Alisa Piekny et al. (2006) Development & Evolution Meeting "Equatorial Accumulation of RhoA-GTP During Cytokinesis"

Michael Glotzer, Alisa Piekny

[Development & Evolution Meeting, 2006]

RhoA GTPase (rho-1) is essential for cytokinesis in early C. elegans embryos as determined by RNAi. Furthermore, regulators of Rho-1 activity, the RhoGEF ect-2 and the RhoGAP cyk-4 also cause failed cytokinesis upon RNAi and genetic depletion. To follow the localization of RhoA during cytokinesis in human cells, we generated a probe consisting of YFP fused to C. elegans RhoA (YFP:RhoA). Unlike YFP:human RhoA, the probe based on the C. elegans protein localizes discretely to the equatorial cortex. In addition, the cortical localization of this probe is enhanced by overexpression of a RhoGEF domain and reduced by overexpression of a RhoGAP domain and likely represents the RhoA-GTP pool. Using this probe we have determined one of the mechanisms that regulate cleavage furrow formation. During anaphase the generation of the active form of RhoA, RhoA-GTP, requires both the human ect-2 homologue (ECT2) and the human cyk-4 homologue. CYK-4 has been well characterized as a member of the centralspindlin complex with the kinesin protein MKLP1 in both C. elegans and human cells. CYK-4 binds ECT2 in a phosphodependent manner both in vitro and in vivo, and they colocalize on the central spindle in human cells. It is likely that CYK-4 may act by relieving the autoinhibition of ECT2. However, disruption of the central spindle by MKLP1 depletion results in variable expansion of the RhoA-GTP domain. Thus, the central spindle redundantly contributes to the localization of active RhoA and we would like to identify factors that contribute to RhoA localization through this redundant mechanism. The differential localization of ceRhoA and hsRhoA can be attributed to a few amino acid changes, which may alter their affinity toward specific binding factors. ceRhoA and hsRhoA are 94% identical in their NH2 terminus, but diverge in the C-terminus, and chimeric constructs verified that differences in their localization patterns are due to C-terminal amino acid changes distinct from the CAAX box. Furthermore, we separated regions required for RhoA-GTP accumulation and cortical localization. Using chemical ligation, lipid-modified ceRhoA and hsRhoA variants are being generated for differential pull down experiments to identify proteins that specifically bind RhoA during its accumulation in anaphase.

Alisa J Piekny et al. (2002) West Coast Worm Meeting "Characterization of nmy-1, a suppressor of mel-11"

Jacque-Lynne F Johsnon, Gwendolyn D Cham, Paul E Mains, Alisa J Piekny

[West Coast Worm Meeting, 2002]

C. elegans elongation, whereby the embryo is transformed from an egg into a vermiform larva, is driven by actin-mediated lateral epidermal cell (seam cell) shape changes, which leads to a decrease in the circumferential axis with a concomitant fourfold increase in the longitudinal axis1 . In higher eukaryotes, non-muscle contractile events such as smooth muscle contraction and focal adhesions are regulated by Rho-binding kinase and myosin phosphatase. Myosin phosphatase acts a block to contraction by counteracting the activity of myosin light chain kinase and removing the positive regulatory phosphate from myosin light chain. Rho-binding kinase removes the block to contraction by down-regulating myosin phosphatase activity. We have shown that the C. elegans homologues let-502 (Rho-binding kinase) and mel-11 (myosin phosphatase) similarly regulate non-muscle contractile events such as elongation, cytokinesis and spermathecal contraction in the worm2-5 .

Alisa J Piekny et al. (2001) International C. elegans Meeting "Genes Mediating Elongation of the C. elegans Embryo"

Gwendolyn D Cham, Alisa J Piekny, Paul E Mains, Jacque-Lynne Johnson

[International C. elegans Meeting, 2001]

