Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins.

Abstract

Abstract. Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-'y-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased.Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.T HE activation of cell surface receptors leads to a multiplicity of responses, such as movement, secretion, mitosis, or phagocytosis. Most of these are accompanied by changes in the organization of the cytoskeleton. In secretory cells, such cytoskeletal reorganizations have been suggested to play a role in granule migration and in regulating the access of secretory vesicles to the plasma membrane, as well as endocytotic events after exocytosis (Burgoyne and Cheek, 1985;Linstedt and Kelly, 1987;Sontag et al., 1988).Cytoskeletal changes after cell activation have been attributed to changes in pH, Ca 2÷ or phosphorylation (Downey and Grinstein, 1989, Del Castillo et al., 1992, Stossel, 1989Downey et al., 1992) and also to the activation of GTPbinding proteins (Hall, 1992). The latter can work indirectly, via the production of second messengers (such as an increase in Ca2+i, cAME cGMP, or activation of protein kinase C) or via the increased turnover of phosphoinositides, which have been shown to bind to and regulate many actin-binding proteins (Lassing and Lindberg, 1988). However, evidence for direct control of microfilaments by GTPases is accumulating. In electropermeabilized neutrophils, GTP-3~-S was found to induce an increase in F-actin content, even when phospholipase C activity was inhibited (Bengtsson et al., 1990;Therrien and Naccache, 1989 cumulates at cell surface projections directed towards the chemoattractant, has sequence similarities with the/3 subunit of heterotrimeric G-proteins (de Hostos et al., 1991). These/3 subunits have also been found to be associated with the cytoskeleton of mouse lymphoma cells (Carlson et al., 1986). More recently, microinjection studies with cultured fibroblasts showed that two ms-related small GTPases, rho and rac, are essential for the ...

Changes in actin network during calcium-induced exocytosis in permeabilized GH3 cells: calcium directly regulates F-actin disassembly

“…Our present findings of a change in F-actin distribution -a reduction in F-actin in the cortical region and an increase in F-actin in the interior region -have also been observed in mast cells and adrenal chromaffin cells (Norman et al 1994, Vitale et al 1995. This change in F-actin was different from the changes observed in both GH4C1 cells and GH3C6 cells.…”

“…The changes in the cytoskeleton during cell stimulation have been studied using a variety of cells, such as adrenal chromaffin cells (Zhang et al 1995), mast cells (Norman et al 1994), leukemia cells (Pfeiffer et al 1985) and pancreatic acinar cells (Muallem et al 1995). F-actin network disassembly has been observed in these cells upon stimulation.…”

“…Many agents, such as protein kinase C (Burgoyne et al 1989, Nishizaki et al 1992, calmodulin (Burgoyne 1991, Cheek 1991, fodrin (Burgoyne & Cheek 1987, Perrin et al 1987, gelsolin (Bader et al 1986, Trifaró & Vitale 1993, scinderin (Rodríguez et al 1990, Vitale et al 1995, calpactin , and 14-3-3 proteins (Roth & Burgoyne 1995) and GTP-binding proteins (Norman et al 1994, Price et al 1995, have been studied as Ca 2+ target proteins in the regulation of exocytosis, though the exact mechanisms by which Ca 2+ regulates exocytosis remain unclear. Immunocytochemical studies showed that the cortical actin network was disassembled upon nicotine stimulation of bovine adrenal chromaffin cells (Cheek & Burgoyne 1986, Trifaró & Vitale 1993.…”

ATP-dependent Membrane Assembly of F-Actin Facilitates Membrane Fusion

“…More difficult to rationalize has been the positive effector function of the actin cytoskeleton, which has been extensively described in the process of exocytosis (Orci et al, 1972;Norman et al, 1994;Muallem et al, 1995;Lang et al, 2000). The results of this study, in conjunction with a parallel study have led us to a testable model (our unpublished results) for one possible mechanism by which membrane-catalyzed actin assembly could facilitate the aggregation of membrane organelles prior to fusion.…”

“…They have also been strongly implicated in the regulation of exocytic fusion (Norman et al, 1994;Komuro et al, 1996;Brown et al, 1998;Gasman et al, 1999). A 60% inhibition was seen when 18 M rhoGDI was added to the fusion mix (Fig.…”