Corkins ME et al. (2019), Divergent roles of the Wnt/PCP Formin Daam1 in ...

XB-ART-56249

PLoS One 2019 Jan 01;148:e0221698. doi: 10.1371/journal.pone.0221698.

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Divergent roles of the Wnt/PCP Formin Daam1 in renal ciliogenesis.

Corkins ME , Krneta-Stankic V , Kloc M , McCrea PD , Gladden AB , Miller RK .

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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 Daam1's effect on ciliogenesis is mediated by the formin-activity of Daam1. Moreover, Daam1 co-localizes with the ciliary transport protein Ift88 and is present in cilia. Interestingly, knocking down Daam1 in Xenopus kidney does not lead to loss of cilia. These data suggests a new role for Daam1 in the formation of primary cilia.

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Species referenced: Xenopus laevis
Genes referenced: cby1 ctnnb1 daam1 dvl2 fmn1 grap2 ift88 lhx1 ran
GO keywords: Wnt signaling pathway

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	Fig 1. Daam1 knockdown results in a loss of primary cilia in MDCKII cells. A) Diagram illustrating the domains within the Daam1 protein. The GBD (DiaphanousGTPase-binding Domain), FH3 (Diaphanous FH3 Domain), FH1 (Formin Homology 1), FH2 (Formin Homology 2) and DAD (Diaphanous Auto-regulatory Domain) are indicated. The location of the I698A mutation within the formin homology 2 domain (FH2) is marked with a red line, and positions corresponding to the shRNAs are marked with a blue line. MDCKII canine kidney epithelial cells were infected with either sh-daam1 or a control construct and grown upon transwell filters. B) Cells were stained with acetylated α-Tubulin antibody (acTubulin) to visualize primary cilia (green), DAPI to label nuclei (blue), and phalloidin to label F-actin (magenta). Confocal imaging was used to analyze the effect of Daam1 depletion on primary ciliogenesis. Scale bars equal to 50 μm. C) Cilia were quantified. Error bars are shown as ± SD and black dots indicate value of each image quatified. D) Cell numbers were quantified as described in S5 Fig. Error bars are shown as ± SD and black dots indicate value of each image quatified. E) Western blot of sh-daam1 MDCKII cell lysates showing depletion of Daam1 protein levels. β-catenin was used as a loading control. * indicates p < 0.05 as compared to sh-scrambled using two tailed t test.
	Fig 2. sh-daam1 depleted MDCKII cells show reduced luminal ciliogenesis in 3D cultures. Control and sh-daam1-depleted cells were cultured in collagen I matrix to form cysts. After 12–14 days in culture, cysts were fixed and stained with an antibody against acetylated α-Tubulin (acTubulin) to visualize primary cilia (green), phalloidin for F-actin (magenta) and DAPI for nuclei (blue). Using confocal imaging we analyzed the effect of Daam1 depletion on ciliogenesis. A) Representative merged confocal images showing reduction of luminal cilia upon Daam1 knockdown. White arrow points to luminal cilia in control cysts. Scale bars equal to 10 μm. B) The graph shows quantification of cysts with luminal cilia for each condition. Twenty randomly chosen cyst per sample were analyzed in three independent experiments. Error bars are shown as ± SD; Significance was calculated using unpaired, two-tailed t-test; ns indicates p > 0.05, * indicates p < 0.05, **p < 0.01 C) Western blot showing Daam1 protein levels in wild-type (control) cells and in cells expressing different shRNAs targeting Daam1. GAPDH was used as loading control.
	Fig 3. The sh-daam1 cilia phenotype can be rescued with wild-type Daam1 but not Daam1(I698A). Stable MDCKII cells infected with either sh-daam1 or a control sh-scrambled construct were transiently transfected with constructs that express either GFP, GFP-Daam1, or GFP-Daam1(I698A) constructs. Cells were grown on transwell filters and stained with acetylated α-Tubulin antibody (acTubulin) to label primary cilia (green), DAPI to label nuclei (blue), and phalloidin to label F-actin (Magemta). Confocal imaging was used to analyze the effects upon primary ciliogenesis. A) Representitive images of ciliated cells stained with acetylated tubulin (green), dapi (blue) and phalloidin (magenta). Scale bars equal to 50 μm. B) Quantification of the amount of ciliation. Error bars are shown as ± SD. Black dots indicate individual values for each image quantified. * indicates p < 0.05 as compared to sh-scrambled using two tailed t test.
