Biology subjects

Kloc, M.

Publications and source records attributed to Kloc, M..

3 recordsLinked to original sources

bioRxiv2020

Cell-Cell Adhesion During Nephron Development Is Driven by Wnt/PCP Formin Daam1

E-cadherin junctions facilitate the assembly and disassembly of cell-cell contacts that drive development and homeostasis of epithelial tissues. The stability of E-cadherin-based junctions highly depends on their attachment to the actin cytoskeleton, but little is known about how the assembly of junctional actin filaments is regulated. Formins are a conserved group of proteins responsible for the formation and elongation of filamentous actin (F-actin). In this study, using Xenopus embryonic kidney and Madin-Darby canine kidney (MDCK) cells, we investigate the role of the Wnt/ planar cell polarity (PCP) formin protein Daam1 (Dishevelled-associated activator of morphogenesis 1) in regulating E-cadherin based intercellular adhesion. Using live imaging we show that Daam1 localizes to newly formed cell-cell contacts in the developing nephron. Furthermore, analyses of junctional F-actin upon Daam1 depletion indicate a decrease in microfilament localization and their slowed turnover. We also show that Daam1 is necessary for efficient and timely localization of junctional E-cadherin, which is mediated by Daam1s formin homology domain 2 (FH2). Finally, we establish that Daam1 signaling is essential for promoting organized movement of renal cells. This study demonstrates that Daam1 formin junctional activity is critical for epithelial tissue organization.

developmental biology↗

bioRxiv2020

CDC6 regulates mitotic CDK1 via cyclin B and not cyclin A and acts through a bona fide CDK inhibitor Xic1

The timing of the M-phase entry and its progression are precisely controlled by a CDC6-dependent mechanism that inhibits the major mitotic kinase CDK1, and, thus, regulates the dynamic of CDK1 during the M-phase. In this paper, we describe the differential regulation of the mitotic CDK1 dynamics by exogenous cyclin A or a non-degradable cyclin B added to the Xenopus laevis embryo cycling extracts. We showed that the variations in the level of cyclin B modify both CDK1 activity and the timing of the M-phase progression, while the cyclin A levels modify only CDK1 activity without changing the timing of the M-phase events. In consequence, CDC6 regulates the M-phase through endogenous cyclin B, but not cyclin A, which we demonstrated directly by the depletion of cyclin A, and the addition of CDC6 to the cycling extracts. Further, we showed, by p9 precipitation (p9 protein associates with Cyclin-Dependent Kinases, CDK), followed by the Western blotting that CDC6, and the bona fide CDK1 inhibitor Xic1, associate with CDK1 and/or another CDK present in Xenopus embryos, the CDK2. Finally, we demonstrated that the Xic1 temoprarily separates from the mitotic CDK complexes during the peak of CDK1 activity. These data show the differential coordination of the M-phase progression by CDK1/cyclin A and CDK1/cyclin B, confirm the critical role of the CDC6-dependent CDK1 inhibition in this process and show that CDC6 acts through the cyclin B- and not cyclin A/CDK complexes. This CDC6- and cyclin B-dependent mechanism may also depend on the precisely regulated association of Xic1 with the CDK complexes. We postulate that the dissociation of Xic1 from the CDK complexes allows the maximal activation of CDK1 during the M-phase.

cell biology↗

bioRxiv2019

The Role of the Wnt/PCP Formin Daam1 in Renal Ciliogenesis

Kidneys are composed of numerous ciliated epithelial tubules called nephrons. Each nephron functions to reabsorb nutrients and concentrate waste products into urine. Defects in primary cilia are associated with abnormal formation of nephrons and cyst formation in a wide range of kidney disorders. Previous work in Xenopus laevis and zebrafish embryos established that loss of components that make up the Wnt/PCP pathway, Daam1 and ArhGEF19 (wGEF) perturb kidney tubulogenesis. Dishevelled, which activates both the canonical and non-canonical Wnt/PCP pathway, affect cilia formation in multiciliated cells. In this study, we investigated the role of the noncanoncial Wnt/PCP components Daam1 and ArhGEF19 (wGEF) in renal ciliogenesis utilizing polarized mammalian kidney epithelia cells (MDCKII and IMCD3) and Xenopus laevis embryonic kidney. We demonstrate that knockdown of Daam1 and ArhGEF19 in MDCKII and IMCD3 cells leads to loss of cilia, and Daam1s effect on ciliogenesis is mediated by the formin-activity of Daam1. Moreover, Daam1 co-localizes with the ciliary transport protein IFT88. Interestingly, knocking down Daam1 in Xenopus kidney does not lead to loss of cilia. This data suggests a new role for Daam1 in the formation of primary cilia.

molecular biology↗