated at the G2-
M transition by abrupt reduction in the activity of Myt1 and Wee1 and an abrupt increase in the activities of the Cdc25 enzymes (see Introduction and Chapter 2). The cyclin B1-
EGFP fusion protein is degraded from metaphase to a point in G1 (Chapters 2, 4), which is likely to be when APC/Cdh1 becomes inactive.
4.4.2 Cyclin B2-EGFP localizes to Golgi apparatus and centrosomes in interphase and translocalizes to the nucleus at the beginning of mitosis
Wild type human cyclin B2 localizes to the Golgi apparatus [170]. Under the fluorescence microscope, cyclin B2-EGFP expressed by p017 cells was observed to localize in the cytoplasm and accumulate at a perinuclear structure with a pattern like
Golgi (data not shown). To confirm that this structure was the Golgi, we examined the localization of cyclin B2-EGFP, expressed the same level as endogenous cyclin B2 by immunostaining microscopy. The concentration of Doxycycline 0.40ng/ml that induced cyclin B2-EGFP equivalent to endogenous cyclin B2 levels in p017 stable cells was determined by immunoblotting with cyclin B2 antibody with p017 cells in the presence
112 of different concentrations of Dox (Figure 4-2B and 4-2D). CellLight Golgi-RFP is a modified baculovirus expressing a fusion construct of a Golgi-specific marker and red fluorescent protein to enable visualization of the Golgi apparatus in live cells.
Transducing p017 cells with CellLight Golgi-RFP in the presence of 0.40ng/ml Dox, and monitoring by confocal microscopy, we observed ectopic cyclin B2-EGFP co- localization with the Golgi region in interphase cells, and dispersion into cytoplasm during M (Figure 4-5). S, G2 phase and mitosis can be distinguished by the morphology of the Golgi, as Golgi begins to ribbon breaking at G2 phase. In mammalian cells, the
Golgi apparatus is composed of stacks of cisternae that are connected to form a continuous membranous system , known as the Golgi ribbon [271]. We found that Golgi was intact in S phase and started disassembly in the G2 phase when Golgi ribbon breakdown into isolated stacks as previously described [272,273]. At prophase, these stacks relocate to the nucleus and undergo unstacking, and then at metaphase the segments are further fragmented and dispersed, appearing as the Golgi “haze”. Our results agreed with the previous studies [274,275,276].
The live cell time lapse imaging experiments show that ectopic cyclin B2-EGFP expressed by p017 in the absence of Dox began to express from G1/S phase and degraded at anaphase like wild type cyclin B2 (Figure 4-6A). What surprised us was that cyclin
B2-EGFP was localized to the centrosomes in the interphase, and underwent a dramatic redistribution into the nucleus at prophase, Cyclin B2-EGFP showed identical subcellular localization of wild type cyclin B1 during mitosis that is association with centrosomes, chromosomes, and the spindle during the mitosis (Figure 4-6A). In
113 metaphase, cyclin B2-EGFP bound to the spindle and to condensed chromatin was observed in some cells, although the concentration on both structures appears to be only slightly higher than the surrounding cytoplasm (Figure 4-6B). The localization of ectopic cyclin B2-EGFP expressed at a level equivalent to endogenous cyclin B2 in the absence of endogenous cyclin B2 was also determined by time lapse microscopy. Similar results were obtained as shown in figure 4-6C and 4-6D. These results varied from previous published observations that wild type human cyclin B2 was primarily located to Golgi apparatus and did not translocate to the nucleus at mitosis [170,180]. However, one of these two studies showed that cyclin B2 did change its localization to diffuse throughout most of the cell prophase [170]. Since these results conflicted with the literature on wild- type cyclin B2, and since EGFP can accumulate in the nucleus (Figure 4-4), we measured the subcellular localization of wild type human cyclin B2 in HeLa Tet-Off cells. The cells were co-immunostained with anti-cyclin B2 and anti-γ-tubulin antibodies and localization of cyclin B2 was determined by confocal microscopy. The results are presented in Figure 4-7. In this experiment, the centrosomes, identified by anti-γ-tubulin fluorescence, were also labeled with anti-cyclin B2 in G2, prophase, prometaphase and metaphase cells, confirming the localization of endogenous cyclin B2 to centrosomes
(Figure 4-7A). In G2 and prophase cells, cyclin B2 showed cytoplasmic and a perinuclear distribution (Figure 4-7A). In prophase cells, cyclin B2 was observed to distribute into nucleus and in prometaphase it was associated with chromosomes, and in metaphase cells, cyclin B2 was found to localize to the metaphase spindle, but association with condensed chromatin was not present (Figure 4-7A). To further clarify whether cyclin B2 moves to the nucleus at mitosis, three-dimensional confocal imaging
114 technique was applied to the mitotic cells. The three-dimensional reconstruction of prophase cell images confirmed that cyclin B2 underwent a redistribution into the nucleus
(Data is not shown). We noticed that different cyclin B2 antibodies were used for cyclin
B2 recognition and different fixation and permeabilization methods were used before antibody staining between us and other groups who did not observe cyclin B2 relocalization to the nucleus. These differences may result in the different results. Some epitopes of cyclin B2 could be masked after nuclear entry. It is also possible that the residence time in the nucleus is short and therefore, only a few cells can be detected at any one time. However, cyclin B2 localized to the nucleus and to similar mitotic structures during mitosis helps to explain the redundancy of cyclin B1 and B2 for mitotic entry and progression. These data support a model whereby cyclin B2 promotes mitotic entry and progression by recognizing cyclin B1 substrates localized in the nucleus in the absence of cyclin B1.
4.4.3 Study the localization of cyclin B1 to the Golgi when cyclin B2 knockdown
The studies in Chapter 3 showed cyclin B1 and B2 are redundant on mitotic entry and progression within the context of co-depletion of endogenous cyclins by siRNA. We were therefore interested in knowing whether cyclin B1 relocalizes to Golgi when cyclin
B2 is absent. Colocalization of wild type cyclin B1 to the Golgi in the presence of endogenous cyclin B2 was first determined by induction of CellLight Golgi-RFP in HeLa
Tet-Off cells and immunostaining the cell with cyclin B1 antibody. It was found that in interphase cells, cyclin B1 staining was in the cytoplasm and most notably in the perinuclear region, and the perinuclear distribution of cyclin B1 was partially localized to the Golgi (Figure 4-8). In prophase cells, the Golgi began to disperse to the nucleus and
115 cyclin B1 associated with Golgi at centrosomes area, and in metaphase cells, the Golgi appeared as “haze” around the chromatin where cyclin B1 was (Figure 4-8).
