-
[
International Worm Meeting,
2021]
Computational methods and tools are a powerful complementary approach to experimental work for studying regulatory interactions in living cells and systems. We demonstrate the use of formal reasoning methods as applied to the Caenorhabditis elegans germ line, which is an accessible model system for stem cell research. The dynamics of the underlying genetic networks and their potential regulatory interactions are key for understanding mechanisms that control cellular decision-making between stem cells and differentiation. We model the "stem cell fate" versus entry into the "meiotic development" pathway decision circuit in the young adult germ line based on an extensive study of published experimental data and known/hypothesized genetic interactions. We apply the reasoning engine for interaction networks tool (RE:IN) to derive predictive networks for control of differentiation. Using RE:IN we simultaneously specify many possible scenarios and experiments together with potential genetic interactions, and synthesize genetic networks consistent with all encoded experimental observations. In silico analysis of knock-down and overexpression experiments within our model recapitulate published phenotypes of mutant animals and can be applied to make predictions on cellular decision making. This work lays a foundation for developing realistic whole tissue models of the C.elegans germline where each cell in the model will execute a synthesized genetic network.
-
[
Development,
2005]
Anillins are conserved proteins that are important for stabilizing and remodeling the actin cytoskeleton. Anillins have been implicated in cytokinesis in several systems and in cellularization of the syncytial Drosophila embryo. Here, we examine the functions of three C. elegans proteins with homology to anillin (ANI-1, ANI-2 and ANI-3). We show that ANI-1 and ANI-2 contribute to embryonic viability by performing distinct functions in the early embryo and gonad, respectively. By contrast, ANI-3 appears to be dispensable for embryonic development. ANI-1 is essential for cortical ruffling and pseudocleavage, contractile events that occur in embryos prior to mitosis. ANI-1 is also required for the highly asymmetric cytokinetic events that extrude the two polar bodies during oocyte meiosis, but is dispensable for cytokinesis following mitotic chromosome segregation. During both meiosis and mitosis, ANI-1 targets the septins, but not myosin II, to the contractile ring and does not require either for its own targeting. In contrast to ANI-1, ANI-2 functions during oogenesis to maintain the structure of the rachis, the central core of cytoplasm that connects the developing oocytes in the syncytial gonad. In ANI-2-depleted worms, oocytes disconnect prematurely from the defective rachis, generating embryos of varying sizes. Our results highlight specialization of divergent anillin family proteins in the C. elegans life cycle and reveal conserved roles for this protein family in organizing syncytial structures and cortical contractility.
-
[
International Worm Meeting,
2005]
Anillins are conserved proteins important for stabilizing and remodeling the actin cytoskeleton. Anillins have been implicated in cytokinesis in several systems and in cellularization of the syncytial Drosophila embryo. Here we examine the functions of three C. elegans proteins with homology to anillin (ANI-1, 2 and 3). We show that ANI-1 and ANI-2 contribute to embryonic viability by performing distinct functions in the early embryo and gonad, respectively. In contrast, ANI-3 appears to be dispensable for embryonic development. ANI-1 is essential for cortical contractile ruffling and pseudocleavage during pronuclear migration, but not for the establishment of polarity. ANI-1 is also required for the highly asymmetric cytokinetic events that extrude the two polar bodies during oocyte meiosis, but is dispensable for cytokinesis following mitotic chromosome segregation. During both meiosis and mitosis, ANI-1 targets the septins, but not myosin II, to the contractile ring and does not require either for its own targeting. In contrast to ANI-1, ANI-2 functions during oogenesis to maintain the structure of the rachis, the central core of cytoplasm that connects the developing oocytes in the syncytial gonad. In ANI-2 depleted worms, oocytes disconnect prematurely from the defective rachis, generating embryos of varying sizes. Our results highlight specialization of divergent anillin family proteins in the C. elegans life cycle and reveal conserved roles for this protein family in organizing syncytial structures and cortical contractility.
-
[
International Worm Meeting,
2011]
Processes that involve cell shape changes such as cytokinesis and morphogenesis are vital for the development of an organism and are driven by the cytoskeleton. Cytokinesis occurs due to the contraction of an actin-myosin ring to form two daughter cells, while morphogenesis describes coordinated actin-myosin cell shape changes within a tissue. Both of these events require controlled regulation of cytoskeletal dynamics by the RhoA GTPase. In C. elegans cytokinesis, the conserved GEF ECT-2 activates RHO-1 (RhoA) to drive actin polymerization through CYK-1 (formin) and activation of MLC-4 (myosin light chain) by LET-502 (Rho kinase) to form and ingress a contractile ring. ANI-1 (anillin) is required for asymmetric furrow ingression and contains conserved actin and myosin binding domains in its N-terminus through which it influences actin and myosin localization. In human cells, the AHD (Anillin Homology Domain), a conserved region in the C-terminus of anillin, binds to RhoA and Ect2. Through these multiple interactions, anillin may function as a scaffold to coordinate cytoskeletal components together with their regulators. We hypothesize that anillin's function may not be restricted to mitotic cells, and that the molecular interactions of the C-terminus of anillin are conserved in the C. elegans homologues, which include ANI-1, ANI-2 and ANI-3. ANI-2 is required for proper gonad formation and recently was shown to antagonize ANI-1 if it is stabilized in the early embryo, and ANI-3 has no known function. ANI-1, ANI-2 and ANI-3 contain the AHD region, but ANI-2 and ANI-3 lack the N-terminal actin and myosin binding domains. We found that the AHD region of ANI-1 and ANI-2 interact with RHO-1 and ECT-2. Residues that are required for the human anillin-RhoA and anillin-Ect2 interactions have been identified and we are performing mutational analyses to determine their conservation. Furthermore, ANI-1 and ANI-2 interact with human RhoA and Ect2, but cannot interact with the Drosophila ECT-2 homologue Pebble. Drosophila anillin interacts with the partner for Pbl, RacGAP50C (CYK-4), and it is possible that evolution has favored an anillin-Ect2 interaction in some species and an anillin-Cyk-4 interaction in others and the reason for this selection is not clear.
