Sun L et al. (2000) East Coast Worm Meeting "daf-5 cloning identifies a Sno oncogene homolog as a TGF-beta signaling output"

Sun L, Patterson GI

[East Coast Worm Meeting, 2000]

The reproductive/dauer developmental decision is controlled by environmental cues, such as food, pheromone and temperature. A TGF-beta-related pathway promotes reproductive over dauer development. Mutations in daf-7 (a TGF-beta-like ligand), daf-1and daf-4 (type I and type II receptor kinases), and daf-8 and daf-14 (Smad transcription factors) induce dauer at restrictive temperature regardless of environmental cues. The Daf-c phenotype of these mutants is suppressed by mutations in daf-3 (a Smad) or daf-5. Epistasis analysis suggests that daf-5 and daf-3 are either antagonized by this TGF-beta-related pathway or acting in a parallel pathway. The similarity of the dauer pathway to other TGF-beta pathways suggested that DAF-5 may represent a new molecule functioning in a variety of TGF-beta pathways. We have cloned daf-5 so as to study it at a molecular level. We show that daf-5 encodes a transcription factor homologous to the human oncogene Sno. Mapping and cosmid rescue placed daf-5 in a 8 kb interval of chromosome II. We PCRed a 3.9 kb region of W01G7 from fifteen daf-5 alleles and digested the products with BstUI; we detected a polymorphism in daf-5 (sa211). This region of W01G7 encodes a Sno homologue. Sequencing of daf-5 alleles is in progess. Recently, vertebrate Sno has been shown to be capable of acting as an inhibitor of TGF-beta pathways that functions by interacting with Smad proteins. The genetic function of daf-5 suggests that the function of Sno homologues in TGF-beta pathways is broader than suggested by the vertebrate tissue culture studies, in that daf-5 is not simply acting to block signaling by the receptors. In addition to function in the TGF-beta-related pathway, daf-5 has been shown by various groups to genetically interact with other dauer genes: daf-11 (encoding guanyl cyclase), daf-21 (encoding Hsp90), daf-2 (encoding an insulin-like ligand), and daf-28. Moreover, some daf-5 alleles have a longer life span than N2, implying daf-5 regulates life span as well as dauer formation. One allele of daf-5 does not affect life span independently; however, it further extends the life span of long-lived daf-2 mutants. These results indicate that daf-5 may control aging in parallel to the daf-2 pathway. We hope to identify the role of daf-5 in integrating information from different pathways that control dauer and other developmental events.

Il Minn et al. (2007) International Worm Meeting "Centrosome Attachment to the Nucleus Requires the SUN-1 Mediated Localization of ZYG-12 to the Outer Membrane of the Nuclear Envelope."

Il Minn, Christian J. Malone

[International Worm Meeting, 2007]

The localization of proteins specifically to the outer membrane of the nuclear envelope is thought to be required for attachment of the centrosome to the nucleus. The outer membrane of the nuclear envelope is contiguous with the ER and has been considered to be identical to the ER. Thus, the mechanism of specific outer nuclear membrane localization is unknown. We and others have recently identified a novel class of proteins with KASH domains that specifically localize to the outer membrane of the nuclear envelope via an interaction with SUN proteins. We have shown that two isoforms of the C. elegans protein ZYG-12, which contain KASH and transmembrane domains, depend on a SUN protein, SUN-1 (also called MTF-1), to localize to the nuclear envelope. ZYG-12 is required for attachment of the centrosome to the nucleus in C. elegans. To map the domains of ZYG-12 necessary for SUN-1 mediated localization, we expressed C-terminal fragments of ZYG-12, which contain KASH and transmembrane domains. These C-terminal fragments are sufficient for nuclear envelope localization in a SUN-1 dependent manner. We have investigated the membrane topology of both ZYG-12 and SUN-1. Both epitope accessibility and fluorescent protease protection assays using in vivo nuclei in combination with proteinase K protection assays using microsomes revealed that ZYG-12 and SUN-1 are type II membrane proteins of the outer and inner membrane of the nuclear envelope, respectively. In support of ZYG-12 being a type II membrane protein of the outer nuclear envelope, ZYG-12 physically interacts with cytoplasmic dynein via the N-terminus of ZYG-12 and is required for the localization of dynein to the nucleus. To investigate the molecular mechanism of SUN-1 dependent ZYG-12 localization, we asked if the proteins physically interact. Our data indicate that SUN-1 directly interacts with the KASH domain of ZYG-12. The interaction occurs between the KASH domain and the internal domain distinct from the SUN domain of SUN-1 as mapped by a membrane based two-hybrid assay and a pull-down assay. ZYG-12 at the outer nuclear membrane is necessary for centrosome attachment. Our results suggest that the inner nuclear membrane protein SUN-1 confines localization of ZYG-12 to the outer nuclear membrane via direct binding in the lumen of the nuclear envelope.

