Caveolar proteins do not accumulate at the plasma membrane lesion site in zebrafish embryos

(A) Caveolar proteins do not accumulate in the lesion patch. Fluorescence images of individual cells expressing Caveolin3-Clover, Clover-Cavin1a, EHD2a-Clover, and TinyDysf-Clover before and after laser-induced damage. The lesioning sites are marked by red arrows; white boxes surrounding the lesion sites mark regions with pronounced intensity changes. Scale bar (applies to all panels), 5 μm.

(B) Caveolar proteins show distinct subcellular distributions after membrane lesioning. Time-dependent fluorescence of caveolar proteins fused to Clover at the lesion site (red) and in the cytoplasm (blue), respectively. Thick lines show averages over multiple cells (cell numbers, N, are given in each panel); shaded regions indicate standard errors of the mean. Fluorescence signals were set to 100 at t = 0.

Caveolar proteins do not accumulate near a lesion inflicted in the C2C12 cell membrane

(A) Representative images of the accumulation of TinyDysf-Clover at the site of a laser-induced lesion (marked by red arrow). Scale bar, 5 μm.

(B) Time-dependent fluorescence of Caveolin3-mGarnet2, Cavin1a-mGarnet2, and TinyDysf-Clover at the lesion site. Thick lines show averages over multiple cells (cell numbers, N, are given in each panel); shaded regions indicate standard errors of the mean. Fluorescence signals were set to 100 at t = 0.

Diffusional dynamics of Caveolin3 and TinyDysf within C2C12 cell membranes before and after laser damage

(A) Conventional epifluorescence image of a C2C12 cell transfected with Caveolin3-mEosFPthermo (upper left). Individual dots correspond to caveolar structures (scale bar, 10 μm). Close-up of the region marked by the white square, obtained by SMLM analysis (upper right, scale bar, 2 μm). Representative examples of mature caveolae (bottom, scale bar, 100 nm).

(B) Example trajectories showing the movement of individual Caveolin3-mEosFPthermo proteins. Color bar, time in the image sequence (0–9 s); scale bar, 500 nm.

(C and D) Average diffusion coefficients, D, of Caveolin3-mEosFPthermo and CAAX-mEosFPthermo before (blue) and within 2 min after (red) lesioning as a function of the distance to the lesion site. Each data point corresponds to D log-averaged over all trajectories recorded on at least five cells on a single day (in total six and four days for Caveolin3 and CAAX, respectively). Boxes indicate mean ± SD, the median is given by the line, and whiskers mark the 95% confidence level. Solid lines indicate the average over the data points shown in corresponding colors, except for the data after lesioning in panel C, which were fitted with an exponential, D(r)=D0exp[−r/r0]+D∞, yielding D0 = 0.029 μm² s⁻¹, r0 = 3.7 μm and D∞ = 0.014 μm² s⁻¹ with a coefficient of determination, R² = 0.98. A line fit, D(r)=mr+D0, is also included as a dashed line, yielding slope m = −2.3 × 10⁻³ μm s⁻¹ and offset D0 = 0.035 μm² s⁻¹, with R² = 0.91.

Area-normalized histograms (PDFs) of the number of mEosFPthermo fusion protein trajectories versus the diffusion coefficient, D, (on a logarithmic scale)

Top (A–C) and bottom (D–F) rows, data taken before and after lesioning, respectively. The data were obtained by MSD analysis of trajectories extracted from TIRF image sequences of C2C12 cells transiently expressing mEosFPthermo-tagged (A, D) Cavin1, (B, E) Caveolin3, and (C, F) TinyDysf. Symbols and bars, data; gray line, sum of log-Gaussians fitted to the histograms; individual log-Gaussian distributions are included in magenta, blue, green, and red.

Area-normalized histograms (PDFs) of the number of mEosFPthermo fusion protein trajectories versus the diffusion coefficient, D, (on a logarithmic scale)

Top (A–C) and bottom (D–F) rows, data taken before and after lesioning, respectively. The data were obtained by MSD analysis of trajectories extracted from TIRF image sequences of C2C12 cells transiently expressing mEosFPthermo-tagged (A, D) LactC2, (B, E) GPI anchor, and (C, F) CAAX motif. Symbols and bars, data; gray line, sum of log-Gaussians fitted to the histograms; individual log-Gaussian distributions are included in magenta, blue, green, red, and dark-red.

Cavin1a knockout zebrafish embryos show decreased abundance of caveolar proteins in the sarcolemma

(A) EM images of slices of muscle tissue in wild-type (top) and Δcavin1a zebrafish embryos (bottom). White arrows point to caveolar structures. Scale bar, 100 nm.

(B) Number of caveolae in muscle and notochord cells of wild-type and Δcavin1a zebrafish embryos. Each data point corresponds to the value determined for one muscle cell.

(C) Representative fluorescence images of wild-type and Δcavin1a strains injected with caveolin3:Clover. Scale bar, 5 μm.

(D) Abundance of caveolar proteins tagged with Clover in the Z-lines, relative to the abundance in the sarcolemma. Error bars represent the SD. The significance was tested using the Bonferroni and Holm t test.

(E) Abundance of TinyDysf-Clover in the Z-lines, relative to the abundance in the sarcolemma.

Δcavin1a mutants have slightly abnormal lesion patches but appear functional

Temporal development of the relative emission intensities of different proteins at the lesion site in the wild-type (black) and cavin1a knockout (red) strains, normalized to the fluorescence measured at the sarcolemma at the site right before wounding.

(A) TinyDysf-Clover.

(B) LactC2-EGFP. (C) BODIPY-Cholesterol.

(D) Annexin2a-mOrange.

(E) Annexin6-mOrange.

(F) GCaMP5a-CAAX. Symbols show averages over multiple cells (cell numbers, N, are given in each panel); shaded regions indicate standard deviations. Fluorescence signals were set to 100% at t = 0 (before damage).