Starr, Daniel A. et al. (2011) International Worm Meeting "UNC-83 and UNC-84 bridge the nuclear envelope to move nuclei."

Ly, Nina, Tapley, Erin, Meyerzon, Marina, Starr, Daniel A., Fridolfosson, Heidi

[International Worm Meeting, 2011]

Nuclear migration is important for a wide variety of cellular and developmental processes. We employ nuclear migration in the embryonic hyp7 precursors as a model to study nuclear migration. We have shown that UNC-84 localizes to the inner nuclear membrane with its conserved SUN domain in the perinuclear space. The SUN domain then interacts with the KASH domain of UNC-83 to recruit it to the outer nuclear membrane. Thus, UNC-83 and UNC-84 form a bridge across the nuclear envelope, connecting the nucleoskeleton to the cytoskeleton. Here we report a series of findings about the function and regulation of this bridge. First, we show that UNC-83 recruits kinesin-1 (UNC-116) and dynein (DHC-1) to the surface of the nucleus using KLC-2, BICD-1, NUD-2, LIS-1, and EGAL-1. Live filming of nuclear migrations in various genetic backgrounds showed that kinesin-1 provides the major force to move nuclei forward while dynein regulates backwards movements to bypass roadblocks. We also showed that the microtubule network in these cells is polarized using EBP-1::GFP. Second, we have identified multiple signals that function to recruit UNC-84 to the inner nuclear membrane. Importantly, mutations in an INM-SM or a novel SUN-NELS disrupt the efficient localization of UNC-84, suggesting that an active process exists to traffic UNC-84 from the peripheral ER to the nuclear envelope. Perhaps this is through a small isoform of IMA-3. Third, using deletion analysis of the lumenal domain of UNC-84, we have defined the minimal region of UNC-84 required for nuclear migration, which is surprisingly small. Together, these three findings have greatly enhanced our understanding of the molecular mechanisms of how UNC-83 and UNC-84 mediate nuclear migration.

Daniel Starr et al. (2001) International C. elegans Meeting "UNC-83 and UNC-84 function at the nuclear envelope where they are required for proper nuclear migration"

William Fixsen, Chris Malone, Greg Hermann, Daniel Starr, Min Han, James Priess, H Robert Horvitz

[International C. elegans Meeting, 2001]

Nuclear migration plays an essential role in the growth and development of a wide variety of eukaryotes. Mutations in unc-83 and unc-84 disrupt nuclear migration in three C. elegans tissues, larval P cells, the embryonic hypodermis, and the developing intestinal primordium. P cell nuclei normally migrate during L1 from a lateral position to the ventral cord where they eventually give rise to the vulva and neurons. In embryos, nuclei in precursors of the dorsal hyp7 migrate across the dorsal mid-line, and during polarization of the intestinal primordium nuclei normally migrate towards the mid-line. Mutations in unc-83 and unc-84 disrupt hyp7 nuclear migrations at all temperatures. P cell nuclear migrations are effected only at higher temperatures, while intestinal nuclear migrations in are more severe at lower temperatures. unc-84 encodes for a novel protein with a predicted trans-membrane domain. UNC-84 also has a C-terminal domain (termed SUN domain) similar to the S. pombe spindle pole body component Sad1p and uncharacterized proteins from human and Drosophila . UNC-84::GFP localizes to the nuclear envelope of most or all somatic cells. Mutations in unc-84 also disrupt nuclear anchorage. We have mapped and cloned unc-83 and find that it encodes for a novel protein with a predicted C-terminal trans-membrane domain. We identified three unc-83 transcripts that are expressed in a tissue-specific manner as confirmed by the identification of DNA lesions in 16 unc-83 alleles and transcript specific RNAi experiments. Monoclonal antibodies against UNC-83 co-localize to the nuclear envelope with lamin and UNC-84. Unlike UNC-84, UNC-83 localizes to only certain nuclei. UNC-83 was first observed to localize to the nuclear envelope of migratory hyp7 precursors in pre-bean embryos. At the bean stage UNC-83 was also observed in migratory intestinal cells and P cells. Later, UNC-83 was detected in a number of other cells including some pharynx and hypodermal cells. UNC-83 fails to localize to the nuclear envelope in unc-84 mutants with lesions in the conserved SUN domain of UNC-84, suggesting that the role of UNC-84 in nuclear migration is to recruit UNC-83 to the nuclear envelope. We have shown that centrosomes remain properly associated with nuclei despite the unc-83 or unc-84 nuclear migration defects. Additionally, mutations in unc-83 or unc-84 do not disrupt the gross structure of the nuclear matrix in migrating cells. We favor a model in which UNC-84 recruits UNC-83 to the nuclear envelope where they function to help transfer force between the cytoskeleton and the nucleus. We are currently attempting to immunolocalize UNC-83 by electron microscopy to further refine our model.