As cyclin B1 is partially co-localized at the Golgi, probably the centrosome, it is difficult to tell the difference between association of cyclin B1 to the Golgi between in the presence and absence of endogenous cyclin B2 by immunofluorescence. Thus, we developed a quantitative assay for measuring the association of cyclin B1 to the Golgi using laser scanning cytometry. This assay takes the advantage of quantitative laser scanning cytometry and a “seeded watershed” segmentation algorithm that could define the amount of cyclin B1 in the area occupied by the Golgi and the entire cell (Figure 4-
9A). The ratio of intensity of cyclin B1 fluorescence on the Golgi to that in the whole cellis is taken as a quantitative measure of association of cyclin B1 to the Golgi. The higher the ratio is, the more cyclin B1 on the Golgi. The ratio was measured by laser scanning cytometry with p015 cells with Dox (mimic the wild type HeLa Tet-Off cells status) or without Dox in the presence or absence of endogenous cyclin B2 in G2 and M phase cells. G2 and M phase cells were defined as by DNA content distribution (R3 region in Figure 4-9Ba). The intensity of cyclin B1 in the whole cell was measured from the plots of green fluorescence intensity (fluorescence/area) for the G2 and M phase cells
(Figure 4-9Bb), and the intensity of cyclin B1 in the Golgi was measured form the plots of green fluorescence intensity within the Golgi, defined by the RFP-Golgi area (Figure
4-9Bc). Our primary data showed that: in three experiments, with Dox (normal cyclin
B1), when cyclin B2 was knocked down, there was an increase in the ratio of B1 in the
Golgi relative to the whole cell (average normalized difference = 12%); without Dox
116 (overexpression of cyclin B1), when cyclin B2 was knocked down, there was an increase after B2 kd (average normalized difference = 3%). The results were not statistically significant. The effort supports the idea that cyclin B1 is resident on the Golgi and increases on the Golgi after B2 knockdown, but this approach needs to be further investigated before conclusions can be made.
4.5 DISCUSSION
Previous studies in Chapter 3 show the similarities of cyclin B1 and B2 in that either of them can promote M phase transition, and the differences of cyclin B1 and B2 on mitotic time regulation. Given that cyclin B1 and B2 regulate mitotic events through forming a complex with Cdk1 to phosphorylate the substrates, we decided to examine localization of cyclin B1 or B2 in the absence of others. In this study, the localization of cyclin B1-
EGFP and B2-EGFP expressing from p015 and p017 cells used in the chapter 3 was examined in the somatic cell cycle by live cell images. The data showed that cyclin B1-
EGFP was localized around the nucleus and centrosomes in the interphase. When cells entered mitosis, cyclin B1-EGFP relocalized to the nucleus and associated with chromosome, centrosomes, spindle and condensed chromosome at metaphase, and degraded after metaphase. All these data suggested that the subcellular localization of cyclin B1-EGFP agrees with the previous observation of wild type cyclin
B1[129,170,180,190,198,251,255,256].
The immunostaining experiments with p017 cells showed that cyclin B2-EGFP was localized on the Golgi similarly to wild type cyclin B2 described early [170,180].
117 However, live-cell imaging experiment showed that cyclin B2-EGFP underwent cell cycle-dependent movement similar to that of cyclin B1-EGFP. Cyclin B2-EGFP was found in nucleus and associated with chromosome, centrosomes, spindle and condensed chromosome at metaphase. To confirm the live-cell imaging data, we determined the localization of endogenous cyclin B2 in HeLa Tet-Off cells by co-immunostaining. In this experiment, association of cyclin B2 to chromosome, centrosomes, and spindle was observed in mitotic cells. The 3-D image of prophase cell further confirmed that cyclin
B2 underwent to nucleus and associated with chromosome at M phase. These results varied from previous observations [129,170]. Our observation of human cyclin B2 underwent to nucleus agrees with that of cyclin B2 in avian [277] and Xenopus oocyte
[278]. Our finding for the localization of human cyclin B2 during mitosis in somatic cells supports the previously reported localization of human cyclin B1 studies. In vitro, cyclin
B1 and B2 have very similar substrate specificities, and in vivo, cyclin B2 acquired the broader properties of cyclin B1 for microtubule reorganization when cyclin B2 was targeted to the cytoplasm by replacing of its N-terminal by that of cyclin B1[180].
Centrosomes and spindle poles are an important sites for G2/M transition and mitotic regulators such as Plk1, Cdc25 and Aurora-A to locate and interact in both animal cells and yeast [171,279,280,281,282,283,284]. In mammalian cells, both cyclin B1 [171] and
Cdk1[279,285] are located in the centrosomes in the interphase. Cyclin B1-Cdk1 complex was first activated at centrosomes and relied on the Plk1-depentdent phosphorylation and Cdc25 –dependent dephosphorylation [198,282]. The centrosomes may accelerate mitotic entry by local accumulation of cyclin B1-Cdk1. Our data indicated that, like cyclin B1, the centrosomal localization of cyclin B2 was observed as
118 soon as cyclin B2 is expressed. Our finding showed that both cyclin B1 and B2 localized in the centrosomes during the interphase and localized in the similar mitotic apparatus during mitosis through cell cycle, suggesting cyclin B2-Cdk1 can access to cytoplasmic and nuclear substrates of cyclin B1-Cdk1. These finding suggest that a single cyclin B can promote mitosis entry and cell cycle progression.
