The von Hippel-Lindau tumor suppressor protein controls ciliogenesis by orienting microtubule growth

Abstract

Cilia are specialized organelles that play an important role in several biological processes, including mechanosensation, photoperception, and osmosignaling. Mutations in proteins localized to cilia have been implicated in a growing number of human diseases. In this study, we demonstrate that the von Hippel-Lindau (VHL) protein (pVHL) is a ciliary protein that controls ciliogenesis in kidney cells. Knockdown of pVHL impeded the formation of cilia in mouse inner medullary collecting duct 3 kidney cells, whereas the expression of pVHL in VHL-negative renal cancer cells rescued the ciliogenesis defect. Using green fluorescent protein-tagged end-binding protein 1 to label microtubule plus ends, we found that pVHL does not affect the microtubule growth rate but is needed to orient the growth of microtubules toward the cell periphery, a prerequisite for the formation of cilia. Furthermore, pVHL interacts with the Par3-Par6-atypical PKC complex, suggesting a mechanism for linking polarity pathways to microtubule capture and ciliogenesis.

Figure 1.

pVHL is localized to renal monocilia. (a) Colocalization of pVHL and acetylated tubulin in primary cilia of MDCK cells. MDCK cells were grown on coverslips at 100% confluence and cultured for 5 d before the experiment to allow full polarization and cilia formation. Localization of pVHL was determined by immunofluorescence using a pVHL-specific antibody (green, rabbit polyclonal; sc-5575) with confocal images captured at the level of the apical membrane. Cells were costained with mouse antiacetylated tubulin antibody (middle), a marker protein for cilia. All images were captured using sequential Cy3 and AlexaFluor488 scans on a laser confocal microscope to eliminate bleed-through signals from the green and red fluorescence. (b) Specific localization of pVHL in primary cilia was confirmed by the use of an additional anti-VHL antibody (goat polyclonal; sc-1535) and blocking with recombinant pVHL. Lower magnification (left) and higher magnification views (middle) of ciliated cells are shown. Incubation of the specific pVHL antibody with a blocking recombinant protein (10-fold excess) resulted in the loss of pVHL staining in cilia, demonstrating specificity of the antibody stain (right).

Figure 2.

Lentivirally mediated reexpression of pVHL rescues the cilia-defective phenotype of VHL-negative renal epithelial cells. (a) Lentiviral vectors containing human pVHL₃₀ cDNA or an empty cassette were generated and used to infect VHL-defective A498 RCC cells. Reexpression of pVHL was confirmed by immunoblotting with VHL-specific antibody. #1 and #2 denote two different pools of transduced cells. (b) Ciliogenesis was quantified by blinded counting of cilia in two independent experiments (three slides each). Error bars represent SD. (c) VHL-defective (left; lentiviral control) and -positive cells (right; lentiviral pVHL) were grown 10 d after confluence to allow epithelial polarization and cilia formation. Red, acetylated tubulin; green, reexpressed V5-tagged pVHL; merge in the far right panels. (d) Doxycycline-induced reexpression of pVHL in tet-inducible A498 cells. Ttrp denotes the lentivirally mediated expression of Tet repressor. Top panel shows a dose-response curve (24 h of incubation). Doxycycline-mediated pVHL expression is shown (bottom) and results in the formation of cilia in pVHL-expressing cells (right). Reexpressed pVHL was stained with an anti-V5 antibody.

Figure 3.

pVHL associates with microtubules but does not affect microtubule growth or stability. (a) pVHL binds microtubules. Microtubules were polymerized in vitro, and supernatants and microtubule pellets were then subjected to SDS-PAGE and immunoblotted for the indicated proteins. (b) β-Tubulin coprecipitates with pVHL. FLAG-tagged pVHL (F.VHL) or a control protein (F.GFP) were expressed in HEK 293T cells and precipitated with anti-FLAG antibody. Western blot analysis was performed with a β-tubulin–specific antibody (top). Expression levels of β-tubulin in the lysates are shown (bottom). (c) End-binding protein 1 (EB1) tagged with GFP tracks microtubule growth in the monocilia of kidney cells, suggesting that this protein is a suitable reagent to study the microtubule formation required for ciliogenesis. GFP-EB1 was expressed in MDCK cells, and fluorescence (1,024 × 1,024 pixels) as well as differential interference contrast (DIC) images were recorded with a confocal scanning microscope. The confocal pinhole was set to achieve an optical slice thickness of 0.9 μm (top). The bottom panel shows a z stack of the same area. (d) GFP-EB1 accumulates at the plus ends of microtubules. GFP-EB1 movements were visualized with high speed confocal microscopy. (e) VHL does not affect the number of growing microtubules at the cell cortex. To estimate the number of growing microtubules at the cell cortex, GFP-EB1–positive microtubules were counted at the cell periphery in defined areas of interest and were compared in VHL-positive and -negative cells (n = 8). Tracking paths were measured in two square fields (256 μm²) per cell positioned in the cytosol adjacent to the plasma membrane. Error bars represent SD. Bars, 5 μm.

Figure 4.

VHL deficiency affects the directed growth of microtubules. (a) Representative trackings of EB1 tagged with GFP (GFP-EB1) in the cytoplasm of VHL-positive and -negative cells. Each trace represents the history of GFP-EB1 movement (microtubule growth) over sequential time frames with an acquisition rate of two images per second. (b) Microtubule growth events were expressed as the deviation from the mean angle (depicted as the sum vector in bold) in VHL-positive and -negative cells. A representative experiment of five independent analyses is shown. (c) Tracking paths were measured in one to two square fields (256 μm²) positioned in the cytosol in a total of 10 independent experiments (VHL negative and positive). As a measure for directed or nondirected movement, the deviation of the individual growth angles from the mean angle was calculated in each square. Statistical analysis was performed for VHL-positive and -negative cells using the two-tailed t test (**, P < 0.01). Error bars represent the SEM.

Figure 5.

pVHL interacts with the Par3–Par6–aPKC polarity complex. (a) VHL coprecipitates with Par6. FLAG-tagged Par6 (F.Par6) or a control protein (F.GFP) were coexpressed with HA-tagged VHL (HA.VHL) in HEK 293T cells and precipitated with anti-FLAG antibody. Western blot analysis was performed with anti-HA antibody (top). Expression levels of HA.VHL in the lysates are shown (bottom). (b) pVHL precipitates the Par3–Par6–aPKC protein complex. V5-tagged Par6 (V5.Par6), Par3 (150-kD isoform; V5.Par3), and aPKC (V5.PKCζ) were coexpressed with FLAG-tagged VHL or a control protein (F.EPS) in HEK 293T cells. pVHL and the control protein were precipitated with anti-FLAG antibody, and coprecipitating Par3, Par6, and PKCζ were visualized with anti-V5 antibody on Western blots (right). Expression levels of all proteins in the lysates are shown (left and bottom). (c) Native pVHL from mouse kidney lysates coprecipitates the Par3–Par6–aPKC protein complex. Freshly isolated mouse kidneys were perfused with ice-cold PBS, lysed, and subjected to immunoprecipitation with control or anti-pVHL antibodies. Precipitated pVHL and coprecipitating proteins were detected with specific antibodies. (d) Colocalization of pVHL and aPKC (PKCζ) in primary cilia of MDCK cells. MDCK cells were grown on Transwell filters (0.4-μm pore size; polyesther; Corning) at 100% confluence and cultured for 5 d before the experiment to allow full polarization and cilia formation.