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

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The Role of the Wnt/PCP Formin Daam1 in Renal Ciliogenesis bioRxiv preprint doi: https://doi.org/10.1101/512533; this version posted January 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 2 3 The Role of the Wnt/PCP Formin Daam1 in Renal Ciliogenesis 4 5 Mark E. Corkins†@, Vanja Krneta‐Stankic†‡§@, Malgorzata Kloc||¶, Pierre D. McCrea§¶, Andrew B. Gladden§¶, 6 Rachel K. Miller†§¶#* 7 † Department of Pediatrics, Pediatric Research Center, UTHealth McGovern Medical School, Houston Texas 77030 8 ‡ MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Program in Genes and Development, Houston Texas 77030 9 § MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Program in Genetics & Epigenetics, Houston, Texas 77030 10 || Houston Methodist, Research Institute, Houston Texas 77030 11 ¶ Department of Genetics, University of Texas MD Anderson Cancer Center, Houston Texas 77030 12 # MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Program in Biochemistry & Cell Biology, Houston Texas 77030 13 @ These authors contributed equally to this work 14 * Correspondence: [email protected]; Tel.: +01‐713‐500‐6537 15 Running Head. Role of Daam1 in primary ciliogenesis bioRxiv preprint doi: https://doi.org/10.1101/512533; this version posted January 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 16 ABSTRACT 17 Kidneys are composed of numerous ciliated epithelial tubules called nephrons. Each nephron functions to reabsorb 18 nutrients and concentrate waste products into urine. Defects in primary cilia are associated with abnormal formation of 19 nephrons and cyst formation in a wide range of kidney disorders. Previous work in Xenopus laevis and zebrafish embryos 20 established that loss of components that make up the Wnt/PCP pathway, Daam1 and ArhGEF19 (wGEF) perturb kidney 21 tubulogenesis. Dishevelled, which activates both the canonical and non‐canonical Wnt/PCP pathway, affect cilia 22 formation in multiciliated cells. In this study, we investigated the role of the noncanoncial Wnt/PCP components Daam1 23 and ArhGEF19 (wGEF) in renal ciliogenesis utilizing polarized mammalian kidney epithelia cells (MDCKII and IMCD3) and 24 Xenopus laevis embryonic kidney. We demonstrate that knockdown of Daam1 and ArhGEF19 in MDCKII and IMCD3 cells 25 leads to loss of cilia, and Daam1’s effect on ciliogenesis is mediated by the formin‐activity of Daam1. Moreover, Daam1 26 co‐localizes with the ciliary transport protein IFT88. Interestingly, knocking down Daam1 in Xenopus kidney does not 27 lead to loss of cilia. This data suggests a new role for Daam1 in the formation of primary cilia. 28 INTRODUCTION 29 Primary cilia are microtubule‐based cellular protrusions that allow a cell to sense its environment (Satir et al., 2010). 30 Many cell types in the body contain cilia, and improper cilia development results in a family of diseases called 31 ciliopathies, including polycystic kidney disease, nephronophthisis, Joubert syndrome and Bardet–Biedel syndrome (Lee 32 and Gleeson, 2011). Although ciliopathies can manifest in a number of different ways, the vast majority of ciliopathies 33 result in kidney abnormalities (Arts and Knoers, 2013). For example, one of the first described ciliopathies is the result of 34 a mutation in the gene IFT88/Polaris, a protein is transported within vesicles to facilitate ciliary biogenesis (Yoder et al., 35 2002; Ding et al., 2017). Loss of IFT88 leads to cystic kidney disease in both mouse and human (Moyer et al., 1994; 36 Onuchic et al., 1995). Additionally, loss of IFT88 in human mesenchymal stem cells leads to increased Wnt signaling 37 (McMurray et al., 2013). 38 The canonical Wnt pathway has many connections to cilia development. In the zebrafish left/right organizer, loss of β‐ 39 catenin, the Wnt co‐transcription factor, results in a reduction of cilia (Zhu et al., 2015). In mouse and Xenopus laevis a 40 protein called Chibby1, which functions to shuttle β‐catenin out of the nucleus, localizes to the cilia and is required for bioRxiv preprint doi: https://doi.org/10.1101/512533; this version posted January 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 41 proper ciliogenesis and kidney development (Lee et al., 2014; Shi et al., 2014). Additionally, mutations in genes that 42 regulate cilia formation typically results in increased sensitivity to Wnt ligands, although the mechanism for this 43 phenomenon is heavily debated (Corbit et al., 2008). Moreover, Dishevelled, a component of both the canonical and 44 non‐canonical Wnt pathway, is required for actin assembly and positioning of the basal bodies on the surface of the cell 45 during ciliogenesis in X. laevis multiciliated skin cells (Park et al., 2008). 