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Keywords:

  • nuclear migration;
  • actin;
  • microtubules

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

The invariant cell division patterns that characterize Caenorhabditis elegans development make it an ideal system to study the mechanisms that control nuclear movement and positioning. Forward genetic screens in this system allowed identification of the key molecular machinery for connecting the nucleus to the cytoskeleton; pairs of protein partners, consisting of a KASH domain protein and a SUN domain protein, bridge the nuclear envelope to connect the nucleus to cytoskeletal components. The C. elegans genome encodes several KASH/SUN pairs, and mutant phenotypes as well as tissue-specific expression patterns suggest a diversity of functions. These functions include moving the nucleus but have been extended to effects on the chromosomes inside the nucleus as well. We review the impact of the C. elegans system in pioneering this field as well as the functions of these KASH/SUN protein pairs across spatial and temporal C. elegans development. Developmental Dynamics 239:1352–1364, 2010. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

Using the power of forward genetic screens in model systems, researchers have made great strides in elucidating the genetic pathways and the molecular machinery controlling basic developmental and cellular processes. For example, capitalizing on one of the most advantageous features of Caenorhabditis elegans as a model organism, its invariant development (Sulston and Horvitz,1977; Sulston et al.,1983), has resulted in pioneering advances in such areas as programmed cell death (Conradt and Xue,2005) and signal transduction control of cell differentiation (Greenwald,2005; Sundaram,2006). C. elegans invariant development has proved to be an extremely valuable model to study nuclear behavior as well.

The end result of C. elegans development is an animal in which each somatic cell and its nucleus have adopted a reproducible position within the body of the animal. Various nuclear migrations and movements during the course of development help to establish this strikingly invariant positioning of nuclei. Forces generated by the cell cytoskeleton are required to dynamically reposition nuclei (Swope and Kropf,1993; Guild et al.,1997; Maniotis et al.,1997; Grolig,1998; Reinsch and Gonczy,1998); the actin filament and microtubule cytoskeleton are associated with most nuclear movements, although intermediate filaments are also involved in some cell types (Martys et al.,1999; Wilhelmsen et al.,2005). The underlying mechanisms that allow the nucleus, specifically the nuclear envelope, to interact with the cellular cytoskeleton had long been of interest to both developmental and cell biologists but had been difficult to elucidate. Only in the last 10 years have researchers uncovered the key conserved molecular players in this process. This leap forward stemmed from the identification of the genes associated with C. elegans mutants that were isolated and first characterized decades ago.

FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

In an effective early screen for mutants with defects in C. elegans post-embryonic development, Horvitz and Sulston stained mutagenized animals with DNA-specific dyes and looked for mutants with abnormal numbers of nuclei in the ventral nerve cord (Horvitz and Sulston,1980). They reported isolation of mutations at two loci, unc-83 and unc-84, that were indistinguishable by phenotype; both mutants caused an uncoordinated phenotype and were defective in vulva and ventral nerve cord development. They found that unc-83 and unc-84 specifically affect nuclear behavior in two cell types: the P cells, which give rise to both vulva precursor cells and some ventral cord neurons, and the dorsal hypodermal cells (Sulston and Horvitz,1981). In another laborious screen in the early 1980s, Hedgecock and Thomson (1982) mounted mutagenized animals on slides, searched for animals with observable defects, and recovered individual mutants of interest. They identified mutations in another gene, anc-1, in which the nucleus and the mitochondria are displaced and unanchored within cells (Hedgecock and Thomson,1982). A functional connection between unc-83, unc-84, and anc-1 was briefly proposed and discussed (Hedgecock and Thomson,1982). However, it would be nearly 20 years before these genes were cloned and characterized molecularly, revealing that unc-83, unc-84, and anc-1 encode sets of protein partners consisting of one KASH domain protein and one SUN domain protein (defined in “KASH and SUN Domain Proteins” section). The KASH and SUN proteins form a complex that spans the nuclear envelope and allows the nucleus to interact with cytoplasmic cytoskeletal elements.

Our understanding of the wider complement of KASH/SUN protein sets in C. elegans was expanded when zyg-12 was cloned (Malone et al.,2003). The zyg-12 mutant was identified decades ago as a naturally occurring temperature-sensitive strain (Wood et al.,1980). The name reflects the zygotic-defective, embryonic lethal phenotype, and the zyg-12 mutant was chosen years later for further study because of the striking centrosome detachment phenotype in the zygote. ZYG-12 was recognized as the third C. elegans KASH domain protein upon cloning. Genome-wide reverse genetic screens, first possible via RNAi in C. elegans, facilitated the search for the predicted SUN partner (Kamath et al.,2003). A SUN domain–encoding gene dubbed sun-1 shared the zyg-12 phenotype and acts as the ZYG-12 partner in the embryo (Malone et al.,2003). This collection of unbiased forward genetic screens was quite effective in revealing a novel protein complex that has since been shown to exert conserved functions in nuclear behavior throughout the eukaryotic kingdom.

KASH AND SUN DOMAIN PROTEINS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

The KASH domain derives its name from a homologous domain found in the proteins Klarsicht (Drosophila), ANC-1 (C. elegans), and Syne1 (human) (Starr and Han,2002; Starr and Fischer,2005). The KASH proteins are C-tail-anchored proteins, a class of type II integral membrane proteins inserted into the outer nuclear membrane post-translationally (Borgese et al.,2007; Starr,2009) (Fig. 1A). The KASH domain, found at the C-terminus, consists of the single transmembrane domain followed by a short stretch of 8 to 35 amino acids that can be quite divergent (McGee et al.,2009; Minn et al.,2009; Starr,2009). Although the N-terminal cytoplasmic regions of KASH proteins share no particular homologous domains, KASH proteins do have N-terminal motifs of similar function. For example, long spectrin-like repeats or helical regions extend the N-terminus of KASH proteins into the cytoplasm where they can interact with the cytoskeleton directly or indirectly through their separate and diverse cytoskeletal interacting motifs (Starr et al.,2001; Starr and Han,2002; Zhen et al.,2002; Malone et al.,2003; Fischer et al.,2004; McGee et al.,2006). Four KASH domain proteins have been reported in C. elegans: ANC-1, UNC-83, ZYG-12, and KDP-1.