Daniel Starr et al. (2001) International C. elegans Meeting "ANC-1 is an over 800 kDa coiled-coil protein required for anchorage of nuclei"

Daniel Starr, Min Han

[International C. elegans Meeting, 2001]

The C. elegans adult has many syncytial cells. In the hypodermis alone, over 100 nuclei are in syncytia. Normally nuclei are anchored in a stereotypical pattern evenly spaced throughout the syncytium. Mutations in anc-1 disrupt this anchorage, and the nuclei move through the syncytia (pushed around by underlying body muscles or forces from crawling) and often clump together (Hedgecock and Thomson, 1982 Cell 30:321-330). Certain mutations in unc-84 , which encodes a nuclear envelope protein that functions in nuclear migration events, also disrupt nuclear anchorage (Malone et al., 1999 Development 126:3171-3181). We mapped the anc-1 gene between unc-73 and let-502 , but were unable to rescue the phenotype by traditional cosmid injections. We therefore fine-mapped anc-1 using single nucleotide polymorphisims (SNPs) to a 100 kb region between two SNPs. We used RNAi to knock out candidates in this small region. RNAi against any one of three regions of a predicted 25 kb open reading frame in this region gave a strong ANC phenotype. This suggests that the anc-1 alleles are null. The anc-1 gene is predicted to encode a 7530 amino acid protein. The anc-1 genomic region contains six 3 kb tandem repeats which are nearly 100% conserved, even in a 123 bp intron, suggesting that there are evolutionary forces to keep the ANC-1 protein as large as possible. We have raised antibodies against the repeat region in rats and western analysis confirms that we have cloned anc-1 . The anti-ANC-1 antibodies localize to most somatic post-embryonic cells. ANC-1 was detected in unorganized, fibrous structures throughout the cytoplasm and was often enriched at the nuclear periphery. We propose two alternative models for ANC-1 function. ANC-1 could form a filamentous network and act as a net to anchor nuclei or ANC-1 could tether nuclei by directly attaching the nucleus to the plasma membrane.

Daniel A Starr et al. (2002) West Coast Worm Meeting "ANC-1 tethers nuclei by connecting the nuclear envelope to the actin cytoskeleton"

Min Han, Daniel A Starr

[West Coast Worm Meeting, 2002]

Little is know about organelle membrane proteins that mediate the anchorage of nuclei and organelles within cells. We show here that ANC-1 functions to position syncytial nuclei and muscle mitochondria by linking the actin cytoskeleton to the nuclear envelope. Mutations in anc-1 disrupt the proper positioning of nuclei and mitochondria in Caenorhabditis elegans. anc-1 was shown to encode an 8546 residue protein consisting of mostly coiled regions and a C-terminal nuclear envelope localization domain (KASH domain) conserved with the Drosophila klarsicht and msp-300 and vertebrate syne-1 and syne-2 genes. The N-terminal regions of ANC-1, Msp-300, Syne-1, and Syne-2 contain two calponin-like domains; the ANC-1 domain bound to filamentous actin in vitro. When overexpessed, the N-terminal region of ANC-1 co-localized with actin filaments, disrupted muscle function, and partially disrupted nuclear anchorage. An actin disrupting mutation in unc-60/cofilin also disrupted mitochondrial anchorage. Antibodies against the large repeat region of ANC-1 were localized cytoplasmically, and were enriched at the nuclear periphery. Localization of ANC-1 to the nuclear envelope was dependent on UNC-84, another nuclear envelope-localized protein required for nuclear anchorage. Overexpression of the KASH domain localized to the nuclear envelope and caused a dominant negative nuclear anchorage defect. We propose that ANC-1 connects nuclei to the cytoskeleton by interacting with UNC-84 at the nuclear envelope and directly binding to actin in the cytoplasm. Similarity in the overall structure between ANC-1, Msp-300, and vertebrate Syne proteins suggests that this novel nuclear anchorage mechanism is conserved. Potential similarities between the mechanisms of ANC-1 and Dystrophin will be discussed.

Daniel A Starr et al. (2003) International Worm Meeting "ANC-1, a C. elegans member of a family of conserved giant proteins, positions nuclei by tethering the nuclear envelope to the actin cytoskeleton."