Our immunostaining experiment data also indicated the difference of cyclin B1 and B2 on the subcellular localization. Using CellLight Golgi-RFP, we confirmed that cyclin B2 was colocalized with the Golgi as previously described [170,180] , while cyclin B1 was partially associated with Golgi. However, the laser scanning cytometry data showed more cyclin B1 recruit to the Golgi apparatus when cyclin B2 knockdown. In mammalian cells, the Golgi apparatus is adjacent to the centrosome in the interphase[274]. It is believed that mitotic Golgi disassembly is linked to the cell cycle progression [286]. Mitotic Golgi fragmentation consists of two steps: at G2 phase, the Golgi ribbon is severed into stacks, and after enter mitosis, the Golgi stacks undergo further disassembly [243]. Inhibition of
Golgi disassembly resulted in cell cycle arrest at G2 phase [245,265], while breaking the ribbon is sufficient to overcome this cell cycle arrest and allow cells to enter mitosis
[273]. Blocking of Golgi fragmentation results in inhibition of the activation of Aurora-
A kinase[287], which is localized in the centrosomes and a positively regulate of cyclinB-
Cdk1 activity and mitotic entry [288]. These findings suggest that “Golgi checkpoint” is prerequisite for mitotic entry[289]. The Golgi checkpoint may monitor the binding of spindles to the ribbon determinants that ensures each daughter cell inherit the Golgi ribbon and thus form the Golgi apparatus [243]. The Golgi fragmentation is regulated by
119 the phosphorylation of Golgi-located proteins such as BARS, GRASP65, and GM130 by various kinases [290]. Cdk1 is one of kinases involved in the regulation of both Golgi ribbon serving and Golgi stacks disassembly [245,271,291,292]. Both cyclin B1- and cyclin B2-Cdk1 can lead to Golgi fragmentation in cell-free system or by microinjecting to the cells[180,291,292], and cyclin B1-Cdk1 was more efficient to regulate the Golgi fragmentation when microinjected to the HeLa cells [180]. As Cdk1 is not active until prophase [172,293], it is unlikely cyclin B-Cdk1 initiate the Golgi fragmentation. We hypothesize that cyclin B-Cdk1 regulates the Golgi fragmentation at onset of mitosis, and thus more cyclin B1 translocate to the Golgi in the absence of cyclin B2 leading to shorter mitotic time. The method we developed to determine the quantity of cyclin B1 on the Golgi after cyclin B2 knockdown will test this hypothesis.
120 CHAPTER 5
SUMMARY AND FUTURE DIRECTIONS
5.1. SUMMARY
The MPF (M phase promoting factor) complex consisting of a mitotic cyclin and Cd2 kinase (Cdk1) is essential to enter and transit mitosis in multiple organisms. In mammalian somatic cells, cyclins A2, B1 and B2 are the Cdk1 binding partners of MPF.
Experiments with B cyclin Null mice have shown that cyclin B1 is essential for the viability of embryos and that cyclin B2 is dispensable [153]. However, in cyclin B1 and
B2 RNA interference (RNAi) experiments with human somatic cell lines, neither cyclin
B1 nor B2 is essential, but a B cyclin is necessary for M phase transition. Cyclin B1 and
B2 co-depletion resulted in G2 arrest and subsequent entry into a 4C cell cycle [197,204].
Although exogenous cyclin B2 can reverse G2 arrest caused by cyclin B1 and B2 co- depletion [197], and cell division does take place in the early embryonic cells in cyclin
B1 Null mice [153] , it is still uncertain whether cyclin B1 and B2 are essentially redundant in somatic cells.
There are two lines of evidence that the cyclin B1 has specific functions that are absent in cyclin B2. First evidence is the work of Draviam, et.al [180], which showed that the N terminal fragment of cyclin B1 induced aster formation and nuclear membrane breakdown in G1 cells when a constitutively active Cdk1 bound to a cyclin B1 (or cyclin
B1-B2 fusion protein) was microinjected, whereas cyclin B2 or B2 fusion complex could
121 not. The second evidence is that cyclin B2 is dispensable for mouse embryogenesis and cyclin B1 is not. For any simple model of B cyclin importance, siRNA experiments from two labs (cite Soni and Balanger) conflict with at least the second line of evidence. If cyclin B1 provides an essential function that cannot be supplied by cyclin B2, the residual level of cyclin B1 after siRNA treatment is a sufficient level only in the presence of cyclin B2. In addition, while cyclin B1 is known as rate limiting for mitotic entry and progression [204], the role of cyclin B2 on mitotic timing has not been defined. The purpose of this thesis was to quantitatively assess the kinetics of G2 and M transition as a function of B cyclin type and levels. The hypothesis is that cyclin B1 and B2 are functionally redundant for mitotic entry and are rate limiting for mitotic progression, but have opposite timing roles. The specific aims of this study were:
1) To create cell lines that could be co-depleted for both B cyclins and at the same time express a measureable exogenous B cyclin at different levels. This is described in
Chapter 2.
2) To use the cell lines to quantitatively study the ability of cyclin B1 or B2 to regulate mitotic entry and progression, to quantitatively examine the ability of cyclin B1 or B2 alone to restore cell cycle kinetics under conditions of cyclin B1 and B2 co-depletion.
This is described in Chapter 3.
3) To determine the localization of cyclin B1- and B2-EGFP, and the localization of cyclin B1 and B2 when the other has been depleted. This is described in Chapter 4.
122 The primary work performed in this thesis was focused on comparing the functions of cyclins B1 and B2 on cell kinetics. The observations presented in Chapter III of this thesis strongly supported our hypothesis. This is a novel finding and has a distinct impact on models of cell cycle control by the B cyclins. A subset of the data has been published in PLoS One (Huang, 2013, In press)
Summary of Chapter 2: For this study, I have employed the Tet-Off gene expression system to successfully establish stable cell lines that inducibly express the cyclin B1-
EGFP and cyclin B2-EGFP recombinant genes in the absence of doxycycline. These recombinant genes have been created with synonymous mutations at the siRNA recognition sites. With this system, I have selected clones that allow tunable expression of siRNA-resistant cyclin B1- or B2-EGFP fusion proteins that confer wild type cyclin
B1 or B2 activity. The Tet-Off gene expression and siRNA resistant gene systems were applied in the studies of function of B cyclins respectively [197,213]. To the best of our knowledge, this is the first study of inducible expression of siRNA resistant cyclin B1- or
B2-EGFP applied to cell cycle kinetic studies, although a related approach has described in the study of EDD1 and GRHL2 down-regulation [294]. The elegance of this approach is that it can be used to quantify the rescue phenotype of siRNA interference and characterize each cyclin’s effect on a large population ofcells.
