TWO SIDES OF THE PLANT NUCLEAR PORE COMPLEX AND A POTENTIAL LINK BETWEEN RAN GTPASE AND PLANT CELL DIVISION
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the
Graduate School of the Ohio State University
By
Xianfeng Xu, B.S.
* * * * * The Ohio State University
2007
Dissertation Committee: Approved by Professor Iris Meier, Advisor Advisor Professor Biao Ding
Professor Rebecca Lamb Professor David Mackey Graduate Program in Plant Biology
ABSTRACT
In eukaryotic cells, the nuclear pore complex (NPC) is vital for macromolecular trafficking and thereby the exchange of information between the nucleus and the cytoplasm. While fungal and mammalian NPC has been well studied by proteomics and detailed functional characterization, both the protein components of and various molecular activities associated with the plant NPC are poorly understood. Recent studies have implicated that the plant NPC plays important roles in several processes, including plant-microbe interactions, hormone signaling, and stress tolerance. This highlights a true need to thoroughly understand NPC-associated molecular activities in plants. Here I have identified and characterized two plant NPC-associated proteins, one residing at the inner and the other at the outer side of the NPC. Analyses of these proteins suggest both the conservation and the divergence of the plant NPC from that of other eukaryotes.
ii I identified an Arabidopsis thaliana protein called NUCLEAR PORE ANCHOR (NUA); it is a plant homolog of Tpr/Mlp1/Mlp2/Megator. Tpr-like proteins are long coiled-coil proteins associated with the inner basket of the NPC and are involved in mRNA export, unspliced RNA retention, telomere organization, spindle pole assembly, docking of SUMO protease, and spatially regulated gene expression in a number of eukaryotic species but hadn’t been analyzed in plants. Four nua lesions comprise an allelic series with increasing severity for several correlating phenotypes, such as early flowering, increased abundance of SUMO conjugates, and altered expression of flowering regulators. Together with the genetic and physical interaction between NUA and ESD4 (a SUMO protease), these data suggest that NUA is a conserved component of NPC-associated steps of desumoylation in plants. Defects in SUMO homeostasis affect signaling events of flowering time regulation and additional developmental processes.
Secondly, I identified a plant-specific family of NPC-associated membrane proteins,
WPP-DOMAIN INTERACTING PROTEINS (WIPs). These proteins bind to the WPP domain of the plant Ran GTPase-activating protein RanGAP, and are responsible for the NPC-association of plant
RanGAP. RanGAP is essential for the asymmetric distribution of RanGTP and RanGDP, involved in nucleocytoplasmic transport, mitotic spindle assembly, cell cycle control, and nuclear envelope formation. Mammalian RanGAP targeting to the NPC during interphase and to the spindle and kinetochores during mitosis requires interaction of its sumoylated C-terminal domain with nucleoporin
Nup358/RanBP2. In contrast, I demonstrated that binding to the coiled-coil domain of WIPs is a plant-unique mechanism for targeting RanGAP to the NPC, which supports a separate evolution of
RanGAP targeting in different kingdoms. Moreover, I presented data to suggest that targeting of plant
RanGAP appears to involve different mechanisms at different stages of cell cycle and of differentiation.
iii Lastly, in light of the increasing evidence indicating that NPC-associated proteins play additional roles during mitosis and cytokinesis, I characterized the localization of Arabidopsis RanGAP1 throughout the cell cycle using immunofluorescence. Arabidopsis RanGAP1 colocalizes with the preprophase band (PPB), concentrates at the kinetochores and spindle midzone, and accumulates at the midline of phragmoplast and/or the nascent cell plate. More strikingly, RanGAP1 was found to be the first marker to demarcate the cortical division site after the PPB disassembles. In addition, the
WPP domain of RanGAP1 appears to be necessary and sufficient for the mitotic targeting of
RanGAP1, although this localization does not depend on the WIPs.
Taken together, by performing detailed studies on two Arabidopsis NPC-associated proteins, I demonstrated two general themes of the plant NPC. First, the plant NPC is an evolutionarily conserved structure with similar protein composition and similar associated molecular activities to those of other eukaryotes, as shown by the conservation of NUA. Second, the plant NPC also diversified to fulfill potential plant-specific cellular and developmental requirements, indicated by the presence of the plant specific NPC-associated WIP family. In addition, a potential link between the
Ran cycle and the plant-unique mode of cytokinesis is proposed based on the mitotic targeting of
Arabidopsis RanGAP1. Together, my research contributes to a better understanding of the plant NPC and a better appreciation of plant cytokinesis.
iv
Dedicated to my family, my love!
v
ACKNOWLEDGMENTS
I was extremely fortunate to get the opportunity to work with my advisor, Prof. Iris Meier. I thank her for the continuous guidance, support and motivation, which will benefit me throughout my future career.
I am grateful to all my committee members, Prof. Rebecca Lamb, Prof. Biao Ding and Prof. David
Mackey for their time, advice and encouragement. I also want to thank Prof. Biao Ding for his generosity in sharing the confocal microscope, without which this dissertation would not have been possible.
I would also like to thank all my lab-mates through the years: Dr. Tomasz Calikowski, Dr. Sun Yong
Jeong, Dr. Shalaka Patel, Dr. Annkatrin Rose, Heather Wang, Qiao Zhao, Dr. Jelena Brkljacic,
Sowmya Venkatakrishnan, Sivaramakrishnan Muthuswamy, Thushani Rodrigo-Peiris, Chao Sylvia He,
Grace Fry, Kelly Lake, Nicholas Holomuzki, and other undergraduate assistants. I am grateful to their warmth, encouragement, and helpful discussion. Also I want to thank Dr. Annkatrin Rose,
Sivaramakrishnan Muthuswamy, Dr. Sun Yong Jeong, Sowmya Venkatakrishnan, and Qiao Zhao for showing some of their data on the collaborative project described in chapter 1. I am thankful to
Thushani Rodrigo-Peiris and Heather Wang for citing some of her unpublished data.
I am sincerely thankful to Dr. Tea Meulia for the delightful collaboration on the electron microscopic studies. I also appreciate all the expertise that she showed me about general microscopy.
vi I am grateful to all the people in the Plant Biotech Center, for critical feed-back on my research. I thank all our former and current staff members, Denise Blackburn-Smith, Jill Hartman, Rene Madsen, and Laurel Shannon from the Department of Plant Cellular and Molecular Biology; Melinda Parker,
Diane Furteny, Dave Long and Scott Hines from the Plant Biotech Center; Joe Takayama from our greenhouse facility; and James Mann, Debbie Crist, Emma Knee, and Luz Rivero from The
Arabidopsis Biological Resource Center (ABRC).
Last but not least, I thank my family and all the people who care about me, for their love and support.
Especially, I am deeply in debt to Chao Sylvia He for all her love, understanding and tremendous help in my research.
vii
VITA
Dec. 28, 1983 ……………………………... Born in Anhui, P.R.China
2002 ………………………………………. B.S in Biotechnology College of Life Science Peking University, Beijing, P.R.China
2002 – present …………………………….. Graduate Teaching/Research Associate Plant Cellular and Molecular Biology The Ohio State University, Columbus, Ohio
PUBLICATIONS
Meier I., Xu X.M., Brkljacic J., Zhao Q., and Wang H-J. Going Green: Plants’ Alternative Way to Position the Ran Gradient. Journal of Microscopy, in press.
Xu X.M., Rose A., and Meier I. NUA Activities at the Plant Nuclear Pore. Plant Signaling & Behavior, in press.
Xu X.M., Meulia T., and Meier I. Anchorage of Plant RanGAP to the Nuclear Envelope Involves a Novel Family of Plant Nuclear Pore-Associated Transmembrane Proteins. Current Biology, 2007, 17:1157–63.
Xu X.M., Rose A., Muthuswamy S., Jeong S.Y., Venkatakrishnan S., and Meier I. NUCLEAR-PORE ANCHOR, the Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator, Is Involved in mRNA Export, SUMO Homeostasis and Affects Diverse Aspects of Plant Development. Plant Cell, 2007, 19(5):1537-48.
FIELD OF STUDY
Plant Biology
viii
TABLE OF CONTENTS
Page
Abstract …………………………………………………………………………………….………….ii
Dedication………………………………………………………………………………………..…….v
Acknowledgments……………………………………………………………………………….……..vi
Vita…………………………………………………………………………………………….……...viii
List of Figures………………………………………………………………………………………...xiv
List of Tables…………………………………………………………………………………….……xvi
Chapters:
1. NUCLEAR PORE ANCHOR, the Arabidopsis homolog of Tpr/Mlp1/Mlp2/Megator, is involved in SUMO homeostasis and affects diverse aspects of plant development……………………………...1
1.1 Abstract……………………………………………………………………………………....2
1.2 Introduction…………………………………………………………………………………..3
1.3 Materials and Methods……………………………………………………………………….6
1.3.1 Plant material and growth conditions……………………………………………..6
1.3.2 Flowering time measurements…………………………………………………….6
1.3.3 PCR-based genotyping of T-DNA insertion lines…………………………………7
1.3.4 Arabidopsis transformation………………………………………………………..7
1.3.5 RT-PCR……………………………………………………………………………8
1.3.6 Sumoylation assays………………………………………………………………..8
1.3.7 Immunolocalization………………………………………………………………..8
ix 1.4 Results………………………………………………………………………………………..9
1.4.1 Identification of NUCLEAR-PORE ANCHOR (NUA), an Arabidopsis protein
similar to mammalian Tpr, Drosophila Megator, and Yeast Mlp1/Mlp2 – (Data generated
by Annkatrin Rose and Sun Yong Jeong) …………………………………………………9
1.4.2 Validation of NUA full-length cDNA…………………………………………….10
1.4.3 NUA is conserved in different plant species……………………………………..10
1.4.4 Characterization of NUA T-DNA insertion lines…………………………………11
1.4.5 NUA T-DNA insertion alleles flower early in long days and short days and have
pleiotropic developmental defects………………………………………………………..12
1.4.6 Subcellular localization of NUA…………………………………………………13
1.4.7 nua-1 esd4-2 double mutant analysis…………………………………………….14
1.4.8 Effects of nua mutants on sumoylation and flowering gene expression…………14
1.4.9 GFP-ESD4 localization is not altered in nua mutants……………………………16
1.5 Discussion…………………………………………………………………………………..16
1.5.1 The first viable null mutant of a Tpr-like protein in a multicellular organism…..16
1.5.2 Connection between nucleoporins and SUMO proteases, comparable but not
identical in all kingdoms…………………………………………………………………17
1.5.3 NUA activities link SUMO homeostasis, RNA metabolism, gene regulation and
plant developmental control……………………………………………………………...18
1.6 Future Outlook………………………………………………………………………………20
1.6.1 NUA and microRNA homeostasis………………………………………………..20
1.6.2 The ultra-structural subcellular location of NUA………………………………...20
1.6.3 Better understanding of the NUA activities by identifying physical and genetic
interactions ………………………………………………………………………………21
x 2. The Kingdom- and Tissue-Specific Anchorage of Plant RanGAP to the Nuclear Envelope
Involves a Novel Family of Plant Nuclear Pore-Associated Transmembrane Proteins……………….37
2.1 Abstract……………………………………………………………………………………...38
2.2 Introduction…………………………………………………………………………………38
2.3 Materials and Methods……………………………………………………………………...42
2.3.1 Protein sequence analyses………………………………………………………..42
2.3.2 Plant materials……………………………………………………………………42
2.3.3 RNA extraction and RT-PCR……………………………………………………..43
2.3.4 Constructs………………………………………………………………………...43
2.3.5 Yeast two-hybrid assays………………………………………………………….44
2.3.6 Antibody development…………………………………………………………...44
2.3.7 Generation of transgenic plants…………………………………………………..44
2.3.8 Co-IP experiments………………………………………………………………..45
2.3.9 Cell culture……………………………………………………………………….46
2.3.10 Immunolabeling and confocal microscopy………………………………………46
2.3.11 Scanning electron microscopy……………………………………………………47
2.3.12 Transmission electron microscopy immunolocalization…………………………47
2.3.13 GUS assays……………………………………………………………………….48
2.4 Results………………………………………………………………………………………48
2.4.1 Identification of an Arabidopsis RanGAP-binding protein family……………….48
2.4.2 Subcellular localization of the WIP protein family and the WIP-RanGAP
interaction………………………………………………………………………………...50
2.4.3 Ultrastructural analyses of WIP1 and RanGAP1 localization at the NE – (Data
generated by Tea Meulia) …………………………………………………………….….51
2.4.4 Localization of the WIP family during cytokinesis………………………………53
2.4.5 Plant NE dynamics during cell cycle……………………………………………..53
xi 2.4.6 Domain requirement for the subcellular targeting of WIP1……………………...54
2.4.7 Role of the WIP family in RanGAP anchoring…………………………………..55
2.5 Discussion…………………………………………………………………………………..57
2.5.1 Different NE-targeting mechanisms for higher eukaryotic RanGAPs…………...57
2.5.2 Why does plant and animal RanGAP accumulate at the NE?...... 59
2.5.3 Do WIPs represent novel plant-specific transmembrane nucleoporins?...... 61
2.5.4 WIP1 as a NE-specific TA membrane protein……………………………………62
2.5.5 Plant NE membrane and cell plate……………………………………………….64
2.6 Future Outlook………………………………………………………………………………64
1.6.1 Other anchoring proteins for plant RanGAP at the NE and cell plate……………64
1.6.2 Unraveling the biological roles of RanGAP concentration at the NE in root tip
cells………………………………...... 65
1.6.3 The function of WIPs and RanGAP during the elongation of the etiolated
hypocotyl ………………………………………………………………………………...66
1.6.4 Better understanding of the sorting signal in WIPs responsible for the
NE/NPC/Cell plate association…………………………………………………………...67
3. The Mitotic Localization of Arabidopsis RanGAP1: Ran Cycle as the Spatial Cue for Future
Division Plane in plants?...... 78
3.1 Abstract……………………………………………………………………………………...78
3.2 Introduction…………………………………………………………………………………79
3.3 Materials and Methods……………………………………………………………………...82
3.3.1 Plant Materials……………………………………………………………………82
3.3.2 Cloning and Transformation……………………………………………………...83
3.3.3 Immunolabeling and Confocal Microscopy……………………………………...84
3.4 Results………………………………………………………………………………………85
xii 3.4.1 Mitotic Localization pattern of Arabidopsis RanGAP1………………………….85
3.4.2 The WPP motif is critical for the mitotic targeting of RanGAP1………………..86
3.4.3 The WPP domain is sufficient for directing the mitotic targeting of RanGAP1…86
3.4.4 Mitotic targeting of RanGAP1 is NOT dependent on WIPs……………………..87
3.4.5 Identification of Arabidopsis mutants underexpressing RanGAP………………..88
3.5 Discussion…………………………………………………………………………………...89
3.5.1 Characterization of RanGAP1 localization during the cell cycle………………...89
3.5.2 The first positive protein marker for the division plane in plant cells……………90
3.5.3 Connection between RanGAP1 and the chromosomal passenger proteins………90
3.5.4 Similarities and differences between the mammalian and plant RanGAP mitotic
localization……………………………………………………………………………….91
3.5.5 Possible modes of action for plant RanGAP in specifying the division plane…...92
3.6 Future Outlook………………………………………………………………………………93
3.6.1 The dynamic mitotic and cytokinetic localization pattern of plant RanGAP with a
higher spatial and temporal resolution…………………………………………………...93
3.6.2 Establishing the potential link between the Ran GTPase and plant cytokinesis…94
3.6.3 Understanding the mitotic anchorage of plant RanGAP1………………………..95
3.6.4 Unraveling the functional significance of plant RanGAP1 during mitosis………96
Bibliography…………………………………………….……………………………………………106
xiii
LIST OF FIGURES
Figure Page
1.1 NUA has a similar domain structure with its homologs from other species……………………..22
1.2 The validation of the full length NUA cDNA……………………………………………………23
1.3 Position of T-DNA insertions and PCR primers………………………………………………….24
1.4 Analysis of NUA transcript in nua mutants………………………………………………………25
1.5 Phenotypic characteristics of nua mutant alleles…………………………………………………26
1.6 NUA is located at the nuclear envelope during interphase and in the vicinity of the spindle during prometaphase…………………………………………………………………………………………..27
1.7 NUA protein revealed by immunofluorescence in all mutant alleles…………………………….28
1.8 NUA is localized at the inner side of nuclear envelope, shown by co-localization with the outer
NE/NPC-localized protein GFP-WIP1………………………………………………………………..29
1.9 NUA is recruited to the newly-formed NE/NPC relatively late during mitosis…………………30
1.10 Genetic interaction between NUA and ESD4……………………………………………………31
1.11 Nua mutant alleles lead to increasing accumulation of SUMO conjugates and altered expression of genes involved in different flowering pathways……………………………………………………32
1.12 The concentration of ESD4 on the nuclear periphery is not abolished in nua mutants…………..33
1.13 Characterization of the nua-4 mutant…………………………………………………………….34
2.1 The coiled-coil domain of WIP1 interacts with the WPP domain of RanGAP…………………..68
2.2 WIP1 and RanGAP1 interact at the outer NE, most likely at the cytoplasmic side of the NPC…70
2.3 The dynamic localization of WIP1, revealed by tracking GFP-WIP1 throughout the cell cycle...71
xiv 2.4 Domain requirement for the subcellular targeting of WIP1……………………………………..73
2.5 Stepwise mutagenesis of the predicted NLS of WIP1……………………………………………74
2.6 In the wip1-1/wip2-1/wip3-1 triple mutant (“triple”), RanGAP1 is dislocated from the NE in undifferentiated root cells (undiff.), while NE targeting in differentiated cells (diff.) is not affected...75
2.7 RanGAP targeting to the NE, but not the cell plate, depends on the WIP family………………..76
2.8 Expression pattern of WIP1 and its family members…………………………………………….77
3.1 The localization of Arabidopsis RanGAP1 during mitosis………………………………………98
3.2 RanGAP1 forms a PPB-like ring circumscribing the nucleus during G2/prophase……………...99
3.3 RanGAP1 positively marks the cortical division site during the anaphase……………………..100
3.4 The WPP motif is critical for the mitotic targeting of RanGAP1……………………………….101
3.5 The WPP Domain is sufficient for the mitotic targeting of RanGAP1…………………………102
3.6 The mitotic targeting of RanGAP1 is NOT dependant on the WIP family……………………..103
3.7 Identification of mutants underexpressing RanGAP……………………………………………104
3.8 Proposed Model on plant RanGAP activity in providing the spatial cue for plant cell division..105
xv
LIST OF TABLES
Table Page
1.1 Flowering time of WT Columbia and nua mutants, given as number of rosette leaves at time of bolting…………………………………………………………………………………………………35
1.2 Flowering time of WT Columbia, nua mutants, and flc-3 null mutant, given as number of rosette leaves at the time of bolting…………………………………………………………………………...35
1.3 Primer sequences used in Chapter 1.……………………………………………………………..36
xvi
CHAPTER 1
NUCLEAR PORE ANCHOR, THE ARABIDOPSIS HOMOLOG OF
TPR/MLP1/MLP2/MEGATOR, IS INVOLVED IN SUMO HOMEOSTASIS AND AFFECTS
DIVERSE ASPECTS OF PLANT DEVELOPMENT1
1 This chapter has been published as: Xu XM, Rose A, Muthuswamy S, Jeong SY, Venkatakrishnan S, Zhao Q, and Meier I. (2007). NUCLEAR PORE ANCHOR, the Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator, Is Involved in mRNA Export and SUMO Homeostasis and Affects Diverse Aspects of Plant Development. Plant Cell 19, 1537-48. 1 1.1 Abstract
Vertebrate Tpr and its yeast homologs Mlp1/Mlp2, long coiled-coil proteins of nuclear pore inner basket filaments, are involved in mRNA export, telomere organization, spindle pole assembly, and unspliced RNA retention. We identified Arabidopsis NUCLEAR PORE ANCHOR (NUA), which encodes a 237 kD protein with similarity to Tpr. NUA is located at the inner surface of the nuclear envelope in interphase and in the vicinity of the spindle in prometaphase. Four T-DNA insertion lines were characterized, which comprise an allelic series of increasing severity for several correlating phenotypes, such as early flowering under both short and long days, increased abundance of SUMO conjugates, and altered expression of several flowering regulators. nua mutants phenocopy mutants of
EARLY IN SHORT DAYS4 (ESD4), an Arabidopsis SUMO protease concentrated at the nuclear periphery. nua esd4 double mutants resemble nua and esd4 single mutants, suggesting that the two proteins act in the same pathway or complex. Our data indicate that NUA is a component of nuclear pore-associated steps of sumoylation in plants and that defects in this process affect the signaling events of flowering time regulation and additional developmental processes.