Matthew McGee et al. (2007) International Worm Meeting "KASH and SUN proteins bridge the nuclear envelope to transfer force from the cytoskeleton to the nucleoskeleton during nuclear positioning."

Matthew McGee, Daniel Starr

[International Worm Meeting, 2007]

Nuclear positioning, which includes both migration and anchorage of the nucleus, is an important process in C. elegans development. The outer nuclear membrane (ONM) KASH proteins UNC-83 and ANC-1 are required for nuclear migration and anchorage, respectively. The inner nuclear membrane (INM) SUN protein UNC-84 is required for both nuclear migration and anchorage. The UNC-83 and ANC-1 KASH domains are thought to interact with the UNC-84 SUN domain in the perinuclear space. This allows force to be transferred from the cytoskeleton to the nucleoskeleton during nuclear positioning. KASH protein localization is dependent on SUN proteins, but SUN protein localization is not dependent on KASH proteins. Using a membrane-bound yeast two-hybrid system (Igor Stagljar, Dualsystems Biotech), we have shown that the KASH domain directly interacts with two separable domains of UNC-84, the SUN and linker domains. This interaction is required for nuclear migration and anchorage. Using a molecular genetics approach, we found that the UNC-84 SUN domain is required for KASH protein localization and function in vivo. We also demonstrated that point mutations in the KASH domain disrupt the function but not the localization of UNC-83. This suggests a stronger interaction, presumably to allow a large transfer of force during nuclear positioning, is required to move or anchor the nucleus than is required to simply localize the KASH proteins to the ONM (McGee, et al. Mol Biol Cell. 2006:1790-801). This KASH and SUN interaction appears to be conserved across eukaryotes. To identify residues in the linker domain that are required for interaction with the UNC-83 KASH domain, we are performing a mutational analysis of the UNC-84 linker domain in the membrane-bound two-hybrid system. We are also using this two-hybrid system in a screen to test whether there are unidentified binding partners with the SUN domain of UNC-84. In order to test the conservation of function between human and C. elegans KASH and SUN domains, we are testing whether the human domains in the C. elegans KASH and SUN proteins localize and are functional. We have found that a human KASH domain in UNC-83 is able to partially rescue UNC-83 function in migrating hyp-7 nuclei and localize to the nuclear envelope. We have also found that this hyp-7 rescue is dependent on the UNC-84 SUN domain. This suggests that the human KASH domain is able to interact with the UNC-84 SUN domain in vivo. Further experiments are testing the functional rescue of human SUN domains in UNC-84.

Aya Sato et al. (2006) Development & Evolution Meeting "The role of the nuclear envelope in homologous chromosome pairing and synapsis during meiosis"

Abby Dernburg, Carolyn Phillips, Aya Sato, Roshni Kasad

[Development & Evolution Meeting, 2006]