Summary of Chapter 3: Through kinetic and time-lapse studies in Chapter 3, I have demonstrated that cyclin B2 depletion resulted a G2 delay and shorter mitotic time. These results support the hypothesis that cyclin B2 is a rate limiting for mitotic entry and
123 progression, but has the effect is opposite of cyclin B1 for mitotic time. Furthermore, the kinetic data of G2 and M transition with over-expression of either cyclin B1- or cyclin
B2-EGFP showed that over-expression of B cyclin did not alter the rates of G2 and M transition, suggesting that endogenous B cyclins are expressed at saturating levels for control of G2 and M kinetics. It was reported that after either cyclin B1 or cyclin B2 depletion, cells can go through mitosis, while cyclin B1 and B2 co-depletion resulted G2 arrest. Cells entered but did not complete mitosis [197,204]. To examine the extent of redundancy for G2 and M phase transitions, I performed kinetic studies when cells expressed single cyclin B1- or B2-EGFP in the absence of endogenous cyclin B1 and B2 through siRNA depletion. These kinetic experiments demonstrated that either cyclin B1-
EGFP or B2-EGFP can restore the cell cycle phenotype caused by the cyclin B1 and B2 co-depletion. In support of these results, single cyclin B1-EGFP or cyclin B2-EGFP were demonstrated to restore the G2 arrest phenotype after codepletion, and this restoration was dose-dependent. In these experiments, cyclin B2-EGFP appears more efficient to restore the phenotype of cyclin B1 and B2 co-depletion, but this could be simply that cyclin B2-EGFP is more active than cyclin B1-EGFP. Taken together, all data in Chapter3 demonstrated that single cyclin B1 or cyclin B2 can restore the phenotype of cyclin B1 and cyclin B2 co-depletion, and supports the idea that cyclin B1 and B2 are redundant for G2 and M transition and progression, using the simplest model
(see below).
Summary of Chapter 4: In Chapter 4, I have determined the localization of ectopic cyclin B1-EGFP and B2-EGFP by fluorescence time-lapse microscopy and fluorescence
124 confocal microscopy. Expression of cyclin B1-EGFP at an endogenous cyclin B1 level in the absence of endogenous cyclin B1 was primarily in the cytoplasm during interphase and was localized to the nucleus in mitosis and associated with chromosomes, centrosomes, and the spindle, and condensed chromatin at metaphase, which is the wild type cyclin B1 phenotype described in the previous studies [129,180,190,251,255,256].
Ectopic cyclin B2-EGFP expressed at a level equivalent to the endogenous cyclin B2 in the absence of endogenous cyclin B2 was localized on the Golgi like the wild type cyclin
B as described earlier [180]. However, like cyclin B1, cyclin B2-EGFP relocalized to the nucleus and associated with chromosome, centrosomes, spindle and condensed chromatin at mitosis. The association with chromosomes and condensed chromatin appeared weak compared to cyclin B1 and equally, the nuclear entry in prophase did not appear as strong as cyclin B1 (although, still quite apparent). Follow-up immunofluorescence studies demonstrated that endogenous cyclin B2 could also be found at the same locations. These findings suggested cyclin B2-Cdk1 is present at the sites of cyclin B1-Cdk1 during mitosis. This idea is supported by the fact that in vitro, B cyclin-Cdk1 appear to have very similar substrate specificities [170]. I have also determined the localization of cyclin
B1-EGFP by laser scanning cytometry when endogenous cyclin B2 was knocked down.
We found that there was an increased localization of cyclin B1 on the Golgi when cyclin
B2 was knocked down. This finding might help to explain why cyclin B2 depletion resulted short mitotic time. In cyclin B2 depleted cells, the decreased mitotic time might result from the possibility that more cyclin B1 on the Golgi satisfy the Golgi disassembly checkpoint quicker, as cyclin B1-Cdk1 was reported to be more efficient than cyclin B2-
Cdk1 to regulate the Golgi fragmentation [170].
125
Models for regulation of the mitotic entry: Based on this study, we propose two possible models for co-operation of cyclin B1 and B2 for regulation of G2 and M transition and progression in human somatic cells. Mode1: cyclin B1 is unnecessary for mitosis. In this model, A threshold level of B cyclin –Cdk1 activity is required to promote mitosis, and cyclin B1 and B2 are functionally redundant since both cyclin B1 and B2 are relocalized to the nucleus and have common substrates. In the cyclin B1 depletions, cyclin B2-Cdk1 promotes G2 and M transition. In the cyclin B2 depletions, cyclin B1-Cdk1 promotes G2 and M transition. Either cyclin is expressed at levels that exceed any threshold level. However, when cyclin B1 and B2 are co-depleted, any low, residual level of either cyclin is below that threshold, so cells arrest in G2. In single knockdown of cyclin B2, more cyclin B1 moves to the Golgi and regulates the Golgi fragmentation more efficiently, and M transition time is decreased. Thus, while the two cyclins are redundant for the ability to promote mitosis, there are subtle behavioral differences. Model 2: cyclin B1 is necessary for mitosis. In this model, cyclin B1 is necessary for somatic cell transitions but cyclin B2 is not. In this model, at least one critical substrate with two B cyclin/Cdk1 phosphorylation sites is proposed.
Phosphorylation of the first site is required before the second site can be phosphorylated.
The first site is a low affinity site that can be phosphorylated by either cyclin B at endogenous levels, and the second site is a cyclin B1 specific, high affinity site that can be phosphorylated by residual levels of cyclin B1 after knockdown when cyclin B2 is present to phosphorylate site one. This model gives a good explanation for phenomenon that single B cyclin depletion can promote mitosis, but double knockdown of cyclin B1
126 and B2 arrest the cell cycle at G2 phase, and the citation of unpublished data that cyclin
B1 knockout results in G2 arrest (cite Pines paper), and the previous knockout mouse data (Hunt paper).
5.2. FUTURE DIRECTIONS
There are several interesting projects to be done as future directions.
5.2.1 To explore whether cyclin B1 is necessary for mitotic entry and progression.
We have proposed two possible models for cyclin B1 and B2 co-operation to regulate G2 and M transition and progression. One is a threshold model if cyclin B1 is not necessary, and another one is a more complex critical substrate model with at least two phospho- epitopes, one non-specific (either cyclin) and a cyclin B1 specific site. This model works if residual cyclin B1 is necessary. So, it is very important to verify whether cyclin B1 is necessary for mitotic entry and progression. This question could be answered by applying
CRISPR interference (CRISPRi) in HeLa cell or by generating Cre/lox conditional knockout mice. CRISPRi is a recently described RNA-based method to target silencing of transcription in bacteria and human cells with up to 99.9% repression on target gene
[295]. Cre/lox recombination system is the most commonly used technique for knocking specific genes in diploid cells [296].
5.2.2 To study the effect of single cyclin B1 or B2 on Golgi disassembly
Current observation of the difference between cyclin B1 and B2 on the kinetics of the G2 and M phase transition is the different role of cyclin B1 and B2 on mitotic timing. Cyclin
B1 depletion lengthens mitotic time while cyclin B2 depletion accelerates mitotic time.
