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Changes in the State of Actin during the Exocytotic Reaction of Permeabilized Rat Mast Cells

Anna Koffer, Peter E. R. Tatham and Bastien D. Gomperts
The Journal of Cell Biology
Vol. 111, No. 3 (Sep., 1990), pp. 919-927
Stable URL: http://www.jstor.org/stable/1614196
Page Count: 9
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Changes in the State of Actin during the Exocytotic Reaction of Permeabilized Rat Mast Cells
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Abstract

The major part of mast cell actin is Triton-soluble and behaves as a monomer in the DNase I inhibition assay. Thus, actin exists predominantly in monomeric or short filament form, though filamentous actin is clearly apparent in the cortical region after rhodamine-phalloidin (RP) staining. The minimum actin content is estimated to be ∼2.5 μ g/106 cells (cytosolic concentration ∼110 μM. After permeabilization of mast cells by the bacterial cytolysin streptolysin-O, ∼60% of the Triton-soluble actin leaks out within 10 min. However, the staining of the cortical region by RP remains undiminished, and the cells are still capable of exocytosis when stimulated by GTP-γ-S together with Ca2+. In the presence of cytochalasin E the requirement for Ca2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by Ca2+-dependent actin regulatory proteins; their presence is indicated by a Ca2+-dependent inhibition of polymerization of extraneous pyrene-G-actin by a Triton extract of mast cells. The effect of cytochalasin E on secretion is similar to that of phorbol myristate acetate, an activator of protein kinase C; both agents enhance the apparent affinity for Ca2+ and cause variable extents of Ca2+-independent secretion. Exposing the permeabilized cells to increasing concentrations of Ca2+ caused a progressive decrease in F-actin levels as measured by flow cytometry of RP-stained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP-γ-S was not required for the Ca2+-dependent cortical disassembly. Thus, since conditions have not yet been identified where secretion can occur in its absence, cortical disassembly may be essential (though it is not sufficient) for exocytosis to occur.

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