Daniel A Starr, Min Han

[International Worm Meeting, 2003]

Little is know about organelle membrane proteins that mediate the anchorage of nuclei and other organelles within cells. Mutations in anc-1 disrupt the proper positioning of syncytial nuclei and muscle mitochondria in C. elegans. We show here that ANC-1 functions to position syncytial nuclei by linking the actin cytoskeleton to the nuclear envelope. anc-1 encodes an 8546 residue protein consisting of mostly helical, possibly spectrin-repeat like, regions and a C-terminal nuclear envelope localization domain (KASH domain) conserved with Drosophila klarsicht and msp-300 and vertebrate syne-1 and syne-2 genes. The N-terminal regions of ANC-1, Msp-300, Syne-1, and Syne-2 contain two calponin-like domains. The ANC-1 calponin domains bound to filamentous actin in vitro. When overexpessed, the N-terminal region of ANC-1 co-localized with actin filaments, disrupted muscle function, which lead to a Pat phenotype, and partially disrupted nuclear anchorage. An actin disrupting mutation in unc-60 cofilin also disrupted mitochondrial anchorage. Antibodies against the large repeat region of ANC-1 were localized cytoplasmically, and were enriched at the nuclear periphery. Localization of ANC-1 to the nuclear envelope was dependent on UNC-84, another nuclear envelope-localized protein required for nuclear anchorage. Over-expression of the KASH domain localized to the nuclear envelope and caused a dominant negative nuclear anchorage defect. We propose that ANC-1 connects nuclei to the actin cytoskeleton by interacting with UNC-84 at the nuclear envelope and directly binding to actin in the cytoplasm. We are currently searching for additional mutations that disrupt positioning of organelles through traditional and RNAi feeding screens. We are also in the process of over-expressing the KASH domains of Msp-300 and Syne-1 in Drosophila and mice respectively. In addition, we are characterizing the positioning of nuclei in Drosophila tissues in a null allele of msp-300. We will present our current progress on these fronts.

Cain, Natalie et al. (2015) International Worm Meeting "Building and switching LINC complexes in vivo."

Cain, Natalie, Starr, Daniel

[International Worm Meeting, 2015]

Nuclear positioning is essential for many cell and developmental processes, and defects in nuclear positioning have been linked to a number of human diseases, including muscular dystrophies, autism, and cancers. Nuclear positioning is facilitated by nuclear envelope bridges, also known as Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, composed of inner nuclear membrane SUN proteins that interact with outer nuclear membrane KASH proteins in the perinuclear space. Many questions remain about LINC complex in vivo structure and regulation of LINC complex formation. Particularly, in cells expressing multiple SUN and KASH proteins, how specific LINC complexes are formed and how that choice is regulated are poorly understood. In C. elegans somatic cells, the SUN protein UNC-84 interacts with either UNC-83, which recruits kinesin and dynein to facilitate nuclear migration, or ANC-1, which interacts with actin to anchor nuclei in syncytia. UNC-83 and ANC-1 are both expressed in many adult nuclei, allowing us to examine the regulatory mechanisms that control how SUN proteins choose KASH partners. Structural studies of human LINC complexes suggest that SUN and KASH domains are covalently linked by a disulfide bond. Interestingly, while the intermolecular disulfide cysteines are conserved in UNC-84 and ANC-1, UNC-83 lacks a cysteine in the correct location. We hypothesize that regulation of disulfide bond formation between UNC-84 and ANC-1 is critical for determining the relative abundance of UNC-84/UNC-83 versus UNC-84/ANC-1 LINC complexes. In support of this hypothesis, UNC-84(C953A) expressed from an extrachromosomal array, which we hypothesize cannot form a disulfide bond with ANC-1, rescues the nuclear migration defect of unc-84(null) worms, but has a partial nuclear anchorage defect. We are confirming this finding by generating KASH and SUN domain mutants using CRISPR/Cas9 genome editing. Investigation of the in vivo structure and regulation of LINC complex formation will provide further insight into the function of LINC complexes.

Herrera, Leslie et al. (2017) International Worm Meeting "Mutations in the CDC-42 GEF cgef-1 enhances unc-84 nuclear migration defects in larval P cells."