2 1.2 Introduction
The nuclear pore complex (NPC) is a large multiprotein complex that is the sole gateway of macromolecular trafficking between the cytoplasm and the nucleus. The mammalian and yeast NPC consists of multiple copies of the approximately 30 different nucleoporins (Nups). Together, they form a channel-like structure with eight-fold symmetry that has been roughly divided into three elements: a nuclear basket, a central pore, and cytoplasmic fibrils. While a small number of Nups are anchored to the nuclear envelope membrane, others form a protein scaffold or line the central pore cylinder with
FG-repeat containing hydrophobic domains. Nuclear import and export receptors traffic through the pore bound to their cargos, and the Ran cycle provides spatial information on the directionality of the transport (reviewed in Tran and Wente, 2006).
Recently, several reports have demonstrated that Nups are involved in functions beyond being building blocks of the NPC. Some Nups are highly dynamic in localization and appear in locations away from the pore (Griffis et al., 2002; Rabut et al., 2004). Several Nups have mitotic functions, for example involvement in kinetochore assembly (reviewed in Chan et al., 2005). Possibly the most exciting “new” function of Nups is their ability to dock specific enzymatic activities to the NPC, thereby providing spatial regulation for the respective activities. An example that has been known for several years is the docking of the mammalian Ran GTPase activating protein RanGAP1 to the outer surface of the NPC by the nucleoporin RanBP2, where it hydrolyzes the RanGTP bound to export complexes (Matunis et al., 1998). Mammalian RanGAP1 requires sumoylation to bind to RanBP2, and subsequently it was found that RanBP2 itself is a SUMO E3 ligase (Matunis et al., 1998; Pichler et al.,
2002).
At the nuclear side of the pore, the nuclear basket is also involved in regulating SUMO modification.
The nucleoporins Mlp1/Mlp2 in yeast (Saccharomyces cerevisiae) and Nup153 in mammals dock a
3 SUMO protease to the NPC (Ulp1 in yeast and SENP2 in mammals) (Panse et al., 2003; Zhang et al.,
2002b). Mlp1 is also a docking site for heterogeneous nuclear ribonucleoproteins (hnRNPs) (Green et al., 2003) and mammalian hnRNPs have recently been shown to be sumoylated (Vassileva and
Matunis, 2004). It has been proposed that Mlps act as a quality control checkpoint for mRNA export
(Galy et al., 2004).
At the outer pore surface, another nucleoporin acts as an anchor/activator of a step in mRNA export.
The nucleoporin Gle1 binds the DEAD box helicase Dbp5 and together with the soluble inositol polyphosphate InsP6 activates the ATPase activity of Dbp5. This leads to a highly localized activation of mRNA remodeling, likely involved in a release step of mRNA export (Alcazar-Roman et al., 2006;
Galy et al., 2004; Weirich et al., 2006). Together, these examples support the emerging picture that precise spatial regulation is crucial for a number of nuclear functions, and emphasize that this spatial regulation can be provided by anchoring enzymatic activities to nuclear pore proteins.
4 While the components of the animal and yeast NPC have been identified in proteomic studies
(Cronshaw et al., 2002; Rout et al., 2000), the plant NPC is still largely a black box. Recently, putative plant nucleoporins have been identified by genetic screens for the seemingly unrelated signaling pathways involved in plant-microbe interactions, cold tolerance, and the action of the plant hormone auxin (Zhang and Li, 2005; Dong et al., 2006; Kanamori et al., 2006; Parry et al., 2006). The identified proteins have convincing sequence similarity to Nup96, Nup160 and Nup133, which are all components of the Nup107-160 complex, suggesting its conservation during evolution. The
Nup107-160 complex is believed to provide a core scaffolding function for NPC assembly in mammals (Harel et al., 2003; Walther et al., 2003a). Partial in vivo depletion of this complex from
HeLa cells via RNAi or in vitro immunodepletion from Xenopus laevis nuclear assembly reactions leads to defects in the assembly of the NPC, which suggests their pivotal roles (Harel et al., 2003;
Walther et al., 2003a). The various phenotypes associated with the mutations of putative plant nucleoporins underline that many cellular and developmental processes involve the communication between the nucleus and the cytoplasm via the NPC.
A nuclear-rim associated activity with developmental functions is desumoylation mediated by the
SUMO protease EARLY IN SHORT DAYS 4 (ESD4). esd4 mutants flower extremely early and have pleiotropic alterations in shoot development (Reeves et al., 2002). The early flowering phenotype is consistent with reduced expression of the flowering repressor FLOWERING LOCUS C (FLC). In line with the in vitro SUMO protease activity of ESD4, esd4 mutants accumulate more SUMO conjugates and have less free SUMO than wild type (WT) plants (Murtas et al., 2003). These data suggest a connection in Arabidopsis between SUMO homeostasis and flowering-time regulation.
Here, we show that mutants of an Arabidopsis protein (NUCLEAR PORE ANCHOR, NUA) with similarity to the inner nuclear basket proteins Tpr (for Translocated Promoter Region), Mlp1/Mlp2
(Myosin-like proteins 1 and 2), and Megator flower extremely early and have several phenotypic
5 characteristics in common with esd4 mutants. NUA is located at the inner nuclear envelope and nua esd4 double mutants resemble nua and esd4 single mutants, indicating that the two proteins might act in a shared pathway or complex. Nua mutant alleles show an increase in SUMO conjugates and reduction of free SUMO, and altered expression of several genes involved in flowering time regulation. We propose that NUA acts as a docking site at the inner nuclear pore for activities required for desumoylation and that disruption of this docking affects the expression of key regulators of plant development.
1.3 Materials and Methods
1.3.1 Plant material and growth conditions
For T-DNA insertion mutants nua-1 (SALK_057101), nua-2 (SALK_069922), nua-3
(SAIL_505_H11), nua-4 (WiscDsLox297300_17E) and esd4-2 (SALK_032317), T3 or T4 bulk seeds were acquired from the ABRC. The nua-1 esd4-2 double mutant was identified in the F2 generation from crosses between nua-1 and esd4-2. Arabidopsis WT and T-DNA lines were grown on soil under standard long-day condition (16 hour light/8 hour dark) or short-day condition (8 hour light/16 hour dark) or on MS plates under constant light.
1.3.2 Flowering time measurements
Plants were grown on soil in short-day or long-day conditions. Flowering time was measured by counting the total number of rosette leaves at the time of bolting. Data shown are the mean value and standard deviation of 11 to 53 samples per line.
6 1.3.3 PCR-based genotyping of T-DNA insertion lines
Genomic DNA was extracted as described (Krysan et al., 1999). Primers were designed using SIGnAL iSect Tools (http://signal.salk.edu/tdnaprimers.html). All primer sequences are summarized in Table
1.3. The exact T-DNA insertion sites were determined by sequencing the PCR products derived from primer combinations of gene-specific primers plus T-DNA-specific primers. In nua-1 and nua-3, primer combinations LP057101.1/LBa1 and CS821281RP/pCSA110-LB were used, respectively. In nua-2, primer combinations RP069922/LBa1 and LP069922/New-RB-primer were used. In nua-4, the insertion site was mapped to nucleotide +2145 (within exon 9) using primer combinations
CS850695FP/p745-primer and CS850695RP/p745-primer.
1.3.4 Arabidopsis transformation
Arabidopsis Columbia WT, nua-1, nua-2, and nua-3 were transformed by floral dipping (Clough and
Bent, 1998) with Agrobacterium strain ABI harboring plasmid pK7WGF2-ESD4. Primary transformants were selected for Kanamycin resistance.
7 1.3.5 RT-PCR
Total RNA was extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from
10-day-old seedlings (a stage at which none of the mutants has bolted yet.) grown on MS plates under long-day condition. After digestion with DNase I (Amplification grade, Invitrogen), cDNA was synthesized with oligo-dT primers from ~3μg total RNA, using the ThermoscriptTM RT-PCR system
(Invitrogen). cDNA templates were PCR amplified with gene-specific primers (see Supplemental
Table 2 online) for quantification of mRNA levels of flowering time integrator genes. 28 cycles were used for tubulin 2, 35 cycles for FLC, 30 cycles for FT, 26 cycles for SOC1, 35 cycles for LFY, and 33 cycles for MYB33 and MYB65.
1.3.6 Sumoylation assays
For the analysis of sumoylation profiles, total protein from 2-week-old seedlings was extracted as described (Thompson et al., 2005). Approximately 100μg protein was separated on a two-layer
SDS-PAGE gel to resolve both the free SUMO and high-molecular-weight conjugates (8% top half,
15% bottom half). After SDS-PAGE, proteins were then transferred to Polyvinylidene Difluoride
(PVDF) membrane (Bio-Rad, Hercules, CA), probed with anti-AtSUMO1 (Abcam, Cambridge, MA) and subsequently with peroxidase-conjugated anti-rabbit secondary antibody (GE healthcare,
Piscataway, NJ, 1:15, 000). For detection, the Supersignal West Pico Chemiluminescent Substrate for
HRP system (Pierce, Rockford, IL) was used.
1.3.7 Immunolocalization
Whole mount immunolocalization in Arabidopsis root tip cells was carried out as described (Friml et al., 2003; Thompson et al., 2005) using anti-NUA (1:100), monoclonal anti-α-tubulin (DM1A,
8 Sigma-Aldrich, 1:100), monoclonal anti-NPC (QE5, recognizing Nup214, Nup153, and p62 in mammals, Covance, Princeton, NJ, 1:250), monoclonal anti-GFP (Molecular Probe, Eugene, OR,
2.5μg/ml) primary antibodies and appropriate secondary antibodies conjugated to Alexa Fluor 488 or
568 (Invitrogen). DNA was counterstained by DAPI (Sigma-Aldrich). The images in Figure 1.6 and
1.9 were collected from a Leica TCS SP2 AOBS Confocal Laser Scanning Microscope equipped with
4 lasers (Red Helium Neon 633nm, Green Helium Neon 543nm, Argon 458/476/488/496/514 nm,
Argon UV). All the other fluorescence images were collected on a PCM 2000/Nikon Eclipse 600 confocal laser scanning microscope as described (Rose and Meier, 2001).
1.4 Results
1.4.1 Identification of NUCLEAR-PORE ANCHOR (NUA), an Arabidopsis protein similar to mammalian Tpr, Drosophila Megator, and Yeast Mlp1/Mlp2 – (Data generated by Annkatrin
Rose and Sun Yong Jeong)
NUA was identified in a targeted phenotypic screen of 36 T-DNA insertion mutants in Arabidopsis genes coding for long coiled-coil proteins that might play a structural-organizational role in the nucleus or the endomembrane system. The genes were selected by the following criteria from the
ARABI-COIL Arabidopsis coiled-coil protein database (Rose et al., 2004): The proteins should be at least 500 amino acids long, with a coiled-coil coverage of at least 50% and either a nuclear localization signal (NLS) or at least one predicted transmembrane domain. For all selected ORFs,
T-DNA insertion lines generated by the Salk Institute Genomic Analysis Laboratory (SIGnAL,
(Alonso et al., 2003; Rose et al., 2004) were acquired from the Arabidopsis Biological Resource
Center (ABRC, The Ohio State University, Columbus, OH) and segregating populations were screened for visible phenotypes to identify proteins with an experimentally approachable biological role. Nua-1 was identified as an extreme early-flowering mutant that was stunted in growth and had phyllotaxy defects in the inflorescence.
9 The translated open reading frame of NUA has significant sequence similarity to mammalian Tpr, an
inner nuclear pore-associated long coiled-coil protein. Figure 1.1 shows the coiled-coil domains in
NUA in comparison to human Tpr, Drosophila (Drosophila melanogaster) Megator, and yeast Mlp1
and Mlp2. The size of the predicted protein, the length of the coiled-coil domain, the presence of a
non-coiled-coil C-terminal tail, and the distribution of predicted NLS are very similar in NUA and the
known Tpr-like proteins (Kuznetsov et al., 2002).
1.4.2 Validation of NUA full-length cDNA
Due to the large size of the predicted NUA coding sequence (> 6kb), the NUA cDNA had been cloned
in four fragments and assembled using the unique AatII, ScaI, and XmaI restriction enzyme sites
(work done by Sun Yong Jeong and Annkatrin Rose). To validate the assembled > 6kb cDNA,
full-length NUA transcript was reverse-transcribed with gene specific primers. After amplification
using BD AdvantageTM 2 PCR enzyme system (BD Biosciences Clontech, NJ), the 6282bp cDNA was fully sequenced and found identical to the assembled cDNA (Figure 1.2).
1.4.3 NUA is conserved in different plant species
By using the full-length NUA protein in WU-BLAST against the plant Gene Indices
(http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/Blast/index.cgi), homologous partial protein sequences with a significant identity and similarity (> 35% and 50%, respectively) were recovered from various plant species, including rice (Oryza sativa), maize (Zea mays), wheat (Triticum aestivum), and potato
(Solanum tuberosum). Hence, NUA is evolutionarily conserved both among and within different kingdoms.
10 Based on the existing EST and microarray data (Zimmermann et al., 2004), NUA appears to be
expressed ubiquitously in all tissues and during all developmental stages. This was also confirmed by
RT-PCR with different tissues including root, stem, flower, silique, and cauline leaf (Performed by
Qiao Zhao; see (Xu et al., 2007b).
1.4.4 Characterization of NUA T-DNA insertion lines
Three additional T-DNA insertion alleles of NUA were identified (Figure 1.3). Flanking sequences of
the T-DNA insertion sites were amplified by PCR and sequenced. The original allele nua-1
(SALK_057101) has an insertion at nucleotide +1855 (with +1 being the A of the ATG) (within intron
8), the nua-2 (SALK_069922) insertion causes a 45 bp deletion between nucleotide +5657 and +5902,
and nua-3 (SAIL_505_H11) has an insertion at nucleotide +7531 (within exon 29). The insertion site
of nua-4 (WiscDsLox297300_17E) was mapped to nucleotide +2145 (within exon 9).
After immunoprecipitation with anti-NUA antibody, no 220 kD protein was detected in nua-1 (note that the short N-terminal fragment potentially expressed in nua-1 would not be detected by anti-NUA).
A weak band of approximately full-length size was detected in nua-2 and a band corresponding to a truncated protein of ca. 100 kD in nua-3. NUA protein was also absent in nua-4 (All the immunoprecipitation data were generated by Sivaramakrishnan Muthuswamy).
Analysis of NUA transcripts by RT-PCR showed that the locus is transcribed both upstream and downstream of the T-DNA insertion in nua-1, nua-3 and nua-4, but that no transcript was detected across the insertion (Figure 1.4 and 1.13B). In contrast, a small amount of full-length transcript in addition to truncated transcripts was detected across the nua-2 insertion (Figure 1.4). The truncated bands were sequenced and indicated that alternative splicing leads to in-frame deletions of 135 bp
(exon 22) or 135 + 99 bp (exons 22 and 23) (Figure 1.3). All PCR products were derived from specific
11 cDNA templates, since they were not present in the control reactions in which reverse-transcriptase
was omitted (data not shown) and since all primer pairs span introns. We speculate that either mRNA
is synthesized through the T-DNA insertion (which is confirmed in the nua-2 mutant) or that a
transcript reads out of the T-DNA into the NUA gene. In any case, no protein in the NUA open reading
frame is made downstream of the T-DNA insertions of nua-1 and nua-4, because it would be
detectable with our antibody. In summary, we conclude that nua-1 and nua-4 are likely null mutations.
In nua-2, a small amount of full-length protein and/or almost-full length proteins with short internal
deletions are present. In nua-3, a partial protein of ca. 100 kD is made.
1.4.5 NUA T-DNA insertion alleles flower early in long days and short days and have
pleiotropic developmental defects
All four lines flowered early in long days (Figures 1.5A, and Table 1.1). nua-1 and nua-4 were the
most extreme alleles and bolted with 4-5 rosette leaves in long days, while nua-2 and nua-3 bolted
with 8-10 and 6-8 leaves, respectively (WT Columbia: 10-12 leaves). Nua-1, nua-2 and nua-3 also
flowered early in short days (Table 1.1), with nua-1 again being the most severe, nua-3 being moderate and nua-2 being a mild allele (nua-4 was not tested). In addition to early flowering, nua-1, nua-4 and nua-3 also showed stunted inflorescences (Figure 1.5 and 1.13A) and smaller, narrower rosette leaves (Figure 1.5), while adult nua-2 plants were indistinguishable from WT.
nua-1 and nua-4 showed several additional developmental alterations not found in nua-2 and nua-3
(Figure 1.5 C-E and 1.13A). The inflorescence of the mutants showed some abnormalities with a reduced number of flower buds on the top of the main inflorescence (Figure 1.5C). Siliques were found at unexpected positions; two or three siliques were positioned at one node or at the top of the main inflorescence. In addition, indeterminate shoots were found in one node together with a silique and a cauline leaf. The size of siliques of the mutants was shorter and more stunted compared to WT 12 (Figure 1.5D), with fewer developed seeds per silique. Furthermore, the majority of flowers did not set seeds. The stamens of the mutants were shorter and the size of petals was slightly smaller than WT
(Figure 1.5E).
Together, we conclude that loss of NUA leads to severe developmental defects and that the isolated
T-DNA insertions comprise an allelic series of increasing severity in the order nua-2, nua-3 and nua-1/nua-4.
1.4.6 Subcellular localization of NUA
We utilized immunofluorescence with anti-NUA to characterize the subcellular localization of NUA in root-tip cells of Arabidopsis seedlings. In interphase cells, NUA is clearly located at the nuclear envelope (Figures 1.6B to 1.6E). During prometaphase, at the onset of spindle formation, the antibody decorated a structure in the vicinity of the spindle, which did not directly co-localize with tubulin
(Figures 1.6B and 1.6C). During metaphase, this signal was less obvious (Figures 1.6D and 1.6E).
During cytokinesis, NUA re-appeared at the nuclear envelope (Figures 1.6D and 1.6E). Figure 1.7 shows that the NE signal is absent in nua-1, indicating that anti-NUA specifically decorates NUA at the NE. To determine if NUA is associated with the inner nuclear envelope, we performed double-labeling in plants expressing a GFP fusion of WIP1, an Arabidopsis outer NE/NPC-associated protein (Xu et al., 2007a). Figure 1.8 shows that the NUA signal could be clearly resolved inside the
GFP-WIP1 signal, indicating that NUA is indeed associated with the inner surface of the nuclear envelope. Consistent with mammalian Tpr being only peripherally attached to the NPC and recruited to the reassembled NPC relatively late during the telophase, NPC assembly and recruitment of other plant nucleoporins like WPP-domain Interacting Protein1 (WPP1) precedes the NPC association of
NUA (Figure 1.9) (Hase and Cordes, 2003; Xu et al., 2007a).
13 1.4.7 nua-1 esd4-2 double mutant analysis
The observed pleiotropic phenotype of nua-1 and nua-4 is reminiscent of the esd4-1 and esd4-2
mutations (Reeves et al., 2002; Murtas et al., 2003). To investigate their genetic interaction,
homozygote nua-1 plants were crossed with esd4-2 (SALK_032317) plants, resulting in WT
phenotype for all plants in the F1 generation. Double mutants in the F2 generation were
indistinguishable from nua-1 and esd4-2 single mutants in terms of flowering time under long day
conditions (Figures 1.10A and 1.10B). The only difference observed was that nua-1 esd4-2 had even
shorter stamens and further reduced fertility than the single mutants (Figure 1.10C and data not
shown). These data suggest that nua-1 and esd4-2 might act in a shared pathway or complex that affects flowering time as well as vegetative and inflorescence development, but that they act
additively in stamen development. This notion is supported by the interaction of NUA and ESD4 in
yeast two-hybrid assays, indicating that ESD4 binds specifically to the N-terminal 533 amino acids of
NUA (Data generated by Sowmya Venkatakrishnan; see Xu et al., 2007b).
1.4.8 Effects of nua mutants on sumoylation and flowering gene expression
ESD4 is a SUMO protease associated with the nuclear envelope (Reeves et al., 2002; Murtas et al.,
2003). Mutations in ESD4 lead to an increase in SUMO conjugates and a decrease in free SUMO
(Murtas et al., 2003). To test if mutations in NUA have a similar molecular phenotype, we investigated
the SUMO conjugation pattern in nua-1, nua-2, nua-3 and nua-4, compared to the T-DNA insertion allele esd4-2 and WT Columbia plants (Figure 1.11A and 1.13C). In esd4-2, the level of high molecular weight SUMO conjugates was increased while the free SUMO level was reduced, as described previously (Murtas et al., 2003). We found that nua-1 and nua-4 phenocopy esd4-2, and that nua-3 leads to an intermediate and nua-2 to the least increase of SUMO conjugates. The level of free
SUMO was reciprocally altered in all alleles.
