Increasing cellular G-actin, using latrunculin B, in either intact or permeabilized rat peritoneal mast cells, caused translocation of both actin and an actin regulatory protein, cofilin, into the nuclei. The effect was not associated with an increase in the proportion of apoptotic cells. The major part of the nuclear actin was not stained by rhodamine-phalloidin but could be visualized with an actin antibody, indicating its monomeric or a conformationally distinct state, e.g. cofilin-decorated filaments. Introduction of anti-cofilin into permeabilized cells inhibited nuclear actin accumulation, implying that an active, cofilin-dependent, import exists in this system. Nuclear actin was localized outside the ethidium bromide-stained region, in the extrachromosomal nuclear domain. In permeabilized cells, the appearance of nuclear actin and cofilin was not significantly affected by increasing [Ca(2+)] and/or adding guanosine 5'-O-(3-thiotriphosphate), but was greatly promoted when ATP was withdrawn. Similarly, ATP depletion in intact cells also induced nuclear actin accumulation. In contrast to the effects of latrunculin B, ATP depletion was associated with an increase in cortical F-actin. Our results suggest that the presence of actin in the nucleus may be required for certain stress-induced responses and that cofilin is essential for the nuclear import of actin.
Abstract. The major part of mast cell actin is Tritonsoluble 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/~g/106 cells (cytosolic concentration ~110 #M.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--y-S together with Ca 2÷. In the presence of cytochalasin E the requirement for Ca 2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by CaE+-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 Ca 2+ and cause variable extents of CaE+-independent secretion.Exposing the permeabilized cells to increasing concentrations of Ca 2÷ caused a progressive decrease in F-actin levels as measured by flow cytometry of RPstained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP--y-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. p ERMEABILIZED rat mast cells provide an excellent system for investigating the mechanism of exocytosis. In response to provision of Ca 2÷ and GTP (or its analogues), they release up to 100% of their contained histamine and hexosaminidase, both of which can be rapidly and sensitively measured (16). The exocytotic process, which may be studied by providing appropriate effect.ors (bypassing the receptor-agonist interaction), involves fusion of the granular and plasma membranes followed by the release of granule contents. Since all mammalian cells contain an actin-rich network in the cortical region (6), it has been suggested that disassembly of the cortical cytoskeleton might be a prerequisite for exocytosis (3,7,26,42). On the other hand, the motile elements of the cytoskeleton are also thought to be involved in the transport of vesicles to the plasma membrane. Several studies have demonstrated increased actin filament assembly after cell stimulation (10,39,41). Controversial conclusions have also been presented on the basis of experiments involving the use of microfilament-specific agents such as phalloidin and cytochalasin (2,13,16,19,29,30,34,42,44). Thus, the exact role (if any) of the cytoskeleton in the...
In mast cells, activation of GTP-binding proteins induces centripetal reorganization of actin filaments. This effect is due to disassembly, relocalization, and polymerization of F-actin and is dependent on two small GTPases, Rac and Rho. Activities of Rac and Rho are also essential for the secretory function of mast cells. In response to GTP-gamma-S and/or calcium, only a proportion of permeabilized mast cells is capable of secretory response. Here, we have compared actin organization of secreting and nonsecreting cell populations. We show that the cytoskeletal and secretory responses are strongly correlated, indicating a common upstream regulator of the two functions. The secreting cell population preferentially displays both relocalization and polymerization of actin. However, when actin relocalization or polymerization is inhibited by phalloidin or cytochalasin, respectively, secretion is unaffected. Moreover, the ability of the constitutively active mutants of Rac and Rho to enhance secretion is also unaffected in the presence of cytochalasin. Therefore, Rac and Rho control these two functions by divergent, parallel signaling pathways. Cortical actin disassembly occurs in both secreting and nonsecreting populations and does not, by itself, induce exocytosis. A model for the control of exocytosis is proposed that includes at least four GTP-binding proteins and suggests the presence of both shared and divergent signaling pathways from Rac and Rho.
These results provide direct evidence that, in mast cells, both Rac and Rho are components of the signalling pathway that leads to secretion.
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