Fig 2: MC-membrane tubules are PACSIN1-, EHD1- and MT-dependent and form in vivo. a Quantification of PACSIN2 positive MC-tubules in cells as in Fig. 6c following siRNAs treatments (siCtrl = 258 cells, n = 5; siPACS1 = 168, siEHD1 = 75, siRAB8A/B = 147 cells, n = 3). b Quantification of GFP-EHD1-positive MC-tubules from triple line treated with siRNAs, starved 3 h and imaged every 2 min for 30 min (siCtrl = 35, siPACS1 = 36, siRAB8A/B = 57 cells, n = 3). c Quantification of MC-tubules from cell lines starved for 3 h and stained with PACSIN2 and CEP164 antibodies (wt -dox = 262, wt +dox = 142, K483E +dox = 177 cells, n = 2). d Schematic of the T181E tubulation defective mutation in the F-BAR domain of mPacsin1 (top). Immunoblot analysis of cells transfected with wildtype- or T181E-mPacsin1 after 6 h of siRNA treatment (bottom left) and ciliation rescue experiment (bottom right, siCtrl = 108, GFP = 71, rescT181E = 85 cells, n = 3). e Images of the 3 h starved triple line taken every minute. Membrane tubules are outlined in green and centrioles in blue (bottom panel, 15 MC-tubules). Single xy planes were smoothed. f Single plane xy epifluorescence images of 3 h starved GFP-EHD1 cells that were CtxB positive (~5% of cells). 53% of GFP-EHD1 MC-tubules contained CtxB (12 MC-tubules). g Images of GFP-EHD1 cells as in f stained with RAB8A, Actub, and CEP164 antibodies (10 cells). Scale bars: 1 µm in (e-g). h Quantification of cells as in b treated with 10 µM Nocodazole (untreated = 38, Nocodazole = 40 cells, n = 3). i Model for intracellular ciliogenesis. DAV distal appendage vesicle, CV ciliary vesicle, IFT intraflagellar transport, TZ transition zone, PM plasma membrane. In a–d and h, means ± SEM and two-tailed t-test analyses are indicated in figure. *P < 0.05, **P < 0.001, non significant (n.s). j Live image of the tail (red box, left schematic) from a 24 hpf embryo showing a tdTom-EHD1 positive MC-tubule (white box, 9 MC-tubules). k Timelapse of MC-tubule formation (centrioles in blue) as in j from embryo injected with rab8 MO (11 MC-tubules). Scale bars: 2 µm in j and k

Fig 3: PACSIN1 functions at the CV stage prior to CP110 loss. a Quantification of CP110 dots in serum starved RPE-1 cells treated with siRNAs for 72 h, serum starved for the last 24 h, and stained with CP110, Actub and CEP164 antibodies. CP110 localization on both centrioles (2 dots) or only the daughter centriole (1 dot) was quantified (left panel, siCtrl = 100, siPACS1 = 63 cells). b Quantification of CEP97 dots in cells treated as in a and stained with CEP97, Actub, and CEP164 antibodies. CEP97 localization on the centrioles was quantified as in a(siCtrl = 201, siPACS1 = 125 cells). c Quantification of SMOM2-GFP centrosomal accumulation in cells treated as in a, and stained with Actub and CEP164 antibodies. “Preciliary” indicates SMOM2-GFP vesicle accumulation at the MC without axonemes (siCtrl = 100, siPACS1 = 67 cells). Scale bar: 2 µm for images in a–c. d Quantification of MC with non-ciliary distal appendages structures from cells treated as in a. Pooled data from 3 independent experiments (siCtrl = 56, siPACS1 = 41 cells). Representative electron micrographs of quantified MCs shown on right. Scale bar: 200 nm. MC mother centriole, DAV distal appendages vesicles, CV ciliary vesicle, or DA non-membrane-associated distal appendages. e–g Quantification of TZ accumulation of TMEM67 (e, siCtrl = 113, siPACS1 = 89 cells), B9D2-GFP (f, siCtrl = 365, siPACS1 = 250 cells), RPGRIP1L (g, siCtrl = 70, siPACS1 = 63 cells) in cells treated as in a. h mIFT20-GFP cells treated as in a, stained with Actub and CEP164 antibodies, and quantified for mIFT20 fluorescence intensity at the MC/BB (siCtrl = 46, siPACS1 = 43 cells). Scale bar: 2 µm for images in e–h. In a–c, and e–h, representative images of quantified cells shown on right and means ± SEM, n = 3 independent experiments. Two-tailed t-test analysis as compared with siCtrl was performed. **P < 0.001, ***P < 0.0001

