Fig 1: EHD2, pacsin2 and EHBP1 stabilize caveolae at the plasma membrane.a Representative PREM example images of wildtype MEFs (wt), EHD2 knockout MEFs (KO), pacsin2 siRNA or EHBP1 siRNA treated MEFs. Triple knockout/knockdown (KO/KD) indicates EHD2 KO MEFs that were treated with pacsin2 and EHBP1 siRNA. Scale bar is 200 nm. b Example PREM image of low, medium and highly curved caveolae. c In PREM images the total caveolae number at the plasma membrane in wt, EHD2 KO, pacsin2 siRNA treated, EHBP1 siRNA treated, or triple KO/KD MEFs was measured (n(wt) = 25 cell regions, n(EHD2 KO) = 13 cell regions, n(pacsin2 siRNA) = 16 cell regions, n(EHBP1 siRNA) = 15 cell regions, n(Triple KO/KD) = 16 cell regions, 3 independent experiments). d Number of individual caveolae types in wt, EHD2 KO, pacsin2 siRNA, EHBP1 siRNA or triple KO/KOD MEFs (n(wt) = 13 cell regions, n(EHD2 KO) = 13 cell regions, n(pacsin2 siRNA) = 17 cell regions, n(EHBP1 siRNA) = 15 cell regions, n(Triple KO/KD) = 16 cell regions, 3 independent experiments). e Caveolae radius (round caveolae domains were assumed) of low, medium and highly curved caveolae in wt, EHD2 KO, pacsin2 siRNA, EHBP1 siRNA or triple KO/KOD MEFs (caveolae number: low: n(wt) = 127, n(EHD2 KO) = 139, n(pacsin2 siRNA) = 163, n(EHBP1 siRNA) = 123, n(Triple KO/KD) = 69; medium: n(wt) = 148, n(EHD2 KO) = 130, n(pacsin2 siRNA) = 146, n(EHBP1 siRNA) = 177, n(Triple KO/KD) = 145; high: n(wt) = 129, n(EHD2 KO) = 157, n(pacsin2 siRNA) = 106, n(EHBP1 siRNA) = 142, n(Triple KO/KD) = 133, 3 independent experiments). Box plots indicate mean values ± SE, whiskers show SD, each replicate is depicted, statistical significance was measured by two-sided t test in normally distributed data sets, otherwise the nonparametric two-sided Mann–Whitney test was applied.
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: PACSIN and EHD proteins accumulate on CPM tubules that contain RAB8A. a Representative 3D volume view images (generated by SlideBook) of GFP-EHD1 cells serum starved for 24 h and stained with antibodies to PACSIN2 and Actub. The z-stack was captured using a SDC microscope and a CMOS camera. b, c SMO-GFP cells imaged as in a and stained with antibodies to PACSIN2 or EHD1, CEP164, and anti-GFP. Contrast enhanced and inverted image in the middle right panel demonstrate the absence of SMO-GFP in PACSIN2/EHD1 tubules. d Representative N-SIM maximum intensity projection images of CPM-associated membrane tubules in ciliated GFP-EHD1 (green) + SMO-tRFP (pseudo-colored blue) cells stained with PACSIN2 antibody (pseudo-colored red). e Epifluorescence projected z-stack images of a GFP-EHD1-positive CPM-tubule in ciliated cells stained with RAB8A and CEP164 antibodies highlighting the presence of endogenous RAB8A in both the ciliary membrane and the CPM tubules (7 cells). f Representative image of CPM-associated membrane tubules in tail cilia of 24 hpf zebrafish embryos expressing tdTom-EHD1 and ARL13B-GFP imaged by SDC microscope with a CMOS camera. Tail region is represented by red box in schematic of zebrafish embryo on the left. g GFP-EHD1 + SMO-tRFP cells were starved for 24 h and imaged live every 2 min (16 ciliated cells). Arrows mark dynamic tubules associated with the CPM over time. h GFP-EHD1 cells transiently transfected with tRFP-RAB8A, starved for 24 h, and imaged live using TIRF-M (upper panels). tRFP-RAB8A signal is shown inverted (middle images) and in red (merged right images). Arrows indicate breaks in membrane tubules. Scale bar: 1 µm. Enlarged regions (lower panels) from upper images showing membrane tubule breaks with additional time-lapse images added (12 cilia). Scale bar: 500 nm. Images in (g) and (h) are single xy planes. Scale bars: 1 µm for all images unless specified
Fig 4: Spatially distribution of EHD2, pacsin2 and EHBP1.a Representative STED-CLEM images for MEFs expressing EHD2-EGFP (labelled with GFP nanobody-Atto647N) or white arrows indicate accumulation of EHD2 around the caveolae center. b EHD2 STED fluorescence profile from the center of caveolae to its edges obtained from STED-CLEM images (a). Each individual caveolae type is depicted (graph shows mean ± SE, caveolae number: n(low) = 72, n(medium) = 50, n(high) = 163, 2 independent experiments). c STED-CLEM showing endogenous EHD2 antibody staining at low curved caveolae (secondary antibody tagged with Atto647N, 2 independent experiments). d,e Representative CLEM images for MEFs expressing either pacsin2-EGFP (d) or EHBP1-EGFP (e) that were labelled with GFP nanobody-Atto647N (cyan). f Quantitative analysis of low or highly curved caveolae by STED fluorescence profiles from the center of caveolae to their edges (indicated by green dashed line, line graphs show mean ± SE, caveolae number: caveolin1: n(low) = 145, n(high) = 178; EHD2: n(low) = 72, n(high) = 163; pacsin2: n(low) = 103, n(high) = 77; EHBP1: n(low) = 70, n(high) = 79, 3 independent experiments). g Percentage of low or highly curved caveolae that were targeted by either pacsin2 or EHBP1. Bar plot indicates mean ± SE, n(pacsin2) = 1357 caveolae/3 cells, n(EHBP1) = 660 caveolae/4 cells, 3 independent experiments. Significant difference was tested by two-sided Mann–Whitney test. Scale bar is 100 nm.
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)
Supplier Page from Proteintech Group Inc for PACSIN2 antibody