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Figure 1. Multiple KASH and SUN domain proteins are expressed in C. elegans. A: The model for KASH/SUN pair bridging of the nuclear envelope. The SUN domain proteins span the inner nuclear membrane (INM) with their N-terminus in the nucleus where it can interact with nuclear lamins or other nuclear components and their SUN domain in the perinuclear space. The KASH proteins span the outer nuclear membrane (ONM) with their C-terminal KASH domain in the perinuclear space. The KASH-SUN interaction in the perinuclear space confines the KASH protein to the outer nuclear membrane (ONM), away from the endoplasmic reticulum (ER). The N-terminus of the KASH proteins extends into the cytoplasm to interact with a cytoskeleton directly or indirectly via motor proteins. B: The pair-wise, spatial and temporal expression profile of KASH and SUN protein pairs in C. elegans development.

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The SUN domain proteins are the partners to the KASH proteins and comprise the inner half of the bridge across the nuclear envelope (Fig. 1A). The SUN domain derives its name from homologous C-terminal regions in Schizosaccharomyces pombe Sad1 and C. elegans UNC-84 (Malone et al.,1999). This domain is remarkably conserved in eukaryotic organisms, as homologous domains were first identified in the human proteins SUN1 and SUN2 (Malone et al.,1999) and subsequently in S. cerevisiae Mps3p (Jaspersen et al.,2006), D. melanogaster Klaroid (Kracklauer et al.,2007), and in the plant OzSAD1 protein (Moriguchi et al.,2005). The SUN domain proteins span the inner nuclear membrane with the C-terminal SUN domain protruding into the perinuclear space and the N-terminus residing in the nucleoplasm (Fig. 1A) (Crisp et al.,2006; Wang et al.,2006; Minn et al.,2009). Interestingly, some SUN proteins, such as C. elegans UNC-84 and human SUN2, require the nuclear lamina for specific nuclear envelope localization (Lee et al.,2002; Crisp et al.,2006), while other SUN proteins including C. elegans SUN-1 and human SUN1 do not (Fridkin et al.,2004; Crisp et al.,2006; Hasan et al.,2006). In C. elegans, SUN-1 and UNC-84 are the only identified SUN domain proteins to date.

As the nuclear envelope–spanning model predicts, the KASH domain directly interacts with the C-terminal region of SUN domain proteins (Fig. 1A). In some cases, this interaction is mediated by a short region prior to the actual SUN domain, while the SUN domain mediates the interaction directly in other protein pairs (Padmakumar et al.,2005; Crisp et al.,2006; Haque et al.,2006; McGee et al.,2006; Stewart-Hutchinson et al.,2008; Minn et al.,2009). C. elegans studies established that KASH protein localization depends on the SUN protein partner. In the absence of UNC-84, both UNC-83 and ANC-1 are expressed but fail to localize to the nuclear envelope (Starr et al.,2001; Starr and Han,2002). Similarly, SUN-1 is required for ZYG-12 localization (Malone et al.,2003). In contrast, the SUN proteins maintain their nuclear envelope localization in the absence of the KASH proteins (Malone et al.,1999). Thus, the KASH/SUN interaction within the perinuclear space specifically sequesters KASH proteins to the outer nuclear membrane and keeps them out of the endoplasmic reticulum membrane (Fig. 1A). This model revealed a solution to a long-standing puzzle in cell biology: how and even if such a specific outer nuclear membrane protein localization pattern could exist apart from a pan-ER pattern. This localization of the KASH proteins is probably important to ensure that forces generated by the cytoskeleton are properly conveyed to the nucleus.

To date, a variety of biological functions have been ascribed to KASH/SUN pairs, first via phenotypic analysis in C. elegans and, subsequently, in various other eukaryotic models. These functions include mediating nuclear migration (Malone et al.,1999; Mosley-Bishop et al.,1999; Starr et al.,2001; Kracklauer et al.,2007), anchoring nuclei in syncytial cells (Malone et al.,1999; Starr and Han,2002; Zhen et al.,2002; Zhang et al.,2007; Lei et al.,2009), tethering the microtubule organizing center to the nucleus (Mosley-Bishop et al.,1999; Malone et al.,2003; Jaspersen et al.,2006; Zhang et al.,2009), and, remarkably, regulating chromosome dynamics (Bupp et al.,2007; Conrad et al.,2007,2008; Ding et al.,2007; Penkner et al.,2007; Bhalla and Dernburg,2008; Jaspersen and Hawley,2009). Our review focuses on the molecular and functional roles of KASH/SUN pairs across temporal and spatial C. elegans development, and we also discuss the impact of the C. elegans studies on mammalian systems. Thorough reviews of the biochemical properties of KASH and SUN proteins and their roles in other organisms are published elsewhere (Starr and Han,2003; Starr and Fischer,2005; Tzur et al.,2006; Wilhelmsen et al.,2006; Schneider et al.,2008; Razafsky and Hodzic,2009).

C. ELEGANS KASH/SUN PAIR EXPRESSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

KASH/SUN pairs exhibit distinct spatial and temporal expression patterns during C. elegans development (Fig. 1B). SUN-1 is proposed to be provided maternally to the embryo (Fridkin et al.,2004) and, together with ZYG-12, provides important biological function in the mitotic cycles of the early embryo (Malone et al.,2003). In later embryogenesis, SUN-1 disappears from the somatic nuclei but remains in the primordial germ cells Z2 and Z3 (Fridkin et al.,2004). A similar detailed description of the temporal expression of ZYG-12 after early embryogenesis is not documented. However, germ line expression of ZYG-12 is detected by immunostaining as early as the first larval stage (K. Zhou, unpublished data). ZYG-12 and SUN-1 are exclusively expressed in the germ line during larval and adult stages (Malone et al.,2003; Fridkin et al.,2004).

