I have developed novel imaging approaches that allow for the visualization cytokinetic ring dynamics in dividing cells with unprecedented spatial and temporal resolution. We combine this 4 dimensional high spatial‐ and temporal resolution imaging with custom quantitative image analysis pipelines to study these dynamics. This work allows as to follow local and global changes in composition and kinetics
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The small GTPase RhoA is a central regulator, activating cortical actomyosin contractility during cytokinesis and other cellular events. It acts as a molecular switch that can alternate between an active and inactive state. The sterile20‐family serine/threonine kinase GCK‐1 and its cofactor CCM‐3 form a conserved complex and suppress RhoA
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I study how the distribution of conserved structural and regulatory elements of the actomyosin cytoskeleton, on rachis bridges and germ cell comparment membranes affect germ cell structure and oocyte formation. To test whether these protein abundance dynamics represented significant increases and decreases, and how abundance dynamics correlate to germ cell development, we used SiZer (SIgnificant ZERo crossing of the derivatives) statistical analysis to identify germline regions with statistically significant changes in rachis bridge perimeter. Subdividing the germline into distinct regions based on the changes in rachis bridge size allows us to identify significant correlations between change in protein distribution and rachis bridge size. Our observations suggest that regulation of germline architecture and oogenesis rely on a balance of distribution of contractile
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During my PhD research in the laboratory of Michael Glotzer I used high resolution confocal microscopy to study cytoskeletal dynamics during establishment of embryonic polarity and cytokines in C.elegans embryos. My research involved developing an experimental approach to genetically separate two redundant pathways involved in cytokines in the same embryo. This approach revealed that these two pathways promote drastically different organization of the actomyosin cytoskeleton both sufficient to drive furrow ingression. Furthermore this research contributed to the identification and characterization of the roles of three different proteins in regulating cortical actomyosin dynamics, spindle positioning and cytokinesis in the one cell C.elegans embryo.
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During my work as a Postdoc in the lab of Brian Mitchel we showed that intracellular effectors interpret polarity to organize cellular morphology in accordance with asymmetric cellular function. We observe that both cellular actin and microtubule networks undergo drastic reorganization, providing differential roles during the polarized organization of cilia. Using computational angular correlation analysis of cilia orientation, we report a graded cellular organization downstream of cell polarity cues. Actin dynamics are required for proper cilia spacing, global coordination of cilia
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The directed movement of cells is a fundamental aspect of tissue morphogenesis. The skin of Xenopus laevis embryos represents an excellent model to study the movement of cell across epithelia. Multiciliated cells (MCCs) and ionocytes (ICs) are specialized cell types that differentiate in a subapical layer of the epidermis. These cells then move in a directed manner toward the outer epithelial cells where they undergo radial intercalation pushing through cell vertices and establishing new junctions while maintaining epithelial integrity. We identified a novel function for both the microtubule-binding protein CLAMP and members of the microtubule-regulating Par complex during intercalation.
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Multiciliated cells represent an interesting variation of centriole duplication in that these cells generate greater than 100 centrioles, which form the basal bodies of their motile cilia. This centriole amplification is proposed to require a structure termed the deuterosome, thought to be capable of promoting de novo centriole biogenesis. I contributed to the characterization of the deuterosome and identify it as a site for the
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Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified a frameshift variant that introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Loss of GAS2L2 in mouse tracheal epithelial cell (mTEC) cultures and in X. laevis
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C21orf59/Kurly (Kur), a cytoplasmic protein with some enrichment at the base of cilia, is needed for motility, proper cilia polarization in the zebrafish kidney and the larval skin of Xenopus laevis. CRISPR/Cas9 coupled with homologous recombination to disrupt the endogenous kur locus in Xenopus resulted in the asymmetric localization of the PCP protein Prickle2 being lost in mutant multiciliated cells. Kur also makes interactions with other PCP components, including Disheveled. This supports a model wherein Kur plays a dual role in cilia motility and polarization.
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AMBMP is a small molecule that has been previously reported to be both a Wnt agonist and a microtubule (MT) regulator. We showed that a detailed analysis of AMBMPs effects on MTs and on MT associated cellular processes including cell polarity, ciliogenesis, and cell migration. Specifically, treatment of Xenopus embryos with AMBMP leads to defects similar to the MT depolymerizing drug nocodazole, including a failure to generate or polarize
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MT-associated proteins (MAPs) have context-dependent roles in regulating their stability and dynamics. The poorly characterized clamp/Spef1 gene encodes a MAP and is expressed in several tissues and cell types. We have shown that CLAMP dynamically interacts with the microtubule latice and promotes promotes centriole positioning and radial intercalation of multiciliated cells by connecting microtubule organization to apico-basal polarity.
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Mamalian cylicin is localized to the cytoskeletal calyx that surrounds part of the nucleus in the head of sperm. The two highly conserved proteins, CYLC-1 and CYLC-2 in C.elegans have been suggested to function in the intracellular compartmentalization of tubulin levels in uterine muscle cells. We generated CRISPR tagged worm strains for both genes and show that both proteins are highly enriched in sperm in both hermaphrodites and males concentrated in puncta presumed to be membranous organelles.
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