Release of myosin II from the membrane-cytoskeleton of Dictyostelium discoideum mediated by heavy-chain phosphorylation at the foci within the cortical actin network

Abstract

Abstract. Membrane-cytoskeletons were prepared from Dictyostelium amebas, and networks of actin and myosin II filaments were visualized on the exposed cytoplasmic surfaces of the cell membranes by fluorescence staining (Yumura, S., and T. . Cell . Addition of ATP caused contraction of the cytoskeleton with aggregation of part of actin into several foci within the network, but most of myosin II was released via the foci. However, in the presence of 10 mM MgCI2, which stabilized myosin II filaments, myosin II remained at the foci. Uttrastructural examination revealed that, after contraction, only traces of monomeric myosin II remained at the foci. By contrast, myosin H filaments remained in the foci in the presence of 10 mM MgCI2. These observations suggest that myosin II was released not in a filamentous form but in a monomeric form.Using [732P]ATP, we found that the heavy chains of myosin II released from membrane-cytoskeletons were phosphorylated, and this phosphorylation resulted in disassembly of myosin filaments. Using ITP (a substrate for myosin 1I ATPase) and/or ATP3tS (a substrate for myosin II heavy-chain kinase [MHCK]), we demonstrated that phosphorylation of myosin heavy chains occurred at the foci within the actin network, a result that suggests that MHCK was localized at the loci. These results together indicate that, during contraction, the heavy chains of myosin II that have moved toward the foci within the actin network are phosphorylated by a specific MHCK, with the resultant disassembly of filaments which are finally released from membrane-cytoskeletons. This series of reactions could represent the mechanism for the relocation of myosin II from the cortical region to the endoplasm. MYOSIS II, which is one of the major components of the cytoskeleton in nonmuscle cells, produces the motive force necessary for cell movements and cytokinesis via interactions with actin filaments. Myosin II isolated from Dictyostelium amebas can assemble into bipolar thick filaments in vitro (28). Immunofluorescence studies and irm'nunoelectron microscopy have shown that myosin II in Dictyostelium amebas, similar to myosin in muscle cells, forms filaments in vivo (30, 31). In addition, most of the myosin II in Triton-Xl00-insoluble cytoskeletons of Dictyostelium amebas is in the filamentous form (4, 27). All these observations suggest that, in Dictyostelium aniebas, actin and myosin II generate the motive force by a mechanism analogous to the sliding-filament model of actomyosin in muscle cells. However, actin and myosin filaments in D/ctyostelium amebas, unlike those in muscle cells, show no evidence of any regular arrangement such as that observed in the sarcomere in muscle cells and, in addition, they do not stay at a single site but can relocate within a cell. For example, myosin H filaments are concentrated at the tail region during locomotion, while they are concentrated in the furrow region to form the contractile ring during cell division (12,30,33). Upon chemotactic stimulation of cells at the aggregation st...

Dictyostelium Myosin II Is Regulated during Chemotaxis by a Novel Protein Kinase C

“…Stimulation by cAMP results in dissociation of myosin II from the cytoskeleton and increase in MHC phosphorylation, possibly as a result of cAMP-induced MHC-PKC translocation to the membrane and phosphorylation of the cortical myosin II. These results are consistent with the results reported by Yumura and Kitanishi-Yumura (31), which indicate that during contraction, the myosin II that have moved toward the foci are phosphorylated by a specific MHCK that is localized at the foci, with the resultant disassembly of filaments which are finally released from membrane cytoskeleton.…”

“…In addition to MHC-PKC, several other MHCKs have been identified in Dictyostelium (8,29,30 Overexpression of MHC-PKC resulted in highly phosphorylated MHC that does not represent new phosphorylation sites. MHC-PKC phosphorylates four sites on MHC in vitro (9); nevertheless, the in vivo levels of phosphate incorporation into MHC are very low (0.05 mol of phosphate/mol of MHC) (3,31). It was proposed that incorporation of a single phosphate to a single myosin II filament is enough for filament disassembly (31).…”

“…MHC-PKC phosphorylates four sites on MHC in vitro (9); nevertheless, the in vivo levels of phosphate incorporation into MHC are very low (0.05 mol of phosphate/mol of MHC) (3,31). It was proposed that incorporation of a single phosphate to a single myosin II filament is enough for filament disassembly (31). It is therefore conceivable that in vivo MHC-PKC phosphorylates small fraction of MHC and/or less then four sites on a single MHC, which is sufficient for filament disassembly.…”

Rapid Translocation of Myosin II in Vegetative Dictyostelium Amoebae during Chemotactic Stimulation by Folic Acid.

“…When number of actin foci began to increase, filaments of myosin II decreased in number. In a previous study, it was shown that myosin II is gathered at the actin foci and released from intact membrane-cytoskeletons when ATP is applied (31). In that study, it was demonstrated that myosin II was phosphorylated at the actin foci, disassembled to monomers,and released from the membrane.…”

Contraction speed of the actomyosin cytoskeleton in the absence of the cell membrane

“…58 The term remodeling is used in a general sense, including bending and buckling of the actin filaments (due to their low flexural rigidity [59][60][61] ), the possible redistribution of the cross-links and depolymerization of filaments along which the myosin heads slide. In our experiments, the actin filaments were stabilized by 0.5% polyethylene glycol in the medium 62 and the time required for the contraction of the wt cytoskeletons on a low adhesiveness substrate was of the order of 15 s, i.e.…”