46 Actin filaments are important for proper ciliogenesis. In motile multiciliated cells, the cilia are connected by an actin 47 network (Werner et al., 2011). Although this actin network is not clearly visible around primary cilia, application of drugs 48 that broadly inhibit or stabilize actin filaments typically results in longer primary cilia (Drummond et al., 2018). Formin 49 proteins act to polymerize actin filaments. However, treatment with the formin inhibitor smiFH2 has been found to 50 decrease the number and length of primary cilia (Copeland et al., 2018). This suggests a complex story in which different 51 sub‐populations of actin can have either a positive or negative affect on ciliogenesis. 52 Daam1 (Dishevelled Associated Activator of Morphogenesis 1) is a Diaphanous‐related Formin homology (DRF) protein 53 that functions through the non‐canonical Wnt/PCP pathway. It is regulated in part by Wnt ligands through Frizzled 54 receptors and Dishevelled (Liu et al., 2008). Daam1 is associated with a number of cellular functions, such as directed 55 cell migration, planar cell polarity (PCP) and endocytosis (Kida et al., 2007; Hoffmann et al., 2014; Luo et al., 2016). 56 Developmentally, Daam1 is necessary for gastrulation and normal kidney development in X. laevis (Habas et al., 2001; 57 Miller et al., 2011; Corkins et al., 2018). Additionally, it is present in the multiciliated epidermal cells of the X. laevis skin 58 where it regulates the actin network that stabilizes cilia. Loss of Daam1 in motile cilia results in their loss of polarity 59 (Yasunaga et al., 2015). 60 The Diaphanous family of formin proteins is regulated by the Rho family of GTPases (Alberts, 2001). Daam1 is known to 61 bind to RhoA, a protein that regulates cytoskeletal dynamics. However unlike other Diaphanous family formin proteins, 62 Daam1 is thought to be more strongly regulated by Dishevelled than Rho (Liu et al., 2008). Wnt signaling through 63 Dishevelled can activate RhoA, a process that is regulated by the Daam1/ArhGEF19 (wGEF) complex (Tanegashima et al., 64 2008). In mice, arhgef19 is expressed mainly in the intestine, liver, heart and kidney (Wang et al., 2004), and loss of 65 either ArhGEF19 or Daam1 in X. laevis leads to kidney malformations (Miller et al., 2011; Corkins et al., 2018). bioRxiv preprint doi: https://doi.org/10.1101/512533; this version posted January 4, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 66 In this study, we find that loss of the PCP component Daam1 negatively regulates ciliogenesis in MDCKII and IMCD3 cells. 67 Daam1 knockdown within these mammalian kidney epithelial cells result in the formation of fewer cilia. Furthermore, 68 knockdown of Daam1 within Xenopus embryonic kidneys does not appear to result in disruption of primary ciliogenesis. 69 Daam1 deficient kidneys have primary cilia; however, we cannot exclude the possibility that their structure, organization 70 and/or function is not affected. Our data indicate that ciliogenesis in MDCKII cells depends on the formin activity of 71 Daam1. Additionally, the Daam1 partner, ArhGEF19, is also required for proper cilia formation in MDCKII cells. Finally, 72 we found that in MDCKII and IMCD3 cells Daam1 localizes in vesicles that carry ciliary components, further supporting its 73 role in ciliogenesis. 74 RESULTS 75 Loss of Daam1 results in a failure of kidney epithelial cells to ciliate 76 To determine whether Daam1 is required for primary ciliogenesis, daam1 was knocked down in polarized MDCKII cells. 77 MDCKII kidney epithelia form primary cilia when plated on a transwell membrane and are allowed to grow until 78 confluency. Loss of Daam1 leads to cilia reduction in MDCKII cells (Figure 1). To ensure that this phenotype is due to loss 79 of Daam1 we used two different shRNAs: one targeting the 3’UTR (#1) and one targeting the coding sequence of daam1 80 (#3). Both shRNAs reduce Daam1 protein expression as visualized by Western blot (Figure 1E). A third shRNA, sh‐daam1 81 #2, failed to reduce Daam1 expression (Figure 2C). Additionally, similar experiments were carried out in IMCD3 cells 82 using a sh‐daam1 #1 construct adapted for mice. These experiments also indicate that knockdown of daam1 disrupts 83 primary ciliogenesis (Figure S1A,B). 84 Not only do sh‐daam1 cells have fewer numbers of cilia, but the cells grow to a higher density and exhibit a tendency to 85 grow on top of each other, rather than forming a cell monolayer.
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