14 esd4 mutations have been shown to decrease the mRNA level of the floral repressor FLC and increase mRNA levels of the floral activators FT and SOC1 (Reeves et al., 2002). To test whether the nua mutants would also phenocopy these effects, we tested mRNA abundance of FLC, FT and SOC1 by semi-quantitative RT-PCR. Indeed, as shown in Figure 1.11B, FLC mRNA level is strongly reduced in nua-1 and nua-4, comparable to esd4-2, and is somewhat reduced in nua-2 and nua-3, with nua-2 showing the weakest effect. Consistently, FT and SOC1 mRNA levels were found inversely affected.
Since nua-1 and nua-4 flower significantly earlier than the flc-3 null mutant (4-5 compared with 10-11 rosette leaves in long day condition, Table 1.2) (Michaels and Amasino, 1999; Michaels and Amasino,
2001), additional factors involved in flowering-time regulation are likely affected in nua mutants.
Additionally, the ESD4 mutation was also suggested to promote flowering both through and independently of FLC (Reeves et al., 2002). To probe into additional effects of NUA and ESD4 mutations on the flowering pathways, we tested mRNA levels of several additional floral repressors and floral integrators. MAF4, one of the FLC paralogs also implied in floral repression (Ratcliffe et al.,
2003) was decreased in nua-1, nua-3, nua-4 and esd4-2 (Figure 1.11B). Furthermore, the phytohormone gibberellin (GA) is known to promote floral transition strongly under short day conditions via activating the floral integrator LEAFY (LFY) by GAMYB transcription factors (Achard et al., 2004; and references therein). When the expression levels of LFY, MYB33 and MYB65 were analyzed, elevation in nua-1 and nua-4 mutants was apparent (Figure 1.11B), again supporting the notion that several flowering pathways were affected in the nua mutants. We noted that LFY, MYB33 and MYB65 expression levels were not significantly altered in esd4-2, supporting an overlapping, but not identical effect of the two gene knockouts on flowering regulator expression.
15 1.4.9 GFP-ESD4 localization is not altered in nua mutants
If NUA acted as a nuclear pore anchor for ESD4, as predicted for the interaction between Ulp1 and
Mlp1 in yeast, depletion of NUA should lead to a release of ESD4 from the nuclear periphery. We
tested this model by investigating the localization of GFP-ESD4 in WT Columbia and the nua mutant
alleles. Figure 1.12A shows that GFP-ESD4 has a nuclear location with enrichment at the nuclear
envelope in root cell files, consistent with previous reports for ESD4-GFP (Murtas et al., 2003).
Figures 1.12B to 1.12D and 1.13D show that no significant changes in this pattern were seen in nua-1, nua-2, nua-3 and nua-4, suggesting that the increase in SUMO conjugates in nua mutants is unlikely to be based on delocalization of ESD4.
1.5 Discussion
1.5.1 The first viable null mutant of a Tpr-like protein in a multicellular organism
Mammalian Tpr (Park et al., 1986) and its closest structural relatives in Drosophila (Zimowska et al.,
1997) and yeast (Strambio-de-Castillia et al., 1999), are large coiled-coil proteins located at the nucleoplasmic side of the NPC (Bangs et al., 1998; Zimowska et al., 1997; Strambio-de-Castillia et al.,
1999; Cordes et al., 1997). While Mlp1/Mlp2 deletion mutants in yeast are viable, knock-out mutants of Megator are embryonic lethal (Qi et al., 2004). Megator depletion via RNAi leads to a reduction of cells going through mitosis (Qi et al., 2004). No Tpr knock-out mutants have so far been reported in vertebrates. We show here that a likely knock-out of the single Arabidopsis Tpr/Megator/Mlp1/Mlp2 homolog NUA is viable. Loss of NUA leads to complex developmental phenotypes including early flowering, stunted growth, defects in stamen and silique development, and changes in phyllotaxy. Our data indicate that loss of NUA affects a number of regulatory pathways, likely by affecting the expression level of key regulators, as shown here for the example of flowering-time regulators.
16 1.5.2 Connection between nucleoporins and SUMO proteases, comparable but not identical in all kingdoms
Yeast Mlp1 and Mlp2 function in anchoring the SUMO protease Ulp1 to the NPC (Zhao et al., 2004).
Mlp1/Mlp2 deletion mutants in yeast exhibit a clonal lethality phenotype caused by an increase of extrachromosomal 2-micron circle DNA and show an enhanced sensitivity to DNA-damaging drugs
(Galy et al., 2000; Kosova et al., 2000; Zhao et al., 2004). This phenotype is consistent with SUMO pathway mutants and can be suppressed by overexpression of Ulp1, whereas deletion of Ulp1 or delocalization of Ulp1 through deletion of its targeting domain mimics Mlp1/Mlp2 deletion mutants
(Zhao et al., 2004). These data indicate that Mlp1/Mlp2 are required for Ulp1 function, most likely through anchoring Ulp1 at the NPC. In contrast, the mammalian SUMO protease SENP2 is anchored to the inner nuclear pore by binding Nup153, the same nucleoporin that is involved in anchoring Tpr to the nuclear basket (Hang and Dasso, 2002; Zhang et al., 2002b).
Our data show that in Arabidopsis NUA and ESD4 are both associated with the nuclear periphery, and the accumulation of sumoylated proteins in nua mutants indicates that NUA is involved in desumoylation. The close similarity of the whole-plant and molecular phenotypes of nua and esd4 knockout mutants, together with their ability to interact in yeast-two-hybrid assays, are consistent with a model in which the two proteins interact at the nuclear periphery, thereby affecting desumoylation and altering gene expression. If the interaction of NUA and ESD4 were analogous to Mlp1/Mlp2 and
Ulp1, we would expect ESD4 to lose nuclear rim localization in a nua null mutant. However, our data show that GFP-ESD4 is still targeted to the nuclear rim in nua-1 and nua-4, suggesting that the desumoylation defect observed in nua mutants is not caused by the delocalization of ESD4. In addition, the interaction between NUA and ESD4 in yeast two-hybrid assays might be indirect or additional proteins might tether ESD4 to the NPC. Further experiments are needed to test these hypotheses. Our current working model for the functional interaction between NUA and ESD4 is
17 therefore that ESD4 and possibly other Arabidopsis SUMO proteases and NUA have to be in close proximity at the NPC for WT-level desumoylation to occur. This could, for example, be envisioned if sumoylated proteins bind to NUA, thereby being presented to localized SUMO protease activity.
1.5.3 NUA activities link SUMO homeostasis, RNA metabolism, gene regulation and plant developmental control
We have identified an allelic series of four T-DNA insertion alleles that have increasing severity of several correlating whole-plant and molecular phenotypes. In the order nua-2, nua-3, and nua-1/nua-4, we have observed an increase in severity of early flowering, accumulation of sumoylated proteins, nuclear accumulation of poly(A)+ RNA (Data generated by Sivaramakrishnan Muthuswamy, see Xu et al., 2007b; (Jacob et al., 2007), and altered expression of several flowering-time regulators. This suggests that these events are connected by the activity of NUA. Molecular characterization of the
NUA insertion alleles indicates that nua-1 and nua-4 are likely null alleles, that nua-2 is a knock-down allele and that nua-3 expresses a ca. 100kD N-terminal fragment of NUA. Nua-3 acts as a functional knock-down of intermediate severity, indicating that part of the NUA activity involved in SUMO homeostasis, RNA metabolism, and flowering time regulation resides in the N-terminal fragment expressed in nua-3.
SUMO modification has been discussed as a possible mechanism to control nucleocytoplasmic transport of proteins (for review see (Pichler and Melchior, 2002). Recently, hnRNPs, RNA helicases and other proteins involved in RNA metabolism were identified as substrates for SUMO modification in mammals (Vassileva and Matunis, 2004; Vertegaal et al., 2004; Li et al., 2004). This suggests a previously unrecognized link between the SUMO pathway and mRNA metabolism. Our data, showing that nua mutants are affected both in SUMO homeostasis and nuclear RNA accumulation, indicate that such a link also exists in plants. However, how these two processes are connected molecularly
18 and mechanistically remains elusive and an exciting direction to be pursued. Several Arabidopsis mutants are available in which either mRNA export is defective, like los4, or SUMO homeostasis is disrupted, such as esd4, siz1, and the SUMO1/2 overexpressor (Murtas et al., 2003; Gong et al., 2005;
Miura et al., 2005; Lois et al., 2003). It will be of great interest to investigate any correlation between
SUMO and RNA homeostasis in these mutants.
Among all the developmental defects in the nua mutants, flowering time regulation was analyzed in the greatest detail. One of the key factors misregulated in nua mutants is FLC, whose expression is controlled via epigenetic silencing and chromatin remodeling in the autonomous pathway. Several autonomous pathway components are involved in RNA binding and RNA processing (Baurle and
Dean, 2006). Our analysis of nua mutants further strengthens the point that RNA metabolism is important for flowering time regulation. Perhaps, a more intriguing speculation is that NUA, like its yeast homologs Mlp1/Mlp2 (Hediger and Gasser, 2002), may be involved in the nuclear architecture and spatial positioning of the genome which affects the transcriptional state of key regulators and/or more directly the epigenetic control of FLC expression.
19 1.6 Future Outlook
1.6.1 NUA and microRNA homeostasis
Loss of NUA leads to a number of developmental defects, the most striking one being extreme early flowering under both long-day and short-day conditions. Early flowering is consistent with a reduction of FLC expression and a concomitant increase in the expression of FT and SOC1, a reduction of
MAF4 expression, as well as an increase in MYB33, MYB65 and LFY. Since MYB33 and MYB65 gene expression is posttranscriptionally regulated by miR159, their upregulation is consistent with the recent report of a reduction of miR159 in a nua mutant (Achard et al., 2004; Jacob et al., 2007; Millar and Gubler, 2005). However, it still remains undetermined whether NUA is involved in miRNA export or processing or both, which can be addressed by separately analyzing the nuclear and the cytoplasmic small RNA fractions as done for hasty mutants (Park et al., 2005).
1.6.2 The subcellular location of NUA
Several immuno-EM studies have suggested that Tpr is located at the nuclear basket of the NPC
(Cordes et al., 1997; Frosst et al., 2002; Krull et al., 2004). Nevertheless, at the light-microscopic level a continuous NE staining was seen in several recent studies of human Tpr (Hase et al., 2001; Hase and
Cordes, 2003; Krull et al., 2004). Similarly, in our system, we do not detect a punctate pattern for
NUA. This does not contradict nuclear pore localization, but simply indicates that a higher level of resolution (such as immuno-gold labeling) is required to investigate the precise ultrastructural location of NUA.
20 1.6.3 Better understanding of the NUA activities by identifying physical and genetic interactions
Our data suggest that the long coiled-coil protein NUA is important for the spatial organization and regulation of mRNA export and sumoylation in plants, and that disruption in these processes affects the signaling events involved in diverse developmental processes. However, it is unclear how mechanistically NUA is involved in these processes. Because of the role of coiled-coil domains in scaffolding via protein-protein interactions, it will be essential to isolate in vivo binding partners for
NUA to fully understand its function at the molecular level. Proteins involved in mRNA export and
targets of sumoylation are conceivable interacting proteins for NUA. Meanwhile, it will certainly
deepen our understanding on NUA activities to take advantage of the power of Arabidopsis genetics
and to screen for genetic interaction partners of NUA, such as suppressors of the severe alleles
(nua1/4) and enhancers of the intermediate allele (nua-3).
21
NUA NLS NLS dimer NPC NLS Tpr NLS Megator NLS MLP1 NLS 200 aa MLP2
Figure 1.1: NUA has a similar domain structure with its homologs from other species. The coiled-coil domain prediction of NUA (At1g79280) using COILS, and comparison with human Tpr, Drosophila Megator, and yeast Mlp1 and Mlp2. Black bars show predicted coiled-coil domains. NLS, nuclear localization signal. The dimerization and NPC association domain of Tpr are also depicted. A scale bar of 200 aa is shown. (Data generated by Annkatrin Rose)
22
12345 kb
7 6 5 4 * 3
* 2
1.6
Figure 1.2: The validation of the full length NUA cDNA. The full length NUA cDNA was amplified from RNA prepared from 10-day-old whole seedlings. Primer combinations used were F1 + R4-1 (lane 1, full length), F1 + R1 (lane 2, nucleotides 1 to 1599), F1 + R2 (lane 3, nucleotides 1 to 3798), and F4-1 + R4-1 (lane 4, nucleotides 3703 to 6282). Size markers are shown on the right. Two asterisks in lane 1 indicate two non-specific bands.
23
NUA-B nua-1 nua-2 nua-3
NUA-A nua-4 NUA-D NUA-E NUA-F NUA-C
T-DNA nua-2 int 21 int 22 int 23 genome ex 21 ex 22 ex 23 45bp WT cDNA 135bp 99bp 300 bp band 200 bp band
Figure 1.3: Position of T-DNA insertions and PCR primers. The confirmed insertion sites in nua-1, nua-2, nua-3, and nua-4 are indicated by vertical arrows above and below the schematic exon-intron structure of the NUA gene. Exons are depicted as grey bars, introns as black lines. Horizontal arrows indicate the positions of PCR primers and brackets indicate the positions of the RT-PCR products shown in figure 1.4. The area surrounding the nua-2 insertion site is shown enlarged and the structure of the ca. 200 bp and ca. 300 bp fragments derived from RT-PCR with “NUA-D” primers in nua-2 are indicated. Ex, exon; int, intron.
24
WT nua-1 nua-2 nua-3 esd4-2
NUA-A
NUA-B
NUA-C
NUA-D short exposure
500bp NUA-D 300bp long 200bp exposure
NUA-E
NUA-F
Tub
Figure 1.4: Analysis of NUA transcript in nua mutants. RT-PCR products derived from primer pairs indicated on the right (as shown in figure 1.2) and plant tissues as indicated on the top. Approximate sizes of the fragments amplified with the primer pair for “NUA-D” in nua-2 are indicated on the left. Two exposures are shown for the fragments amplified with “NUA-D” primers. The primer combinations in each reaction were: NUA-A, F1 + LP115409; NUA-B, F1 + SALK_057101LP; NUA-C, RP057101.1 + R1(1599); NUA-D, RP069922 + LP069922; NUA-E, CS821281FP + R2(3798); NUA-F, RP079795 + R3(4993). All primer sequences are listed in Table 1.3. Tub, β-tubulin 2 (At5g62690).
25
A
WT nua-1 nua-2 nua-3 B
WT nua-1 nua-2 nua-3
C D nua-1 WT WT nua-1 EE
WT nua-1
Figure 1.5: Phenotypic characteristics of nua mutant alleles. (A) Seedlings at 25 days after germination (DAG), grown under long-day condition. (B) Plants at 34 DAG, grown under long-day condition. (C) Phyllotaxy defects of nua-1. (D) Silique phenotype of nua-1. (E) Flower phenotype of nua-1. Data in C-E were generated by Sun Yong Jeong.
26
A E I
B F J
C G K
D H L
Figure 1.6: NUA is located at the nuclear envelope during interphase and in the vicinity of the spindle during prometaphase. Immunofluorescence images of root tip cell files in interphase (A through D and I through L), prometaphase (A through D), metaphase (E through H), and late cytokinesis (I through L). Green, anti-NUA; red in D, H, and L, and magenta in B, C, F, G, J, and K, anti-tubulin; blue in D, H, and L, DAPI. A, E, and I show the green channel only, B, F, and J show the red channel only (false colored in magenta). C, G, and K show the red and green channels, and D, H, and L show the red, green, and blue channels. Arrowhead in A, interphase nuclear envelope; arrow in A, spindle-like structure in prometaphase. Scale bars in D, H, and L: 10μm.
27
WT nua-1 nua-2 nua-3 nua-4 NUA QE5 Overlay Profile Fluorescence
Figure 1.7: NUA protein revealed by immunofluorescence in all mutant alleles. Immunofluorescence in WT and nua-1, nua-2, nua-3, and nua-4 root tip cells. Anti-NUA (Green) and NPC marker QE5 (magenta) was used to show that NUA is located on the nuclear envelope. The fluorescence profiles of the dotted lines in overlay images were illustrated at the bottom. Note that the green peaks correlate perfectly with the magenta peaks in WT, but not in nua-1, nua-3, and nua-4. Also note the weak NUA signal at the NE in nua-2. The seemingly higher cytoplasmic signal from anti-NUA seen in mutants likely is non-specific. In order to show the much weaker immunofluorescence decoration on the NE in nua-2 and the complete absence of the NE decoration in other mutants, a much higher gain-setting than for wild type was used for those images. Scale bars: 10μm.
28
Figure 1.8: NUA is localized at the inner side of nuclear envelope, shown by co-localization with the outer NE/NPC-localized protein GFP-WIP1. NUA and GFP-WIP1 were detected by rabbit anti-NUA antibody (A) and mouse anti-GFP antibody (B), respectively, with appropriate secondary antibodies. The overlay image is shown in C (green, GFP-WIP1; magenta, NUA). The dashed box in C was enlarged and the green and magenta fluorescence profiles were analyzed in D. Scale bar: 5μm.
29
NUA GFP-WIP1 DAPI Overlay
Figure 1.9: NUA is recruited to the newly-formed NE/NPC relatively late during mitosis. Immunofluorescence in GFP-WIP1 transgenic Arabidopsis root tips with anti-NUA and anti-GFP antibodies. DNA was stained with DAPI. The arrow indicates the reforming daughter nuclear envelope; the arrowhead points to the cell plate.
30
A
WT nua-1
esd4-2 nua-1 esd4-2 B
nua-1 WT nua-1 esd4-2 esd4-2
C
nua-1tpr-1/ esd4-2 WTWT nua-1tpr-1 esd4-2 esd4-2esd4-2
Figure 1.10: Genetic interaction between NUA and ESD4. The nua-1 esd4-2 double mutant resembles nua-1 and esd4-2 in flowering time and stunted growth characteristics, while additive effects exist for stamen length. (A) WT, nua-1, esd4-2, and nua-1 esd4-2 seedlings at 21 DAG, gown under long-day condition. (B) Plants after 34 days in long-day condition. (C) Close-up for open flowers. Arrows indicate top of stamens.
31
A WT nua-1 nua-2 nua-3 esd4-2 kD 250 150
100
75 50
* 30
15
B WT nua-1 nua-2 nua-3nua-4 esd4-2 Tub
FLC
MAF4
FT
SOC1
LFY
MYB33
MYB65
Figure 1.11: Nua mutant alleles lead to increasing accumulation of SUMO conjugates and altered expression of genes involved in different flowering pathways. (A) Protein extracts from 10-day-old seedlings were probed with an anti-SUMO1 antibody. Free SUMO is reduced (arrowhead) and the amount of SUMO conjugates increased (bracket) in nua mutants, like previously shown for esd4 mutants. The effect increases in severity in the order of nua-2, nua-3, and nua-1. Asterisk, putative SUMO dimer. (B) RT-PCR of tubulin 2 (Tub), FLC, MAF4, FT, SOC1, LFY, MYB33, and MYB65 mRNAs in Arabidopsis WT, nua-1, nua-2, nua-3, nua-4 and esd4-2.
32
A B
WT nua-1 C D
nua-2 nua-3
Figure 1.12: The concentration of ESD4 on the nuclear periphery is not abolished in nua mutants. GFP fluorescence was observed at the nuclear periphery, in root cell files of Arabidopsis seedlings transformed with GFP-ESD4. (A) WT background; (B) nua-1 background; (C) nua-2 background; (D) nua-3 background. Scale bar: 10μm.
33
A D
C WT nua-4 kD 250
WT nua-4 WT nua-4 150 100 B Tub NUA-A NUA-B NUA-C NUA-D NUA-E NUA-F 75 WT nua-4 WT nua-4 WT nua-4 WT nua-4 WT nua-4 WT nua-4 WT nua-4 2kb 30 1kb *
0.5kb
0.2kb 15
Figure 1.13: Characterization of the nua-4 mutant. (A) Phenotypic comparison between 35-day-old WT and nua-4 plants. (B) RT-PCR analysis in nua-4. The condition and fragments tested were the same as in Figure 1D. The expected bands are depicted by white arrowheads and size markers are shown on the left. (C) Similar to nua-1 and esd4-2, the nua-4 mutant accumulated a high level of SUMO conjugates (bracket) with a decreased free SUMO level (arrowhead). The putative SUMO dimer band was marked with an asterisk. (D) GFP-ESD4 is still concentrated around the nuclear periphery in the nua-4 mutant. GFP-ESD4 driven by the 35S promoter is stably expressed in nua-4 root cells. Scale bars: 10μm.
34
Columbia nua-1 nua-2 nua-3 nua-4 Long Day 12.2 ± 1.0 4.0 ± 0.0 8.0 ± 0.9 6.1 ± 0.6 4.2 ± 0.4 (45 plants) (53 plants) (16 plants) (16 plants) (53 plants) Short Day 50.6 ± 2.0 7.4 ± 0.5 45.6 ± 1.8 33.6 ± 1.2 ND (11 plants) (53 plants) (10 plants) (6 plants)
Table 1.1: Flowering time of WT Columbia and nua mutants, given as number of rosette leaves at time of bolting. ± standard deviation; Long day:16 hours light, 8 hours dark, 75-125 μmol s-1 m-2. Short day: 8 hours light, 16 hours dark, 85-95 μmol s-1 m-2. ND: Not Determined.
