Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins.

Abstract

Rat, but not human, erythrocytes undergo fusion promoted by the membrane-mobility agent 2-(2-methoxyethoxy)-ethyl cis-8-(2-octylcyclopropyl)octanoate (A2C). The difference in behavior is correlated with rat erythrocyte membrane protein degradation caused by Ca2+-activated proteases. The human erythrocyte is deficient in such protease activity. Membrane protein degradation is a necessary, but not sufficient, requirement for membrane fusion. Membrane protein degradation probably releases membrane components from certain constraints. In addition, the motion of membrane components precedes fusion and must be promoted by reagents such as A2C, leading to the creation of fusion-potent lipid areas. This sequence of chemical and physical events occurs in other fusion processes.Membrane fusion is a ubiquitous cellular process, mediating such phenomena as fertilization, exocytosis, endocytosis, phagocytosis, and turnover of intracellular organelles (1, 2). Fusion can be induced experimentally by certain viruses (3) and chemical agents (4, 5). Membrane-mobility agents are long-chain esters designed to promote motion of molecules in cell membranes. Membrane-mobility agents, which enter cells via the small particles formed on dispersion in aqueous medium, promote the lateral mobility of certain ligand-membrane receptor complexes and inhibit cytokinesis in some types of cells (5-9). Membrane-mobility agents, especially 2-(2-methoxyethoxy)ethyl cis-8-(2-octylcyclopropyl)octanoate (A2C), are efficient promoters of cell fusion; the results obtained using a defined agent such as A2C have allowed the identification of stages in the overall fusion process and the formulation of a molecular mechanism for membrane fusion (10-13).We have previously found differences in the occurrence of A2C-induced fusion among various mammalian non-nucleated erythrocytes as well as among avian nucleated erythrocytes (12, 13): erythrocytes of some species fuse easily, whereas those of others do not. "Mixed fusion," that between fusible erythrocytes from different species, is promoted by A2C. Moreover, the potential for fusion is a transferable characteristic, fusion being induced by A2C in otherwise nonfusible cells if mixed with fusible erythrocytes (13). The molecular basis for the differences observed among the erythrocytes of various species and for the transfer of fusibility has not yet been clarified.We now show that the difference between fusible rat erythrocytes and the nonfusible human cells is correlated with proteolytic activity; degradation of rat cell membrane proteins is caused by a Ca2'-activated cytoplasmic protease(s). The human erythrocyte is deficient in this protease activity. Membrane protein degradation is shown to be a necessary, but not sufficient, prerequisite for membrane fusion. Release of membrane components from constraints imposed by the cytoskeleton, allowing membrane component mobility, is proposed as one of the steps in the fusion process. Furthermore, the mobility of the membrane components must be promoted by...

Fusion of rat erythrocytes by membrane-mobility agent A2C depends on membrane proteolysis by a cytoplasmic calpain

“…Rat erythrocytes fuse easily when treated with A2C and Ca2+, whereas human cells do not fuse under these conditions [12]. We have found the difference in fusibility to be correlated with the occurrence of Ca2+-induced membrane protein degradation [13]. Using rat erythrocyte ghosts we have found membrane protein degradation in the presence of Ca2+ and hemolysate.…”

“…Using rat erythrocyte ghosts we have found membrane protein degradation in the presence of Ca2+ and hemolysate. A2C promotes fusion in these ghosts [13].We have now partially purified and defined the protease involved in the membrane proteolysis associated with A2C-induced fusion. In the intact rat erythrocyte, membrane proteolysis (Ca2+ -dependent) or membrane proteolysis and fusion (Ca' + and A2C-dependent) are prevented by inhibitors to Ca2 +-activated, thiol proteases and by thiol alkylation.…”

Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability

“…Cell-membrane fusion processes, including myoblast fusion, involve destabilization of cell membrane and cytoskeletal framework, allowing the creation of membranefusion-potent domains [1][2][3]. A limited membrane-protein degradation appears to be required for cell fusion, a degradation that would allow membrane disorganization in fusing cells [1][2][3][4][5][6]. It has been proposed that calpain (Ca# + -dependent intracellular protease) is involved in the fusion-associated protein degradation [4][5][6][7][8][9][10][11].…”

“…A limited membrane-protein degradation appears to be required for cell fusion, a degradation that would allow membrane disorganization in fusing cells [1][2][3][4][5][6]. It has been proposed that calpain (Ca# + -dependent intracellular protease) is involved in the fusion-associated protein degradation [4][5][6][7][8][9][10][11]. Most cells, in addition to having one or more of the known calpain isoenzymes, contain the specific inhibitor calpastatin, with ratios of calpain to calpastatin varying among different tissues and cell types [12,13].…”

“…Most cells, in addition to having one or more of the known calpain isoenzymes, contain the specific inhibitor calpastatin, with ratios of calpain to calpastatin varying among different tissues and cell types [12,13]. Using red cells as an experimental model, it was found that the cell fusibility depended on the ratio of calpain to calpastatin [4,6]. In a study on rat L8 myoblast fusion, we found that the µ-calpain and m-calpain levels did not change significantly during myoblast differentiation, whereas calpastatin diminished markedly prior to myoblast fusion and reappeared after fusion [10].…”