The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Abstract

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4~ in a stabilization solution (60). These extracts gelled on warming to 25~ and contracted in response to micromolar Ca ++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis(/3-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis[2-ethane sulfonic acid] (PIPES), 1 mM MgCI2, 1 mM ATP, and 20 mM KCI at pH 7.0 (relaxation solution), while micromolar levels of Ca ++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca ++, pH >7.0, cytochalasin B, elevated concentrations of KC1, MgCl2, and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca ++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material squeezed out the contracting extract; (c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4-to 10-fold.In the absence of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absence of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding purified Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++-regulated gelation.These results indicated: (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel,

Regulation of intracellular pH in eukaryotic cells

“…The contractile activity of purified preparations of actin and myosin has been shown to be dramatically influenced by comparatively small changes in pH (Condeelis & Taylor, 1977) with low pH reducing the contractility. Also, microtubule assembly and disassembly is affected by pH with an increased disassembly at alkaline pH (Regula et al, 1981).…”

Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes.

“…Purification of a 67-kDa Protein (p67) from a Contracted Actin-Myosin Complex Contracted actin-myosin complex was prepared from cells starved for 12 h essentially as described previously (Condeelis and Taylor, 1977;Fechheimer and Taylor, 1984;de Hostos et al, 1991). Washed cells were resuspended at 4°C in an equal volume of homogenization buffer [5 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), pH 6.8, 5 mM ethylene glycol-bis(,B-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail (PIC) consisting of leupeptin, bestatin, pepstatin A, antipain, 10 ,ug/ml each] and disrupted by nitrogen decompression in a Parr bomb (800 psi), followed by gentle agitation with a loose fitting Dounce homogenizer.…”

Isolation of a high molecular weight actin-binding protein from baby hamster kidney (BHK-21) cells.

“…11). On the other hand, gelation of purified ABP and actin, or of extracts from several cell types, is temperature and calcium sensitive (10,(13)(14)(15). The different observations may be due to changes in the HMWP during isolation, such as partial denaturation or proteolysis, or to loss of required regulatory factors.…”

“…HMWPs that interact with actin have been implicated in cytoplasmic consistency changes (3,10,11,(13)(14)(15)19). Macrophage ABP and smooth muscle filamin have been isolated and appear to be similar (7)(8)(9)(10).…”

“…Actin-binding protein (ABP) from macrophages (5) and leukocytes (6) and filamin from smooth muscle (7)(8)(9) have similar, but not identical, physical and biological characteristics, such as size (two identical 250,000-dalton subunits), amino acid composition, and their ability to participate with purified actin in a gelation reaction (5)(6)(7)(8)(9)(10)(11). Electrophoretic or immunological analyses have resulted in identification of proteins similar to ABP and filamin in extracts of many other cells (12)(13)(14)(15)(16)(17), including cultured mammalian cells (18)(19)(20). In addition to the high molecular weight actin crosslinkers, several low molecular weight gelation factors have been isolated from Acanthamoeba (21).…”