127 We speculate that more cyclin B1 relocalization to the Golgi during cyclin B2 depletion accelerates mitotic time as cyclin B1 is more efficient at satisfying the Golgi disassembly checkpoint. Phosphorylation of Golgi-associated proteins by various kinases including
Cdk1 has been implicated in the control of the Golgi disassembly [243]. GRASP65 is a cis Golgi membrane protein, phosphorylation of which promotes Golgi stack disassembly
[247,291]. The peripheral Golgi protein, GM130, is another critical factor in the regulation of Golgi fragmentation. Cdk1-dependent phosphorylation of GM130 is required for Golgi vesiculation and mitotic progression [297]. It would be interesting to explore the level of single B cyclin as a function of Golgi disassembly quantification,
Golgi disassembly time, and phosphorylation of GM130/GRASP65.
5.2.3 To explore the residues that regulate human cyclin B2 nuclear localization
We find in this study that, like human cyclin B1, cyclin B2 is also moved to the nucleus at mitosis. Less is known about the regulation of nuclear localization of cyclin B2. For the cyclin B1, phosphorylation of four serine residues at the cytoplasmic retention sequence (CRS) is required for cyclin B1-Cdk1 to rapidly localize to the nucleus
[177,242,254]. The cytoplasmic retention signal is highly conserved among B-type cyclins in higher eukaryotes[254]. The region aa89-aa126 of cyclin B2 is homologous to the CRS of cyclin B1[254]. In this region, ser92, thr94, ser99 are identified as the phosphorylation sites by phosphoproteomics [298,299,300]. Thus these three residues are the potential candidates whose phosphorylation may regulate cyclin B2 nuclear localization.
128 5.2.4 To identify distinct sequences in cyclin B2 that bind to the chromosome, centrosomes, and spindle during mitosis
In Chapter 3, our kinetic experimental results indicate that cyclin B1 and B2 are redundant for G2 and M phase. In Chapter 4, our image data have shown that cyclin B1 is translocated into nucleus and associates with chromosome, centrosome, spindle and condensed chromatin during mitosis. All these data suggest that the functional redundancy of cyclin B1 and cyclin B2 in promoting the G2 and M transition and progression may rely on their similar subcellular localizations. While cyclin B1-Cdk1 is known to promote mitotic events such as chromosome condensation, nuclear membrane breakdown and spindle assembly [180], less is known about the function of cyclin B2 for mitotic regulation. To identify the sequences in cyclin B2 that mediate its localization to specific mitotic structures will be important to understand the function of cyclinB2-Cdk1 during G2 and M transition and progression. Utilizing time-lapse microscopy and fluorescence microscopy to image constructs expressing different cyclin B1 fragments and EGFP fusion protein, we could determine the distinct sequences in cyclin B1 associated with specific mitotic structures.
5.2.5 To study the role of B cyclin –Cdk1 in regulation of chromosome segregation
Accurate chromosome segregation during mitosis is critical to maintain genome stability and prevent aneuploidy. In our lab and in another group’s studies, maloriented chromosomes were observed in the cyclin B1 depletions, and cyclin B1 depletions compromised nocodazole-induced spindle checkpoint, suggesting that cyclin B1-Cdk1
129 play a role in faithfully segregating chromosome and maintaining spindle checkpoint
[204,255].
Chromosome segregation in mitosis is mediated by dynamic interaction of spindle microtubules and kinetochore [195]. In early mitosis (prometaphase), kintochores attach to the spindle microtubules with errors [301], and then move to the spindle pole in a dynein-dependent process [302]. How attachment error correction of kinetochore- microtubule (K-fibres) is achieved remains unknown. Several studies have indicated that dynein and kinesin family proteins such as centromere protein E (CENP-E) [303] are critical in kinetochore congression regulation, spindle formation and error correction.
Phosphorylation of CENP-E by cyclin B-Cdk1 may promote CENP-E ability to drive the movement of kinetochore to the metaphase plate [304]. In mouse liver cells, depletion of CENP-E cause lagging chromosomes in anaphase, which is reminiscent of cyclin B1 depletion. Therefore, it will be interesting to study the role of B cyclin-Cdk1 in regulation of chromosome segregation. For example, experiments can be done to explore the effect of cyclin B on lagging chromosomes, K-fibres stabilities, and chromosome attachment error correction by live cell and fluorescence imaging. Effect of cyclin B on phosphorylation of kinesin protein CENP-E can also be addressed.
5.2.6 To explore the co-operation and redundancies of cyclin A2 and B cyclins on G2 and M transition
Cyclin A2 is another mitotic cyclin in mammalian cells which is detectable slight early and are degraded during prometaphase [129]. Cyclin A2 can bind both Cdk1 and Cdk2. It shuttles between the cytoplasm and nucleus, and is primarily nucleus [129]. Cyclin A2
130 and cyclin B1 have shown some functional redundancy and cooperation in early mitotic events. RNAi experiments indicated that cyclin B1-Cdk1 can restore chromatin condensation in cyclin A2 knockdown cells [305]. Cyclin A2 regulates NEB through activation of cyclin B1-Cdk1 complex [223]. To determine the relationship of cyclin A2, cyclin B1 and cyclin B2 on the G2 and M phase transition will help to understand how these cyclins coordinate during mitosis, and how much redundancy exists between these cyclin–Cdk complexes.
131
Fig 1-1: The phase of cell cycle and the checkpoint of cell cycle. Cell cyclin is composed of interphase , which consists of G1, S and G2 phase, and M phase, which consists of mitosis and cytokinesis. Cell cycle control system triggers DNA replication, mitosis and cytokinesis when cell cycle reaches specific points: G1 checkpoint, G2 checkpoint and metaphase checkpoint.
Adapted from Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and W, P. Molecular Biology of The Cell (Fourth edition)
132
Fig 1-2: Two models for the Cdk control of cell cycle. The quantitative model: in fission yeast, a single cdk and cyclin B (cdc13) can promote both S phase and M phase. A moderate level of kinase activity triggers S phase and a high level is required for mitosis. The qualitative model: in higher eukaryotes, different Cdks form heterodimers with different cyclins that regulate the different cell-cycle stages.
Adapted from Uhlmann, F., Bouchoux, C. and Lopez-Aviles, A., A quantitative model for cyclin-dependent kinase control of the cell cycle: revisited. Phil. Trans. R. Soc. B 2011 366, 3572-3583
133
Fig 1-3: positive feedback loop that regulates cyclinB-Cdk1 activity. Myt1 and Wee1 kinases phosphorylate Cdk1 on T14 and Y15, thereby inhibits cyclinB-Cdk1 activity. T14 and Y15 phosphorylations can be removed by Cdc25 phosphatase. Thus cyclin B- Cdk1 is activated. Once activated, cyclin B-Cdk1 inhibits Wee1 and Myt1, and activates Cdc25, which further amplifying Cdk1 activity.