Pairing and recombination between homologous chromosomes are essential for accurate segregation of chromosomes during meiotic prophase. Specific chromosome sites known as Pairing Centers (PCs) are essential for homolog pairing and synapsis in C. elegans. Our laboratory has identified a family of four related proteins, HIM-8, ZIM-1, ZIM-2, and ZIM-3, that bind to the PCs on each chromosome and mediate homologous synapsis. Interestingly, foci of these proteins are associated with the nuclear envelope during early meiotic prophase, suggesting that they might tether the chromosomes to the nuclear envelope. We have found that two nuclear envelope proteins in C.elegans, SUN-1 and ZYG-12, form distinct patches at the nuclear envelope in transition zone nuclei in which homologous chromosomes are pairing, and that these SUN-1/ZYG-12 patches co-localize with HIM-8, ZIM-1, -2, and -3. To dissect the role of the nuclear envelope in homologous chromosome pairing and synapsis, we have carried out functional analysis of SUN-1 and ZYG-12 by genetic, cytological, and biochemical approaches. SUN-1 is a germline enriched nuclear envelope protein. ZYG-12 displays SUN-1-dependent localization at the nuclear envelope during meiosis, similar to its localization requirement in mitosis.4 We also found that sun-1 mutations genetically interact with X chromosome PC deficiencies, and that cosuppression of zyg-12 results in a Him phenotype. Both sun-1 and zyg-12 deletion mutants are sterile and show severe defects in germline mitosis, as well as meiosis. However, HIM-8 foci are still associated with the nuclear envelope in these mutants, suggesting that the role of SUN-1 and ZYG-12 is not simply to tether chromosomes to the envelope. We are currently investigating whether these proteins interact with other cellular components to mediate their roles in meiosis.

Hunter CP et al. (1991) International C. elegans Meeting "NON-AUTONOMY OF HER-1 FUNCTION IMPLICATES CELL INTERACTIONS IN C. ELEGANS SEX DETERMINATION."

Hunter CP, Wood WB

[International C. elegans Meeting, 1991]

Activity of the her-l gene is required for normal male development in C. elegans. Loss-of-function her-l mutations cause feminization of XO animals (normally male) to produce fertile hermaphrodites, while gain-of-function her-l mutations cause masculinization of XX animals ( normally hermaphrodite) to produce pseudo-males. Genetic analysis indicated that her-l functions early in the hierarchy of regulatory interactions among the major sex determination genes (1). We have analyzed her-l genetic mosaics to determine which cells must express her-l for wild-type male development. If her-l functions cell- autonomously, then in XO animals her-l (+) cells will follow male fates, and her-1(-) cells will follow hermaphrodite fates. If her-l or a her-l regulated activity functions non-autonomously, then the her-l genotype of a cell may not determine its sexual phenotype. The phenotypes of XO her-l genetic mosaics are variable, but clearly show that both her-1(-) and her-l (+) cells can follow either hermaphrodite or male fates. We conclude that her-l is neither necessary nor sufficient to cell-autonomously determine male sexual phenotypes and, therefore, that her-l or a her-l regulated activity must be able to function non-autonomously. The observed patterns of non-autonomous effects suggest that many or all cells express and respond to her-l activity. These findings implicate cell interactions in C. elegans sex determination. Earlier genetic analysis indicated that her-l might directly control tra-2 activity (1). Molecular characterization of the gene products of her-l and tra-2 (see abstracts by M. Perry et al. and P. Kuwabara et al) is consistent with a model in which a secreted ligand encoded by her-l interacts with a cell-surface receptor encoded by tra-2. We will discuss the possible role of such an interaction in coordinating the sex determination decision.

Erin C. Tapley et al. (2007) International Worm Meeting "Characterizing the UNC-83/UNC-84 interaction in vitro.."

Erin C. Tapley, Daniel A. Starr

[International Worm Meeting, 2007]

