ensure sealing of nondamaged areas. Rapid binding
of ESCRT proteins may also prevent extension of
the wound and enhance cell survival. Although
these mechanisms would be sufficient to explain
localized membrane repair, it may be associated
with other mechanisms described previously, such
as patching of membrane gaps with intracellular
vesicles (fig. S2A). The size of the gap may dic-
tate the choice of the mechanism involved for
repair because the patching model has clearly
been demonstrated for larger wounds and because
we observed critical involvement of ESCRT-III
proteins to repair wounds smaller than 100 nm.
These wounds were rather long-lasting as compared
to what has been observed for larger wounds.
In conclusion, plasma membrane integrity is
essential for cell homeostasis and survival. We
propose that ESCRT-III proteins play a central
role in repairing local injuries by ensuring extra-
cellular shedding of damaged areas. Plasma mem-
brane shedding, sometimes positive for ESCRTs,
has been observed in several normal and path-
ological conditions (35, 36), and it will now be
important to test whether this is linked to local
plasma membrane damage and whether ESCRT-
III proteins are involved in these processes.
References and Notes
1. J. F. Nabhan, R. Hu, R. S. Oh, S. N. Cohen, Q. Lu,
Formation and release of arrestin domain-containing
protein 1-mediated microvesicles (ARMMs) at plasma
membrane by recruitment of TSG101 protein.
Proc. Natl. Acad. Sci. U.S.A. 109, 4146–4151 (2012).
doi: 10.1073/pnas.1200448109; pmid: 22315426
Fig. 6. ESCRT-positive cell surface budding upon
wounding. HeLa CHMP4B-EGFP cells were used for
these experiments. Wounds and time-lapse acquisition
was performed on a spinning disc microscope equipped
with a UV laser. (A) Time-lapse observation of the
recruitment of CHMP4B-EGFP in wounded cells. (B)
Lateral view of the cell 4 min after wounding. (C)
Correlative image of the cell fixed and processed
for SEM (representative of six cells analyzed). (D
and F) Higher magnification of the areas pointed in
(A) and (C). (E) Fluorescence to SEM alignment based
on cell morphology criteria; see movie S4. (G) Recruitment of cytoplasmic CHMP4B-EGFP at blebs
generated at the wounded site. The resorption of the
bleb is accompanied by concentration of CHMP4B at
the wounding site. Lateral views correspond to an
orthogonal projection where the z images were separated by 5 pixels and intrapolated. White arrows
point at the wound site where CHMP4B is recruited.
Scale bars, 10 mm (A, C, and G); 1 mm (D to F).
Fig. 7. Energy-independent ESCRT recruitment and energy-dependent shedding of
wounded membrane. HeLa CHMP4B-EGFP cells
were wounded and imaged on a spinning disc
microscope equipped with a UV laser. (A) Shedding
of ESCRT-positive particles from wound site pointed by
the arrow. For corresponding lateral view, see movie
S5. (B and C) Cells were incubated in phosphate-buffered saline (PBS) + PI supplemented with glucose,
glucose + sodium azide, or deoxyglucose + sodium
azide, as indicated. Cells were wounded as previously
described. PBS containing deoxyglucose and sodium
azide was washed out in certain assays [(C) and fig.
S17] and replaced with PBS-glucose 10 min after
wounding (WO). (D and E) PI entry (D) and CHMP4B
(E) recruitment were quantified over time and
averaged. Arrows point at the wound site where
CHMP4B is recruited. Empty arrows point at CHMP4B-
positive shedding particles. Error bars correspond to
half 95% confidence intervals. Scale bars, 10 mm.