	Fig 4. Daam1 localizes to ciliary vesicles and cilia. A) MDCKII cells were co-transfected with constructs that express mCherry-Daam1 and GFP-Daam1(I698A) then imaged via confocal to analyze the localization in live cells. B) MDCKII cells were co-transfected with constructs that express mCherry-Daam1 and Ift88-GFP than imaged via confocal in live cells to determine the subcellular localization. Scale bars equal to 10 μm. C) MCDKII Cells were transfected with mCherry-Daam1 along with GFP, GFP-Ift88, or GFP-Daam1(I698A), then colocalization was measured using both the Pearson and Manders equations. Error bars are shown as ± SD and black dots indicate colocalization value for each cell measured. D) Wildtype and sh-daam1 (#1) cells were cells were ciliated, fixed, then stained for Daam1 (Proteintech) and acTubulin (Sigma). E) MDCKII cells were subconfluently grown then fractionated for subcellular components. “Total” indicates whole cells lysated isolated prior to fractionation. Lysates were ran on Western blot and probed for Daam1, secretory components (Rab11), ciliary vesicles (Ift88), and cytoplasmic + vescular components (GAPDH). F) Cilia was isolated from IMCD3 cells and lysates were ran on a Western blot. “Total” indicates whole cell lysated isolated prior to deciliation. The blot was probed for the Daam1, a ciliary marker Ift88, and a cellular marker GAPDH.
	Fig 5. Arhgef19 knockdown results in loss of primary cilia in MDCKII cells. A) Diagram of the domains within the Arhgef19 (WGEF) protein. The corresponding position of the shRNA is marked with a blue line. B) MDCKII cells were infected with either a control construct or a construct that targets Arhgef19 and then polarized on transwell filters. Cells were stained with acetylated α-Tubulin antibody (acTubulin) to visualize primary cilia (green), DAPI to label nuclei (blue), and phalloidin to label F-actin (magenta). Confocal imaging was used to analyze the effects Arhgef19 depletion upon primary ciliogenesis. Scale bars equal to 50 μm. C) The number of cilia was counted and cell numbers were quantified as described in S5 Fig. * indicates p < 0.05 as compared to sh-scrambled. Error bars are shown as ± SD and black dots indicate value of each image quatified.
	Fig 6. Daam1-depletion does not cause absence of cilia within Xenopus embryonic kidneys. Because knockdown of Daam1 in Xenopus kidney leads to kidney defects, we analyzed the effect of Daam1 knockdown on renal ciliogenesis. We injected Daam1 or Standard (Control) morpholino in combination with a membrane-tagged red fluorescent protein (mRFP) mRNA as a lineage tracer, into a Xenopus blastomere fated to the nephric anlagen. Embryos were fixed at stage 39–40 and stained with an antibody against acetylated α-Tubulin (acTubulin) to label cilia (green), anti-mRFP lineage tracer (magenta) and lectin to label the proximal tubule (blue). A) Stereoscope brightfield imaging shows the gross morphology of Control and Daam1-morpholino injected embryos. Confocal fluorescent imaging of boxed regions (green) shows magnified views of corresponding kidneys. Kidney tubules displaying primary cilia are outlined in white. Neurons (n), multiciliated epidermal cells (mcc) and multiciliated cells within nephrostomes (ns) are immunostained with acetylated α-Tubulin (acTubulin) antibody. Scale bars equal to 20 μm. B) The graph represents the percentage of Control (n = 32 embryos) and Daam1-depleted (n = 34 embryos) kidneys with primary cilia. C) Western blot showing Daam1 protein expression levels in uninjected wild-type, control and Daam1 morphant embryos.