Columbia nua-1 nua-4 flc-3 11.7 ± 0.7 4.7 ± 0.7 4.5 ± 0.6 10.7 ± 0.6 (23 plants) (22 plants) (21 plants) (22 plants)
Table 1.2: Flowering time of WT Columbia, nua mutants, and flc-3 null mutant, given as number of rosette leaves at the time of bolting. ± standard deviation; Long day:16 hours light, 8 hours dark, 75-125 μmol s-1 m-2.
35
Table 1.3: Primer sequences used in Chapter 1.
36
CHAPTER 2
THE KINGDOM- AND TISSUE-SPECIFIC ANCHORAGE OF PLANT RANGAP TO THE
NUCLEAR ENVELOPE INVOLVES A NOVEL FAMILY OF PLANT NUCLEAR
PORE-ASSOCIATED TRANSMEMBRANE PROTEINS2
2 This chapter has been published as:
Xu XM, Meulia T, Meier I. (2007). Anchorage of Plant RanGAP to the Nuclear Envelope Involves
Novel Nuclear-Pore-Associated Proteins. Curr. Biol. 17, 1157-63.
37 2.1 Abstract
Ran GTPase controls multiple cellular processes including nucleocytoplasmic transport, spindle assembly, cell-cycle control, and nuclear envelope (NE) formation. Its roles are accomplished by the asymmetric distribution of RanGTP and RanGDP enabled by the specific locations of the Ran
GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1. Mammalian RanGAP1 targeting to the NE during interphase and the spindle and kinetochores during mitosis requires interaction of its sumoylated C-terminal domain with the nucleoporin Nup358/RanBP2. In contrast,
Arabidopsis RanGAP1 is associated with the NE during interphase and the cell plate during cytokinesis, mediated by an N-terminal, plant-specific domain (WPP domain). In the absence of
RanBP2 in plants, the mechanism of spatially sequestering plant RanGAP is unknown. Arabidopsis
WPP-domain Interacting Protein 1 (WIP1) was identified, which interacts with RanGAP1 in vivo and co-localizes with RanGAP1 at the NE and cell plate. WIP1 and its two homologs WIP2 and WIP3 contain an extended coiled-coil domain necessary and sufficient for RanGAP1 binding and a
C-terminal transmembrane domain region necessary and sufficient for NE targeting. Immuno-gold labeling indicates that WIP1 is associated with the nuclear pore. In a wip1-1/wip2-1/wip3-1 triple mutant, RanGAP1 is dislocated from the NE in undifferentiated root tip cells, while NE targeting in differentiated root cells and targeting to the cell plate remain intact. We propose that WIP family members are novel plant nucleoporins involved in RanGAP1 NE anchoring in specific cell types. Our data support a separate evolution of RanGAP targeting mechanisms in different kingdoms.
2.2 Introduction
Ran, a small GTPase of the Ras superfamily, has been shown to play essential roles in several cellular events, such as nucleocytoplasmic transport of macromolecules, spindle formation, mitotic regulation and post-mitotic nuclear envelope (NE) re-assembly (Arnaoutov and Dasso, 2003; Arnaoutov and
38 Dasso, 2005; Dasso, 2002; Hetzer et al., 2002; Quimby and Dasso, 2003). Ran accomplishes its roles by switching between a GTP- and a GDP-bound form. Like all small GTP-binding proteins, Ran has a very low intrinsic GTP hydrolysis activity, which is stimulated by the Ran GTPase activating protein
RanGAP together with the accessory factor RanBP1 (Bischoff et al., 1994; Seewald et al., 2003;
Bischoff et al., 1995b). The nucleotide exchange factor RCC1 replaces the GDP from Ran-GDP with
GTP, thus regenerating Ran-GTP (Bischoff and Ponstingl, 1991).
During interphase, Ran is predominately GTP-bound in the nucleus and GDP-bound in the cytoplasm, due to nuclear RCC1 and cytoplasmic RanGAP activity, respectively (Izaurralde et al., 1997). The
Ran-GTP concentration gradient across the NE provides the information for the subcellular compartment identity and establishes the directionality of active nucleocytoplasmic transport
(Izaurralde et al., 1997; Kalab et al., 2002). Specifically, Ran-GTP promotes the loading and unloading of transport receptors in a manner that is appropriate to the nucleus or cytoplasm (Gorlich and Kutay, 1999a; Gorlich et al., 2003). During mitosis, in vertebrates, chromatin-associated RCC1 generates a high concentration of Ran-GTP around the chromosomes, promoting the spindle formation by releasing factors required for microtubule polymerization from transport receptors (Gruss et al.,
2001; Nachury et al., 2001; Wiese et al., 2001). Meanwhile, spindle checkpoints are responsive to
Ran-GTP levels, which are also important for the definition of kinetochore fibers and chromosome segregation at anaphase (Arnaoutov and Dasso, 2003; Arnaoutov and Dasso, 2005). Furthermore, during telophase, local concentration of Ran-GTP and its hydrolysis around the daughter chromosomes are important for NE and nuclear pore complex (NPC) re-assembly (Hetzer et al., 2000;
Walther et al., 2003b; Zhang and Clarke, 2000; Zhang et al., 2002a). Taken together, the distribution of
Ran-GTP and Ran-GDP, determined by the spatial positioning of RCC1 and RanGAP, is critical for the various functions of Ran. Hence, understanding the subcellular localization of the Ran cycle regulators, specifically RanGAP, is crucial to appreciate the control and functions of the Ran pathway.
39 In vertebrates, RanGAP1 is cytoplasmic during interphase, with a large fraction concentrated on the
outer NE, specifically around the cytoplasmic side of the NPC (Mahajan et al., 1997; Matunis et al.,
1996). The concentration of RanGAP1 on the NE requires the interaction between RanGAP1 and
RanBP2/Nup358, depending on the SUMO1 modification of a Lysine residue in the C-terminal
domain of RanGAP1 (Mahajan et al., 1997; Mahajan et al., 1998; Matunis et al., 1998; Saitoh et al.,
1997; Saitoh et al., 1998). In vitro experiments have demonstrated that RanBP2-associated RanGAP1 is required for nuclear import and cytosolic RanGAP1 cannot substitute for this requirement (Mahajan et al., 1997). However, in vivo evidence and computer simulation have suggested that the concentration of RanGAP1 close to the NPC is largely dispensable for simple Ran-driven import or export processes (Bernad et al., 2004; Bernad et al., 2006; Forler et al., 2004; Gorlich et al., 2003;
Hutten and Kehlenbach, 2006; Walther et al., 2002). Sumoylated RanGAP1 and RanBP2/Nup385 also form a stable complex during mitosis and localize in the vicinity of the spindle and at kinetochores, where they are essential for microtubule-kinetochore interactions and chromosome segregation
(Arnaoutov and Dasso, 2005; Joseph et al., 2002; Joseph et al., 2004; Matunis et al., 1996; Salina et al.,
2003).
Unlike the situation in mammalian cells, yeast RanGAP (Rna1p) is predominantly cytoplasmic without a significant concentration on the NE and does not possess the sumoylation domain (Hopper et al., 1990; Melchior et al., 1993). In Aspergillus nidulans, RanGAP is also predominantly cytoplasmic during interphase, with no association with the NE, and disperses throughout the cell during mitosis (De Souza et al., 2004). Similar to mammalian RanGAP and unlike fungal RanGAP, plant RanGAP is concentrated at the NE (Pay et al., 2002; Rose and Meier, 2001). However plant
RanGAP lacks the sumoylated C-terminal domain, and instead, contains an N-terminal plant-specific domain (WPP domain, named after a highly conserved Trp-Pro-Pro motif) (Meier, 2000). The WPP domain is necessary and sufficient for the targeting of plant RanGAP to the NE during interphase and to the cell plate during cytokinesis (Jeong et al., 2005; Rose and Meier, 2001). Fungi, in contrast to
40 plants and animals, undergo closed mitosis (defined as mitosis occurring inside an intact nuclear membrane), and purely or predominantly cytoplasmic RanGAP would theoretically be sufficient for maintaining the asymmetric distribution of Ran-GTP required for nucleocytoplasmic transport.
Consequently, the specific RanGAP targeting observed in animals and plants might be more crucial to mitotic than to interphase Ran gradient functions.
No evidence exists for the presence of a RanBP2/Nup358 homolog in plants. Together with the kingdom-specificity of the plant and mammalian RanGAP targeting domains, this suggests that separate mechanisms have evolved in different higher eukaryotes for targeting RanGAP to its subcellular positions in interphase and during cytokinesis. While mammalian RanGAP appears to traffic bound to the nucleoporin RanBP2/Nup358, the interaction partner(s) of plant RanGAP are unknown. Due to its different targeting domain and mitotic localization, we have postulated that plant
RanGAP utilizes a protein or proteins fundamentally different from RanBP2/Nup358 (Jeong et al.,
2005).
Here, we have identified a family of plant specific membrane proteins that interact with the WPP domain and that are required for the accumulation of plant RanGAP at the NE. Plant RanGAP targeting appears to involve different binding partners in different cell types. We provide the first evidence on a multicellular level that concentration of RanGAP at the NE is dispensable for normal growth and development. In addition, targeting of plant RanGAP to the NE in interphase and the cell plate during cytokinesis appears to involve separate mechanisms.
41 2.3 Materials and Methods
2.3.1 Protein sequence analyses
To identify putative homologs of WIP1, first, GenBank was searched using PSI-BLAST and
PHI-BLAST. After six rounds of iteration, proteins with significant similarity to WIP1 were identified
from rice and wheat. Hits from non-plant species had similarity only within the coiled-coil domain,
indicating that they were not true homologs. Second, a WU-BLAST search was performed with the
TIGR Unique Gene Indices (http://tigrblast.tigr.org/tgi/). Again, only plant sequences were found with significant similarity. Hits representing full-length and partial protein sequences were used to construct the alignments in Figures 2.4B and 2.5A using MegAlign (DNASTAR). The coiled-coil and transmembrane domain prediction was performed using COILS
(http://www.ch.embnet.org/software/COILS_form.html) and TMPRED
(http://www.ch.embnet.org/software/TMPRED_form.html), respectively. The amino acid sequence identity and similarity shown in Figure 1C was determined by bl2seq
(http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) (Tatusova and Madden, 1999) using the
BLOSUM62 matrix.
2.3.2 Plant materials
Arabidopsis seedlings (Columbia ecotype) used for all analyses were grown in soil under standard
long-day condition (16 hour light and 8 hour dark) or on MS (Caisson Laboratories, Rexburg, ID)
plates under constant light. For T-DNA insertion mutants wip1-1 (SAIL_390_A08) and wip2-1
(SALK_052226), T3 or T4 bulk seeds were acquired from the Arabidopsis Biological Resource
Center (ABRC, The Ohio State University, Columbus, OH). Mutant wip3-1 (459H07) was obtained from the GABI-Kat collection (Rosso et al., 2003). Homozygous insertion plants were identified by
PCR genotyping (http://signal.salk.edu/tdnaprimers.2.html).
42 2.3.3 RNA extraction and RT-PCR
Total RNA was extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from 6-10 day
old seedlings or tissues from 30-day-old Arabidopsis plants grown on soil. After digestion with DNase
I (Amplification grade, Invitrogen, Carlsbad, CA), cDNA synthesis was performed using oligo-dT
primers and the ThermoscriptTM RT-PCR system (Invitrogen). cDNA templates were PCR amplified with gene-specific primers for cloning or quantification of mRNA levels.
2.3.4 Constructs
The WIP1, WIP2a and WIP3 cDNAs were cloned by RT-PCR from total seedling RNA into the pENTR/D-TOPO vector (Invitrogen). The inserts were confirmed by sequencing. To construct the
N-terminal GFP fusion proteins, each cDNA was moved into pK7WGF2 (Karimi et al., 2002) by LR recombination cloning (Invitrogen). All GFP fusions with WIP1 fragments were generated the same way. WIP1, WIP2a and WIP3 cDNAs were moved into the ProQuest® Y2H vectors pDEST22 and pDEST32 (Invitrogen) by LR recombination to express the GAL4 AD and BD fusion proteins.
AD-WIP1N and AD-WIP1cc were generated the same way. RanGAP1, RanGAP1ΔN, RanGAP1ΔC,
WPP1, WPP2 and WPP3 (Patel et al., 2004; Rose and Meier, 2001) were first subcloned into pENTRTM3C as an EcoRI fragment and then recombined into pDEST22 and pDEST32.
BD-RanGAP2 was made by moving the ORF of RanGAP2 from pENTR/D-TOPO to pDEST32. For
constructs used in BiFC analysis, WIP1, WIP2a and WIP3 were cloned into pSY736 (Bracha-Drori et
al., 2004) as a SpeI fragment, generated by PCR. RanGAP1 and WPP1 were subcloned into pSY738
as an NcoI fragment from corresponding pRTL2 vectors (Rose and Meier, 2001). For promoter-GUS
constructs, 749bp, 1809bp and 1611bp genomic fragments upstream of the ATG of WIP1, WIP2 and
WIP3, respectively, were cloned into pENTR/D-TOPO and moved by LR recombination into
pMDC162 (Curtis and Grossniklaus, 2003). For mutagenesis, starting with WIP1ΔTDF in
43 pENTR/D-TOPO, point mutations were introduced by QuickChange® Site-Directed Mutagenesis kit
(Stratagene, La Jolla, CA) and confirmed by sequencing. The mutant versions of WIP1ΔTDF were then moved into pK7WGF2 to express GFP fusion proteins under the control of the 35S promoter.
2.3.5 Yeast two-hybrid assays
All plasmid pairs were transformed into the yeast strain PJ694A (James et al., 1996) according to published protocols (Dohmen et al., 1991). Handling of yeast cultures and plate growth assays were as described in the Clontech Yeast Protocols Handbook (1996).
2.3.6 Antibody development
For anti-WIP1 antibody production, a partial protein consisting of the N-terminal 322 aa was expressed as a His-tag fusion protein from pDEST17 (Invitrogen). After purification on a Ni-NTA resin and preparative SDS-PAGE, a rabbit antiserum (OSU 159) was produced by Cocalico
Biologicals (Reamstown, PA).
2.3.7 Generation of transgenic plants
Plasmids expressing GFP-WIP1, GFP-WIP2a, GFP-WIP3, GFP-WIP1ΔN, GFP-WIP1ΔTDF,
GFP-TDFWIP1, GFP-WIP1ΔVVPT and GFP-RanGAP2 were mobilized into the Agrobacterium strain ABI by triparental mating with the helper plasmid pRK2013 (Ditta et al., 1980). Transconjugants were selected on LB plates containing 50μg/ml kanamycin, 34μg/ml chloramphenicol, and 50μg/ml spectinomycin. Arabidopsis Columbia wild type and wip1-1/wip2-1/wip3-1 were transformed by floral dipping (Clough and Bent, 1998) and selected by Kanamycin resistance. RanGAP1-GFP and
RanGAP1ΔC-GFP transgenic Arabidopsis were previously described (Jeong et al., 2005).
44 2.3.8 Co-IP experiments
RanGAP1-GFP, GFP and GFP-WIP1 expressing Arabidopsis were grown in constant light for 14 days.
Whole seedlings were collected and ground into fine powders. Extracts for co-IP were prepared at 4oC
in a buffer containing 50mM Tris–HCl pH 7.5, 150mM NaCl, 0.5% NP-40, 1mM EDTA, 3mM
dithiothreitol (DTT), 1mM phenylmethylsulphonyl fluoride (PMSF), Protease Inhibitor Cocktail
(1:100, Sigma, St Louis, MO). The immunoprecipitates were prepared using monoclonal anti-GFP
antibody (A11120, Molecular Probes, Eugene, OR) bound to Protein A-sepharose beads (GE
healthcare, Piscataway, NJ) with a 3-hour binding. The precipitates were then analyzed by SDS-PAGE,
transferred to nitrocellulose membranes and probed with anti-WIP1 (1:2000) or anti-RanGAP1 antibody (1:3000).
45 2.3.9 Cell culture
Maintenance of Nicotiana benthamiana cell culture, protoplast isolation and transfection by
electroporation were performed essentially as described (Qi and Ding, 2002). 5-10 μg of each plasmid
DNA was used per electroporation. After overnight incubation, protein expression was visualized by
confocal microscopy.
2.3.10 Immunolabeling and confocal microscopy
Whole-mount immunolocalization in Arabidopsis roots was carried out as described (Friml et al.,
2003). Polyclonal anti-RanGAP1 (1:100 to 1:200), monoclonal anti-tubulin (DM1A, Sigma, 1:100),
monoclonal anti-NPC (QE5, Covance, Princeton, NJ, 1:150) primary antibodies and appropriate secondary antibodies conjugated with Alexa Fluor 488 or 568 were used. Propidium iodide (Sigma),
SYTOX Orange (Invitrogen) and DAPI (Sigma) were used in H2O at the final concentrations of
10μg/ml, 1μM and 1μg/ml, respectively. Images including DAPI staining were collected from a Leica
TCS SP2 AOBS Confocal Laser Scanning Microscope equipped with 4 lasers (Red HeNe 633nm,
Green HeNe 543nm, Argon 458/476/488/496/514 nm, Argon UV). All other fluorescence images were collected on a PCM 2000/Nikon Eclipse E600 confocal laser scanning microscope as described (Rose and Meier, 2001).
46 2.3.11 Scanning electron microscopy
200 μm thick samples from wild-type Arabidopsis root callus tissues were pressure frozen in the presence of 20% BSA using the Bat-Tec HMP instrument, and then transferred using the VCT100 transfer (shuttle) device to the Bal-Tec MED020 freeze-fracturing instrument. Samples were fractured and etched in the MED020 at –1050C, rotary shadowed with Pt/C at a 450 angle with stage rotation at
80rpm. Samples were then transferred using the VTC shuttle to the Hitachi S-4700 FE-SEM equipped
with the Emitech1250 cryostage, and imaged at 2.0kV using the secondary detector.
2.3.12 Transmission electron microscopy immunolocalization
Samples from callus tissues developed from roots of wild-type Arabidopsis were cryopreserved in the
presence of 20% BSA as filler using the Bal-Tec HPM instrument, and freeze-substituted to ethanol in
the Leica AFS freeze-substitution system. Samples were transferred directly to the substitution
temperature (-900C), then warmed up to room temperature at 100C/hour, infiltrated and embedded in
LR White resin (EMS cat#14383) following the standard procedures.
Thin sections (80nm) from 2 separate blocks, were collected on formovar and carbon coated 200 mesh nickel grids. Grids were wetted with double-distilled water (ddH20), and blocked with the Aurion Goat
Blocking solution (EMS cat#25596). Antibody incubations were performed overnight at 40C with a
1/50 dilution of the primary antibody, and for 4 hours at room temperature with a 1/100 dilution of the secondary antibody in PBS (10mM potassium phosphate, 150 mM NaCl, pH7.2) containing 10mM
NaAzide, and 0.5% BSA-c (EMS cat#25557). The following antibodies were used: rabbit polyclonal anti-WIP1, rabbit polyclonal anti-RanGAP1, the corresponding pre-immune sera, and
F(ab’)2-goat-anti-rabbit coupled to 10nm gold particles (EMS cat#25365) as secondary antibody.
After each antibody incubation, grids were washed 8 times with incubation buffer, and then 3 times
47 with ddH20, prior post-fixation with 1% glutaraldehyde in ddH20 and subsequent 5 washes with ddH20.
Sections were stained with 2% aqueous uranyl acetate for 8 minutes and Raynold’s lead citrate stain for 2 minutes, and viewed on the Hitachi H-7500 transmission electron microscope.
Gold particles on the nuclear perimeter were counted on at least three separate grids from three separate experiments. The nuclear perimeter was measured using ImageJ (Abramoff et al., 2004), and the amount of labeling was reported as number of gold particles per unit of NE. On average, 50-60 nuclei were counted for each specimen. Background immunogold labeling was estimated by comparing the labeling on the mitochondria with the preimmune and immune sera. As mitochondria appear of different sizes and shapes, gold particles on mitochondria of a defined size (average size of
0.42 square microns as measured using ImageJ) within a grid section were counted, and the number of gold particles per organelle calculated. The labeling from three separate experiments was counted. For mitochondrial labeling we observed an increase of 56% for RanGAP1, and 20% for WIP1, while for the nuclear envelope labeling we observed an increase of 888% for RanGAP1, and 162% for WIP1, with RanGAP1 and WIP1 antiserum relative to the corresponding pre-immune serum.