In somatic tissues, the SUN domain protein UNC-84 is expressed ubiquitously and localizes to the inner nuclear membrane (Malone et al.,1999; McGee et al.,2006). UNC-84 recruits two KASH domain proteins, UNC-83 and ANC-1, to the outer nuclear membrane. UNC-83 is expressed in a subset of somatic cells while ANC-1 is expressed in all larval and adult somatic cells (Starr et al.,2001; Starr and Han,2002). UNC-83 and ANC-1 are known to connect the nucleus to the microtubule and actin cytoskeleton, respectively (Starr et al.,2001; Starr and Han,2002). The most recently identified KASH protein KDP-1 is broadly expressed in the embryo, the germ line, and the soma (McGee et al.,2009). In the germ line, KDP-1 requires SUN-1 to localize to the nuclear envelope. However, the SUN domain partner for KDP-1 in the somatic tissue where SUN-1 is not expressed is intriguingly not yet identified, as UNC-84 is not its putative binding partner (McGee et al.,2009). The tissue-specific expression patterns of the KASH/SUN pairs suggest distinct functions and experimental evidence confirms various activities.

THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

The ZYG-12/SUN-1 pair expressed in the earliest embryonic stages acts to tether the centrosomes to the nucleus (Fig. 2). A single centrosome is introduced to the embryo with the male pronucleus at sperm entry. This centrosome duplicates, and the two centrosomes move to opposite sides of the male pronucleus. The centrosomes continue a complicated dance of vigorous migrations and movements during the first mitotic division of the one-celled embryo. Notably, as forces pull the centrosomes this way and that, they always remain in close contact with the nucleus (Fig. 2A) (Bornens,1977; Nadezhdina et al.,1979; Kuriyama and Borisy,1981). This contact is lost in the zyg-12 mutants, allowing the centrosomes to drift freely in the cytoplasm (Fig. 2A) (Malone et al.,2003). The female and male pronuclei also fail to migrate and meet when ZYG-12 KASH protein function is compromised. Ultimately, improper chromosome segregation occurs and the embryos die. ZYG-12 function in centrosome attachment can be compromised via mutations in ZYG-12 that affect ZYG-12 self-interaction or via elimination of SUN-1, resulting in no recruitment of ZYG-12 to the nuclear envelope (Malone et al.,2003; Minn et al.,2009). A ZYG-12 isoform that lacks the KASH domain localizes separately to the centrosome, and homotypic interactions between the N-terminus of nuclear envelope-localized and centrosome-localized ZYG-12 isoforms are proposed to mediate centrosome attachment (Fig. 2B) (Malone et al.,2003). This model has yet to be validated.

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Figure 2. ZYG-12 and SUN-1 tether centrosomes to the nucleus in the early embryo. A: In wild-type embryos (top), the centrosomes remain attached to the male pronucleus while pronuclei migrate and meet and a normal spindle is set up after the nuclear envelope breaks down. In the embryo of zyg-12 mutants (bottom), centrosomes detach from the nucleus, and the two pronuclei fail to migrate or meet, resulting in abnormal spindle formation and aberrant chromosome segregation. Micrographs adapted from Malone et al. (2003). B: The working model for ZYG-12 in centrosome attachment. SUN-1 dimers or multimers at the INM recruit ZYG-12 to the ONM, which interacts with centrosome-localized ZYG-12 to tether the centrosome to the nucleus. ZYG-12 also recruits dynein to the nuclear envelope, which plays a minor role in centrosome attachment but a more important role in maintaining gonad architecture (Fig. 4).

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Indirect ZYG-12 interactions with the cytoskeleton do play a role in centrosome attachment but appear to be secondary to ZYG-12-ZYG-12 interactions. The N-terminus of ZYG-12 also interacts with DLI-1, dynein light intermediate chain, recruiting the dynein motor to the surface of the nucleus (Fig. 2B). Because RNAi of DLI-1 or dynein component DHC-1 causes a mildly penetrant centrosome detachment phenotype (Gonczy et al.,1999; Yoder and Han,2001), dynein is proposed to facilitate attachment whereas ZYG-12 is more strictly required. Although dynein may not be key to centrosome attachment in the early embryo, the DHC-1 and DLI-1 RNAi studies demonstrate that dynein is required for pronuclear migration, as is ZYG-12. It may be that the dynein recruited to the female pronucleus can act to pull the female pronucleus towards the microtubule-organizing center (i.e., the centrosome) attached to the male pronucleus. However, this has not been directly examined, and the role of the dynein that is recruited to the nuclear envelope by ZYG-12 in the early embryo has not been specifically investigated. ZYG-12-mediated recruitment of dynein to the nuclear envelope is critical in the germ line (Zhou et al.,2009) (see “The Germ Line” section).

LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

ZYG-12 and SUN-1 expression are lost from the soma in later embryogenesis while UNC-83 and UNC-84 become expressed. The UNC-83 and UNC-84 pair mediates nuclear movements in migrating and polarizing cells. An early example of this function is seen in the development of the dorsal syncytial hypodermal cell hyp7 (Sulston and Horvitz,1981). At the start of hyp7 formation during embryonic morphogenesis, two groups of epithelial cells align along the dorsal midline and then intercalate by elongating and crossing the midline (Fig. 3A). The nuclei in adjacent cells then migrate in contralateral directions. After the nuclear migration, these cells fuse and form the dorsal syncytial hypodermal cell, hyp7. Other cells fuse to hyp7 during the course of development, resulting in a syncytium with over 100 nuclei (Sulston et al.,1983). Mutations in unc-83 and unc-84 cause an identical failure during the nuclear migration stage of hyp7 formation; the hyp7 nuclei initiate but fail to complete the migration to the contralateral position (Fig. 3B). As a result of failed migration, nuclei end up abnormally positioned on the dorsal midline (Sulston and Horvitz,1981; Malone et al.,1999; Starr et al.,2001).

