Mastronardi, Karina et al. (2017) International Worm Meeting "Studying Ran-GTP regulation of cytokinesis."

Ozugergin, Imge, Williams, Brittany, Piekny, Alisa, Mastronardi, Karina, Beaudet, Daniel

[International Worm Meeting, 2017]

Cytokinesis physically divides a cell into two daughters at the end of mitosis and must be highly robust to avoid aneuploidy and cell fate changes. A RhoA-dependent actomyosin contractile ring ingresses to divide the appropriate genetic and cell fate determinants into each daughter. The prevailing dogma in the field is that the mitotic spindle regulates contractile ring assembly and ingression. However, microtubule-independent mechanisms also regulate cytokinesis, and may be crucial in polarized cells. During metaphase, a Ran-GTP gradient forms around chromatin where it mediates mitotic spindle assembly by releasing importins a/ beta complexes from microtubule-binding proteins and motors. Our data suggests that the Ran-GTP gradient persists in early anaphase where it affects the localization of contractile proteins for cytokinesis in human cells. Importantly, one of the Ran targets may be anillin, which is a scaffold that binds to and coordinates actin, myosin, RhoA and its upstream regulators, microtubules, and phospholipids. We found that human anillin contains a C-terminal nuclear localization signal (NLS) that binds to importin- beta . Point mutations in the NLS delay anillin's recruitment to the cortex, and the contractile ring oscillates and subsequently fails to ingress in a subset of cells rescued with this mutant. Interestingly, mutant anillin's localization is strongly demarcated by astral microtubules, suggesting that the mitotic spindle dominantly regulates cytokinesis in these cells. We hypothesize that the Ran-GTP regulation of cytokinesis is conserved among metazoans, and the strength of this pathway is proportional to ploidy, cell geometry and/or cell volume. To test this, we are determining if the Ran pathway regulates cytokinesis in early C. elegans embryos. Studies showed that there is a negative correlation between the rate of furrow ingression and cell volume as cells decrease in size through the first four rounds of divisions (Carvalho et al., 2009). We are determining how perturbing the Ran pathway alters the rate of furrow ingression and the localization of contractile proteins during these divisions. Also, we are determining if ANI-1 (C. elegans anillin), which is highly conserved with human anillin, contains an NLS that may function in the same way as human anillin. We also will determine if the Ran pathway regulates other well-known cytokinesis components. Carvalho et al. (2009) Cell 137: 926-37.

Mastronardi, Karina et al. (2015) International Worm Meeting "Non-muscle myosin contractility in the neuroblasts influences epidermal morphogenesis."

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

[International Worm Meeting, 2015]

Epidermal morphogenesis is essential for the development of metazoans and occurs due to the coordinated migration, adhesion and shape changes of epidermal cells. Studies in Drosophila have shown that epidermal morphogenesis also relies on extraembryonic amnioserosal cells, which lie underneath the epidermal cells, and coordinated contractility in both tissues is required for epidermal sheets to cover the dorsal surface of the embryo. However, it is not known if analogous events occur in other organisms. We study C. elegans ventral enclosure, part of epidermal morphogenesis, when the ventral epidermal cells migrate over neuronal precursor cells, the neuroblasts, to encase the embryo in a single layer of epidermis. Non-muscle myosin has not been studied in ventral enclosure, and it was not clear if contractility contributed to the process. Genetic studies revealed that key regulators of myosin contractility, including RHO-1/RhoA, its activator ECT-2/RhoA guanine nucleotide exchange factor (GEF) and LET-502/Rho kinase, are required for ventral enclosure. ECT-2 localizes to foci in patterns reminiscent of myosin in the epidermal cells and neuroblasts, and is required for the accumulation of myosin foci in both cell types, suggesting that the foci in both cell types are contractile. Using SeedWater Segmenter software, we found that a subset of neuroblasts underneath the migrating ventral epidermal cells undergo re-arrangements; starting out as two columns, then re-organizing to share a common vertex enriched with myosin. These cells may be connected via the adherens junction component HMP-1, which we hypothesize may coordinate myosin networks in the neuroblasts to help mediate closure of the overlying epidermal tissue. We are investigating the potential role that adherens junctions play in the neuroblasts during ventral enclosure. These studies should shed light on the interplay between tissues during embryonic development.

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.

Richard, Victoria et al. (2021) International Worm Meeting "Coordinating neuronal signaling pathways with anterior epidermal cell migration"

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

[International Worm Meeting, 2021]

We study the cell patterning that regulates anterior morphogenesis in C. elegans embryos. We recently described how the epidermal, pharyngeal and neuroblast cells must be properly coordinated for development of the anterior lumen. However, the signaling events that coordinate these cell types are still not well understood. We found that soon after the ventral epidermal cells meet at the ventral midline, PAR-6 (polarity protein) foci associated with different subsets of cells form distinct patterns in the anterior of the embryo. Specifically, we observed two pentagons, a larger focal point and a more ventrally positioned semi-circle of foci. While the larger focal point corresponds to the arcade cells (anterior most cells of the pharynx), the pentagons appear to correspond to neuronal precursor and/or glial cells (UNC-119, MIR-228), while the semi-circle of foci may correspond to neuronal precursor cells (UNC-119, HLH-16), although some of these could come from the epidermis. The dorsal foci within each pentagon move in concert with the dorsal epidermal cells, while the semi-circle of foci move with the ventral epidermal cells. Ventral epidermal cells migrate using F-actin-rich projections, and we observed that these projections come close to, but do not cross the semi-circle of foci suggesting they are guided by nearby cues. We found that blocking neuroblast cell division and disrupting the PAR-6 patterns caused delays in epidermal cell migration by decreasing the number of F-actin projections. Based on these findings, we propose that signals associated with the neuronal and/or glial cells control anterior epidermal cell migration. To identify these signals, we are performing tissue-specific RNAi to guidance cues and their receptors including ephrins (enf-1/2/3), netrin (unc-6), slit (slt-1) and sax/robo (sax-3), as well as Wnt (lin-44 , mom-2, mig-5, etc), and determining how they disrupt either 1) the pentagon or semi-circle of foci and/or, 2) migration of the anterior epidermal cells. This work will continue to provide new insights on the mechanisms underlying the multi-tissue cooperation required for successful anterior morphogenesis of C. elegans embryos.

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.