2.3.13 GUS assays
Histochemical staining of GUS expression was performed essentially as described (Jefferson et al.,
1987).
2.4 Results
2.4.1 Identification of an Arabidopsis RanGAP-binding protein family
To understand the anchoring mechanism of RanGAP1 at the plant NE, a yeast two-hybrid screen was performed to identify proteins that interact with the WPP domain of Arabidopsis RanGAP1.
48 WPP-domain Interacting Protein 1 (WIP1, At4g26450) was found to bind full-length RanGAP1 as
well as the WPP domain, but not RanGAP1 without the WPP domain (Figure 2.1D). It is a previously
undescribed protein of 489 amino acids with a single predicted transmembrane domain at the
C-terminus and an adjacent coiled-coil domain (Figure 2.1A). No further conserved motifs were
identified except for a putative bipartite NLS (Nuclear Localization Signal) (Figures 2.1A and 2.5).
To confirm the interaction between RanGAP1 and WIP1 in vivo, we performed co-immunoprecipitation (co-IP) experiments from either RanGAP1-GFP or GFP-WIP1 expressing transgenic Arabidopsis plants (Figure 2.1B). When RanGAP1-GFP was precipitated with an anti-GFP antibody, WIP1 could be detected in the precipitates, while no signal was detected from a plant expressing free GFP (Figure 2.1B, upper panel). Consistently, RanGAP1 was co-precipitated with
GFP-WIP1, but not free GFP (Figure 2.1B, lower panel).
WIP1-like proteins were identified from rice, tomato, wheat, maize, and several other higher plant species (see Figure 2.4B). In Arabidopsis, At5g56210 (WIP2) and At3g13360 (WIP3) encode the most closely related proteins (Figure 2.1C). All WIP-like proteins shared similar domain structures, including the NLS, the coiled-coil domain, and the putative transmembrane domain. Available cDNA sequences (GenBank accession number: AY735735 and AY735734) indicate that two WIP2 alternative splicing forms exist. WIP2a contained all the domains conserved in the whole family while WIP2b lacked the N-terminal 220 amino acids including the NLS (see Figure 2.6A). No WIP-like proteins were identifiable from non-plant species.
To test WIP2a and WIP3 for interaction with RanGAP1, map the interaction domain of WIP1, and test the binding of WIP family members to other Arabidopsis WPP-domain proteins, yeast two-hybrid interactions were assayed. Two WIP1 fragments were generated, representing the N-terminal 312 amino acids (WIP1N) and the coiled-coil domain (WIP1cc) (aa 313-459). Two previously described 49 fragments of RanGAP1 were tested, RanGAP1ΔC (aa 1-119, representing the WPP domain) and
RanGAP1ΔN (aa 120-535, RanGAP1 without the WPP domain) (Rose and Meier, 2001). Arabidopsis expresses five WPP-domain proteins. In addition to RanGAP1 these are the second RanGAP
(RanGAP2) and three short proteins with similarity to the N-terminus of RanGAP, WPP1, WPP2, and
WPP3. WPP1 and WPP2 associate with the NE, while WPP3 is cytoplasmic and nuclear. Knock-down mutants of the WPP gene family have root growth defects (Patel et al., 2004).
All yeast two-hybrid results are summarized in Figure 2.1D. The RanGAP1 WPP domain
(BD-RanGAP1ΔC) and the WIP1 coiled-coil domain (AD-WIP1cc) are necessary and sufficient for
RanGAP1-WIP1 interaction. WIP2a and WIP3 bind the WPP domain of RanGAP1, but not full-length
RanGAP1. RanGAP2 binds WIP1 and WIP2a, but not WIP3, and the WIP1 coiled-coil domain is necessary and sufficient for the interaction with RanGAP2. WPP1 and WPP2 bind all three WIP full-length proteins and the WIP1 coiled-coil domain, while WPP3 does not bind WIP family members.
WIP2a does homodimerize as well as heterodimerize with WIP1, but not with WIP3. The WIP1 coiled-coil domain is necessary and sufficient for heterodimerization with WIP2. WIP3 neither homodimerizes nor heterodimerizes with WIP1 or WIP2a. (Because WIP1, WIP1N and WIP1cc self-activate and could only be tested as GAL4 activating domain fusions, no data exist about WIP1 homodimerization.)
2.4.2 Subcellular localization of the WIP protein family and the WIP-RanGAP interaction
To determine the localization of WIP1 and its family members, N-terminal GFP fusion proteins were visualized in transiently transformed Arabidopsis protoplasts (data not shown) and in transgenic
Arabidopsis roots. GFP-WIP1, GFP-WIP2a and GFP-WIP3 clearly associated with the NE (Figure
2.2A). Co-localization of WIP1 and RanGAP1 at the NE was confirmed in GFP-WIP1 transgenic plants by double immunolabeling with a mouse GFP antibody and rabbit RanGAP1 antibody (Figure
50 2.2B, upper panel). Because the punctuate staining observed for GFP-WIP1 was reminiscent of nuclear pores, especially at a low expression level, double labeling with a nuclear pore marker (QE5, recognizing Nup214, Nup153, and p62 in mammals) (Pante et al., 1994) was performed. The good correlation between the GFP and QE5 staining patterns indicated a possible association of GFP-WIP1 with the NPC (Figure 2.2B, lower panel).
The yeast two-hybrid data showed that WIP2a and WIP3 interact with the WPP domain of RanGAP1 but not full length RanGAP1 (Figure 2.1D), reflecting either lower affinities or different specificities.
To probe into all protein-protein interactions in vivo, we employed Bimolecular Fluorescence
Complementation (BiFC) (Hu et al., 2002), which also allows determination of the subcellular location of the interaction (Bracha-Drori et al., 2004). The N-terminal half of YFP (YN) was fused to
WIP1, WIP2a and WIP3 and the C-terminal half of YFP (YC) to RanGAP1 and to WPP1 as a representative of the WPP family. Fluorescence was detected when either RanGAP1-YC or WPP1-YC was co-expressed with any of the three YN-WIP constructs (Figure 2.2C). None of the constructs showed fluorescence when transfected into protoplasts alone or when co-transfected with an empty
YN or YC vector (data not shown). Fluorescence was mainly at the NE, with some fluorescence also detected in the cytoplasm and in cytoplasmic aggregates, possibly reflecting over-expression of the fusion proteins. These data indicate that all three WIP family members are capable of interacting with
RanGAP1 and with WPP1 at the NE.
2.4.3 Ultrastructural analyses of WIP1 and RanGAP1 localization at the NE – (Data generated by Tea Meulia)
Mammalian RanGAP1 and its NE anchor RanBP2/Nup358 have been mapped ultrastructurally at the cytoplasmic filaments of the NPC (Mahajan et al., 1997; Matunis et al., 1996; Walther et al., 2002).
The functional conservation between metazoan and plant RanGAP suggests that plant RanGAP might
51 also be at the cytoplasmic side of the NE and enriched around the NPC (Bischoff et al., 1995a; Pay et al., 2002). A series of z-stack confocal images were taken, spanning half of the nucleus in GFP-WIP1 expressing root callus cells. When three-dimensional maximal projection was constructed, the GFP signal appeared in a dotted pattern (Figure 2.2E, left panel), which closely resembled the NPCs, illustrated by scanning electron microscopy on cryo-preserved Arabidopsis root callus nuclei (Figure
2.2E, middle and right panel).
Subcellular localization of plant RanGAP1 and WIP1 was further investigated using transmission electron microscopy (TEM) post-embedding immunogold labeling. The gold labeling is consistent with RanGAP1 accumulating at the cytoplasmic side of the NE, mostly in association with nuclear pores (Figure 2.2F, top panel). The anti-WIP1 antibody gave a lower amount of signal compared to
RanGAP1, which was however still 2.6 fold above the preimmune background (Figures 2.2G and
2.2H, for background quantification see Methods). Gold particles were detected at the outer surface of the NE, mostly in association with nuclear pores (Figure 2.2F, lower panels). Taken together, both the fluorescence microscopy and TEM data support the idea that WIP1 is an outer NE protein, most likely located at the nuclear pore.
52 2.4.4 Localization of the WIP family during cytokinesis
Like animal cells, plant cells undergo open mitosis with NE breakdown during prophase and re-assembly during telophase (Jurgens, 2005). However, cytokinesis differs between plant and animal cells. While animal cells divide by furrow ingression and scission, plant cytokinesis involves the assembly of new plasma membrane and cell wall (called the cell plate) via the phragmoplast, a microtubule-based structure possibly analogous to the spindle midzone/midbody (Otegui et al., 2005).
Arabidopsis RanGAP1 was shown to associate with the nascent cell plate during cytokinesis, suggesting an additional role for the Ran cycle during plant cell division (Nachury et al., 2001). Here, we show that GFP-WIP1 and GFP-WIP2a co-localize with RanGAP1 at the cell plate in dividing root tip cells while GFP-WIP3 appears at the re-forming nuclei, but not the cell plate (Figure 2.2D).
2.4.5 Plant NE dynamics during cell cycle
WIP1 is the first available plant protein to track the fate of the plant NE during cell cycle. We therefore followed the dynamics of GFP-WIP1 at a higher resolution during mitosis and cytokinesis
(Figure 2.3). During late preprophase, first ruptures could be seen in an otherwise continuous
GFP-WIP1-decorated NE. In metaphase, when chromosomes were aligned at the mid-plate,
GFP-WIP1 appeared on a tubular-vesicular network distant from the chromosomes, towards the spindle pole regions. During anaphase, the GFP-WIP1 signal was further vesiculated, with aggregations starting to form at the outer core regions of the daughter chromosomes. At telophase, when the phragmoplast formed, GFP-WIP1 partially enclosed the daughter chromosomes consistently only at the outer region. Simultaneously with fully encircling of the daughter nuclei, GFP-WIP1 also appeared at the cell plate. Using lamin B receptor-GFP and GFP-emerin, it was shown that in HeLa cells assembly of the NE started at late anaphase to very early telophase, just after the contractile ring started pinching the plasma membrane and that several NPC components (RanBP2, Nup153, p62)
53 reconstituted around chromosomes early in telophase prior to the recovery of nuclear import activity
(Haraguchi et al., 2000). Our data show that similarly plant NE re-assembly commences in anaphase
and appears complete concomitant with early cell plate formation.
2.4.6 Domain requirement for the subcellular targeting of WIP1
If WIP1 is an anchor for plant RanGAP at membrane systems, we would predict that its own targeting
to the NE and cell plate is independent of the RanGAP1 binding domain, and likely dependent on the
transmembrane domain. To determine which domain is responsible for targeting WIP1 to the NE and
cell plate, different deletion constructs were fused to GFP and tested both in transiently transfected
Nicotiana benthamiana protoplasts (data not shown) and transgenic Arabidopsis plants (Figures 2.4A).
The N-terminal domain of WIP1 (aa 1-312) was not required for NE targeting (Figure 2.4A). Without
the C-terminal 36 amino acids, containing the predicted transmembrane domain, GFP-WIP1ΔTDF lost
NE targeting and became predominantly nuclear (Figure 2.4A), dependent on the bipartite NLS in the
N-terminal domain (see figure 2.5). If the transmembrane domain-containing fragment (TDF, aa
453-489) was fused to GFP, the fusion protein (GFP-TDFWIP1) was specifically targeted to the NE
(Figure 2.4A), indicating that the TDF is necessary and sufficient for NE targeting of AtWIP1.
Likewise, the TDF was necessary and sufficient for cell plate targeting, with GFP-WIP1ΔTDF accumulating in the re-forming nuclei and GFP-TDFWIP1 accumulating at the cell plate (Figure 2.4C).
When GFP was fused to the C-terminus of WIP1, the fusion protein accumulated in the cytoplasm
(data not shown), indicating that the free C-terminus is required for NE targeting, indicative of WIP1 being a tail-anchored (TA) protein (Borgese et al., 2003).
All WIP1-like proteins have a highly conserved 4-amino acid motif at the very C-terminus, adjacent to the conserved transmembrane domain (Figure 2.4B). When the last 4 amino acids (VVPT) of WIP1 were deleted, the specificity of NE targeting was significantly impaired compared to GFP-WIP1
54 (Figure 2.4A), indicating that this motif is involved in the selective association of WIP1 with the NE.
Together, these data indicate that WIP1 is targeted to the NE and cell plate via its C-terminus, likely
by direct membrane association, consistent with the features postulated for a RanGAP anchor.
2.4.7 Role of the WIP family in RanGAP anchoring
To determine the role of WIP1 and its family members in plant RanGAP anchoring, homozygous
T-DNA insertion lines in each locus were identified (Figure 2.6A). Figure 2.6B shows that in wip1-1
neither WIP1 RNA nor WIP1 protein could be detected, consistent with a knockout mutation and that
in wip3-1 no WIP3 RNA could be detected. Because of the two alternative splicing variants of WIP2, the line wip2-1 has an insertion in the first exon of AtWIP2a and the first intron of AtWIP2b. This line
was a knockout for AtWIP2a and a severe knockdown for AtWIP2b, based on RNA level (Figure
2.6B). Individual homozygous insertion lines were tested by immunofluorescence for RanGAP1
localization and no difference from wild-type plants was found (data not shown). Lines were therefore
crossed to obtain all double mutant combinations and the triple mutant. No difference in RanGAP1
localization was found in the double mutants (data not shown).
Strikingly however, both endogenous RanGAP1 and RanGAP1-GFP were entirely absent from the NE
in root tip cells of wip1-1/wip2-1/wip3-1 triple mutant lines (Figures 2.6C and 2.6D). While RanGAP1
NE association was entirely lost in root tip cells, the association was unchanged in cells from the root
differentiation zone (defined by the appearance of root hairs, see (Ishikawa and Evans, 1995). QE5
decoration of the NE was not altered in wip1-1/wip2-1/wip3-1, indicating the absence of gross
alterations in NPC or NE assembly. Figure 2.6D shows that a GFP fusion protein of the N-terminal
RanGAP1 WPP-domain also lost association with the NE in root tips of wip1-1/wip2-1/wip3-1 triple
mutants, indicating that loss of NE targeting reflects loss of WPP-domain binding. Again, NE
association was not lost in differentiated cells (note that this fusion protein also enters the nucleus due
55 to its small size). Similarly, in wip1-1/wip2-1/wip3-1 triple mutants RanGAP2 lost its concentration on
the NE in the root tip zone but not in differentiated cells, shown by a GFP fusion protein (Figure 2.6D).
As control, RanGAP1(AAP)-GFP with the WPP motif mutated to AAP, lost the association with the
NE in all cell types (Figure 2.6D).
To confirm that the lack of RanGAP1 NE association in wip1-1/wip2-1/wip3-1 was indeed due to the absence of the three proteins and that WIP1, WIP2a and WIP3 are redundant in anchoring RanGAP1 to the root tip NE, we separately retransformed wip1-1/wip2-1/wip3-1 with GFP fusions of each protein. Figure 2.7A shows that in lines expressing a single GFP fusion protein in the wip1-1/wip2-1/wip3-1 mutant background, RanGAP1 NE association was re-established in root tips.
This clearly demonstrates that any single WIP paralog is sufficient for RanGAP1 NE targeting in root tip cells and that the GFP fusion proteins are functional. In addition, consistent with the results that the coiled-coil domain of WIP1 interacts with RanGAP and the N-terminal domain is dispensable for the targeting of WIP1 to the NE, GFP-WIP1ΔN is sufficient to recruit RanGAP1 and cause an accumulation of RanGAP1 around the NE (Figure 2.7A).
Finally, we investigated the mitotic localization pattern of RanGAP1 in the wip1-1/wip2-1/wip3-1 root meristem. Figure 2.7B shows that RanGAP1 association with the cell plate was indistinguishable from wild-type plants at both early and late stages of cell plate formation. This suggests that unlike NE association, concentration of RanGAP1 at the cell plate does not depend on the WIP family.
56 2.5 Discussion
2.5.1 Different NE-targeting mechanisms for higher eukaryotic RanGAPs
Ran as well as its regulators are evolutionarily conserved in all eukaryotes and the Ran cycle has been studied extensively in yeast and mammalian systems. Relatively few studies have been conducted to address the plant Ran system; however, the available data indicate that its function in nucleocytoplasmic transport is conserved (Ach and Gruissem, 1994; Haizel et al., 1997; Kim and
Roux, 2003; Wang et al., 2006; Zhao et al., 2006). Despite this functional conservation, at least three classes of RanGAPs exist based on their specific locations and the presence or absence of targeting domains. Yeast RanGAP (Rna1p) is the simplest form, appearing predominantly cytoplasmic throughout the cell cycle (Hopper et al., 1990; Melchior et al., 1993). Yeast RanGAP consists of a leucine-rich repeat important for its GTPase stimulating activity, and an acidic domain recently recognized to be important for microtubule organization during mitosis (De Souza et al., 2004;
Melchior et al., 1993; Seewald et al., 2003). Along with these two domains, both vertebrate and plant
RanGAPs contain additional kingdom-specific domains necessary for anchoring RanGAP to the outer
NE during interphase (Jeong et al., 2005; Mahajan et al., 1998; Matunis et al., 1996; Matunis et al.,
1998; Meier, 2000; Rose and Meier, 2001; Saitoh et al., 1997). In vertebrates, the sumoylated
C-terminal domain of RanGAP is required for binding to the nucleoporin RanBP2/Nup358, thereby tethering RanGAP at the NE (Joseph et al., 2004; Mahajan et al., 1998; Matunis et al., 1996; Matunis et al., 1998; Saitoh et al., 1997; Saitoh et al., 2006). The N-terminal NE-targeting domain of plant
RanGAP has no similarity to the C-terminus of vertebrate RanGAP and there is no evidence showing that it is a target for sumoylation. No homolog of RanBP2/Nup358 can be detected in the completed plant genomes, indicating that it is either not conserved or has evolved beyond the detection limits of sequence similarity searches. In addition, there are profound differences in the subcellular localization of RanGAP during mitosis, with plant RanGAP being targeted via its N-terminal domain to the growing cell plate, a structure unique to plant cytokinesis. The only other class of proteins with
57 similarity to the plant RanGAP N-terminus (WPP proteins) appear to be strictly plant-specific and share the locations of RanGAP both at the NE and the cell plate, suggesting that they use the same targeting mechanisms (Patel et al., 2004). Together, these findings have let us to propose that plant and animal RanGAP might have separately acquired kingdom-specific protein interaction domains and have adapted interactions with different proteins for the interphase and mitotic subcellular anchoring that are required to establish the functional Ran gradients of the respective organisms.
58 In support of this hypothesis, we report here a family of plant-specific NE anchors for plant RanGAP.
WIP family members have no similarity with RanBP2. Through their coiled-coil domains, WIP family members specifically interact with the WPP domain of plant RanGAP and other
WPP-domain-containing proteins. Their C-terminal transmembrane domain together with a short
C-terminal tail is necessary and sufficient for NE association. We have demonstrated that the three
WIP family members are necessary and sufficient for the association of RanGAP1 to the Arabidopsis root tip NE. Interestingly, the WIP family is not required for RanGAP1 NE association in other cell-types, including differentiated root cells and hypocotyl cells. All available studies on animal
RanGAP NE anchoring were performed at the single-cell level and therefore did not address potential differences between tissues or developmental stages (Joseph et al., 2002; Mahajan et al., 1997;
Mahajan et al., 1998; Matunis et al., 1996; Matunis et al., 1998; Saitoh et al., 1997; Saitoh et al., 1998).
It is therefore currently unclear whether tissue-specific differences in RanGAP targeting are unique to plants, or whether such differences (and additional anchoring activities besides RanBP2) also exist in animals. Our data indicate that while at the Arabidopsis root tip, anchoring of RanGAP at the NE depends on the WIP family, in differentiated root cells and in hypocotyl cells additional players are likely involved. While those additional players are currently unknown, we have found that the WPP domain of RanGAP1 has affinity for a number of additional coiled-coil proteins in Arabidopsis (data not shown). It is possible that the level of redundancy of RanGAP NE anchoring differs in different cell types, or that an unknown protein (or proteins) takes over the role of the WIP family in different tissues. The dependence of RanGAP anchoring on the WIP family at the root tip is consistent with the high expression of all three WIP genes in this region (Figure 2.8).
2.5.2 Why does plant and animal RanGAP accumulate at the NE?
Mammalian and plant RanGAP is concentrated at the NE while yeast RanGAP is not. This has led to the questions: what is the function of this local concentration in some organisms, and for which of the
59 currently known functions of the Ran cycle is it important. In terms of nuclear import and export, a pore-located RanGAP could potentially steepen the Ran-GTP concentration gradient across the nuclear pore. It has been estimated that the concentration difference between free nuclear and cytoplasmic Ran-GTP was at least 200-fold during interphase in cell free Xenopus system (Kalab et al.,
2002). Because the 22 kD Ran-GTP could diffuse from the nucleus and prematurely dissociate import complexes at the cytoplasmic side, a "GTP-hydrolysis guard" at the pore could be advantageous.