Genes mediating elongation of the C. elegans embryo Alisa J. Piekny, Jacque-Lynne Johnson, Gwendolyn D. Cham and Paul E. Mains Genes and Development Research Group and Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada Morphogenesis of the C. elegans embryo results from an actin-mediated contraction of the epidermal cells that transforms a ball of cells into a long, thin worm. We have previously identified two genes, let-502 and mel-11 , which are essential to this process 1 . These genes also function during other contractile events including cytokenesis (see abstract by Piekny and Mains) and in the somatic gonad 2 . Based on our genetic results and analogies to smooth muscle contraction, we proposed that contraction (elongation) occurs when myosin light chain kinase phosphorylates myosin light chain. Myosin phosphatase (MEL-11) removes the activating phosphate, blocking contraction, and so loss of mel-11 results in hypercontraction. Elongation is triggered when Rho induces Rho-binding kinase (LET-502) to inhibit myosin phosphatase (MEL-11), thereby removing the MEL-11 brake to contraction. Consistent with this, let-502 mutants hatch (and arrest) with minimal elongation . However, the double null phenotype of let-502 and mel-11 appears to be near-normal elongation, implying that a redundant pathway can mediate morphogenesis 3 . To identify more genes involved in elongation, candidates similar to genes involved in contractile events in other systems were tested for genetic interactions with let-502 and mel-11 2,3 . We also constructed triple mutants to determine how the genes functioned during elongation: those acting downstream or in a pathway redundant with let-502 or mel-11 would block the near normal elongation of let-502; mel-11 while mutations of upstream genes would have no effect. We found that daf-2 and age-1 (insulin pathway) and mig-2 (Rho/Rac-like) act upstream. unc-73 (Rho/Rac GEF) and fem-2 (PP2C phosphatase) appear to act in parallel to let-502 - mel-11 , while mlc-4 (myosin light chain) acts downstream 3 . More recently, we have found that the Rac-mediated pathway required for engulfment of cell corpses ( ced-2, ced-5 and ced-10) may function upstream of let-502 - mel-11 . We are also characterizing several new genes isolated in a screen for mel-11 suppressors. We have generated polyclonal antibodies to LET-502 and MEL-11 and examined their expression throughout development. LET-502 and MEL-11 co-localize at cleavage furrows during early cell divisions. During morphogenesis LET-502 and MEL-11 are expressed in the epidermis, pharynx and Z2/Z3 cells. In adults, they are expressed throughout the developing germ line and spermatheca. Therefore, the expression of LET-502 and MEL-11 are consistent with their mutant phenotypes during different stages of the life cycle. 1 Wissmann et al. (1997) Genes Develop. 11:409; 2 Wissmann et al. (1999) Dev. Biol. 209:111; 3 Piekny et al. (2000) Genetics 156:1671

Alisa J Piekny et al. (2001) International C. elegans Meeting "The role of C. elegans Rho-binding kinase and myosin phosphatase in cytokinesis"

Alisa J Piekny, Paul E Mains

[International C. elegans Meeting, 2001]

Alisa J Piekny et al. (2003) International Worm Meeting "A new target for the Rho-binding kinase pathway during Caenorhabditis elegans embryonic elongation"

Alisa J Piekny, Paul E Mains

[International Worm Meeting, 2003]

Rho-binding kinase (ROK) and myosin phosphatase targeting subunit (MYPT) regulate nonmuscle contractile events in higher eukaryotes. Genetic evidence indicates that the C. elegans homologs, LET-502/ROK and MEL-11/MYPT mediate embryonic elongation by regulating the actin-mediated epidermal cell shape changes that transform the spherical embryo into a long, thin worm. Here we describe the physical mechanism of how LET-502 and MEL-11 mediate these cell shape changes. LET-502 localizes with actin/myosin filaments during contraction where it could trigger this event. MEL-11, which inhibits contraction, transiently associates with these structures only prior to elongation, and then becomes sequestered to the plasma membrane. Mutations in the nonmuscle myosin heavy chain gene, nmy-1, were isolated as suppressors of the mel-11 hypercontraction phenotype. Like let-502 and nonmuscle myosin light chain, mlc-4, nmy-1 mutants display elongation defects. Based on genetic and molecular data, we propose that NMY-1 is a nonmuscle myosin heavy chain that partners with MLC-4 during embryonic elongation. However, the nmy-1 null allele displays elongation defects less severe than the mlc-4 null mutant. This results because nmy-1 is partially redundant with another nonmuscle myosin heavy chain, nmy-2, which was previously known only for its role in anterior/posterior polarity and cytokinesis in the early embryo.

Loloyan, Michael et al. (2011) International Worm Meeting "Molecular characterization of the C. elegans anillin isoforms."

Piekny, Alisa, Loloyan, Michael

[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.

