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Vol. 11, Issue 12, 4339-4346, December 2000
and
*Department of Molecular and Cell Biology, University of
California, Berkeley, California 94720-3200; and We hypothesized that the requirement for Ca2+-dependent
exocytosis in cell-membrane repair is to provide an adequate lowering of membrane tension to permit membrane resealing. We used laser tweezers to form membrane tethers and measured the force of those tethers to estimate the membrane tension of Swiss 3T3 fibroblasts after
membrane disruption and during resealing. These measurements show that,
for fibroblasts wounded in normal Ca2+ Ringer's solution,
the membrane tension decreased dramatically after the wounding and
resealing coincided with a decrease of ~60% of control tether force
values. However, the tension did not decrease if cells were wounded in
a low Ca2+ Ringer's solution that inhibited both membrane
resealing and exocytosis. When cells were wounded twice in normal
Ca2+ Ringer's solution, decreases in tension at the second
wound were 2.3 times faster than at the first wound, correlating well
with twofold faster resealing rates for repeated wounds. The
facilitated resealing to a second wound requires a new vesicle pool,
which is generated via a protein kinase C (PKC)-dependent and brefeldin A (BFA)-sensitive process. Tension decrease at the second wound was
slowed or inhibited by PKC inhibitor or BFA. Lowering membrane tension
by cytochalasin D treatment could substitute for exocytosis and could
restore membrane resealing in low Ca2+ Ringer's solution.
Laser
Microbeam and Medical Program, Beckman Laser Institute and Medical
Clinic, University of California, Irvine, California 92697
Corresponding author. E-mail
address: rsteinha{at}socrates.berkeley.edu.
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