Likewise, RanGAP at the NPC could assure that the dissociation of export complexes takes place locally and might facilitate the export process (Hutten and Kehlenbach, 2006).
At least for the Arabidopsis root tip, we can conclude that RanGAP association with the NE is dispensable, because normal growth and development (and therefore by inference nuclear import and export) are unchanged in a wip1-1/wip2-1/wip3-1 triple mutant (data not shown). Several studies in mammalian systems have also suggested that RanBP2/Nup358, and therefore RanGAP-NE association, are largely dispensable for protein nuclear import and export (Bernad et al., 2004; Bernad et al., 2006; Hutten and Kehlenbach, 2006; Walther et al., 2002). In addition, computer simulation has argued that the primary goal of RanGAP concentration on the NE is unlikely the steepening of the Ran gradient across the NE (Gorlich et al., 2003). Nevertheless, none of these studies (including our own) address whether NPC-associated RanGAP provides a small increase in nucleocytoplasmic transport efficiency that might not be resolved by the employed assays, but that might still provide a selective advantage.
Is it possible that the advantage of RanGAP at the NE is greater in larger than in smaller cells because a large cytoplasm otherwise "dilutes out" RanGAP? Our triple mutant might then show no obvious defects because the RanGAP delocalization occurs only in the small, undifferentiated cells of the root tip. Modeling suggests that it is the total amount of cytoplasmic RanGAP (in contrast to the concentration) that determines the cytoplasmic RanGTP levels (Gorlich et al., 2003), indicating that at
60 a constant expression level, small and large cells should harbor the same cytoplasmic RanGTP hydrolysis capacity. Nevertheless, a more recent study discussing the effect of cell shape and size for diffusion-controlled gradients of cellular effectors highlights that cellular geometry imposes an important variable on such systems (Meyers et al., 2006). Nuclear association might therefore provide a more constant geometry for the interphase Ran gradient in a multicellular organism with vastly varying cell shapes and sizes.
2.5.3 Do WIPs represent novel plant-specific transmembrane nucleoporins?
Despite the fact that mammalian NPCs (~125MD) are much larger in size than yeast NPCs (~50MD), proteomic analyses have revealed a similar number of protein constitutes (nucleoporins) (Cronshaw et al., 2002; Rout et al., 2000). Although the three-dimensional organization of NPC is quite similar between yeast and vertebrates, the primary sequences of the nucleoporins have limited similarity.
However, a number of nucleoporins from all subcomplexes of the nuclear pore share recognizable homologs in all extant eukaryotic lineages, indicating a shared origin. In contrast, kingdom-specific anchoring nucleoporins exist in fungi and animals, which indicates that they are a relatively recent innovation and might have replaced an ancestral anchoring system (Bapteste et al., 2005; Mans et al.,
2004). There are three known integral membrane nucleoporins in S. cerevisiae (Pom34p, Pom152p,
Ndc1p) and three in mammalian cells (pom121, gp210, NDC1) (Madrid et al., 2006; Stavru et al.,
2006a; Stavru et al., 2006b). Among these proteins, only NDC1 appears to be conserved between the two kingdoms. Putative gp210 and NDC1 homologs can be found in the Arabidopsis genome by sequence similarity searches (Mansfeld et al., 2006; Stavru et al., 2006a), but neither Pom34p or
Pom152p nor pom121 is conserved.
Based on the localization pattern of WIP1 revealed by fluorescence and electron microscopy, the presence of its C-terminal transmembrane domain necessary and sufficient for NE localization, and its
61 functional similarity with Nup358/RanBP2, we propose that WIP1 might be a novel, plant-specific
nucleoporin. The presence of a coiled-coil domain in the WIP family supports this notion, given that
among the rather limited number of fold compositions of nucleoporins the coiled-coil domain is a
prominent fold (Devos et al., 2006; Schwartz, 2005). Studies have suggested extensive redundancy in
protein-protein interactions within the NPC and a robustness and fault-tolerance of the NPC assembly
process (Stavru et al., 2006a; Stavru et al., 2006b). This is consistent with the non-essential nature of
the WIP family in Arabidopsis. Indeed, of the three yeast transmembrane nucleoporins Pom152p,
Pom34p, and Ndc1p, only Ndc1p is essential, which might be connected to its second function as a spindle pole body component (Madrid et al., 2006). Functional mammalian NPCs can assemble in cells that are devoid of gp210 and severely depleted of pom121 (Stavru et al., 2006b). Further studies in Arabidopsis such as stacking the WIP family triple mutant with knockout mutations in the putative gp210 and NDC1 genes will allow us to investigate the genetic interaction of these putative plant transmembrane nucleoporins.
2.5.4 WIP1 as a NE-specific TA membrane protein
TA membrane proteins are a class of proteins that are targeted post-translationally to various organelles where they play diverse roles. They share several distinct structural features, including an
N-terminal cytosolic domain that represents the majority of the protein, a single hydrophobic segment located near the C-terminus, and a short C-terminal tail sequence that protrudes into the organelle lumen (Kutay et al., 1993). In general, the organelle-specific targeting signal resides within the transmembrane domain and the tail region with unique physicochemical properties and sequence-specific characteristics. Although there are numerous, functionally important TA proteins, the mechanism of their transmembrane insertion and the basis of their membrane selectivity are still unclear (Borgese et al., 2003; Hwang et al., 2004; Wattenberg and Lithgow, 2001).
62 Based on our ultrastructural study, the position of the predicted transmembrane domain, and the known topology of RanGAP-NE association, we predict that WIP1 is a TA protein with an N-terminus that resides in the cytoplasm/nuclear pore, a membrane-spanning alpha helix, and a very short lumenal tail. The last 36 amino acids of WIP1 contain all information for the selective association with the NE, the first example, to our knowledge, for such a targeting domain. This specificity requires a highly conserved C-terminal four-amino-acid motif. While the molecular machinery involved in interpreting the information in TA proteins has not yet been identified, an involvement of membrane lipid composition has recently been indicated (Borgese et al., 2003; Brambillasca et al., 2005; Ceppi et al.,
2005). It is currently not clear if the lipid environment is different between the NE and ER, but a very recent study reporting a spectral shift of the lipid fluorescent dye FM4-64 at the NE is suggestive of such a difference (Zal et al., 2006). The WIP1 NE-targeting domain can now serve as a valuable tool to analyze membrane-targeting specificity for the NE.
63 2.5.5 Plant NE membrane and cell plate
Plants undergo open mitosis and the plant NE breaks down at the onset of mitosis and re-assembles at its end. Although nothing is known about the mechanism of plant NE re-assembly, studies in vertebrates have demonstrated that NE assembly is a vesicle fusion event (Hetzer et al., 2000; Zhang and Clarke, 2000; Zhang et al., 2002a). Plant cytokinesis involves the emergence and the subsequent centrifugal growth of the cell plate at the division plane, which have been shown to be mediated by the fusion of Golgi- and endosome-derived vesicles (Dhonukshe et al., 2006; Jurgens, 2005;
Segui-Simarro et al., 2004). Both RanGAP1 and the short WPP-domain protein WPP1 are targeted to the NE during interphase and to the cell plate during cytokinesis, which suggests an unrecognized mechanistic connection between these two membrane rearrangement events (Jeong et al., 2005; Patel et al., 2004). The data presented here, which indicate that two putative TA membrane proteins, WIP1 and WIP2a also reside both on the NE and the cell plate, strengthen the idea that the two membrane compartments share some identity.
2.6 Future Outlook
2.6.1 Other anchoring proteins for plant RanGAP at the NE and cell plate
In a wip1-1/wip2-1/wip3-1 triple mutant, two possibilities may be attributable to the unchanged
RanGAP1 concentration at the cell plate during cytokinesis and at the NE in differentiated root cells.
First, it is formally possible that a severely reduced amount of WIP2b in the triple mutant is sufficient for RanGAP1 anchoring at the cell plate and at the NE in some cell types. To test this hypothesis, it will be essential to isolate a WIP2 null allele for both splicing variants. In the absence of T-DNA insertion mutants which can potentially knock out both WIP2a and WIP2b, TILLING is a feasible approach to identify WIP2 alleles removing both WIP2 isoforms (Till et al., 2003). The other, more likely explanation for the remaining RanGAP1-NE/Cell plate association in the triple mutant is that
64 other unidentified proteins function redundantly with WIPs as anchors for RanGAP1 at the NE and the cell plate. Hence, investigating other RanGAP1-interacting proteins, especially proteins interacting with the WPP domain may uncover new anchors for RanGAP1. Other candidate proteins from the yeast two-hybrid screen that led to the identification of WIP1 remain to be investigated. In addition, it will be helpful to identify additional in vivo interaction partners of RanGAP1 using Tandem Affinity
Purification (TAP) strategies developed for plant protein complex isolation (Rubio et al., 2005; Rohila et al., 2004).
2.6.2 Unraveling the biological roles of RanGAP concentration at the NE in root tip cells
Based on our analysis of RanGAP targeting in the wip1-1/wip2-1/wip3-1 triple mutant, we concluded that RanGAP association with the NE in root tip cells is dispensable for plant growth and development under standard laboratory conditions. However, it is formally possible that RanGAP accumulation at the NE is only essential for processes related to plant responses to certain abiotic and biotic stresses.
Hence, it is critical to perform a systematic phenotypic analysis on the wip1-1/wip2-1/wip3-1 triple mutant at both macroscopic and microscopic levels. Although a standard root-elongation assay with the triple mutant indicates no change in auxin sensitivity (data not shown; see (Lincoln et al., 1990), it may still be worthy to analyze the sensitivity to other hormones in the triple mutant. In light of the recent reports on the involvement of plant RanGAP in Resistance (R) gene-mediated viral resistance
(Tameling and Baulcombe, 2007; Sacco et al., 2000), it will be interesting to investigate any change in the disease resistance response in the triple mutant.
Because of the prevalent function of RanGAP as the activator of Ran GTP hydrolysis, a local concentration of RanGAP at the cytoplasmic side of the NPC may enable a high rate of Ran GTP hydrolysis and also maintain a high RanGDP/RanGTP ratio at this specific site. The highly localized
Ran GTP hydrolysis can potentially facilitate a more rapid dissociation of the exportin-RanGTP-cargo
65 ternary complex and promote a more efficient nuclear export. At the same time, the high
RanGDP/RanGTP ratio at the outer side of the NPC may favor the assembly of the protein import complex and prevent a premature release of the import cargo before its translocation through the NPC.
Hence, the delocalization of RanGAP from the NE in the triple mutant may affect the efficiency of both the nuclear export and the nuclear import. Monitoring the nucleocytoplasmic shuttling of fluorescent marker proteins in the triple mutant with approaches like FRAP (Fluorescence Recovery after Photobleaching) and FLIP (Fluorescence Loss in Photobleaching) can shed light on the potential roles which the RanGAP NE association may play during the nucleocytoplasmic transport (Tillemans et al., 2006).
2.6.3 The function of WIPs and RanGAP during the elongation of the etiolated hypocotyl
Some preliminary data suggest that there is an about 30% reduction of the hypocotyl length in 3 and
5-day-old etiolated wip1-1/wip2-1/wip3-1 seedlings. Together with the expression of WIPs and
RanGAP1 in the upper part of the hypocotyls specifically from etiolated seedlings (Figure 2.8I and data not shown), the short-hypocotyl phenotype may indicate a role for WIPs and RanGAP during cell expansion and/or elongation in the dark-grown hypocotyls. Because RanGAP1-GFP is still concentrated at the NE in the hypocotyls of dark-grown wip1-1/wip2-1/wip3-1 mutants (data not shown), it seems unlikely that the short-hypocotyl phenotype is based on the delocalization of
RanGAP. Further studies, for example, analyzing the hypocotyl length of the wip1-1/wip2-1/wip3-1 triple mutant expressing GFP-WIP1, GFP-WIP2a or GFP-WIP3, are needed to confirm that the observed defect in etiolated hypocotyl elongation is caused by mutations in WIPs.
66 2.6.4 Better understanding of the sorting signal in WIPs responsible for the NE/NPC/Cell plate association
The last 36 amino acids of WIP1, which contain the transmembrane helix and the short tail region, are necessary and sufficient for targeting WIP1 to the NE and cell plate. We have also shown that the last four amino acids of the tail region are critical for the efficient targeting of WIP1 to the NE. It remains undetermined whether these four amino acids are essential for the association of WIP1 with the cell plate. Although some commonality between the NE/NPC and the cell plate is suggested by the targeting of WIP1, WIP2a, and RanGAP1 to both membrane compartments, an interesting observation is that WIP3 is associated with the NE/NPC but not the cell plate. Hence, there must be some sorting information residing in WIP3 that can distinguish the membrane identity of these two compartments.
To test if the membrane-distinguishing activity comes from the TDF or other domains in WIP3, deletion studies similar to those performed on WIP1 are required. A close examination on the alignment with the TDFs from different WIPs also indicates several other highly conserved elements.
Mutagenesis studies on these amino acids will facilitate our understanding of the sorting signals which specifically direct putative TA membrane proteins to the NE/NPC and cell plate.
67
A D AD-WIP1 AD-WIP2a AD-WIP3 AD-WIP1N AD-WIP1cc AD 1 75 101 323 462 489 BD-RanGAP1 + - -+-- B Input IP P P BD-RanGAP1ΔC 1GF 1GF GAP GAP + + -+-+ Ran GFP Ran GFP WIP1 70kD BD-RanGAP1ΔN ------Input IP BD-RanGAP2 P1 P1 + + -+-- WI P WI P GFP GF GFP GF BD-WPP1 RanGAP1 70kD + + -+-+ C BD-WPP2 Rice Rice Wheat + + -+-+ WIP2a WIP3 BAD08716 BAD46344 CAJ19339 BD-WPP3 WIP1 40 (51) 31 (47) 24 (43) 24(43) 24(40) ------WIP2a - 31 (45) 23(37) 23(42) 25(41) BD-WIP2a WIP3 -- 21(37) ND 26(41) + + -+-- Rice --- 42(56) 41(55) BD-WIP3 BAD08716 ------Rice - - - - 61(71) BD BAD46344 ------
Figure 2.1: The coiled-coil domain of WIP1 interacts with the WPP domain of RanGAP. (A) Domain structure of WIP1. WIP1 has an extended coiled-coil domain (sky blue) and a single, C-terminal transmembrane domain (red). A bipartite NLS is the only recognizable motif (pink) (see Figure 2.5A) amino terminal (dark blue) of the coiled-coil domain. Numbers above the bar indicate amino acid positions. (B) WIP1 and RanGAP1 are in the same complex in vivo, shown by co-IP. Samples immunoprecipitated (IPed) with the anti-GFP antibody from RanGAP1-GFP and GFP expressing lines were probed with anti-WIP1 antibody (top). Samples IPed with the anti-GFP antibody from GFP-WIP1 and GFP lines were probed with anti-RanGAP1 antibody (bottom). Inputs are shown on the left. (C) Percent identity and similarity (in parentheses) on amino acid sequence level among WIP1 family members and WIP-like proteins from rice (Oryza sativa) and wheat (Triticum aestivum). ND, no similarity detected. (D) Interaction between the WIP1 family and Arabidopsis WPP-domain-containing proteins in yeast two-hybrid assays. Fusion proteins are schematically shown below the construct names. AD, GAL4 activation domain; BD, GAL4 DNA binding domain. Plus (+), positive interaction, (-), no interaction.
68
Figure 2.2: WIP1 and RanGAP1 interact at the outer NE, most likely at the cytoplasmic side of the NPC. (A) GFP-WIP1, GFP-WIP2a and GFP-WIP3 are targeted to the NE in Arabidopsis root tip cells, while free GFP is distributed throughout the nucleus and cytoplasm. Confocal images were taken from transgenic lines expressing 35S promoter-controlled fusion proteins with cell walls stained with propidium iodide (magenta). (B) Double immunofluorescence of GFP-WIP1 and RanGAP1 or the NPC marker QE5 in Arabidopsis root tip cells, with DNA counterstained with DAPI. (C) RanGAP1 and WPP1 interact with WIP1, WIP2a and WIP3 at the NE in Nicotiana benthamiana protoplasts, as shown by BiFC. YN, N-terminal domain of YFP; YC, C-terminal domain of YFP. None of the constructs showed fluorescence when transfected into protoplasts alone or when co-transfected with an empty YN or YC vector (data not shown). Position of the nucleus was judged by bright field images (not shown). (D) GFP-WIP1 and GFP-WIP2a, but not GFP-WIP3 (green), co-localize with RanGAP1 (magenta) at the cell plate, revealed by immunofluorescence in transgenic Arabidopsis root tip cells. GFP-WIP3 is targeted to the daughter NE but not to the cell plate during cytokinesis. Scale bars in A-D: 10μm. (E) Three-dimensional maximal projection of confocal images spanning half of the nucleus from root callus cells expressing GFP-WIP1 (left panel) and nuclei with nuclear pores from Arabidopsis root callus cells visualized by Scanning Electron Microscopy (middle and right panels). The density of dotted signal from GFP-WIP1 is similar to the density of NPCs. Scale bars: 1μm. (F) Micrographs showing representative images of post-embedding immuno-gold labeling of RanGAP1 (top panel, WT/anti-RanGAP1) and WIP1 (bottom panels, WT/anti-WIP1), in wild-type Arabidopsis root callus tissues. Scale bars: 100nm. (G) Quantification of the gold labeling. Nb gold part./10μm: number of gold particles counted per 10μm of the NE. The distribution of the gold particles was shown for inside of the NE, outside of the NE, and between the double membranes. Total nucl. perimeter: total length of the nuclear perimeter (envelope) measured for each specimen using ImageJ. Nb nuclei counted: number of nuclei counted for each specimen. (H) Graphical representation of the numbers shown in G.
69 A C YN-WIP1 YN-WIP2a YN-WIP3 GFP-WIP1 GFP-WIP2a GFP-WIP3 GFP RanGAP1-YC
B GFP-WIP1 RanGAP1 DAPI Overlay WPP1-YC
D Anti-GFP Anti-RanGAP1 Overlay GFP-WIP1
GFP-WIP1 QE5 DAPI Overlay E GFP-WIP2a GFP-WIP3
F H
WT/ anti-RanGAP1
WT/ anti-WIP1 G Gold part. Gold part. Gold part. Gold part. Total nucl. Nb nuclei Antibody /10μm /10μm /10μm /10μm perimeter counted Total Inside Outside Between (μm) pre-RanGAP1 2.02 0.58 0.74 0.70 954.4 74 anti-RanGAP1 19.97 0.31 18.28 1.38 763.1 52 pre-WIP1 1.00 0.28 0.35 0.37 1198.4 92 anti-WIP1 2.62 0.40 1.78 0.44 748.9 54
Figure 2.2: WIP1 and RanGAP1 interact at the outer NE, most likely at the cytoplasmic side of the NPC. (See preceding page for legend.)
70
GFP-WIP1 Tubulin DAPI Merge Pre-prophase Metaphase Anaphase Telophase Cytokinesis
Figure 2.3: The dynamic localization of WIP1, revealed by tracking GFP-WIP1 throughout the cell cycle. In transgenic Arabidopsis root tips, GFP-WIP1 (green) and tubulin (red) were immunolabeled with polyclonal anti-GFP and monoclonal anti-α-tubulin antibodies, respectively. DNA was counterstained with DAPI (blue). The different cell cycle stages are listed on the left. All scale bars: 10μm.
71
Figure 2.4: Domain requirement for the subcellular targeting of WIP1. (A) Confocal images were taken from transgenic Arabidopsis root tips expressing fusion proteins indicated on the top with cell wall stained with propidium iodine (magenta). Green channels were shown on the top, with overlay images at the bottom. (B) Alignment of the WIP1 TDF with the C-termini of WIP1 homologs from different plant species: tomato (Lycopersicon esculentum), grape (Vitis vinifera), barrel medic (Medicago truncatula), columbine (Aquilegia formosa x Aquilegia pubescens), poplar (Populus deltoides), wheat (Triticum aestivum), rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare), sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum) and pine (Pinus taeda). Identical and functionally similar amino acids are shaded in dark and light grey, respectively. The predicted transmembrane helix is underlined and the well conserved last four amino acids are boxed. (TC accession numbers from TIGR Unique Gene Indices, others from GenBank.) (C) The TDF is necessary and sufficient for cell plate targeting, with GFP-WIP1ΔTDF accumulating in the re-forming nuclei and GFP-TDFWIP1 accumulating at the cell plate. Transgenic Arabidopsis root tips were immuno-stained with GFP antibody (green), α-tubulin antibody (red) and counterstained with DAPI (blue). All scale bars: 10μm.