Fig 4: PACSIN1 and -2 are required for ciliogenesis. a Western analysis of PACSINs depletion in RPE-1 cells treated with siCtrl or siPACSINs (sequences in Supplementary Table 1). b Quantification of ciliated cells treated as in a (n = 4 independent experiments, siCtrl = 801, siPACS1 = 543 cells in total; n = 3, Ctrl = 215, siPACS2 = 128 cells; n = 3, siCtrl = 107, siPACS3 = 85 cells; n = 3, siCtrl = 106, siPACS1+2 = 75 cells). Images are in Supplementary Figure 1a. c Quantification of ciliation in RPE-1 cells treated with siPACSIN1 for 6 h, rescued with indicated constructs and starved at 48 hpf (n = 3, siCtrl = 108, GFP = 71, GFP-mPacs1 = 39, GFP-PACS2 = 75, GFP-PACS3 = 55 cells). d Quantification of ciliation in PANC1 cells treated as in b (n = 3, siCtrl = 273, siPACS1 = 194, siPACS2 = 175 cells). e Western analysis of PACSINs expression in RPE-1 and PANC1 cells. f Western analysis of Pacsins expression in 3 days post fertilization (dpf) CRISPR mutants (gRNA sequences shown in Supplementary Table 1). Arrow indicates Pacsin2 band. Schematic of organs of interest (Otic vesicle: OV, and Olfactory placode: OP). g Quantification of abnormal OV from embryos as in f and rescued with human PACSIN RNAs. Cas9, 38 OVs, n = 5; pacs1bCRISPR, 37 OVs, n = 4; pacs1bCRISPR + hPACSIN1, 20 OVs, n = 3; pacs1bCRISPR + hPACSIN2, 16 OVs, n = 3; pacs2CRISPR, 23 OVs, n = 3; pacs2CRISPR + hPACSIN2, 20 OVs, n = 3; pacs1b/2CRISPR, 15 OVs, n = 2; pacs1b/2CRISPR + hPACSIN2, 5 OVs, n = 1. Images are shown in (i) (scale bar: 5 µm). h Quantification of abnormal OP in embryos injected as in g. Cas9, 37 OPs, n = 5; pacs1bCRISPR, 25 OPs, n = 4; pacs1bCRISPR + hPACSIN1, 17 OPs, n = 3; pacs1bCRISPR + hPACSIN2, 14 OPs, n = 3; pacs2CRISPR, 18 OPs, n = 3; pacs2CRISPR + hPACSIN2, 19 OPs, n = 3; pacs1b/2CRISPR, 15 OPs, n = 2; pacs1b/2CRISPR + hPACSIN2, 6 OPs, n = 1. Images are shown in j (scale bar: 10 µm). Images in i and j are maximum intensity projections of deconvolved z-stacks. Means ± SEM. Two-tailed t-test analyses as compared with siCtrl in b and d or as indicated in figure. *P < 0.05, **P < 0.001, non significant (n.s)

Fig 5: PACSIN and EHD proteins co-localize on dynamic MC-tubules during ciliogenesis. a Representative N-SIM images of SMO-tRFP cells transiently expressing GFP-PACSIN1, serum starved for 3 h, and stained with CEP164 antibody. b Representative N-SIM images of SMO-GFP cells serum starved for 3 h and stained with CEP164 and PACSIN2 antibodies. The xz images (bottom panels) in a and b show orthogonal views at the position of the arrow indicated in the xy plane (top panels). Scale bars: 500 nm. c Representative images of RPE-1 cells serum starved for 3 h and stained with CEP164, Actub and PACSIN2 antibodies. Images were taken by epifluorescence microscopy using a 63× objective. Maximum intensity projections of deconvolved z-stacks are shown. d Quantification of PACSIN2, EHD1, or GFP-EHD1-positive MC tubules in RPE-1 cells, serum starved at 0 and 3 h and stained with PACSIN2, EHD1 antibodies, or observed in GFP-EHD1 cells imaged as in c (PACS2 0 h = 79, PACS2 3 h = 140, EHD1 = 67 cells, pooled from n = 2; GFP-EHD1 = 100 cells, pooled from n = 3). Means ± SD. e Graph representing the length of PACSIN2 and GFP-EHD1-positive tubules in cells treated as in (c) (25 tubules per condition). f GFP-EHD1 cells serum starved for 3 h, stained with PACSIN2, Actub (Alexa 305 nm), and CEP164 (Alexa 647) antibodies, and imaged by epifluorescence microscopy using a 63× objective. Z-stack images were deconvolved and a single xy plane is shown. Note the co-localization of PACSIN2 and GFP-EHD1 in MC-associated tubules (25 cells). g, h HPNE (g) and NIH3T3 (h) cells serum starved for 3–6 h and stained with antibodies for PACSIN2, CEP164, and Actub. Images were taken with a 100× objective and are maximum intensity projections of deconvolved z-stacks. i Triple line starved for 3 h, labeled with 300 nM SNAP-Cell647-SiR substrate for the last hour, washed, and imaged live every 10 min. Images are single xy planes (15 cells). Scale bars: 1 µm for (c, f–i)