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Figure 3. UNC-83/UNC-84 and ANC-1/UNC-84 pairs mediate nuclear migration and anchorage, respectively. A: Wild-type hyp7 development. Two rows of epithelial cells (hyp7 precursors) on the dorsal side of the embryo (shown as the surrounding oval) undergo elongation, intercalating at the dorsal midline to form a single row of cells. The nucleus in each cell subsequently migrates (arrows) to the opposite end of the cell. Illustrations adapted from Malone et al. (1999) and Starr et al. (2001). B: The contralateral nuclear migrations fail in unc-83 and unc-84 mutants, resulting in nuclei abnormally residing in the dorsal midline. C: The working model for nuclear migration in hyp7 precursors. The hyp7 precursor cells are proposed to polarize, adopting opposite microtubule orientations in adjacent cells. UNC-84 (red) at the INM recruits UNC-83 (blue) to the ONM. UNC-83 interacts with the plus-end-directed microtubule motor kinesin, which pulls the nucleus towards the plus end (arrows) of the microtubule array. This model has yet to be validated. D: ANC-1/UNC-84 mediate nuclear anchorage in hyp7. In wild-type animals (left) the hyp7 nuclei (arrowheads) adopt a regularly aligned, evenly spaced configuration within the hyp7 syncytium. This anchorage is lost in anc-1 (right) and unc-84 (not shown) mutants, resulting in free-floating nuclei that often cluster as the animal flexes (arrowheads). Micrographs adapted from Hedgecock and Thomson (1982). ANC-1 is a large protein that is recruited to the nuclear envelope by UNC-84 and that extends into the cytoplasm to bind to the actin cytoskeleton.

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How nuclei in adjacent hyp7 cells migrate in opposite directions is an intriguing puzzle that is beginning to be deciphered. The UNC-84 SUN domain protein at the inner nuclear membrane recruits UNC-83 KASH domain protein to the outer nuclear membrane (McGee et al.,2006; Meyerzon et al.,2009a). Reminiscent of ZYG-12, which recruits dynein motor, UNC-83 recruits KLC-2, the light chain of the plus-end-directed motor kinesin-1, to the nuclear envelope (Meyerzon et al.,2009a). In fact, expression of a KLC-2::KASH chimera can partially rescue the unc-83 null phenotype (Meyerzon et al.,2009a). The current model for the contralateral migrations proposes that the hyp7 cells polarize during the elongation stage with adjacent cells adopting opposing microtubule orientations (Fig. 3C). The kinesin motor, thus, moves the nucleus from one end of the cell to the other in opposite directions in adjacent cells along the polarized microtubule networks (Meyerzon et al.,2009a). Consistent with this model, microtubules are known to be actively rearranged into parallel arrays along the long axis of the hyp7 cells during hyp7 cell intercalation, and microtubules are required for hyp7 nuclear migration (Williams-Masson et al.,1998). However, whether the orientation of the microtuble arrays alternates in adjacent cells has not been examined.

Another set of nuclear migrations takes place during embryogenesis in the intestinal primordium. Initially, two groups of endodermal cells align along the intestinal midline where the future intestinal lumen will form (Leung et al.,1999). Then nuclei of both groups move toward the intestinal midline and take up positions near the apical surface of the cells. This nuclear migration also fails in unc-83 mutants; instead of moving to the apical surface, the nuclei remain in the middle of each cell (Starr et al.,2001). Surprisingly, the unc-83 effect appears related to temperature, as embryos raised at 25°C show a less severe defect than embryos grown at 15°C. As in the hyp7 cell, how the nuclei migrate in a specific direction is an intriguing question. While UNC-83 is known to recruit the plus end–directed motor kinesin-1, the role of this motor in intestinal nuclear migration is unknown. Furthermore, a plus end–directed motor is not the predicted motor to mediate migration to the apical side of an intestinal cell. In mammalian systems, a mature, polarized epithelial cell contains non-centrosomal microtubule arrays with their minus ends directed to the apical surface (Bacallao et al.,1989; Bartolini and Gundersen,2006). Similarly, microtubules appear to have an organized microtubule array emanating from the apical surface of the C. elegans intestinal epithelial cells, and the apical surface of these cells was proposed to act as a microtubule organizing center (Leung et al.,1999). However, it remains possible that the array is reversed relative to mammals since orientation of minus and plus ends has not been examined directly. On the other hand, a motor other than KLP-1 may be recruited by another isoform of UNC-83 in this tissue. The unc-83 locus encodes several isoforms that differ at their N-terminus and at least one of the isoforms with an extended N-terminus is required for intestinal nuclear migration (Starr et al.,2001). Further characterization of the cellular cytoskeleton in these cells as well as closer analysis of the expression of the UNC-83 isoforms will help us to understand the molecular basis of this nuclear migration.

LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

During the course of development, many cells migrate from their birth site to a final functional site. In general, the behavior of the nucleus appears to be coupled to the motion of cells during the course of a cell migration. For example, in a cultured fibroblast wound-healing assay, the nucleus undergoes a characteristic series of movements. Upon the addition of growth factor, the nucleus first moves away from the advancing edge of the cell, positioning the centrosome ahead of the nucleus. This forward positioning of the centrosome may be required to generate force to pull the nucleus in conjunction with the extension of the leading edge in this cell type (Gomes et al.,2005). The C. elegans P cells move during the first larval stage from a lateral position to the ventral midline where they give rise to ventral cord neurons and vulval precursor cells (Sulston,1976; Sulston and Horvitz,1977). While the process of P-cell migration is distinct from that of fibroblasts in a wound-healing assay, characteristic behaviors of the nucleus are similarly coordinated with the overall cellular movements. During P-cell movement, the leading edge of the P-cell body initially extends to a ventral position while the cell body and the nucleus remain in a lateral position. The nucleus subsequently migrates through the cellular extension to the leading edge on the ventral side. After the nucleus adopts the ventral position, the rest of the cell body follows. In both unc-83 and unc-84 mutants, the nucleus initiates its prescribed movement but arrests midway through migration and then returns to its original position (Malone et al.,1999). The cell subsequently dies. Because of the death of P cells, the animals lack some neurons in the ventral cord as well as the vulva precursor cells and exhibit both uncoordinated and egg-laying defective phenotypes (Horvitz and Sulston,1980; Malone et al.,1999; Starr et al.,2001). As observed in intestinal nuclear migration, temperature affects the penetrance of the unc-83 and unc-84 P-cell nuclear migration defects. However, the P-cell temperature effect is opposite to the cold-sensitive phenotype observed in intestinal development. In all tested unc-83 and unc-84 alleles, 50–90% of cell nuclei fail to migrate at 25°C but approximately 90% of nuclei successfully migrate at 15°C (Malone et al.,1999; Starr et al.,2001). While the mechanisms at play here are still mysterious, the fact that the nucleus initiates movement but then fails to complete its migration may be consistent with the presence of a weak and easily severed attachment to the cytoskeletal elements providing the force for movement when UNC-83 or UNC-84 are missing.

