Functional genomic screen for modulators of ciliogenesis and cilium length

Abstract

Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell-cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.

Figure 1. Regulators of actin dynamics modulate cilium assembly

a, b, GSN knockdown reduced ciliated cell numbers. a, d, ACTR3 knockdown increased the number of cells with longer cilium (> 6 μm). c, e, Western blots showing protein levels (^†GSN siRNA#1 was cytotoxic). f, ACTR3 knockdown induced ciliogenesis without serum starvation. g, CytochalasinD treatment for 8 hrs increased ciliated cell numbers without serum starvation. h, Ciliogenesis defect by GSN knockdown was rescued by 8 hr cytochalasinD treatment. i, Live imaging of htRPE-SmoEGFP cells after 60 hr siRNA transfection. Arrowheads, the pericentrosomal preciliary compartment (PPC) at the ciliary base. j, Morphology of PPC. Gamma-tubulin labels the centrosome. k, Live imaging of a PPC positive cell in serum-free medium. l, Live cell imaging in serum-free medium containing DMSO or cytochalasinD. Values, mean ± SD [n = 4 (b,d), 2 (f) and 3 (g,h)]. Student's t-test: *P < 0.05, **P < 0.01. Scale bars, 10 μm (a); 5 μm (i,l); 2.5 μm (j,k).

Figure 2. Endocytic recycling pathway is linked to ciliogenesis

a, PPC overlapping with endocytosed transferrin-alexa594 and Rab11 (arrowhead). b, PTPN23 knockdown decreased the number of ciliated cells. c, PTPN23 knockdown caused an accumulation of SmoEGFP on EEA1-positive early endosomes. d, PTPN23 immunofluorescence was detected at the ciliary base. e, PLA2G3 knockdown increased the number of cells with longer cilia (> 6 μm). f, PLA2G3 knockdown induced ciliogenesis without serum starvation. g, Pericentriolar localization of PLA2G3. h, PLA2G3 knockdown increased the number of cells exhibiting recycling endosomes concentrated at high levels around the centrosome (arrowheads). Values, mean ± SD [n = 4 (b,e) and 3 (f)]. Student's t-test: *P < 0.05, **P < 0.01. Scale bars, 2.5 μm (a,d,g); 5 μm (c); 20 μm (h).

Figure 3. Pharmacological rescue of ciliary defect on IFT88 mutant cells

a, Cytochalasin D treatment for 16 hrs partially rescued cilium elongation defect caused by hypormorphic IFT88 mutation. b, Percentage of cells with the primary cilium longer than 1.5 μm. Values, mean ± SD (n = 3). Student's t-test: *P < 0.05. Scale bar, 5 μm.