Nuclear migration and positioning is important to a wide variety of eukaryotes. For example, mammalian skeletal muscles must specialize nuclei at the neuromuscular junction, and in the developing human brain nuclei must migrate toward the cortex. Using C. elegans, we have identified two proteins that function in nuclear migration: UNC-83 and UNC-84. UNC-84 is a member of a family of proteins that share a conserved C-terminal SUN domain and that localize to the inner nuclear membrane (INM). UNC-83 is a member of a family of proteins that share a conserved C-terminal KASH domain and that localize to the outer nuclear membrane (ONM). Using genetic methods, we have shown that both the SUN and KASH domains are essential for nuclear migration in vivo and that a stronger SUN-KASH interaction is required for nuclear migration than for nuclear localization. Membrane bound yeast two hybrid (MBYTH) assays show that the SUN domain of UNC-84 directly interacts with the KASH domain of UNC-83 in the perinuclear space and that two independent domains, SUN and linker, of UNC-84 interact with UNC-83s KASH domain (for further details see McGee et. al 2006). We propose that the SUN-KASH interaction creates a structural bridge across the nuclear envelope that allows the transfer of force from the nuclear lamina to the cytoskeleton during migration. To further characterize the KASH-SUN and KASH-SUN-linker interactions biochemically, we have expressed and purified several MBP-6His fusion proteins from E. coli. Previous transmembrane (TM) prediction programs suggest that UNC-84 has one TM domain located at residues 512-532, which is consistent with our model that the N-terminus of UNC-84 is nucleoplasmic and the C-terminus resides in the perinuclear space. The largest fusion protein, which contains H568-V1111 is insoluble. TopPred2, a TM prediction program, suggests that UNC-84 may have as many as five TM domains. Three of these TM domains reside within the previously described linker domain: 685-705, 719-739, and 763-783. Previous data from our MBYTH suggest that the region between 742-780 is important for membrane targeting. Smaller fusion proteins, D739-V1111 and R783-V1111, which lack either one or both of the last two predicted TMs have also been expressed and purified. Both R783-V1111 and the SUN domain fusion protein T931-V1111 are completely soluble. Soluble SUN-linker and SUN fusion proteins will be used to test the interaction with a custom synthesized UNC-83 KASH peptide in vitro. As the linker domain may be vital for force transfer, it is essential to narrow down its functional domain. To do this, we first need to clearly establish where UNC-84s TM domains are located. Protease protection assays using Promegas TNT ® T7 Quick for PCR , S35 methionine and canine microsomes are currently being performed.

Cain, Natalie et al. (2013) International Worm Meeting "UNC-84 spans the nuclear envelope and connects the nucleoskeleton to KASH proteins at the outer nuclear membrane."

McDonald, Kent, Lorton, Ben, Starr, Daniel, Cain, Natalie, Bone, Courtney, Tapley, Erin

[International Worm Meeting, 2013]

Nuclear positioning is central to many cellular and developmental events. A bridge of inner nuclear membrane (INM) SUN proteins and outer nuclear membrane (ONM) KASH proteins spans the nuclear envelope. In C. elegans, the SUN protein UNC-84 recruits the KASH protein UNC-83 to the ONM where it targets dynein and kinesin-1 to move nuclei. However, it remains unknown how SUN proteins span the perinuclear space of the nuclear envelope and how forces transferred across the bridge are dissipated at the nucleoskeleton. To elucidate mechanisms of how mechanical forces are transferred from the bridge to the nucleoskeleton, we performed a yeast two-hybrid screen with the nucleoplasmic domain of UNC-84 as bait and identified an interaction with the lamin B gene LMN-1. lmn-1(RNAi) animals had a hyp7 nuclear migration defect. Moreover, the LMN-1 interaction was significantly weakened by an UNC-84 P91S mutation that was previously identified in genetic screens with a partial loss-of-function hyp7 nuclear migration defect. These data support a model where UNC-84 interacts with LMN-1 to dissipate force from the bridge to the nucleoskeleton. We identified a new player in this network, the integral INM protein SAMP-1 (T24F1.2); samp-1(RNAi) animals had a weak nuclear migration defect. To test the hypothesis that UNC-84 spans and regulates the space between the INM and ONM, we examined the morphology of the nuclear envelope by electron microscopy in unc-84(null) mutants. We observed extreme separation of the ONM from the INM in unc-84(null) L1 muscle nuclei. Surprisingly, a deletion of ~300 residues between the trans-membrane and SUN domains of UNC-84 remained functional, thus, the majority of this linker domain is not required for nuclear migration. However, a 60 amino acid span immediately upstream of the SUN domain, and the SUN domain itself are required for function. Additionally, mutations in the SUN domain of UNC-84 predicted to interact with KASH peptides from human SUN structures disrupt hyp7 nuclear migrations. In our model the nucleoplasmic domain of UNC-84 directly binds lamin to transfer forces from the bridge to the nucleoskeleton. UNC-84 is also required for even nuclear envelope morphology.