The distal tip cell (DTC) is another C. elegans cell type that undergoes a dramatic and dynamic migration. DTCs are hermaphrodite somatic gonad cells that lead the growth of the gonad at both tips as the rapid proliferation of the germ line takes place during the third larval stage. They have a characteristic migration pattern away from the gonad midline toward the head and the tail until the end of the third larval stage. Then they turn dorsally and then proximally, migrating back to the gonad midline by the middle of the fourth larval stage, resulting in the formation of an anterior and a posterior U-shaped gonad arm (Hirsh et al.,1976; Kimble and Hirsh,1979). unc-84 mutants display morphological abnormalities in gonad arms, indicative of an altered migration path of the DTCs. Interestingly, the rate of gonad outgrowth is the same as in wild type, indicating that only the migrating path of the DTC is affected. Like P-cell nuclear migration, the DTC migration of unc-84 mutants is also sensitive to temperature; animals raised at 15°C have a normal gonad while a severe phenotype is observed when animals are raised at 25°C (Malone et al.,1999). A similar DTC migration defect has also been reported for unc-83 mutants (Malone et al.,1999). While the behavior of the nucleus presumably underlies the DTC migration disturbances, the mechanisms at play here are unknown and could reveal new functions for the KASH/SUN complex.

NUCLEAR ANCHORAGE ACROSS DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

The multinucleate syncytial cells in C. elegans provide a striking example of regulation of nuclear positioning. The hypodermis is a syncytial system consisting of several cells that each contains multiple nuclei in a common cytoplasmic pool. Similarly, the germ cell nuclei reside in a large syncytium. Interestingly, nuclei in these syncytia are anchored at specific locations. KASH/SUN pairs function throughout development and adulthood to prevent movement of these nuclei in the cytoplasm.

The hyp7 cell discussed previously is the largest of the hypodermal cells covering the C. elegans body. In wild-type animals, the more than 100 hyp7 nuclei are evenly spaced and anchored in position (Fig. 3D). A specific nucleus may shift slightly as the body of the animal flexes during movement, but each will resume a normal position soon after the muscle relaxes (Hedgecock and Thomson,1982). In anc-1 mutant animals, neither nuclei nor mitochondria are anchored as expected within the cytoplasm. Mutation of anc-1 affects syncytial cells in general, but is most striking and most easily observed in the large hyp7 cell; the many hyp7 nuclei drift freely in the syncytial cytoplasm, often pile together (Fig. 3D), and are dramatically pushed around during body flexion (Hedgecock and Thomson,1982). ANC-1 functions both embryonically and postembryonically as the nuclei of newly hatched larva are not anchored, and the nuclei that join the syncytium via seam cell fusion at the transition to adulthood also drift freely after fusion (Hedgecock and Thomson,1982).

ANC-1 is a large protein comprised of 8,546 amino acid residues. Like ZYG-12 and UNC-83, ANC-1 also associates the nucleus with the cytoskeleton (Fig. 3D). The N-terminus of ANC-1 contains a calponin domain that binds to actin (Starr and Han,2002). This domain is followed by six repeats of a large coiled-coil region and the C-terminal KASH domain, which targets ANC-1 to the outer nuclear membrane. As expected, the outer nuclear membrane localization of ANC-1 depends on the somatically expressed SUN protein UNC-84 (Starr and Han,2002,2003). The bulky size of ANC-1 allows it to stretch into the cytoplasm and tether the nucleus to the actin cytoskeleton.

THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

While UNC-84 recruits KASH proteins to the nuclear envelope in the adult soma, SUN-1 serves this function in the germ line, recruiting ZYG-12 and KDP-1. Both ZYG-12 and SUN-1 RNAi induce a dramatically disorganized gonad with a reduced number of germ cells that cluster abnormally. A sun-1 deletion mutant and the zyg-12(ts ct350) mutant share this phenotype (Malone et al.,2003; Fridkin et al.,2004; Penkner et al.,2007). The ZYG-12/SUN-1 pair at the nuclear envelope in the germ line appears to have at least two functions. One role is in maintaining the structure of the germ line syncytium and the second is in chromosome dynamics during meiosis.

Architectural Stability

Each germ line arm of an adult hermaphrodite gonad is a large syncytium comprised of many nuclei that are maintained in an incomplete cell membrane compartment (Fig. 4A). The incomplete compartment has a bridge connecting to a central cytoplasmic pool called the rachis (Hirsh et al.,1976; Hubbard and Greenstein,2005). Each germ cell nucleus localizes to the periphery of its compartment in an evenly-spaced alignment within the gonad (Fig. 4A). This arrangement may be important for communication between germ cells and the surrounding somatic gonad, for regulation of meiotic progression, and for the physical movement of germ cells toward the proximal gonad where oogenesis occurs. Cytoskeletal-derived forces are probably required to maintain this global gonad architecture, and a KASH/SUN bridge plays a critical role.

