Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4°C in a stabilization solution (60). These extracts gelled on warming to 25°C 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(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'bis[2-ethane sulfonic acid] (PIPES), 1 mM MgCl2, 1 mM ATP, and 20 mM KCl 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 KCl, 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 -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, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producing contraction suggests that the regulation of contraction by the gel is structural in nature.
ASJC Scopus subject areas
- Cell Biology