Ozugergin, Imge et al. (2021) International Worm Meeting "The Ran pathway uniquely regulates cytokinesis in cells with different fates in the early C. elegans embryo"

Mastronardi, Karina, Ozugergin, Imge, Piekny, Alisa

[International Worm Meeting, 2021]

Cytokinesis occurs through the ingression of a contractile ring that cleaves the daughter cells. This process is tightly controlled to prevent fate changes or aneuploidy, and the core cytokinesis machinery is highly conserved among metazoans. The central dogma is that spindle-dependent pathways regulate ring assembly, but studies have shown that spindle-independent pathways do so as well. In human cells, we found that active Ran is a chromatin-associated cue that forms an inverse gradient with importins that can control protein function near the cortex. We found that importin-binding is required for the cortical localization and function of anillin, a ring scaffold. To study how requirements for different cytokinesis pathways vary with cell type, we studied cytokinesis in the differently fated AB and P1 cells of the early embryo. We found that AB and P1 cells have different assembly kinetics supported by different levels and cortical patterning of equatorial non-muscle myosin II (NMY-2). The ring assembles rapidly in AB cells, which has higher midplane levels and cortical patches of NMY-2. In contrast, the levels of NMY-2 are lower and there are no/few patches in P1 cells, which have slower ring assembly. Depleting polarity regulators (PAR-1, -3 or -6) equalized assembly kinetics, indicating that kinetics depend on cell fate. By generating stable tetraploid strains, we show that differences in kinetics also depend on cell size. Diploid AB-sized tetraploid P1 cells had faster ring assembly supported by higher levels of NMY-2. However, ring assembly was slower in tetraploid vs. diploid AB cells, which had very high levels of NMY-2. This suggests that ideal NMY-2 levels support ring assembly, which is hindered when levels are too low or high, and that P1 cells operate close to a minimum threshold. Next, we determined if the chromatin pathway controls differences in AB and P1 cell ring assembly. We found that depleting the RanGEF (RAN-3/RCC1) caused ring assembly to occur equally and rapidly in both AB and P1 cells, which may be due to increased importin-regulation of cortical proteins. In support of this, the faster kinetics were suppressed by co-depletion of ECT-2 (RhoGEF). Interestingly, ANI-1 (anillin) suppressed kinetics in AB cells but not P1, suggesting that the chromatin pathway functions differently in these cell types. Collectively, our results show that there are different pathway requirements in AB and P1 cells that supports differences in cytokinesis.

Grimbert, Stephanie et al. (2021) International Worm Meeting "C. elegans Anterior Morphogenesis: A Tale of Three Tissues"

Piekny, Alisa, Grimbert, Stephanie, Mastronardi, Karina, Richard, Victoria

[International Worm Meeting, 2021]

Morphogenesis encompasses the biological events driving tissue and organ development. The need to build tissues with precision relies on the spatiotemporal coordination of cell shape changes, migration and adhesion. As complex structures are derived from multiple tissue types, studying their organization in vivo is essential for a comprehensive understanding and yet is extremely challenging. In this study we show how cells from three different tissues are coordinated to give rise to the anterior lumen. While most aspects of epidermal morphogenesis and several aspects of pharyngeal morphogenesis have been well-described in C. elegans, it is less clear how cells from the pharynx, epidermis and neuroblasts coordinate to define the location of the anterior lumen and supporting structures. Using various microscopy and software approaches, we found that interactions between pharyngeal, epidermal and neuronal cells are required for anterior morphogenesis. Specifically, to define the position and timing of the anterior lumen and future mouth of the worm. We characterized the movements and patterns of these three cell types and defined key events associated with anterior morphogenesis. We found that the first visible marker of the location of the future anterior lumen is provided by projections from the anterior-most pharyngeal cells (arcade cells) and facilitates patterning of the surrounding neuroblasts. These neuroblasts organize into distinct patterns which subsequently influences the rate of epidermal cell migration towards the anterior. Conversely, the epidermal cells ultimately reinforce the position of the future lumen, as they must join with the pharyngeal cells for their successful epithelialization. We described the crucial involvement of junction (HMP-1, AJM-1), polarity (PAR-6 and PAR-3) and contractility (NMY-2) proteins in this precise patterning. Thus, we have established an innovative model system to explore the in-depth molecular regulation of these different patterns and interactions between multiple cell-types.

Fotopoulos, Nellie et al. (2011) International Worm Meeting "Roles for anillin (ani-1) in regulating cell shape changes during C. elegans embryogenesis."