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Figure 4. The ZYG-12/SUN-1 pair has at least two biological functions in the germ line. A: Schematic illustrating C. elegans gonad structure and germ line arrangement. The germ line nuclei are maintained in partially cellularized compartments at the periphery of the gonad. The mitotic zone at the gonad distal tip contains germ line stem cells. As cells leave the mitotic zone, they initiate meiosis. Successive stages of meiosis are encountered in the transition zone, pachytene zone, and the loop region, culminating in formation of mature oocytes in the most proximal region of the gonad. The fluorescence micrograph (left) shows a region spanning the transition and pachytene zones. Membranes are marked with SYN-4::GFP (green) (Jantsch-Plunger and Glotzer,1999) and nuclei are stained blue with DAPI. In zyg-12(ct350) mutants (right), the nuclei become displaced, entering the central rachis, and the membranes are disorganized. B: Model for ZYG-12/SUN-1 function in germ line architecture. Each germ cell is partially surrounded by membrane but retains a bridge to the shared cytoplasmic pool called the rachis. The maintenance of the separate germ cell compartments depends on ZYG-12 and SUN-1 on the nuclear envelope. Dynein localizes to the nuclear envelope by interacting with the N-terminus of ZYG-12. γ-tubulin (green) at the plasma membrane nucleates microtubules that are captured by dynein on the nuclear envelope. The tension generated helps to pull the membrane around the nucleus and maintain the overall gonad architecture. Diagrams in A and B adapted from Zhou et al. (2009). C: Model for ZYG-12/SUN-1 function in meiosis. The KASH/SUN pair mediates interactions between chromosome pairing centers and the microtubules to mediate chromosome movement. The molecular connection between SUN-1 and the chromosome is not fully understood. ZYG-12 recruitment of dynein in the cytoplasm allows force to be used to pull pairing chromosomes apart. Homologous pairs are proposed to withstand this opposing force while non-homologous pairing is more easily disrupted, allowing chromosomes to continue to search out the correct partner.

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The germ line of zyg-12(ct350 ts) animals is disorganized when raised at 25°C (Fig. 4A). Both germ cell membrane and microtubule organization undergo dramatic rearrangement within 4 hr after a shift to the restrictive temperature. This immediate effect of temperature excludes a mitotic defect as a possible cause of the gonad disorganization (Zhou et al.,2009). It is not known if ZYG-12 and SUN-1 are required for germ cell mitosis as they are for early embryonic mitoses. Interestingly, another zyg-12 temperature-sensitive allele, or577, displays normal gonad architecture when raised at 25°C, even though it has an identical detached centrosome phenotype in embryogenesis. Molecular analysis reveals that zyg-12(or577) retains the ability to interact with dynein light intermediate chain DLI-1 and recruits dynein motor to the nuclear envelope, while the zyg-12(ct350 ts) animals do not (Malone et al.,2003; Zhou et al.,2009). This difference in molecular activities between alleles implicates interactions between the nucleus and the microtubule cytoskeleton in maintaining the overall gonad architecture (Fig. 4A). It was demonstrated that γ-tubulin is nucleated from the partial germ cell membranes and it was proposed that the dynein on the nuclear envelope captures these non-centrosomal microtubules and, thus, provides the tension to pull the membrane around each nucleus to maintain the compartments (Fig. 4B) (Zhou et al.,2009).

Chromosome Dynamics

SUN domain proteins play essential roles in the formation of the meiotic chromosome bouquet, an aggregation of chromosome telomeres at the nuclear envelope, in fission and budding yeast as well as mice (Chikashige et al.,2006; Conrad et al.,2007; Ding et al.,2007). The disrupted germ line phenotype caused by the zyg-12 and sun-1 alleles would be expected to mask any similar functions of ZYG-12 and SUN-1 in the later stages of germ line development, making similar analysis in C. elegans difficult. However, a weak sun-1 allele and RNAi studies have revealed meiotic defects. Unlike the sun-1 deletion mutants, the EMS-induced sun-1 allele jf18 is not a molecular null; sun-1(jf18) recruits detectable but greatly reduced amounts of ZYG-12 to the nuclear envelope (Penkner et al.,2007). Moreover, the germ line of sun-1(jf18) mutants appears normal in size and morphology, making it possible to examine the role of this SUN protein in later stages of germ line development (Penkner et al.,2007).

Germ line development occurs in a reproducible temporal and spatial progression in a wild-type animal (Fig. 4A). Germ cells at the distal end of the gonad retain a stem cell fate and divide mitotically. The mitotic zone is followed by a transition zone, where a crescent-shaped aggregation of the chromosomes appears close to the nuclear periphery (Crittenden et al.,2006). In the transition zone, the homologous chromosomes pair and establish synapses. Transient non-homologous chromosome alignment occurs at some frequency during this time but can be recognized and displaced to permit an appropriate alignment (Couteau and Zetka,2005; Martinez-Perez and Villeneuve,2005).

The sun-1(jf18) mutant germ line lacks a distinct transition zone. Additionally, homologous chromosome pairing is reduced and extensive non-homologous synapses are established (Penkner et al.,2007). Closer analysis of the ZYG-12 and SUN-1 expression patterns provided further evidence for a role for this KASH/SUN pair in meiosis. The arrangement of the ZYG-12 and SUN-1 proteins in the nuclear envelope is actively reorganized during meiotic prophase; aggregations of ZYG-12 and SUN-1 coalesce into large patches (Penkner et al.,2009; Sato et al.,2009). The patches are dynamic, and association of the aggregated chromosomes in the transition zone with the nuclear periphery occurs at or near the patches. Unlike in other organisms where the telomere mediates attachment to the nuclear envelope during bouquet formation, a unique region of each C. elegans chromosome called the pairing center is in closest proximity to the membrane (Phillips et al.,2009). The chromosome-pairing centers co-localize with the ZYG-12/SUN-1 patches, suggesting a functional relationship between the KASH/SUN bridge and the pairing chromosomes (Penkner et al.,2009; Sato et al.,2009). This linkage may be achieved via a protein complex that includes the zinc finger family of HIM and ZIM proteins that bind the C. elegans chromosome-pairing centers (Phillips et al.,2009) (Fig. 4C). However, no direct interaction between these proteins and SUN-1 has been reported, and other unknown proteins might facilitate the interaction between the pairing center binding proteins and the ZYG-12/SUN-1 bridge.