	S1 Fig. Loss of Daam1 results in a reduction of primary cilia and mCherry-Daam1 localizes to vesicles carrying Ift88 in IMCD3 cells. Murine inner medullary collecting duct (IMCD3) were infected with either sh-daam1 or a control construct then ciliated on glass coverslips. A) Cells were stained with acetylated α-Tubulin antibody (acTubulin) to label primary cilia (green), DAPI to label nuclei (blue), and phalloidin to label F-actin (magenta). Confocal imaging was used to analyze the effects Daam1 depletion upon primary ciliogenesis. Scale bars equal to 20 μm. B) Western blot of sh-daam1 IMCD3 cell lysates showing depletion of Daam1 protein levels. GAPDH was used as a loading control. C) Cells were co-transfected with constructs that express mCherry-Daam1 and Ift88-GFP than imaged in live cells. Colocalization analysis was performed on individual cells using both Pierson and Manders formulas. Error bars are shown as ± SD and black dots indicate each image quatified. D) Representitive images of mCherry-Daam1 and Ift88-GFP in IMCD3 cells. Scale bars equal to 5 μm
	S2 Fig. Phenotypes derived from control and sh-daam1 knockdown. Daam1-depleted 3D MDCKII cyst were scored for the presence of (1) non-luminal cilia–cilia that do not protrude into central lumen, (2) multiple lumens and (3) hollow lumens-luminal clearance. Twenty cysts were randomly selected for analysis in three independent experiments. A) The graph indicates the relative percentage of cyst for each phenotype. Error bars are shown as ± SD; Significance was calculated using unpaired, two-tailed t-test; ns indicates p > 0.05, * indicates p < 0.05, **p < 0.01 B) Representative images of cysts with non-luminal cilia phenotype. In Daam1-depleted cysts, white arrows point at cilia protruding out into extracellular matrix. Scale bars equal to 10 μm.
	S3 Fig. Daam1 localiation at cilia and vesicles. A-B) Murine inner medullary collecting duct (IMCD3) cells were transfected with mCherry-Daam1 along with either Cby1-GFP or α-Tubulin-GFP. Cells were grown to confluency and serum starved to ciliate. Then cells were analyzed via confocal for colocalization of Daam1 and these ciliary markers. White boxes outline the ciliary transition zone in Cby images and cilia in α-Tubulin images. Scale bars equal to 10 μm. C-D) IMCD3 cells were ciliated fixed with glyoxal then stained for Ift88 and Daam1 using two diferent Daam1 antibodies. E) IMCD3 cells transfected with mCherry-Daam1 construct were grown to confluency and puncta were imaged using Airyscan super-resolution system. Vesicles are circled with a yellow dotted line.
	S4 Fig. Daam1-depletion does not lead to the absence of cilia during development of Xenopus embryonic kidneys. To further analyze the effect of Daam1 depletion on ciliogenesis, we fixed 8-cell Daam1 and Standard (control) morpholino injected embryos during early stages of kidney morphogenesis (stage 30). mRFP mRNA was used as a lineage tracer and coinjected with morpholinos. Stage 30-fixed embryos were immunostained with an antibody against anti-mRFP to visualize tracer (magenta) together with an Lhx1 antibody to label nephric progenitor cells (blue) and acetylated α-Tubulin antibody to label primary cilia (green). Subsequently, embryos were analyzed using a confocal laser-scanning microscope and representative maximum projections of Z-stack sections are shown. Acetylated α-Tubulin antibody stains primary cilia (white arrows), neurons (n) and multiciliated epidermal cells (mcc). Scale bar is equal to 50 μm.
	S5 Fig. Quantification methodology. A) To obtain unbiased quantitation of cell numbers in MDCKII depletion experiments (Figs 1 and 5), DAPI images were divided into a 4 x 4 grid. Nuclei were counted within the 4 indicated and the number of cells was averaged. This number was multiplied by 16 to obtain the approximate number of cells per image. B) Cilia labeled with using acetylated Lys40 tubulin antibody were counted manually. All cilia within an image were counted as shown in red. C) The lumen of 3D cysts were scored either for presence or absence of cilia. The lumens of cysts are marked with yellow dashed lines.

References [+] :

Alberts, Identification of a carboxyl-terminal diaphanous-related formin homology protein autoregulatory domain. 2001, Pubmed