-
[
International Worm Meeting,
2013]
Ventral enclosure (VE) is a key part of epidermal morphogenesis, where the ventral surface of the C. elegans embryo is enclosed in a layer of epithelial cells. VE requires the coordinated migration of ventral epidermal cells and their adhesion at the ventral midline. Known regulators of VE include RhoGTPases, nucleators of F-actin and the catenin/cadherin complex. There likely are additional proteins that regulate F-actin for cell shape change, migration and/or adhesion that have not yet been identified. One candidate is anillin, ANI-1, a multi-domain scaffolding protein that coordinates actomyosin contractility in the early embryo. We show that
ani-1 is required for epidermal morphogenesis. In
ani-1 RNAi embryos expressing AJM-1:GFP (adherens junctions marker), ventral epidermal cells fail to meet and adhere at the ventral midline. Although they migrate at a rate comparable to control embryos, they often are not properly aligned with their contralateral neighbors. Interestingly, ANI-1 does not localize to adherens junctions or epidermal F-actin, but is present in HAM-1-expressing neuroblasts. Neuroblasts lie underneath the epidermis and are hypothesized to serve as a substrate for ventral epidermal cell migration, likely by providing chemical cues for their guidance. ANI-1 localizes to the cleavage furrows of dividing neuroblasts, which fail to divide upon ANI-1 depletion, suggesting that ANI-1 regulates neuroblast cytokinesis. In support of ANI-1's non-autonomous regulation of VE,
ani-1 RNAi enhances VE phenotypes caused by hypomorphic alleles of the catenin/cadherin complex. Also, ANI-1 and alpha-catenin co-suppress one another when one is over-expressed and the other is mutated or depleted by RNAi. Therefore, strengthening the junctions between cells (e.g. by alpha-catenin over-expression) could make the substrate partially redundant and likewise, strengthening the cytoskeleton of the substrate (e.g. by ANI-1 over-expression) could make junctions partially redundant. These data support the model that mechanotransduction between multiple tissues in the developing embryo is essential for epidermal morphogenesis.
-
[
International Worm Meeting,
2015]
Cytokinesis is the last step required to separate the daughter cells after mitosis. In the germline of most animals, cytokinesis often fails, leading to the formation of a stable intercellular bridge and, eventually, a syncytium. Whereas the C. elegans adult germline is syncytial, the regulators implicated in its nucleation remain unknown. ANI-2, a non-canonical form of the scaffold protein ANI-1/Anillin, is specifically expressed at the cortex of the last germline blastomere, P4, during C. elegans embryogenesis. During the division of P4 into the two primordial germ cells (PGCs), Z2 and Z3, ANI-2 is redistributed and accumulates at the midbody between the daughter cells and is enriched throughout the subsequent stages of larval development. The presence of ANI-2 in the C. elegans germline is essential, its depletion leading to a loss of stable intercellular bridges, a severe gonad disorganization and sterility, suggesting an important role for ANI-2 in the nucleation of the syncytial germline. To address this, we first used immunofluorescence to determine which genes are implicated in ANI-2 cortical loading to P4, using conditions that perturb contractility/cytokinesis regulators (RNAi or conditional mutants). Our results show that the guanine exchange factor ECT-2, the GTPase activating protein CYK-4/MgcRacGAP and the GTPase RHO-1/RhoA are required for ANI-2 loading to the P4 cortex, whereas other contractility regulators, such as NMY-2/non-muscle myosin II and the actin filament nucleator CYK-1 are not. Further, we found that, unlike many components of the cytokinetic machinery undergoing shedding during cellular abscission, the presence of ANI-2 at the PGC midbody is stable during gastrulation and extends in size during embryonic elongation. We found that ECT-2, CYK-4 and RHO-1 are important for the proper accumulation and localization of the ANI-2 focus during gastrulation. Moreover, we found that other midbody markers, such as NMY-2, also remain at the midbody of the PGCs and undergo changes in shape similar to ANI-2 during embryogenesis, and that ANI-2 is important for NMY-2 persistence at the PGCs' midbody. Our results suggest that different sets of genes regulate ANI-2's loading to the cortex of P4 and its subsequent redistribution to the midbody of the PGCs. Furthermore, ANI-2 reorganization during elongation could be an important step to stabilize contractility regulators at the intercellular bridge and control to syncytium formation.