Investigation of the roles of cytoskeletal elements in patch formation and pairing revealed that microtubules are required for assembly of the larger aggregations of patches as well as for chromosome pairing (Sato et al.,2009). These data suggest a functional mechanism similar to the models for KASH/SUN pair function in other tissues. ZYG-12 and SUN-1 are suggested to bridge the nuclear envelope in meiotic germ cells to allow forces generated by the microtubule cytoskeleton to be applied to the chromosomes within the nucleus (Fig. 4C). Because chromosomes in sun-1 and zyg-12 mutants make non-homologous synapses, the proposed model suggests that the resulting “shaking” of the chromosomes separates mispaired, non-homologous chromosomes, allowing chromosomes to make another attempt to find their homologous partners. When homologous pairing occurs, affinity between homologous chromosomes is proposed to be sufficient to resist the disruptive forces. Thus, homologous pairing is preserved and synapsis can be established (Sato et al.,2009). The selective disruption of non-homologous pairs during this stage of meiosis may represent a conserved mechanism (Koszul et al.,2008; Koszul and Kleckner,2009).

Microtubules are required for any chromosome pairing to occur. Yet, dynein, which as described previously is recruited to the nuclear envelope by ZYG-12, mediates only some of the pairing-related functions of the microtubules (Sato et al.,2009). When dynein function is knocked down, pairing is delayed but not blocked. However, a new phenotype emerges; paired chromosomes fail to form synapses in the absence of dynein. This block in synapse formation is removed if SUN-1 is also depleted. Thus, the model was proposed that the SUN-1 protein actively inhibits synapsis in a manner that can be overcome by dynein. While this model appears to fit the available data, no clues about possible mechanisms for such an interaction are currently available.

By examining the role of SUN-1 in meiosis, C. elegans researchers have also pioneered in the area of how the function of a KASH/SUN nuclear envelope bridge might be regulated. Aggregation of ZYG-12/SUN-1 complexes with the chromosome-pairing centers and dispersal of the aggregates are triggered at defined points in the meiotic cell cycle. How is this regulation accomplished? Cell cycle–regulated phosphorylation and dephosphorylation of a group of serine residues in the nuclear-localized N-terminus of SUN-1 may play a role (Penkner et al.,2009). It remains to be seen if post-translational modifications could regulate the function of other KASH/SUN pairs. However, this revelation about SUN-1 brings up interesting questions about regulation of KASH/SUN pair function in other tissues. Will post-translational modifications impact KASH/SUN function in other tissues? For example, does anchorage have to be disrupted by down-regulating ANC-1 to permit nuclear migration, perhaps via regulated engagement of UNC-83?

NEW KASH DOMAIN PROTEIN: KDP-1

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

The KASH domain protein KDP-1 was identified as an interacting partner of the SUN-1 protein by the yeast-two-hybrid assay (McGee et al.,2009). Unlike the other KASH proteins in C. elegans, KDP-1 is expressed and localized to the nuclear envelope in both the germ line and somatic tissues. Intriguingly, the nuclear envelope localization of KDP-1 depends on SUN-1 in the germ line but does not depend on UNC-84 in somatic tissue, raising the question of whether there are one or more unidentified SUN proteins in C. elegans. The divergent nature of the KASH domain makes a bioinformatic approach to identifying the entire protein family difficult, again raising the question of whether there are unidentified KASH proteins.

KDP-1 appears to be essential throughout development and is proposed to regulate cell-cycle progression, as mutants are delayed in mitotic entry in the embryo. KDP-1 is required for normal germ line progression as well; large chromatin masses and extra chromatin bodies, possibly caused by mitotic or meiotic defects, can be observed in the kdp-1(RNAi) gonad (McGee et al.,2009). Nonetheless, the molecular basis of how KDP-1 functions is not well understood. More detailed phenotypic analysis and identification of proteins that interact with KDP-1 will help to decipher its function.

IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

As we have outlined, genetic and molecular studies in C. elegans resulted in development of the KASH/SUN model for connecting the nucleus to the cytoskeleton. This model has significantly reshaped the field of nuclear migration and has provided solutions to some long-standing puzzles in mammalian muscle formation and neurobiology.

For example, the hundreds of nuclei in a multinucleate muscle fiber are anchored in an evenly spaced, non-random pattern along the fiber, and a small cluster of nuclei are intriguingly anchored under the neural muscular junction (Bruusgaard et al.,2003). However, the mechanisms responsible for arranging or anchoring these nuclei have long been mysterious. Development of the KASH/SUN model allowed researchers to envision a mechanism, and targeted studies to examine the function of the mammalian SUN and KASH proteins revealed roles for both in positioning nuclei in murine muscle. Syne1 (Nesprin1) and Syne2 (Nesprin2) are mammalian orthologs of ANC-1 (Starr and Han,2002,2003); each contains an actin-binding domain, an extended spectrin repeat region, and a KASH domain. Targeted knock-out and dominant-negative studies in mice demonstrated that like ANC-1, Syne1 is critical for anchoring all muscle cell nuclei and Syne2 may contribute to the process (Grady et al.,2005; Zhang et al.,2007).

If a mammalian KASH protein is involved in nuclear anchorage, a SUN partner is likely important. Mammals have 4 SUN proteins, SUN1, SUN2, SUN3, and SPAG4 (Malone et al.,1999; Shao et al.,1999; Hodzic et al.,2004; Crisp et al.,2006). UNC-84 is most closely related to mammalian SUN1 and SUN2, whereas C. elegans SUN-1 is distantly related to all four mammalian SUN proteins (Jaspersen et al.,2006). SUN1 is critical for anchoring nuclei at the neural muscular junction in mice, whereas the SUN1 and SUN2 proteins act redundantly in anchorage of the other muscle nuclei (Lei et al.,2009). As expected Syne1 and Syne2 localization depend on the presence of SUN1 and SUN2 (Zhang et al.,2007; Lei et al.,2009). Similar to the studies in muscle, the demonstrated role of UNC-83/UNC-84 in C. elegans nuclear migration informed investigations into mechanisms of neuronal nuclear migration. Targeted transgenic mice experiments revealed that SUN1 and SUN2 and the Syne2 KASH protein play roles in the cell cycle–dependent, cyclical migration of neuronal epithelial stem cell nuclei from one side of the cell to the other. They also mediate centrosome attachment in cultures of migrating glial cells (Zhang et al.,2009).