Adapted from Shackelford, R.E., Kaufmann, W.K., and Paules, R.S., Cell cycle control, checkpoint mechanisms, and genotoxic stress. Environmental Health Perspectives, Vol 107, 5-24.1999
134
Figure 2-1. Schematic of gene regulation in the Tet-Off Systems. The Tet response element, which consists of seven copies of the 19-bp tet operator sequence (tetO) is located upstream of the minimal immediate early promoter of cytomegalovirus (PminCMV), which lacks the enhancer that is part of the complete CMV promoter. PminCMV is silent in the absence of activation. tTA regulator protein of Tet-Off system binds the TRE—and thereby activates transcription of Gene X (e.g. cyclin B1- or B2-EGFP) in the absence of tetracycline or doxycycline
Adapted from BD Biosciences, BD Tet-Off and Tet-On gene expression systems user manual
135 Figure 2-2. Schematic representation of Tet-response constructs containing EGFP, cyclinB1-EGFP and cyclin B2-EGFP. (a), pTRE2-EGFP(NheI/NotI) (pTRE2-001), (b), pTRE2-EGFP (BamHI/NotI) (pTRE2-002), (c), pTRE2-cyclin B1-EGFP (pTRE2-006), (d), pTRE2-cyclin B2-EGFP (pTRE2- 010) and (e), pTRE2-cyclin B2-EGFP (pTRE2- 008) constructs and nucleotide sequence between cyclins and EGFP gene. Tet-responsive
PhCMV*-1 promoter contains the Tet response element (TRE) and minimal CMV promoter (Pmin
CMV). Consequently, PhCMV*-1 is silent in the absence of binding of tTA to the tetO sequences. Note that the EGFP gene is at C terminal of cyclins and cyclin and EGFP are in the same open reading frame. All constructs contain hygromycin and ampicillin resistance genes for direct selection of stable transformants.
136
Figure 2-3. Transient expression of the tet-response constructs containing cyclin B1- or B2- and EGFP genes in HeLa Tet-Off cells in the presence of siRNA. A: pTRE2- 006 and pTRE2-p015 constructs and pTRE2-001 control construct were transfected into HeLa Tet-Off cells with control siRNA or 5ng/ml (+), 10ng/ml (++), and 15ng/ml (+++) cyclin B1 siRNA. After 24 hr post-transfection, equal amounts of lysates were separated on SDS-PAGE, and immunoblotted with anti-cyclin B1, anti-cyclin B2 and anti-cyclin A2. B. pTRE2-010, pTRE2-p017 constructs and PTRE2-001 control construct were treated as above, except siRNA specific for cyclin B2 was used.
137
Figure 2-4. Detection of doxycycline regulated cyclinB1- or B2-EGFP expression in stable cells under immunofluorescence microscopy. A clone of p015 or p017 was cultured with or without Dox for 24 hr. Differential imaging contrast (DIC) and cyclin B1-EGFP (A) or cyclin B2-EGFP(B) fluorescence were imaged under fluorescence microscopy.
138
Figure2-5. induction of cyclin B-EGFP in selected HeLa Tet-Off clones in the absence of Doxycycline. Selected, stable clones of HeLa Tet-Off cyclin B1- or B2-EGFP were grown in the presence or absence of dox for 24 hr. Cells were fixed and stained for DNA content. Cyclin B-EGFP fluorescence was measured by flow cytometry. A. a sample of bivariate plots of cyclin B-EGFP versus DNA content in the presence or absence of Dox. B. Expression of B1- or B2-EGFP fluorescence in different clones of p006, p015, p016, p010, p017 and p023 stable cell lines in the absence of Dox.
139
Figure2-6. Effect of fixation and permeabilization on intensity of cyclin B-EGFP expression in the stable cell lines. P006 cells were grown with the indicated concentration of Dox for 24 hr. EGFP fluorescence was measured with living cells (non fixed ) or after fixation with Formaldehyde and permeabilization with methanol (fixed) by flow cytometry.
140
Figure 3-1. Expression of cyclin B1- and cyclin B2-EGFP is reduced and tightly regulated by Dox. HeLa stable cell p006-14, p015-4, p016-4, p010-5, p017-4, p023-1, and p001-1 were cultured without or with 10ng/ml Dox for 24hr. A: Immunoblot with anti-GFP or anti-β-tublin (loading control) antibodies. B: Cultured cells were fixed and subjected to flow cytometry to measure the EGFP fluorescence. Bivariate histograms show EGFP fluorescence versus side scatter (SS) of indicated cell lines without and with Dox.
141
Figure 3-2. Time and concentration dependence of doxycycline action on a Tet-Off stable cell line. A. Time course of doxycycline action on a stable cell line. p006-14 cells were incubated in the absence or presence of doxycycline for 24 hr. Then 10ng/ml Dox was added or removed from the cells at 1hr intervals for another 18hr. Cells were fixed and EGFP fluorescence was measured by flow cytometry. B. Dose-response curve for a stable cell line. p006-14 cells were incubated with Dox at final concentrations from 0- 10ng/ml for 24 hr, then subjected to flow cytometry to measure EGFP fluorescence. In both figures, the maximum EGFP fluorescence was set to 100, and all the EGFP fluorescence data was normalized to the maximum.
142
Figure 3-3. Effect of doxycycline and siRNA on the ectopic cyclin B-EGFP expressed by stable cells. A: p006-14, p015-4, p016-4 cells and HeLa Tet-Off control cells were transfected siRNA specific for cyclin B1 or scrambled siRNA in the absence or presence of Dox for 18 hr. Cell lysates were separated by SDS-PAGE and blotted with anti-cyclin B1, Cdk1, β-tublin antibodies to detect endogenous cyclin B1 and ectopic cyclin B1-EGFP. B: p010-5, p017-4, p023-1 and HeLa Tet-Off control cells were treated as above, except siRNA was specific for cyclin B2 and anti-cyclin B2 was used.