Starr, Daniel A., Herrera, Leslie

[International Worm Meeting, 2017]

Nuclear migration is essential for cellular migration. A cell's ability to successfully migrate through a constricted space is limited by the ability of the large and rigid nucleus to deform during migration. Caenorhabditis elegans larval P-cell nuclei (~3-4 um in diameter) must migrate through a constricted space between body wall muscles and the cuticle (~125 nm) while moving from the lateral to the ventral side of the animal. If the nuclei are unable to squeeze through this constriction, migration fails and P-cells die, which leads to uncoordinated (Unc) and egg-laying deficient (Egl) adult animals. C elegans raised at 25 deg C with an unc-84 null mutation have severe P-cell nuclear migration defects, but develop normally when raised at 15 deg C. We hypothesize there is an enhancer pathway acting parallel to the UNC-84/UNC-83 microtubule-based nuclear migration pathway. We performed a forward genetic screen to find mutants in the enhancer of the nuclear migration defect of unc-84 (emu) pathway. To date, we have isolated 8 emu lines from this screen and used a whole genome sequencing approach to clone emu genes. We previously found that toca-1 and fln-2 are in the emu pathway, suggesting the mechanism of nuclear migration is actin based. Here we report that one of our emu mutations, yc3, is a G-to-A substitution causing a premature stop codon in amino acid Trp (345) of cgef-1. To confirm that cgef-1 is an enhancer of the unc-84 nuclear migration defect, we injected fosmids WRM0625dG08 and WRM0622bA03 into the UD285 (yc3; unc-84(n369) oxIs12 X) ?strain for a rescue experiment. We count GABA neurons labeled with Punc-47::GFP to assay P-cell nuclear migration defects. Wild-type C elegans have 19 GABA neurons, unc-84 (null) animals have 17.4 plus or minus 0.2 (mean plus or minus SEM) and cgef-1(yc3); unc-84 (null) 11.7 plus or minus 0.3 GABA neurons at 15 deg C. The fosmids WRM0625dG08 and WRM0622bA03 rescue yc3; the rescued strain had 18.1 plus or minus 0.1 GABA neurons at 15 deg C. To further investigate cgef-1 and the interactions with other hypothesized emu genes, we are in the process of using CRISPR/Cas9 to make a CGEF-1::GFP fusion protein.

Chang, Yu-Tai et al. (2011) International Worm Meeting "TOCA-1 and actin polymerization contribute to P-cell nuclear migration."

Starr, Daniel A., Chang, Yu-Tai

[International Worm Meeting, 2011]

Moving the nucleus to an intracellular location is essential for a wide variety of cell and developmental processes, including the formation of polarized cells, fertilization, differentiation, and cellular migration. Defects in nuclear migration block development and lead to disease. However, the mechanisms of how nuclei are moved are poorly understood. We employ larval P-cells in C. elegans as a model for nuclear migration. 12 P-cell nuclei migrate from lateral to ventral positions during the mid-L1 stage. They subsequently divide to form neurons and the vulva. Null mutations in unc-83 or unc-84 inhibit nuclear migration by disrupting interactions between nuclei and microtubule motors. Nuclear migration defects lead to the death of P-cells resulting in uncoordinated (Unc) and egg-laying defective (Egl) animals missing P-cell derived lineages. Interestingly, the unc-83 and unc-84 P-cell nuclear migration defect is temperature sensitive. P-cell nuclear migration is disrupted in unc-83 or unc-84 null animals at 25 deg C, but at 15 deg C, P-cell nuclear migration occurs normally. We therefore hypothesize that an additional pathway functions in part redundantly to the unc-83/unc-84 pathway to migrate P-cell nuclei at 15 deg C. To test our hypothesis, we carried out forward genetic screens and isolated nine emu (enhancer of the nuclear migration defect of unc-83 or unc-84) alleles at 15 deg C in unc-84 null animals in the background of an unc-84 rescuing array. To quantify the severity of the P-cell nuclear migration defect of emu alleles, we scored the number of UNC-47::GFP-positive GABA neurons in adults. Compared to unc-84 null animals, emu; unc-84 double mutants had significantly fewer GABA neurons at all temperatures. The emu alleles are recessive and have been partially mapped. Using whole-genome sequencing, we have determined that the yc20 allele is a lesion in toca-1 and are currently sequencing other alleles. toca-1(RNAi) phenocopied yc20, suggesting that TOCA-1 functions in parallel to the UNC-84/UNC-83 pathway to move P-cell nuclei. TOCA-1 is conserved in flies and mammals. It consists of an F-bar domain that binds to curved membranes and two domains that recruit cdc42 and WAVE to induce F-actin polymerization. Thus, TOCA-1 functions to regulate actin dynamics at the membrane-cytoskeleton interface; previous studies show that TOCA-1 facilitates clathrin-mediated endocytosis (Giuliani et al., 2009. PLoS Genetics). That same study also had data suggesting that TOCA-1 localizes to the nuclear envelope of oocytes. We are currently investigating the subcellular localization of TOCA-1 in P-cells. Together, our results implicate a novel mechanism for nuclear migration based on the nucleation of actin filaments.