Piekny, Alisa, Chen, Yun, Fotopoulos, Nellie, Makil, Neetha

[International Worm Meeting, 2011]

Changes in cell shape involve reorganization and contraction of the actin-myosin cytoskeleton. During C. elegans embryogenesis, both cytokinesis and elongation use Rho-mediated shape changes. Elongation is where lateral epidermal cells (seam cells) change shape from cube-like to cylindrical, which is transmitted to the ventral and dorsal epidermal cells, transforming the ovoid embryo into the long, thin worm. In the seam cells, actin filaments become highly organized and are shortened by myosin contraction via the Rho kinase LET-502. Myosin phosphatase (MEL-11) inactivates myosin in the dorsal and ventral cells and is sequestered to cell boundaries in the seam cells, which limits its interaction with myosin. LET-502 is likely regulated by RHO-1, but the lack of rho-1 alleles precluded studying its role in elongation. Using a deletion allele generated by the Knockout Consortium, we found that rho-1 is required for elongation and other morphogenetic events, including ventral enclosure. Cytokinesis is the final stage of cell division and requires the Rho-mediated formation and ingression of an actin-myosin ring. The same genes regulate both elongation and cytokinesis, suggesting they use analogous shape change mechanisms. In other eukaryotes, anillin is required for cytokinesis by coordinating actin and myosin filaments. It can also bind to RhoA, scaffolding the upstream regulator to actin and myosin. ani-1 (anillin in C. elegans) is required for polar body extrusion during meiosis and asymmetric furrow ingression during cytokinesis. Since elongation and cytokinesis may use similar shape change mechanisms, we hypothesize ani-1 functions in elongation. Using ani-1 RNAi, we observed phenotypes consistent with elongation defects. We also performed zygotic-specific ani-1 RNAi (by rescuing maternal RNAi) and observed elongation defects, suggesting this phenotype does not arise from earlier defects in meiosis or cytokinesis. We found genetic interactions between ani-1 and genes that function in elongation using zygotic alleles for rho-1 and mlc-4. Furthermore, using GFP:ANI-1 (gift from A. S. Maddox), we observed non-cytokinetic localization patterns for ANI-1 during embryogenesis. These findings may be the first evidence to support developmental-specific functions for anillin outside cytokinesis in any organism.

Wernike, Denise et al. (2015) International Worm Meeting "Neuroblasts mechanically influence epidermal morphogenesis."

Chen, Yun, Wernike, Denise, Mastronardi, Karina, Piekny, Alisa

[International Worm Meeting, 2015]

Tissue morphogenesis is a highly complex event that is poorly understood, as it involves multiple cellular events such as migration, adhesion and shape change. Studies of epidermal morphogenesis and gastrulation in Drosophila revealed that these events also involve the coordination of different cell types, yet it was not known how conserved this is in other organisms. C. elegans is an ideal model to study tissue morphogenesis, since most tissues are formed from a relatively small number of cells in comparison to other organisms. We study ventral enclosure, part of epidermal morphogenesis when epidermal cells migrate to cover the ventral side of the embryo using cues from the underlying neuroblasts. During this process, F-actin accumulates into a supracellular ring along the margins of the ventral epidermal cells, which was hypothesized to close by non-muscle myosin contractility. Consistent with this hypothesis, we found that myosin (nmy-2) is required for ventral enclosure, and localizes to foci that form a pattern reminiscent of F-actin. Using modified TIRF (total internal reflection fluorescence) microscopy, we found that myosin also localizes to dynamic foci that form intercellular networks in the neuroblasts, a tissue that directly underlies the epidermis. We used tissue-specific RNAi to show that myosin is required in the neuroblasts for ventral enclosure. In the area underlying the posterior ventral epidermal cells, a subset of neuroblasts organizes into a rosette-like pattern, and their surface area also appears to decrease as the epidermal cells are drawn together. Interestingly, both rosette formation and changes in surface area fail to occur in mutants with altered neuroblast cell shape or contractility. In addition, myosin distribution is altered in the overlying epidermal cells when neuroblast shape is altered, suggesting that there could be changes in tension that are sensed by the overlying epidermal cells, which affects the distribution of myosin via mechanosensing. We propose that contractility in the neuroblasts may be coordinated with constriction of the supracellular actin-myosin ring in the overlying epidermal cells to facilitate ventral enclosure. This work emphasizes the importance of the interplay between different cell types for tissue development, where contractility could also be contributed by non-epidermal tissues.