143
Figure 3-4. Bcl2 is phosphorylated as a function of cyclin B1- and B2-EGFP expression. P006-14, p015-4, p016-4, p010-5, p017-4 and HeLa-Tet-Off cells were treated with control or cyclin B1 or B2 specific siRNA in the presence or absence of 5ng/ml Doxycycline for 24 hr. Cells were fixed, stained immunofluorescently for phospho-S10-histone H3 (PHH3) and phospho-T56-Bcl2, and then analyzed by flow cytometry. A: expression of the two phospho epitopes by cytometry; R2 = mitotic cells; R5 = G1 cells. B: Mean phospho-T56-Bcl2 fluorescence of mitotic cells in the indicated cell lines when cells express cyclin B1- or B2-EGFP and endogenous cyclins (Control), express only endogenous cyclin B1 or B2 (Dox), express only ectopic cyclin B1- or B2- EGFP (siRNA), or express neither endogenous nor exogenous B cyclins (Dox+siRNA).
144
Figure 3-5. G2 and M phase transitions after cyclin B2 knockdown. HeLa or Dox treated p017-4 cells were transfected with control or cyclin B2 siRNA (B2 kd) for 18 hours, then continuously labeled with BrdU. Cells were then trypsinized, fixed, and immuno-stained for BrdU, PHH3, (and cyclin A2 in one experiment) and DNA content, then measured by flow cytometry. The frequencies of unlabeled G2 and M cells were quantified as previously described [204] and plotted versus time. The distance between the origin and the t1/2 point of the G2 curve (circles) = the average G2 time and the difference between the t1/2 of the M (squares) and G2 curves = the average M phase time. The values calculated from these data are presented in Table 2 (p017 and HeLa).
145
Figure3- 6. Over-expression of B cyclins does not alter G2 and M phase transition times. P006-14, p015-4 and p010-1, p017-4 cells were cultured with BrdU in the presence (Control) or absence of Dox (Over-express) to test the ability of B cyclin over- expression to decrease G2 transit and affect M transit. Cells were fixed, stained, and measured as in Figure 5. A, C: Fractions of unlabeled G2 (circles) or M (squares) cells as a function of time in the absence (white symbols) or presence of Dox (black symbols). B, D: two experiments with p006-14, p015-4, p010-1, and p017-4 were analyzed as in A, C and the transit times calculated. To normalize the data the differences between Dox treated and untreated G2 and G2+M t1/2 values (dotted lines) were divided by the t1/2 for Control cells (Y axis). The data for G2 (Delta G2/G) and G2+M (Delta G2+M/G2+M) are denoted on the X axis. The results are not statistically different from 0 for any data set.
146
Figure3-7. B cyclin-EGFP expression rescues the G2 arrest phenotype of B cyclin co-depletion. Cyclin B2-EGFP conditional expressing p017-4 cells (A,B) and cyclin B1 p015-4 cells (C,D) were transfected with control siRNA and cultured in the presence of Dox (white symbols) or transfected with both B cyclin siRNAs and cultured without Dox (gray symbols). Cells were continuously labeled with BrdU, and at timed intervals, fixed and stained for cell cycle analysis. A, C: Decay curves of the unlabeled G2 fractions. B, D: Decay curves of the unlabeled G2+M fraction. The experiment was performed in duplicate; the mean values are plotted. Regressions were performed on samples from time 0 - 4 hr.
147
Figure 3-8. B cyclin co-depletion induces G2 arrest. HeLa-p015-4 and HeLa-p017-4 cells were transfected with control siRNA or B cyclin siRNAs in the presence or absence of Dox for 24-30 hr. Cell lysates were then subjected to electrophoresis and immunobloting (A) or fixed and stained for flow cytometry (B). A: Immunoblot analysis of cyclin B1 (cycB1), cyclin B2 (cycB2), cyclin A2 (cycA2), Cdk1, and β-tubulin under noted conditions. B, C: example cyclin B1-EGFP expression for p015 in the absence (B) or presence (C) of Dox. D, E: DNA content histograms of control cells without Dox (D) or co-depleted cells with Dox (E). Arrows point to 4C cells. Cells depicted in E are both co-depleted for endogenous cyclins and repressed (Dox treated) for exogenous cyclins.
148
Figure 3-9. Single B cyclin-EGFP can rescue the G2 arrest of endogenous cyclin B1 and B2 co-depletion. Two clonal cell lines for each B cyclin and two independent experiments were performed as in Figure 8. The percentage of 4C cells in control cells (Endo) were normalized to 1. Co-depletion increases G2 cells by 2 to 3 times (None). Expression of B cyclin-EGFP in the presence of endogenous cyclins did not change G2 fractions (Endo+Exo). Expression of single B cyclin-EGFP in the presence of endogenous cyclin co-depletion restored G2 fractions to normal cycling levels (Exo).
149
Figure 3-10. Reanalysis of the primary data of the experiments presented in Figure 9. Each data file was gated (R2) as shown in A. Cell cycle analysis was performed as in B. The 4 C fractions were plotted for the p015 (C) and p017 (D) cells for the treatments that resulted in expression of B cyclin-EGFPs. Rescue of the knockdown phenotype is shown in E and F as a function of EGFP expression levels. The slope for the data in E is significant (p = 0.01); the slope for the data in F is not (p=0.06). The 4C cell ratio is the ratio of the 4C cells in co-depleted cultures (Endo + Exo: B specific siRNA without Dox) to those in rescued cultures (Exo: control siRNA without Dox).
150
Figure 3-11. Single B cyclin EGFP completely rescues the G2 arrest of cyclin co- depletion as a function of dose. p015-4 and p017-4 cells were transfected with B cyclin specific in the presence of Dox at stepped levels for 24-30 hr. EGFP fluorescence and the fraction of 4C cells were measured by flow cytometry (siRNA + Dox). Controls were transfected with scrambled siRNA and expressed endogenous and exogenous B cyclins (- Dox) or only endogenous cyclins (+Dox).