72 A GFP-WIP1 GFP-WIP1ΔN GFP-WIP1ΔTDF GFP-TDFWIP1 GFP-WIP1 ΔVVPT
B WIP1 WIP2 WIP3 Tomato EF426861 Potato TC121630 Grape TC33581 Barrel medic TC78542 Columbine TC18180 Poplar CX173481 Poplar TC33219 Wheat CAJ19339 Wheat TC238289 Wheat TC238290 Wheat TC271927 Rice BAD08716 Rice BAD46344 Rice TC268818 Maize BG320937 Maize TC263633 Maize TC283595 Maize TC290918 Maize TC294978 Barley TC134396 Barley TC151811 Sorghum TC103318 Sorghum TC105075 Sugarcane TC58942 Sugarcane TC60457 Pine TC68912
C GFP-WIP1ΔTDF Tubulin DAPI Overlay
GFP-TDFWIP1 Tubulin DAPI Overlay
Figure 2.4: Domain requirement for the subcellular targeting of WIP1. (See preceding page for legend.)
73
A NT NN TN WIP1 WIP2a WIP3 Tomato EF426861 Tomato BT014210 Potato TC124840 Potato TC115096 Grape TC33581 Barrel medic TC78542 Oilseed rape CD827632 Lettuce BQ865917 Pepper TC6859 Cotton CO118618 Wheat CAJ19339 Wheat TC238289 Rice BAD08716 Rice BAD46344 Maize CC640757 Maize TC313436 2 1 3
B GFP-WIP1ΔTMF GFP-WIP1ΔTMF 1 GFP-WIP1ΔTMF 1/2 GFP-WIP1ΔTMF 1/3 GFP-WIP1ΔTMF 1/2/3
N N N N
N
Figure 2.5: Stepwise mutagenesis of the predicted NLS of WIP1. (A) Alignment of the putative NLS sequences of WIP1 homologs from different plant species. Identical amino acids are shaded in black and functionally similar amino acids are shaded in gray. Gaps are indicated by dotted lines. Species shown in addition to those in Figure 2.4B are oilseed rape (Brassica napus), lettuce (Lactuca sativa), pepper (Capsicum annuum), and cotton (Gossypium hirsutum). Mutations tested in B are marked by red boxes. Exchanged amino acids are indicated on the top and motifs are numbered at the bottom. (B) Localization of GFP-WIP1ΔTDF and mutants of motif 1 (GFP-WIP1ΔTDF 1), motif 1 and 2 (GFP-WIP1ΔTDF 1/2), motif 1 and 3 (GFP-WIP1ΔTDF 1/3), and motif 1, 2 and 3 (GFP-WIP1ΔTDF 1/2/3), transiently expressed in Nicotiana benthamiana protoplasts. N, position of nucleus in the corresponding bright field image. All scale bars: 10μm.
74
C RanGAP1 QE5 Overlay Profile A
wip1-1 WIP2a wip2-1 WIP2b WT undiff. wip3-1
B WT wip1-1 WT wip2-1 WIP2a WIP1 triple undiff. WIP2b ACT ACT
WT wip1-1
WT wip3-1 diff. WT WIP1 WIP3
Loading ACT triple diff.
D WT triple WT triple WT triple WT undiff. diff.
RanGAP1-GFP RanGAP1ΔC-GFP GFP-RanGAP2 RanGAP1(AAP)-GFP
Figure 2.6: In the wip1-1/wip2-1/wip3-1 triple mutant (“triple”), RanGAP1 is dislocated from the NE in undifferentiated root cells (undiff.), while NE targeting in differentiated cells (diff.) is not affected. (A) Schematic representation of T-DNA insertions in wip1-1, wip2-1 and wip3-1. Blue boxes, open reading frames; black lines, introns; yellow boxes, untranslated regions; red vertical arrowheads, T-DNA insertion sites; black horizontal arrowheads, sites of RT-PCR primers. (B) Top left panel, wip1-1 RT-PCR analysis; bottom left panel, wip1-1 immunoblot analysis with the anti-WIP1 antibody. Top right panel, wip2-1 RT-PCR analysis; bottom right panel, wip3-1 RT-PCR analysis. ACT, Actin 2; “Loading”, Coommassie Brilliant Blue staining of a replica gel. (C) Immunofluorescence localization of RanGAP1 and the NE marker QE5 in WT and wip1-1/wip2-1/wip3-1 triple mutant. Undiff., undifferentiated root tip cells (meristem and elongation zone); diff., differentiated root cells (diff.). Profile, fluorescence intensity profile of dotted lines in overlay images. In all cases except the undifferentiated cells from the triple mutant, peaks of green signal (RanGAP1) correlate with peaks of magenta signal (QE5) representing the NE. (D) Root cells of transgenic WT and wip1-1/wip2-1/wip3-1 lines expressing GFP fusions of RanGAP1, RanGAP1ΔC (see Figure 1D), RanGAP2, and RanGAP1(AAP) carrying a WPP to AAP mutation in the RanGAP1 targeting domain (Rose and Meier, 2001), which leads to loss of NE targeting in all cell types. Cell walls were counterstained with propidium iodide (magenta). All scale bars: 10μm.
75
A B GFP-WIP1 GFP-WIP1ΔN GFP-WIP2a GFP-WIP3 Early cytokinesis Late cytokinesis GFP WT RanGAP1 triple Overlay
Figure 2.7: RanGAP targeting to the NE, but not the cell plate, depends on the WIP family. (A) GFP-WIP1, GFP-WIP1ΔN, GFP-WIP2a and GFP-WIP3 rescue RanGAP1 NE targeting in undifferentiated wip1-1/wip2-1/wip3-1 root tip cells. GFP (green) and RanGAP1 (magenta) antibodies were used for immunofluorescence in wip1-1/wip2-1/wip3-1 mutants stably expressing the corresponding GFP fusion protein driven by the 35S promoter. (B) RanGAP1 (green) is concentrated at the cell plate in wip1-1/wip2-1/wip3-1 mutants during early and late cytokinesis, tested by immunofluorescence. DNA was visualized by SYTOX Orange (magenta). All scale bars: 10μm.
76
E A g B CD lin r d e e f w m t e a o te o s le fl s ro
WIP1
WIP2a F G H I
WIP3
ACT
J KLMN O
P Q R STU
Figure 2.8: Expression pattern of WIP1 and its family members. (A) All three members of the WIP family were expressed in all tissues tested by RT-PCR. Primers were as in Figure 3A. (B-I) Expression pattern of WIP1 revealed by promoter-GUS lines. The WIP1 promoter was active during the early stage of Arabidopsis seedling development (one-day-old seedling, B) and shows a patchy expression pattern in the vascular tissue of cotyledons (C). In 3-6 day old seedlings, expression was seen at the root tip (D), the vascular tissue around the shoot apex and in young leaf primodia (E). In adult plants, WIP1 was expressed mainly in the stamens (F), and very weakly at the senescence region of developing siliques (arrow in G). The expression of WIP1 was below the detection limit in hypocotyls of light grown seedlings (H), but could be detected in the upper part of hypocotyls from dark-grown seedlings (I). (J-O) Expression pattern of WIP2 revealed by promoter-GUS lines. (P-U) Expression pattern of WIP3 revealed by promoter-GUS lines. One-day-old seedlings (J, P), cotyledons (K, Q), root tips (L, R) and vascular tissue around the shoot apical meristem (M) from 3-6 day old seedlings. Flowers (N, T), cauline leaves (S) and siliques (O, U) from adult plants.
77
CHAPTER 3
THE MITOTIC LOCALIZATION OF ARABIDOPSIS RANGAP1: THE RAN CYCLE AS
THE SPATIAL CUE FOR FUTURE DIVISION PLANE IN PLANTS?
3.1 Abstract
During plant cell division, the determination of future division plane and its accurate execution is pivotal for plant growth and development. The preprophase band (PPB), a cytoskeletal structure made of cortical microtubules and microfilaments, faithfully predicts the future cortical division site. The
PPB is a transient structure which disassembles upon the formation of the mitotic spindle. Hence, it is generally thought that PPB must leave certain molecular imprints for directing the future fusion between the new cell plate and the mother cell wall. However, the signal transduced from PPB remains unknown. Here I characterized the localization pattern of Arabidopsis RanGAP1 throughout mitosis and cytokinesis. During preprophase, RanGAP1 colocalizes with mature condensed PPB.
Despite the disassembly of PPB during prometaphase, RanGAP1 remains marking the position of the former PPB until the completion of cytokinesis. During metaphase, RanGAP1 also concentrates around the kinetochore regions. As the cells progress into anaphase, RanGAP1 accumulates within the spindle midzone. During cytokinesis, RanGAP1 is concentrated at the midline of the phragmoplast and/or the nascent cell plate. The WPP domain appears to be sufficient for the mitotic targeting of
RanGAP1 and specific point mutations on the WPP motif disrupt the targeting. Moreover, the mitotic targeting of RanGAP1 does not depend on its known NE/NPC anchors, the members of the WIP family. Taking all the data together, I identified the first protein demarcating the cortical division site
78 after the PPB disassembles. Based on the well-established role of RanGAP as a regulator for the Ran cycle, I propose that a Ran-GTP gradient might provide the spatial cue for defining the future division plane.
3.2 Introduction
Plant cells are surrounded by a rigid cell wall and thus lack cell migration. Hence, the determination of division plane is pivotal for organ morphogenesis and the overall architecture of the plant body.
Unlike animal cells, in which the division plane is determined during mitosis by the position of the mitotic spindle, plant cells decide their division plane before the onset of mitosis. The preprophase band (PPB), a cortical cytoskeletal structure made of microtubules and microfilaments, circumscribes the nucleus and faithfully predicts the future division site. The PPB is a transient structure which arises at the G2/prophase transition and completely disassembles during prometaphase (Jurgens, 2005;
Smith, 2001). Hence, it is generally believed that the PPB must leave behind molecular tracks to direct the future fusion between the new cell wall (called the cell plate) and the parental wall. However, the signal transduced from PPB remains mysterious.
Upon the disassembly of the PPB, cortical F-actin is depleted from the former location of the PPB, leaving behind an actin-depleted zone (ADZ) which persists throughout mitosis and cytokinesis
(Jurgens, 2005; Smith, 2001). Coincident with the PPB and the ADZ, a novel marker was identified recently; a plasma-membrane-localized Arabidopsis kinesin, KCA1, is excluded from the cortical division site during mitosis and cytokinesis (Vanstraelen et al., 2006). Both the ADZ and KDZ (for the
KCA-depleted zone) might indicate, or even be required to maintain, the presence of certain molecules at the division site that function as positive landmarks (Smith, 2001). However, no protein marker of the predicted division site has been described to date.
79 During late anaphase, a plant-unique cytoskeletal structure, the phragmoplast, develops from the spindle midzone microtubules between the two sets of daughter chromosomes. The phragmoplast contains F-actin filaments and two antiparallel sets of co-aligned microtubules with their plus ends lying in the plane of division. The phragmoplast facilitates the formation of the cell plate by directing the trafficking of Golgi- and endosome-derived vesicles, which contain the cell plate building blocks.
After the phragmoplast is initiated between the daughter nuclei, it grows centrifugally leading the lateral expansion of the cell plate and eventually attaches the cell plate to the mother wall at the division site. How the developing phragmoplast and cell plate are guided to the division site remains unclear, although some pharmacological and genetic evidence suggests that actin and myosin might be involved in the guidance (Molchan et al., 2002; Holweg and Nick, 2004).
Several mutants have been isolated that help unravel the mechanisms governing the establishment and the accurate execution of the division plane in plant cells. Mutations in TONNEAU2 (ton2), which encodes a putative regulatory subunit of type 2A protein phosphatase, abolish the PPB, which leads to irregularly oriented cell divisions and abnormal cell morphology and arrangement (Traas et al., 1995;
Camilleri et al., 2002). The maize tangled (tan) mutant and Arabidopsis pok1/pok2 (for
PHRAGMOPLAST ORIENTING KINESIN 1/2) double mutant have defects in the phragmoplast guidance to the site previously occupied by the PPB (Walker and Smith, 2002; Smith et al., 1996;
Smith et al., 1995; Cleary and Smith, 1998; Muller et al., 2006; Smith et al., 2001). Despite the well described mutant analyses, the molecular roles of these proteins in the spatial control of cytokinesis remain unresolved.
Both animal and plant cells undergo open mitosis with the NE and NPC breaking down during prophase and reassembling during telophase. During the disassembly of the NE and NPC, the NPC associated protein RanGAP might traffic to different subcellular locations. Indeed, during mitosis, mammalian RanGAP1 is targeted to the kinetochores and mitotic spindles. This targeting is achieved
80 through SUMO-1 conjugation and involves interactions with RanBP2/Nup358, the same mechanism
as for mammalian RanGAP targeting to the NPC (Joseph et al., 2004; Molchan et al., 2002; Joseph et
al., 2004; Mahajan et al., 1997; Mahajan et al., 1998; Matunis et al., 1996; Molchan et al., 2002). The
kinetochore-associated RanGAP1-RanBP2 complex appears to be important for the stable
kinetochore-microtubule interaction and chromosome segregation possibly through regulating Ran
GTPase and its effector protein crm1/XPO1 (Arnaoutov et al., 2005; Joseph et al., 2004; Molchan et
al., 2002).
The localization pattern of plant RanGAP during the cell cycle has been studied to some extent.
However, the two published studies were somewhat contradictory, with one reporting RanGAP
association with the spindle and phragmoplast and the other suggesting RanGAP concentration on the
nascent cell plate (Jeong et al., 2005; Pay et al., 2002). In addition, neither report addressed RanGAP
targeting during all stages. Hence, I sought to re-examine more thoroughly the Arabidopsis RanGAP1
behavior throughout the cell cycle.
Here, I demonstrate that Arabidopsis RanGAP1 co-localizes with the PPB during prophase and with
the kinetochore during metaphase. Subsequently, RanGAP1 concentrates in the spindle midzone during anaphase and on the nascent cell plate or midline of the phragmoplast during cytokinesis. More importantly, after the PPB disappears RanGAP1 labels the cortical division site until the end of the cytokinesis. Based on the observation that RanGAP1 always demarcates the plane of plant cell division, I propose that RanGAP1 (and by extension, the Ran GTPase cycle) might provide spatial cues directing the division plane.
81 3.3 Materials and Methods
3.3.1 Plant Materials
Arabidopsis seedlings (Columbia and Wassilevskiya ecotype) were grown in soil under standard long-day condition (16 hour light and 8 hour dark) or on MS (Caisson Laboratories, Rexburg, ID) plates under constant light. For T-DNA insertion mutants rg2-1 (SALK_032721) and rg2-2
(SALK_006398), T3 or T4 bulk seeds were acquired from the Arabidopsis Biological Resource
Center (ABRC, The Ohio State University, Columbus, OH). Mutant rg2-3 (FLAG_184A06) was obtained from the Versailles T-DNA lines collection (Bouchez et al., 1993; Bechtold et al., 1993).
Homozygous insertion plants were identified by PCR genotyping
(http://signal.salk.edu/tdnaprimers.2.html). Mutant wip1-1/wip2-1/wip3-1 and transgenic plants expressing RanGAP1(AAP)-GFP and RanGAP1ΔC-GFP were described in Xu et al. (2007a).
82 3.3.2 Cloning and Transformation
The coding sequence for N-terminal HA tagged Ran1 was cloned into pENTR/D-TOPO by introducing the DNA sequence for the HA epitope into the forward primer for Ran1. Point mutations were introduced with the QuickChange® Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) with all inserts confirmed by sequencing. HA-Ran1, HA-Ran1T27N and HA-Ran1Q72L were then recombined into the destination vector pMDC7 for estrogen-inducible expression in plants (Curtis and
Grossniklaus, 2003). The constructs were then transformed into GFP-TUB6 plants through
Agrobacterium by floral dip method. A HindIII-EcoRI fragment containing the 35S promoter,
Gateway cassette, and NOS terminator was generated by partial digestion of p4-GW-mCherry (Song et al., 2007). This fragment was then ligated into vector pFGC5941 digested with the same enzyme combination, leading to a new binary vector pFGC-GW-mCherry. RanGAP1 coding sequence without the stop codon was introduced into pENTR/D-TOPO and then recombined with pFGC-GW-mCherry using LR recombination, which results the construct expressing RanGAP1-mCherry.
RanGAP1-mCherry was transformed into GFP-TUB6 and ton2-13 (hetero)/MBD-GFP plants.
83 3.3.3 Immunolabeling and Confocal Microscopy
Whole-mount immunolocalization in Arabidopsis roots was carried out as described (Friml et al.,
2003). Polyclonal anti-RanGAP1 (1:100 to 1:200) and monoclonal anti-tubulin (DM1A, Sigma, 1:100) antibodies and appropriate secondary antibodies conjugated with Alexa Fluor 488 or 568 were used.
SYTOX Orange (Invitrogen) and DAPI (Sigma) were used in H2O at the final concentrations of 1μM and 1μg/ml, respectively. Images including DAPI staining were collected from a Leica TCS SP2
AOBS Confocal Laser Scanning Microscope equipped with 4 lasers (Red HeNe 633nm, Green HeNe
543nm, Argon 458/476/488/496/514 nm, Argon UV). All other fluorescence images were collected on a PCM 2000/Nikon Eclipse E600 confocal laser scanning microscope as described (Rose and Meier,
2001).
84 3.4 Results
3.4.1 Mitotic Localization pattern of Arabidopsis RanGAP1
To best characterize the mitotic localization pattern of RanGAP1, indirect immunofluorescence was performed in Arabidopsis root tip cells with an antibody specifically recognizing Arabidopsis
RanGAP1. Mitotic cells were tracked by staining α-tubulin. During preprophase, RanGAP1 was found colocalized with the mature, condensed PPB. At this stage, RanGAP1 concentration at the NE was still evident, although NE breakdown could already be observed (Figure 3.1 and 3.2). Once the cell entered prometaphase, the PPB disappeared. However, RanGAP1 stayed at the position of the former PPB site until the final stage of cytokinesis (Figure 3.3). When the chromosomes were aligned at the metaphase plate, RanGAP1 accumulated in several bright dots on the chromosomes, resembling the kinetochores (Figure 3.1). As the cells progressed into anaphase, RanGAP1 was found to be concentrated around the spindle midzone (Figure 3.1). When the microtubules from the spindle midzone assembled into the phragmoplast, RanGAP1 was concentrated at the midline of the phragmoplast or the cell plate (Figure 3.1). Along with the outward growth of the cell plate, RanGAP1 was always concentrated at the position of the nascent cell plate but not the mature cell plate.
85 3.4.2 The WPP motif is critical for the mitotic targeting of RanGAP1
Plant RanGAP has a unique domain at the N-terminus, named the WPP domain after a highly conserved tryptophan-proline-proline motif. The WPP motif is critical for Arabidopsis RanGAP1 targeting to the NE during interphase and cell plate during cytokinesis. With the three amino acids
‘WPP’ mutated to ‘AAP’, the targeting of Arabidopsis RanGAP1 to the NE and cell plate is abolished
(Rose and Meier, 2001; Jeong et al., 2005). When a RanGAP1(AAP)-GFP fusion protein was tracked through mitosis, the protein was diffusely distributed throughout the cytoplasm without concentration on the PPB, kinetochore region, or cortical division site (Figure 3.4). This indicates that the WPP motif is necessary for the mitotic targeting of RanGAP1 and likely involved in interacting with some protein partners.
3.4.3 The WPP domain is sufficient for directing the mitotic targeting of RanGAP1
The WPP domain of RanGAP1 has been demonstrated to be sufficient for targeting RanGAP1 to the
NE in interphase and the cell plate during cytokinesis (Rose and Meier, 2001; Jeong et al., 2005). To investigate whether the WPP domain is also sufficient for directing the RanGAP1 targeting during mitosis, the fusion protein between the WPP domain and GFP (RanGAP1ΔC-GFP) was stably expressed in transgenic Arabidopsis with its mitotic localization assessed by immunofluorescence.
Similar to the full-length RanGAP1, RanGAP1ΔC-GFP is targeted to the PPB, kinetochore, cortical division site, and cell plate (Figure 3.5), indicating that the WPP domain is the targeting domain for
RanGAP1 throughout cell cycle.
86 3.4.4 Mitotic targeting of RanGAP1 is not dependent on WIPs
As demonstrated in chapter two, the RanGAP1 concentration around the NE/NPC in
non-differentiated root tip cells depends on the WIP family, while cell plate targeting during
cytokinesis does not. I decided to test the dependence of RanGAP1 mitotic localization on the WIP
family by immunostaining RanGAP1 in the wip1-1/wip2-1/wip3-1 triple mutant. The results in figure
3.6 illustrates that the trafficking of RanGAP1 during mitosis is not dependent on WIPs.