Ellis RE et al. (1991) International C. elegans Meeting "PROGRESS IN CLONING fog- 1"

Kimble JE, Ellis RE

[International C. elegans Meeting, 1991]

The fog-l gene plays an important role in the decision of germ cells to differentiate as either spermatocytes or oocytes (Doniach 1986, Barton and Kimble 1990). In fog-l mutants, all germ cells develop into oocytes. This is true for both males and hermaphrodites. However, these fog-l mutations do not appear to affect other aspects of sexual development. Furthermore, the fog-l mutant phenotype is not suppressed by mutations in any of the other genes known to control sex- determination. Thus fog-l might function to initiate spermatogenesis in response to other genes that regulate general sexdetermination. There are 42 fog-l mutations. These mutations arise at a frequency typical for knockout mutations in other C. elegans genes, and four of these fog-l alleles were isolated from a screen capable of recovering deletions. Thus it is likely that these mutations cause a loss of fog- l function. In order to study the mechanisms by which fog-l functions, and to determine how fog-l expression is regulated by the genes that control sex-determination, we are now cloning this gene. The fog-l gene is located on the left arm of LGI, between sup-ll and unc-ll, a region spanned by a large contig of about one megabase in size. By mapping DNA polymorphisms with respect to fog-l, we have narrowed down the region in which fog-l must be located, and are now injecting YAC DNA that covers this region in an attempt to rescue fog-l mutants.

Christian J Malone et al. (2005) International Worm Meeting "Centrosomal attachment to the nucleus."

Yi Chen, Cheryl Keller, Zhizhen Gao, Christian J Malone, Il Minn

[International Worm Meeting, 2005]

The centrosome and nucleus are intimately associated during interphase in a variety of organisms and cell types, yet the importance and mechanism of the attachment are just beginning to be understood. Mutations in the C. elegans gene zyg-12 specifically perturb the attachment of the centrosome to the nucleus during early embryogenesis. zyg-12 encodes multiple isoforms of Hook-like proteins. Hook proteins mediate the interaction of membrane-bound organelles such as the Golgi with the microtubule cytoskeleton. ZYG-12 isoforms localize to both nuclear envelope and centrosomes if they include a transmembrane domain or just centrosomes if they do not. ZYG-12 localization to the centrosomes, but not the nuclear envelope, requires microtubules. Conversely, ZYG-12 localization to the nuclear envelope, but not the centrosome, requires sun-1, a second gene required for centrosomal attachment. SUN-1 localizes to the nuclear envelope and includes a conserved SUN domain. We are working to characterize both the mechanism of attachment and how ZYG-12 localizes to the nuclear envelope via a SUN-1 dependent mechanism.

Alexandra Penkner et al. (2008) Development & Evolution Meeting "Matefin/SUN-1 Is Required for Homologous Chromosome Pairing in C. elegans Meiosis"

Pawel Pasierbek, Verena Jantsch, Alexandra Penkner, Antoine Baudrimont, Alexandra Fridkin, Yosef Gruenbaum, Jiradet Gloggnitzer

[Development & Evolution Meeting, 2008]

Faithful chromosome partitioning during meiosis relies on the correct recognition and stable pairing of parental homologues during meiotic prophase I. The identification of a highly specific mutation in the germ line-restricted nuclear envelope protein matefin/SUN-1 allowed us to uncover the active contribution of the nuclear envelope to the process of homologous chromosome alignment and pairing in C. elegans meiosis [1]. The inner nuclear envelope protein MTF-1/SUN-1 contains a conserved SUN-domain that presumably interacts with the outer nuclear envelope protein ZYG-12, thereby establishing a proteinaceous bridge across the nuclear envelope that connects components in the nucleus to cytoplasmatic structures. This link is possibly facilitating chromosome movement and homologous chromosome search [2]. Remarkably in leptotene/zygotene, right at the stage when meiotic chromatin reorganizes and homologous chromosome contacts are initiated, specific changes in the characteristics of MTF-1/SUN-1 can be observed. We will report on the analysis of those as well as their impact on homologous chromosome pairing. Our data so far support a model in which the properties of the nuclear envelope are modified during leptotene/zygotene and that these modifications are facilitating homolog sorting.