151
Figure 3-12. Two optional models for cyclin B1 and B2 co-operation to regulate G2 and M transition and progression. A: cyclin B1 in not necessary for G2 and M transition and progression. A threshold level of B cyclin -Cdk1 activity is required for mitotic entry and progression. B: residual level of cyclin B1 is necessary for G2 and M transition and progression. There are two phosphorylation sites on the mitotic critical substrate. Site 1 is the low affinity for cyclin B1 and B2 binding site. Site 2 is the high affinity for cyclin B1 only binding site
152
Figure 4-1. Localization of ectopic cyclinB1-EGFP expressed in p015 through the cell cycle. P015 cells were transient transfected with H2B-mCherry and applied time lapse microscopy after 24 hr. Still images from time lapse experiments present a cell in the interphase, prophase, metaphase and anaphase when it overexpressed cyclinB1- EGFP.Arrows: centrosomes or spindle asters. Green: cyclin B1-EGFP. Red: H2B-cherry. Bar=5.00um
153
Figure 4-2: Titrations to determine optional concentration of doxycycline to obtain ectopic cyclinB1/B2-EGFP level equivalent to endogenous cyclin B1/B2 in p015 and p017 stable cells. (A) p015 cells were incubated with indicated concentration of Dox for 24 h. the expression level of ectopic cyclin B1-EGFP and endogenous cyclin B1 were assessed by immunoblotting with anti-cyclin B1 antibody. (B) p015 cells were incubated with indicated concentration of Dox for 24 hr. the expression level of ectopic cyclin B1- Cdk1 and endogenous cyclin B1 were assessed by immunoblotting with anti-cyclin B1 antibody. The amount of cyclinB1-EGFP and endogenous cyclin B1 in (A), and cyclin B2-EGFP and endogenous cyclin B2 in (B) were analyzed and quantified by Image J software. The ratio of ectopic cyclin B1-EGFP to endogenous cyclin B1 is shown in (C) and the ratio of ectopic cyclin B2-EGFP to endogenous cyclin B2 is shown in (D) with indicated concentration of Dox. The data is given as mean ± SEM from three independent experiments. Dash line: the ratio of ectopic cyclin B-EGFP to endogenous cyclin B is 1.
154
Figure 4-3: Localization of ectopic cyclin B1-EGFP when expressed in a level equivalent the endogenous cyclin B1 in the absence of endogenous cyclin B1. P015 cells in the 0.20ng/ml Dox and cyclin B1 specific siRNA were transient transfected with H2B-mCherry and applied time lapse microscopy after 24 hr. A. Still images from time lapse experiments present a cell in the interphase, prophase, prometaphase, metaphase and anaphase. B. images present a cell at metaphase . The ectopic cyclinB1-EGFP was associated with spindle and chromatin. Green: cyclin B1-EGFP. Red: H2B-cherry. Bar=5.00um
155
Figure 4-4: Localization of transient transfected EGFP protein in HeLa Tet-Off cells during cell cycle. PTRE2-EGFP construct was transient transfected into HeLa Tet- Off cells and time lapse microscopy was performed after 24hr. Four images were present and time stamps (hh:mm) indicate the time of each image captured. Transfected EGFP protein is expressed in the whole cell though the whole cycle including in the interpahse (A), mitosis (B), telaphase (C) and G1 phase (D) as arrow indicated. Bar=15.0um
156
Figure 4-5: Localization of ectopic cyclin B2-EGFP to the Golgi determined by confocal microscopy. CellLightR○ Golgi-RFP was introduced into P017 cells expressing cyclin B2-EGFP in a level equal to endogenous cyclin B2 for ≥16 hr, and then cells were fixed. The localization of cyclin B2-EGFP to the Golgi was examined by confocal in S phase, G2 phase, prophase and metaphase cells. Green: cyclin B2-EGFP. RED: Golgi- RFP. Bar=5um
157
Figure 4-6: Localization of ectopic cyclinB2-EGFP expressed in p017 through the cell cycle. P017 cells were transient transfected with H2B-mCherry and applied time lapse microscopy after 24 hr. Still images from time lapse experiments present a cell in the interphase, early prophase, late prophase, prometaphase, metaphase, anaphase and telaphase (A) when it overexpressed cyclinB2-EGFP. (C) when it expressed cyclin B2- EGFP in a level equivalent to the endogenous cyclin B2 and in the absence of endogenous cyclinB2. B2-EGFP was localized to the condensed chromatin in the metaphase when the cell (B) over expressed cyclin B2-EGFP and (D) expressed cyclin B2-EGFP in a level equivalent to the endogenous cyclin B2 and in the absence of endogenous cyclinB2. Arrows: centrosomes or spindle asters. Green: cyclin B1-EGFP. Red: H2B-cherry. Bar=5.00um
158
Figure 4-7: Localization of wild type of cyclin B2 to the tubulin (centrosomes) and chromosome determined by indirect immunofluorescence. HeLa Tet-Off cells were fixed and stained for anti-cyclin B2 and anti-γ-tubulin antibodies and performed confocal microscopy. Localization of wild type cyclin B2 to centrosomes was examined in G2 phase, prophase, prometaphase, metaphase and anaphase cells. Green: cyclin B2. Red: γ- tubulin. Bar=10um Green: cyclin B2. Blue: DNA.
159
Figure 4-8: Localization of wild type cyclin B1 to the Golgi determined by indirect immunofluorescence. HeLa Tet-Off cells were introduced with CellLightR○ Golgi-RFP for ≥16 hr, and then cells were fixed and stained with anti-cyclinB1 antibody. The localization of wild type cyclin B1 to the Golgi was examined by confocal in S phase, G2 phase, prophase and metaphase cells. Green: cyclin B2. RED: Golgi-RFP. Bar=5um.
160
Figure 4-9: Determine the intensity of cyclin on the Golgi and in the whole cell by laser scanning cytometry.. (A). LSC images of G2 and M phase cells with segmentation on cyclin B1 (Green), Golgi (Red) and DNA (Blue). (B). a). A plot of cyclin B1 versus DNA content distribution in the control cell and in cells. R3 is gated as G2 and M phase cell. b). The intensity of cyclin B1 in the whole cell (R2) is measured from histogram of the cyclin B1 per whole cell area gated from R3. c). The intensity of cyclin B1 on the Golgi (R6) is measured from histogram of the cyclin B1 per Golgi area gated from R3.
161 TABLES
Table 3-1. Synonymous base changes
Table 3-2. G2 and M phase times; effect of cyclin B2 knockdown
Controls Effect of Kd Cyclin Cell Experiment t (h) t (h) t (h) t (h) Kd Line G2 M G2 M p006 1 2.4 2.0 p006 2 1.8 1.6 p015 1 2.2 2.1 p016 1 2.0 1.9 p010 1 2.1 2.4 B2 p010 2 1.7 2.4 1.3 -0.2 p017 1 1.5 2.9 B2 p017 2 2.3 2.6 1.3 -0.4 B2 HeLa 1 2.5 2.2 1.1 -0.3 B2 HeLa 2 2.6 1.8 0.9 -0.5 Means 2.1 2.2 1.1 -0.37
tG2 = tG2 (B2 Kd) - tG2 (Control); MG2 = MG2 (B2 Kd) - MG2 (Control). Cell line controls were treated with Dox to eliminate expression of exogenous B cyclin-EGFP. Rows with blank cells represent experiments without knockdown treatment and are included to better describe averages for tG2 and tM.
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