87 3.4.5 Identification of Arabidopsis mutants underexpressing RanGAP
To better understand the function of plant RanGAP, a reverse genetic strategy was taken. A T-DNA insertion line (SALK_058630) for RanGAP1 was identified and named rg1-1. Despite being without any apparent phenotypes, rg1-1 is a null mutant shown by protein level analysis (Figure 3.7A). Three
T-DNA insertion mutants for RanGAP2 were isolated. The insertion sites for all three lines were mapped to the 5’ UTR region, nucleotide -447 (with +1 being the A of the ATG) for SALK_032721
(rg2-1), nucleotide -369 for SALK_006398 (rg2-2), and nucleotide -186 for FLAG_184A06 (rg2-3)
(Figure 3.7B). Based on the position of the insertion and the RNA and protein level analysis, rg2-1 and rg2-2 are likely knock-down mutants and rg2-3 is a null mutant (T. Rodrigo-Peiris and I. Meier, unpublished data). All the insertion lines for RanGAP2 appear phenotypically indistinguishable from the corresponding wild type with the SALK lines being in Columbia ecotype and the FLAG line being in Wassilevskiya ecotype. Because of the high sequence similarity between RanGAP1 and RanGAP2
(63% identity and 80% similarity on the protein level) as well as the very similar expression pattern derived from both the microarray data (Zimmermann et al., 2004) and promoter-GUS analyses (H.
Wang and I. Meier, unpublished data), it is probable that the two genes are highly redundant. Hence, rg1-1 was crossed into the three RanGAP2 mutants and the analysis on the double mutants is in progress. In a parallel approach, downregulating the expression of RanGAP1 in rg2-3 and RanGAP2 in rg1-1 using inducible RNAi (Wielopolska et al., 2005) is currently in progress.
88 3.5 Discussion
3.5.1 Characterization of RanGAP1 localization during the cell cycle
Pay et al. (2002) reported that tobacco RanGAP co-localized with spindle microtubules during anaphase and with the microtubular phragmoplast during telophase in BY2 cells, using immunolabeling with an antibody developed against the Arabidopsis RanGAP1. Caution should be taken when interpreting the cross-reacting signal in a heterologous system as being from the real homolog, especially when tobacco RanGAP sequence information is missing. In contrast, Jeong et al.
(2005) observed a RanGAP concentration on the nascent cell plate using live or fixed transgenic BY2 cells overexpressing GFP-tagged Arabidopsis RanGAP1. Besides the potential problem with a heterologous expression system, the relatively high cytoplasmic fluorescence signal coming from the overexpression decreases the resolution of imaging, making it possible to miss certain information.
Hence, I believe that the analysis on the endogenous RanGAP1 mitotic localization pattern using immunofluorescence in Arabidopsis root tip cells might reflect the real behavior of RanGAP more faithfully compared with previous studies.
89 3.5.2 The first protein marker for the division plane in plant cells
In plants, the PPB marks the cortical site where the cell plate will fuse with the parental wall during cytokinesis. The PPB disassembles before metaphase, so its existence is separated temporally from cytokinesis; therefore some mechanisms must be utilized to ‘remember’ the division plane. To date no positive markers have been identified to mark the cortical division site in the absence of the PPB. Here,
I have shown that Arabidopsis RanGAP1 co-localizes with the PPB and persists at the cortical division site after the PPB disappears. Furthermore, all three other locations where RanGAP1 accumulates, the kinetochores during metaphase, spindle midzone during anaphase, and phragmoplast midline/nascent cell plate during cytokinesis, appear on or close to the future division plane. Hence RanGAP1 might be involved in defining the spatial identity of the future division plane.
3.5.3 Connection between RanGAP1 and the chromosomal passenger proteins
The chromosomal passenger proteins (CPPs), including Survivin, Aurora B, INCENP and Borealin, form one or more protein complexes and play important roles in early and late mitotic events, like chromosome alignment and segregation, histone modification, as well as cytokinesis (reviewed in
(Adams et al., 2001; Vagnarelli and Earnshaw, 2004; Vader et al., 2006). The hallmark of CPPs is their dynamic pattern of subcellular localization during mitosis: they associate with the centromeric region of chromosomes from prophase to metaphase, relocate to the spindle midzone and equatorial cortex during anaphase and finally accumulate in the midbody during cytokinesis. The only CPP characterized in plants so far is the enzymatic core of the CPP complex, Aurora B kinase (AtAurora) which has three paralogs in Arabidopsis (Demidov et al., 2005; Kawabe et al., 2005). Different
AtAurora paralogs have different localization patterns through the cell cycle. The subcellular structures occupied by at least one AtAurora protein include the nuclear envelope, spindle, centromere/kinetochore, spindle midzone and cell plate (Demidov et al., 2005; Kawabe et al., 2005).
90 AtAurora has been indicated functioning during cytokinesis (van Damme et al., 2004). With the exception of the PPB/cortical division site, the localization pattern of RanGAP1 closely recapitulates that of the AtAurora kinases, which might suggest a potential link between RanGAP (likely the Ran cycle) and the CPP complex activity.
3.5.4 Similarities and differences between the mammalian and plant RanGAP mitotic localization
Mammalian RanGAP1 has been shown to migrate from the NE/NPC to associate with the spindle and concentrate on kinetochores during mitosis. Association with kinetochores persists until late anaphase and is lost coincident with nuclear envelope assembly in telophase (Joseph et al., 2002). Mitotic
RanGAP1 localization in mammalian cells depends on sumoylation, a post-translational modification and association with RanBP2/Nup358, a vertebrate-specific nucleoporin which also anchors mammalian RanGAP1 to the NPC during interphase (Joseph et al., 2002; Joseph et al., 2004; Mahajan et al., 1997; Matunis et al., 1998). Thus, mammalian RanGAP1 appears to utilize one unified mechanism for trafficking to different subcellular locations through the cell cycle. In contrast, the targeting of plant RanGAP1 appears to be more complex. Similar to its mammalian counterpart, plant
RanGAP1 is concentrated at foci near kinetochores. The exact identity of these foci remains elusive and will be confirmed by co-localization study with a centromere marker, GFP fusion with the centromere-specific histone H3 (HTR12-GFP) (Fang and Spector, 2005). Nevertheless, the kinetochore targeting might be the only similarity between the mitotic behaviors of mammalian and plant RanGAP1. More importantly, plant RanGAP1 likely utilizes different protein partners for mitotic targeting from the ones for NE targeting during interphase in root tip cells. Plant RanGAP1 targeting to additional mitotic structures and the involvement of new mitotic anchor proteins might reflect its adaptation to the plant unique mode of cytokinesis.
91 3.5.5 Possible modes of action for plant RanGAP in specifying the division plane
Ran GTPase controls the directionality of active nucleocytoplasmic transport by providing the subcellular compartment identity for the appropriate loading and unloading of the transport factors
(reviewed in Gorlich and Kutay, 1999b; Stewart, 2007). With high affinity to importins, Ran-GTP dissociates importin-cargo complexes leading to the release of cargo proteins in the nucleus.
Conversely, interaction of Ran-GTP with exportins promotes its association with export cargo proteins.
The GTP hydrolysis of Ran-GTP activated by RanGAP destabilizes the export complex and results in the unloading of the export cargo into the cytoplasm. These same mechanisms have been employed in the cells to fulfill other functions of Ran GTPase throughout the cell cycle. During vertebrate mitosis,
Ran-GTP again interacts with importins, displacing factors from the inhibited complexes with importins and promoting spindle assembly (Gruss et al., 2001; Nachury et al., 2001; Wiese et al.,
2001). At the same time, through binding to exportins, Ran-GTP promotes the recruitment of molecules to the kinetochore regions of chromosomes, which is consistent with the kinetochore concentration of RanGAP1 likely involved in Ran-GTP hydrolysis and protein unloading (Arnaoutov et al., 2005). A similar mode of action can be adapted to explain how plant RanGAP1 works during mitosis (Figure 3.8). If I assume that plant RanGAP1 does not change its fundamental biochemical properties during the cell cycle, it is possible that it is involved in the disassembly of a
RanGTP-exportin-cargo ternary complex. Thus, I propose that the targeting of Arabidopsis RanGAP1 to various locations during mitosis and cytokinesis might regulate the Ran cycle and deliver downstream signaling molecules to maintain the identity of these compartments.
Together, I identified plant RanGAP1 as the first protein associated with the PPB and marking the cortical division site after PPB is disassembled. I propose that RanGAP1, through regulating the Ran cycle, might function in providing spatial cues to define specific mitotic structures, for example the cortical division site.
92 3.6 Future Outlook
3.6.1 The dynamic mitotic and cytokinetic localization pattern of plant RanGAP with a higher spatial and temporal resolution
It is certainly advantageous to investigate the localization of a protein at the endogenous level.
However, the whole mount immuno-labeling technique involves fixation, digestion, permeabilization, and vigorous washing, which may all attribute to the damage of cell integrity and the rather low signal to noise ratio. Besides, immunofluorescence on dead cells limits full appreciation of the dynamics in mitotic RanGAP1 localization. Several interesting questions remain unaddressed using the current method, such as (i) whether RanGAP1 starts to co-localize with the PPB during its initiation or at the maturation stage, (ii) when RanGAP1 dissociates from the kinetochores, and (iii) how statically or dynamically RanGAP1 associates with every subcellular structure. To circumvent the technical issues as well as increase the spatial and temporal resolution of my analysis of RanGAP1 targeting through cell cycle, a construct has been made expressing a RanGAP1-mCherry fusion protein under the control of the 35S promoter. I set to co-express RanGAP1-mCherry and the microtubule marker
GFP-TUB6 (Nakamura et al., 2004) in Arabidopsis for time-lapse live cell imaging. As of now, the T1 transgenic plants are being screened. Only the plants expressing RanGAP1-mCherry at a level close to the endogenous RanGAP1 level will be selected for further analysis.
Monitoring RanGAP1 targeting with live cell imaging may bring another convenience, the relative ease to investigate different cell types in different tissue. Investigating the RanGAP1 mitotic localization pattern in tissues other than the root, for example during the stomata development, may reveal either tissue-commonality or tissue-specificity in the mitotic behavior of plant RanGAP and, by extension, the establishment of the Ran gradient.
93 3.6.2 Establishing the potential link between the Ran GTPase and plant cytokinesis
The fact that RanGAP1 is always concentrated in the plane of division during mitosis and cytokinesis suggests a potential role in establishing and/or ‘remembering’ the division plane. Since RanGAP acts as an important regulator in the Ran cycle, I decided to investigate the possible involvement of the
Ran GTPase during plant cytokinesis. I will inducibly express the HA epitope tagged Arabidopsis
Ran1 (At5g20010) protein and its two mutant forms, the GDP-locked Ran1T27N and the GTP-locked
Ran1Q72L. These point mutations were specifically chosen based on numerous studies on yeast and animal Ran proteins and the extreme conservation of Ran across different kingdoms (Klebe et al.,
1995; Bischoff et al., 1994; Haizel et al., 1997). I will express these proteins in plants together with
GFP-TUB6 (Nakamura et al., 2004) which will enable us to investigate any defects of the mitotic cytoskeletal structures or effects on the mitotic progression. I also plan to assess any effects from the inducibly overexpressed Ran and its mutant proteins on the division plan establishment. Specifically, I will stain the roots with propidium iodide for visualizing the highly ordered root cell files and with aniline blue for visualizing the callose in the cell plate (van Damme et al., 2006).
94 3.6.3 Understanding the mitotic anchorage of plant RanGAP1
A number of intriguing questions are posed by my characterization of RanGAP1 mitotic targeting.
First of all, among all the known elements involved in plant mitosis and cytokinesis, what is required
for RanGAP1 mitotic targeting? Specifically, I would like to know whether RanGAP1 targeting is
dependent on microtubules or microfilaments by using inhibitors such as oryzalin and cytochalasin D
to disturb the cytoskeleton (Wodnicka et al., 1992; Anthony and Hussey, 1999). These
pharmacological studies will address several important questions. Disruption of the microtubular
phragmoplast can potentially clarify the association of RanGAP1 with the cell plate membrane or the
plus ends of the microtubules located at the midline of the phragmoplast. Disruption of the cortical
actin filaments and elimination of the ADZ will tell us whether the ADZ is important for the retention
of RanGAP1 on the cortical division site. I can also investigate the mitotic localization pattern of
RanGAP1 in Arabidopsis mutants defective in mitosis and cytokinesis (Sollner et al., 2002; and the
references therein). Among these, two mutants, ton2 (Camilleri et al., 2002; Traas et al., 1995) and ple
(PLEIADE/AtMAP65-3) (Muller et al., 2004) might give extremely interesting insights into RanGAP1 mitotic targeting. Since the PPB as detected by tubulin is completely absent in ton2, dissecting
RanGAP1 mitotic localization in ton-2 will reveal whether targeting of RanGAP1 to the cortical division site is dependent on the PPB. With an unusually expanded phragmoplast midline, ple might
help to address whether RanGAP1 is associated with the cell plate or the plus ends of the
phragmoplast microtubules.
Another aspect of RanGAP1 mitotic targeting to investigate is the anchoring mechanism for
RanGAP1 during mitosis. Since RanGAP1 traffics normally during cell cycle in
wip1-1/wip2-1/wip3-1 mutants, presumably some mitosis-specific partners are responsible for
tethering RanGAP1 to various mitotic locations. Identification of cell cycle-specific interactions has
long been hindered by the quite low mitotic index in whole plants or cell cultures. However,
95 combining the recently developed tandem affinity purification-based platform in Arabidopsis cell
suspension culture (Van Leene et al., 2007) and the improved cell culture synchronization technique
(Menges and Murray, 2002), it is perceivable to isolate mitotic interacting partner(s) of RanGAP1.
Moreover, using the same strategy, potential cell cycle dependent modification(s) of RanGAP1 might
be uncovered.
3.6.4 Unraveling the functional significance of plant RanGAP1 during mitosis
The ultimate goal of characterizing the pattern and mechanism of RanGAP1 mitotic targeting is to
unravel what roles plant RanGAP1 might play during mitosis. The reverse genetic approach by
isolating mutants underexpressing RanGAP will be a key. However, disturbance of the RanGAP
function in nucleocytoplasmic transport during interphase might be deleterious for cells. Hence, other
strategies must be considered for understanding the function of RanGAP, specifically during mitosis.
Microinjection of the specific anti-RanGAP1 antibody into dividing cells in the rg2-3 mutant can
potentially be an excellent way to perturb RanGAP activity without causing severe side effects on
RanGAP function during interphase. In the microinjected cells, I can investigate the potential effects
on the mitotic cytoskeletal structures or the mitotic progression by labeling the cytoskeletal structures
with co-injected dyes or transgenic marker proteins. Since RanGAP1 resides on the cortical division
site coincident with the ADZ, it will be interesting to know whether RanGAP1 is important for ADZ
maintenance.
I can also think of interfering with the endogenous RanGAP1 targeting or activity by inducibly
expressing partial or mutant versions of RanGAP1 and hope for a dominant negative effect. Since
RanGAP1ΔC has the same localization pattern as full length RanGAP1 presumably using the same anchors and lacks the domains for GAP enzymatic activity, overexpressed RanGAP1ΔC can potentially displace endogenous RanGAP1 from its mitotic subcellular locations. Overexpressed
96 RanGAP1(AAP), which retains the GAP activity but loses the correct targeting, may diminish the highly localized RanGAP1 activity derived from endogenous RanGAP. Should RanGAP1 function without the help of other proteins, another interesting experiment would be to investigate any effects on division plane determination by artificially targeting RanGAP1 to the plasma membrane constitutively.
If I assume that during mitosis plant RanGAP1 does not change its fundamental activity as the activator for Ran-GTP hydrolysis, a localized reduction in Ran-GTP concentration would be predicted around the PPB, kinetochores, spindle midzone, and cortical division site (Figure 3.8A and 3.8B). It will be intriguing to actually visualize the Ran-GTP gradient during plant mitosis and cytokinesis using Ran conformation biosensors similar to those developed in mammalian systems (Kalab et al.,
2006; Kalab et al., 2002). Furthermore, based on the analogous studies in mammals (Arnaoutov et al.,
2005), I can hypothesize that the specifically localized Ran-GTP hydrolysis regulated by mitotic
RanGAP1 might transmit signals through the Ran-GTP effector proteins, like Exportin (XPO1)
(Figure 3.8D). Hence, it will also be of great interest to investigate the mitotic localization of
Arabidopsis XPO1.
97
RanGAP1 Tubulin DAPI Overlay Preprophase Metaphase Anaphase
* Cytokinesis
Figure 3.1: The localization of Arabidopsis RanGAP1 during mitosis. Immunofluorescence in dividing Arabidopsis root tip cells with anti-RanGAP1 (green) and anti-tubulin (red) antibody. DNA is counterstained with DAPI (blue). Cell cycle stages are listed on the left. The arrowhead, arrow, bracket and asterisk in the overlay images indicate the PPB, kinetochore, spindle midzone and the phragmoplast midline/cell plate, respectively. All scale bars: 10μm.
98
Figure 3.2: RanGAP1 forms a PPB-like ring circumscribing the nucleus during G2/prophase. Z-series of confocal images taken every 0.15μm from Arabidopsis root tip cells immuno-stained with anti-RanGAP1 antibody (green) and with Sytox Orange (red) labeling the DNA. The arrowheads indicate the PPB-like ring. Scale bar: 10μm.
99
Figure 3.3: RanGAP1 positively marks the cortical division site during the anaphase. Z-series of confocal images taken every 0.15μm from Arabidopsis root tip cells immuno-stained with anti-RanGAP1 antibody (green) and with Sytox Orange (red) labeling the DNA. The arrowheads indicate the concentration of RanGAP1 on the cortical division site. Scale bar: 10μm.
100
RanGAP1 (AAP)-GFP Tubulin DAPI Overlay Preprophase Metaphase
*
* Cytokinesis
Figure 3.4: The WPP motif is critical for the mitotic targeting of RanGAP1. Immunofluorescence in transgenic Arabidopsis root tips expressing RanGAP1(AAP)-GFP with anti-GFP (green) and anti-tubulin antibody (red). DNA is stained with DAPI (blue). Cell cycle stages are listed on the left. The arrowhead and asterisks in the overlay images indicate the PPB and the phragmoplast midline/cell plate, respectively. All scale bars: 10μm.
101
RanGAPΔC -GFP Tubulin DAPI Overlay Preprophase Metaphase
* Telophase
* Late Cytokinesis
Figure 3.5: The WPP Domain is sufficient for the mitotic targeting of RanGAP1. Immunofluorescence in transgenic Arabidopsis root tips expressing RanGAP1ΔC-GFP with anti-GFP (green) and anti-tubulin antibody (red). DNA is stained with DAPI (blue). Cell cycle stages are listed on the left. The arrowheads, arrow and asterisks in the overlay images indicate the PPB/cortical division site, kinetochore and the phragmoplast midline/cell plate, respectively. All scale bars: 10μm.
102
RanGAP1 Tubulin DAPI Overlay Preprophase Metaphase Anaphase
* Cytokinesis
Figure 3.6: The mitotic targeting of RanGAP1 is not dependent on the WIP family. Immunofluorescence in wip1-1/wip2-1/wip3-1 mutant root tips with anti-RanGAP1 (green) and anti-tubulin antibody (red). DNA is stained with DAPI (blue). Cell cycle stages are shown on the left. The arrowheads, arrow, bracket, and asterisks in the overlay images indicate the PPB/cortical division site, kinetochore, spindle midzone and the phragmoplast midline/cell plate, respectively. All scale bars: 10μm.
103
A WT rg1-1 70kD RanGAP1
Coommassie B rg2-1 rg2-3
rg2-2
Figure 3.7: Identification of mutants underexpressing RanGAP. (A) Immunoblot analysis with the anti-RanGAP1 antibody on 10-day-old seedlings showing rg1-1 is a null mutant for RanGAP1. Coommassie brilliant blue staining of a replica gel is shown at the bottom. (B) Schematic representation of the T-DNA insertions in RanGAP2. Dark grey boxes stand for the untranslated regions and light grey box represents the open reading frame. black line shows the intron. Triangles point to the T-DNA insertion sites in the 5’UTR.
104
Figure 3.8: Proposed Model for plant RanGAP providing a spatial cue for plant cell division. The only established biochemical function for RanGAP is to activate the GTP hydrolysis by Ran. The concentration of plant RanGAP at the NE and preprophase band during G2/prophase transition (A) and at the cell plate and cortical division site during cytokinesis (B) enables a highly localized Ran GTP hydrolysis activity at these sites. The dark green represents areas defined by a high rate of Ran GTP hydrolysis facilitated by RanGAP. Analogous to the well elaborated model on the action of RanGAP during protein export from the nucleus (C), a model is proposed for the plant RanGAP activity at the cortical division site (D). Ran GTP hydrolysis catalyzed by RanGAP destabilizes the RanGTP-Exportin (XPO)-cargo ternary complex, facilitating the release of a cargo protein. A similar mechanism could be implemented for delivering proteins essential for the establishment and maintenance of the cortical division site. 105
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