-
[
J Cell Biol,
2014]
Cytokinesis generally produces two separate daughter cells, but in some tissues daughter nuclei remain connected to a shared cytoplasm, or syncytium, through incomplete cytokinesis. How syncytia form remains poorly understood. We studied syncytial formation in the Caenorhabditis elegans germline, in which germ cells connect to a shared cytoplasm core (the rachis) via intercellular bridges. We found that syncytial architecture initiates early in larval development, and germ cells become progressively interconnected until adulthood. The short Anillin family scaffold protein ANI-2 is enriched at intercellular bridges from the onset of germ cell specification, and ANI-2 loss resulted in destabilization of intercellular bridges and germ cell multinucleation defects. These defects were partially rescued by depleting the canonical Anillin ANI-1 or blocking cytoplasmic streaming. ANI-2 is also required for elastic deformation of the gonad during ovulation. We propose that ANI-2 promotes germ cell syncytial organization and allows for compensation of the mechanical stress associated with oogenesis by conferring stability and elasticity to germ cell intercellular bridges.
-
[
Cell Rep,
2023]
During cytokinesis, a contractile ring consisting of unbranched filamentous actin (F-actin) and myosin II constricts at the cell equator. Unbranched F-actin is generated by formin, and without formin no cleavage furrow forms. In Caenorhabditis elegans, depletion of septin restores furrow ingression in formin mutants. How the cleavage furrow ingresses without a detectable unbranched F-actin ring is unknown. We report that, in this setting, anillin (ANI-1) forms a meshwork of circumferentially aligned linear structures decorated by non-muscle myosin II (NMY-2). Analysis of ANI-1 deletion mutants reveals that its disordered N-terminal half is required for linear structure formation and sufficient for furrow ingression. NMY-2 promotes the circumferential alignment of the linear ANI-1 structures and interacts with various lipids, suggesting that NMY-2 links the ANI-1 network with the plasma membrane. Collectively, our data reveal a compensatory mechanism, mediated by ANI-1 linear structures and membrane-bound NMY-2, that promotes furrowing when unbranched F-actin polymerization is compromised.
-
[
Dev Biol,
2013]
The formation of tissues is essential for metazoan development. During Caenorhabditis elegans embryogenesis, ventral epidermal cells migrate to encase the ventral surface of the embryo in a layer of epidermis by a process known as ventral enclosure. This process is regulated by guidance cues secreted by the underlying neuroblasts. However, since the cues and their receptors are differentially expressed in multiple cell types, the role of the neuroblasts in ventral enclosure is not fully understood. Furthermore, although F-actin is required for epidermal cell migration, it is not known if nonmuscle myosin is also required. Anillin (ANI-1) is an actin and myosin-binding protein that coordinates actin-myosin contractility in the early embryo. Here, we show that ANI-1 localizes to the cleavage furrows of dividing neuroblasts during mid-embryogenesis and is required for their division. Embryos depleted of
ani-1 display a range of ventral enclosure phenotypes, where ventral epidermal cells migrate with similar speeds to control embryos, but contralateral neighbors often fail to meet and are misaligned. The ventral enclosure phenotypes in
ani-1 RNAi embryos suggest that the position or shape of neuroblasts is important for directing ventral epidermal cell migration, although does not rule out an autonomous requirement for
ani-1 in the epidermal cells. Furthermore, we show that
rho-1 and other regulators of nonmuscle myosin activity are required for ventral epidermal cell migration. Interestingly, altering nonmuscle myosin contractility alleviates or strengthens
ani-1's ventral enclosure phenotypes. Our findings suggest that ventral enclosure is a complex process that likely relies on inputs from multiple tissues.
-
[
Mol Biol Cell,
2011]
Assembly of a cytokinetic contractile ring is a form of cell polarization in which the equatorial cell cortex becomes differentiated from the polar regions. Microtubules direct cytokinetic polarization via the central spindle and astral microtubules. The mechanism of central spindle-directed furrow formation is reasonably well understood, but the aster-directed pathway is not. In aster-directed furrowing, cytoskeletal factors accumulate to high levels at sites distal to the asters and at reduced levels at cortical sites near the asters. In this paper, we demonstrate that the cytoskeletal organizing protein anillin (ANI-1) promotes the formation of an aster-directed furrow in Caenorhabditis elegans embryos. Microtubule-directed nonmuscle myosin II polarization is aberrant in embryos depleted of ANI-1. In contrast, microtubule-directed polarized ANI-1 localization is largely unaffected by myosin II depletion. Consistent with a role in the induction of cortical asymmetry, ANI-1 also contributes to the polarization of arrested oocytes. Anillin has an evolutionarily conserved capacity to associate with microtubules, possibly providing an inhibitory mechanism to promote polarization of the cell cortex.