Caveolae internalization repairs wounded cells and muscle fibers
- University of Maryland, United States
- Brazil Ministry of Education, Brazil
- National Institute on Deafness and Other Communication Disorders, National Institutes of Health, United States
Figures
Figure 1 with 1 supplement
Caveolae-like vesicles accumulate in cells exposed to SLO and sphingomyelinase.
(A) Cryo-immuno EM with anti-ceramide in NRK cells untreated or exposed to SLO or SM for 30 s. Bars: 100 nm. Arrows: patches of ceramide staining near the PM. (B) Quantification of anti-ceramide …
Figure 1—figure supplement 1
Transcriptional silencing of ASM inhibits intracellular accumulation of caveolae-like vesicles after SLO injury.
(A) TEM of control and ASM siRNA-treated HeLa cells incubated or not with SLO for 60 s. Arrows: <80 nm profiles. Bars: 100 nm. (B) Number of <80 nm vesicular profiles/µm in H. All vesicles (127–216) …
Figure 2
SLO is internalized in Cav1-positive caveolae-like vesicles that separate from the PM.
(A) Cryo-immuno EM localization of GFP-SLO and Cav1 in NRK cells. 5 nm gold: anti-GFP (arrowheads); 10 nm gold: anti-Cav1 (arrows). Bars: 100 nm. (B–D) Quantification of the relative amount of …
Figure 3
Internalized SLO colocalizes with Cav1.
(A) HeLa cells were pre-incubated with 3 µg/ml Alexa 488-SLO (green) for 5 min at 4°C, washed and either kept at 4°C (0 s) or incubated at 37°C in DME+Ca2+ (180 s), followed or not by anti-Alexa …
Figure 4
SLO enters cells associated with Cav1-positive carriers.
(A) Confocal optical section at the bottom surface of a HeLa cell transfected with mRFP-Cav1 (red) and stained with anti-Cav1 antibodies (green). (B) Time-lapse images of HeLa cells expressing …
Figure 5
EHD2-Cav1 colocalization is decreased after caveolar endocytosis triggered by SLO or sphingomyelinase.
(A) TIRF images of Cav1 and EHD2 immunostaining in HeLa cells treated or not with SLO or SM for 30 or 60 s. Bars : 50 µm. Arrows: Cav1 positive, EHD2 negative puncta corresponding to internalized …
Figure 6
PM repair and endocytosis of SLO in caveolar vesicles are Cav1-dependent.
(A) Western blot with anti-Cav1 and anti-actin (loading control) in NRK cell lysates treated with control or Cav1 siRNA. (B) Live imaging of FM1–43 influx in NRK cells treated with control or Cav1 …
Figure 7
Depletion in dynamin-1 and -2 does not inhibit PM repair and SLO or SM-induced internalization of caveolar vesicles.
(A) Western blot with anti-dynamin-2 and anti-actin (loading control) in lysates of NRK cells treated with control or Dyn2 siRNA. (B) Quantification of transferrin uptake in NRK cells treated with …
Figure 8 with 1 supplement
Caveolae accumulate at sites of mechanical wounding, and Cav1 is required for mechanical wound repair.
(A) TEM of NRK cells wounded with glass beads and stained with ruthenium red during fixation. Numerous caveolae-like vesicles (arrows, lower magnified image) are visible near the wound, identified …
Figure 8—figure supplement 1
Clusters of caveolae are observed next to sites of mechanical wounding.
TEM of NRK cells injured with glass beads for 30 s (A) or 60 s (B and C) and stained with ruthenium red after fixation to label sites of PM injury. Bars: 500 nm. Large arrows: injury sites; small …
Figure 9
Cav3 expression, Ca2+-dependent sarcolemma repair, lysosomal exocytosis and endocytosis in C2C12 myoblasts/myotubes.
(A) Western blot with anti-Cav3 or anti-tubulin (loading control) antibodies showing that differentiation of C2C12 myoblasts induced by serum starvation leads to a gradual enrichment of the cultures …
Figure 10
Caveolae-like vesicles accumulate in C2C12 myotubes exposed to sphingomyelinase and SLO, and resealing after injury depends on Cav3.
(A) TEM of myotubes untreated (control) or exposed to SLO or SM. Arrows: caveolae-like vesicles. Arrowheads: merged caveolae-like vesicles. Wide arrow: clathrin-coated pit. Inset: higher …
Figure 11
Primary muscle fibers are sensitive to SLO permeabilization and reseal in the presence of Ca2+.
Flexor digitorum brevis mouse muscle fibers treated or not with 400 ng/ml SLO or 50 mU/ml SM in the presence or absence of Ca2+ and stained with PI (red) after 30 s. Small arrows point to PI …
Figure 12 with 1 supplement
Caveolae accumulate in primary mouse muscle fibers after exposure to sphingomyelinase or sarcolemma injury.
(A–C) TEM of flexor digitorum brevis fibers untreated (A) or exposed for 300 s to SLO (B) or SM (C). Three examples are shown for each. Arrows: single or merged caveolae-like vesicles. Bars: 100 nm. …
Figure 12—figure supplement 1
Accumulation of caveolae-like vesicles in mechanically injured flexor digitorum brevis muscle fibers.
(A) Sequential TEM images along the whole periphery of a fiber fixed shortly after dissection. Caveolae-like vesicles are more abundant in the vicinity (#21–24) of the wound site (#23). Bars: 100 …
Figure 13
Model for PM repair mediated by caveolar endocytosis.
Permeabilization with transmembrane toxin pores (A) or mechanical wounding (B) triggers Ca2+ influx, exocytosis of lysosomes, release of ASM, and generation of ceramide at the PM outer leaflet, a …
Videos
Video 1
Internalization and lateral movement of SLO/Cav1 carriers, (related to Figure 4).
HeLa cells expressing mRFP-Cav1 (red) were pre-incubated for 5 min with 800 ng/ml of GFP-SLO (green) at 4°C and transferred in cold DMEM+Ca2+ to a live imaging chamber at 37°C, to allow for …
Video 2
Internalization and trafficking of SLO/Cav1 carriers (related to Figure 4).
HeLa cells were treated as in video 1. The video shows a Cav1 positive structure on the PM that accumulates SLO before being internalized and moving rapidly into the cell. Video is displayed at 6.67 …
Video 3
Intracellular merger and rapid internalization of SLO/Cav1 carriers (related to Figure 4).
HeLa cells were treated as in video 1. The video shows two separate SLO and Cav1 positive carriers close to the PM (arrows) that merge before rapidly moving further into the cell. The same cell …
Video 4
RNAi-mediated silencing of Cav1 expression inhibits PM repair, allowing sustained FM1–43 influx into SLO-permeabilized NRK cells (related to Figure 6).
NRK cells treated with control or Cav1 siRNA were left untreated (no SLO) or pre-incubated with SLO at 4°C and transferred to a live imaging chamber at 37°C in the presence or absence of Ca2+, …