Palmer, Lira et al. (2015) International Worm Meeting "Using C. elegans as a model to study Laminopathies."

Bone, Courtney R., Starr, Daniel, Palmer, Lira

[International Worm Meeting, 2015]

Lamin, the major component of the nucleoskeleton, supports the nuclear envelope and provides strength and structure to the nucleus. Lamin is also required for many nuclear migration events. Three to four lamin proteins are found in vertebrates. Mutations in human LaminA are associated with broad class of disorders termed laminopathies including Hutchinson-Gilford progeria syndrome and Emery-Dreifuss muscular dystrophy. The molecular underpinnings that lead to the varied pathologies associated with laminopathies are poorly understood. C. elegans provides a realtively simplistic model to study lamin proteins in vivo because it contains only a single lamin, LMN-1. Previous studies using C. elegans as a model for studying mutant forms of LMN-1 had two major limitations. First, mutant forms of LMN-1 were overexpressed from extrachromosomal arrays. Second, the mutant forms were always expressed in the wildtype LMN-1 background. We are taking advantage of CRISPR/Cas9 genome editing to precisely introduce point mutations in heterozygous lmn-1 backgrounds at endogenous loci. Our ultimate goal is to simultaneously express wild type and mutant forms of LMN-1 with different fluorescent tags for live imaging. We first attempted to tag LMN-1 at its C-terminus with GFP. The field has previously relied on a LMN-1::GFP protein for live imaging of the nuclear lamina. We were able to generate the GFP tag at the endogenous lmn-1 locus by CRISPR/Cas9. The GFP localized to the nuclear envelope as expected, but we could not homozygous LMN-1::GFP, suggesting it is not fully functional. We are currently using CRISPR/Cas9 to introduce GFP into a number of different positions within LMN-1 with the hope of generating a functional tagged protein. Once such a reagent is generated, mutations in conserved residues will be introduced and their functions tested in vivo. .

Murphy, Shaun P. et al. (2013) International Worm Meeting "Analysis of novel pathways for nuclear migration in C. elegans."

Starr, Daniel A., Murphy, Shaun P., Chang, Yu-Tai

[International Worm Meeting, 2013]

Moving the nucleus to an intracellular location facilitates many cell and developmental processes. To investigate the behavior of migrating nuclei in-vivo, we utilize live-cell imaging to analyze the behavior of larval P-cell nuclei in. During the mid-L1 stage, P-cell nuclei migrate from the lateral side into the ventral cord through a cytoplasm that is only 150 nm between the cuticle and body wall muscles. Transgenic lines of animals expressing fluorescent nuclear, cytoskeletal, and cell boundary markers driven by a P-cell-specific promoter reveal that cytoskeletal actin, nucleation sites of microtubules, intermediate filaments, and cellular shape are dynamically rearranged within P-cells during nuclear migration. Null mutations in unc-83 or unc-84, which encode KASH and SUN proteins, inhibit nuclear migration by disrupting interactions between the nucleoskeleton and the cytoskeleton at 25 deg C. However, at 15 deg C, P-cell nuclear migration in KASH/SUN null animals occurs normally. We hypothesize that additional pathway(s) functions synthetically to the KASH/SUN pathway to move P-cell nuclei at 15 deg C. Supporting our hypothesis, genetic screens for enhancers of the nuclear migration defect of unc-83/84 (emu) mutants have been carried out. Whole-genome sequencing and RNAi approaches have identified toca-1 and fln-2 as emu genes; here we focus on the divergent filamin fln-2. Filamins are large proteins involved in actin regulation. Significantly, no role for filamins has previously been shown in nuclear migration. Both unc-84(n369); fln-2(RNAi) and unc-84(n369); fln-2(tm4687) animals have severe defects in P-cell nuclear migration. Additionally, we can rescue the P-cell nuclear migration defect of unc-84(n369); fln-2(tm4687) animals using a fln-2::gfp extrachromosomal array. The organization of actin filaments in P-cells appears normal prior to nuclear migration in unc-84(n369); fln-2(tm4687) worms, however, the actin cytoskeleton is disrupted at the time of nuclear migration. We hypothesize that FLN-2 is involved in actin regulation during nuclear migration in C. elegans larval P-cells.