Finally, ZYG-12 and UNC-83 mediate attachments to the cytoskeleton by binding microtubule-dependent motors. ZYG-12 and UNC-83 are not closely related to the Syne1/2 proteins outside of the KASH domain. However, the established interactions with motors in the C. elegans system prompted a search for similar interactions in the murine model. Examination of Syne1 and Syne2 interactions in vivo revealed association with dynein, reminiscent of ZYG-12, and Syne2 associates with kinesin, reminiscent of UNC-83 (Zhang et al.,2007). Mammals have at least two additional KASH domain proteins, Nesprin3 and Nesprin4, and functional studies continue to demonstrate conserved functions in nuclear positioning and cytoskeletal interactions (Wilhelmsen et al.,2005; Ketema et al.,2007; Roux et al.,2009).

PERSPECTIVES ON THE C. ELEGANS STUDIES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES

Tremendous progress has been made in elucidating the functions of KASH and SUN domain proteins using the C. elegans model. These and other studies have led to the prevailing model that KASH/SUN pairs form a bridge across the nuclear envelope, mediating critical communications between the cytosolic and nuclear compartments (Starr,2009). In particular, these complexes connect the nucleus to the cytoskeleton (Fig. 5) to achieve diverse but conserved functions including mediating nuclear migration, centrosome attachment, nuclear anchorage, and chromosomal dynamics.

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Figure 5. The KASH/SUN pairs in C. elegans connect to a diversity of motors and cytoskeletal elements to move and anchor nuclei. Additional connections and functions remain to be elucidated.

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Early embryogenesis is unique as the only C. elegans developmental stage that requires a KASH/SUN pair to tether the centrosome and nucleus. Thus, other mechanisms probably mediate centrosome attachment in other tissues (Zhou et al.,2009). Nonetheless, the role of KASH/SUN pairs in centrosome attachment is conserved. The KASH/SUN role in centrosome attachment is clearly not the full answer to how centrosomes associate with nuclei. While little if any data exists to shed light on alternative centrosome-tethering mechanisms, there are elements of the embryonic centrosome attachment mechanism that we have yet to understand. RNAi of F40H6.6, a gene of unknown function, causes a detached centrosome phenotype in the embryo (Brauchle et al.,2009). Intriguingly, this gene is predicted to encode a type II integral membrane protein, reminiscent of the KASH domain proteins. The size of the nucleus also has an impact on centrosome attachment (Meyerzon et al.,2009b), and potent extrageneic suppressors of the zyg-12 centrosome detachment phenotype have been isolated (Y. Chen and C. J. Malone, unpublished data). These observations illustrate that current models for centrosome attachment may be simplistic. It remains to be determined if other KASH/SUN complexes are also involved.

In terms of primary sequence, there is no homology among the KASH proteins outside the KASH domain and the KASH domain itself can be quite divergent (McGee et al.,2009; Minn et al.,2009; Starr,2009). Nonetheless, the KASH proteins share the same structural features: a single transmembrane-containing KASH domain that allows the proteins to localize to the nuclear envelope by stretching into the perinuclear space and interacting with a SUN protein, a central coil-coiled region that may form dimers or multimers, and a cytosolic N-terminus that interacts with the cytoskeleton directly or indirectly through motor proteins (Fig. 5). Why do different KASH proteins evolve to accomplish the task of connecting the nucleus and the cytoskeleton? Most likely, the need to interact with different cytoskeletal elements in different cell types and/or at different times in development necessitates the use of multiple KASH proteins. The C. elegans germ line and the hypodermis illustrate the need to link to different cytoskeletal elements. Though both tissues are large syncytia, the germ line nuclei are partially enclosed in a membrane while nuclei in the hypodermis are not. The extended structure of ANC-1 can stretch up to 0.5 μm and is long enough to connect to the actin cytoskeleton and anchor the nuclei in place in the large hyp7 cell. In the germ line, tension on membrane-nucleated microtubules is thought to pull the plasma membrane around the nucleus. This might be an important mechanism to secure the integrity of individual germ cells in the overall syncytial environment that ensures only one germ cell can enter the oogenesis process at a time.

The divergent nature of the KASH domain and the lack of identification of the binding partner for KDP-1 in the adult soma suggest that additional SUN and/or KASH proteins remain unidentified. While identification of additional family members and binding partners for KASH proteins is certain to be enlightening in terms of the cytoskeletal elements at play, a complimentary approach that has not been widely applied in C. elegans is to examine the cytoskeletal organization and polarity in additional cell types. For example, the model for hyp7 cell migration defects proposes that adjacent cells have opposite orientation microtubule polarity. Analysis of cytoskeletal organization in this cell type and others would do much to help us decipher how KASH/SUN protein complexes mediate the application of force to the nucleus. The exciting new findings implicating KASH/SUN complex function in application of cytoskeletal forces to components inside the nucleus as well as the complex phenotypes associated with the most recently identified C. elegans KASH protein suggest that we may have just scratched the surface in elucidating functions for KASH/SUN pairs.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FORWARD GENETIC SCREENS IN C. ELEGANS IDENTIFY NUCLEAR MIGRATION AND ANCHORAGE MUTANTS
  5. KASH AND SUN DOMAIN PROTEINS
  6. C. ELEGANS KASH/SUN PAIR EXPRESSION
  7. THE EARLY EMBRYO: ZYG-12 AND SUN-1 TETHER CENTROSOMES TO THE NUCLEUS
  8. LATE EMBRYOGENESIS: NUCLEAR MIGRATIONS IN HYP7 AND INTESTINAL CELLS
  9. LARVAL DEVELOPMENT: COUPLING THE BEHAVIOR OF THE NUCLEUS WITH CELL MIGRATION IN P CELLS AND DTCS
  10. NUCLEAR ANCHORAGE ACROSS DEVELOPMENT
  11. THE GERM LINE: ARCHITECTURAL STABILITY AND CHROMOSOME DYNAMICS
  12. NEW KASH DOMAIN PROTEIN: KDP-1
  13. IMPACT OF THE KASH/SUN STUDIES IN THE C. ELEGANS MODEL ON OUR UNDERSTANDING OF MAMMALIAN CELL BIOLOGY
  14. PERSPECTIVES ON THE C. ELEGANS STUDIES
  15. Acknowledgements
  16. REFERENCES