A method for the morphological identification of contractile filaments in single cells

195

153

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,

Section: Measurement Ofmentioning

confidence: 99%

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

Condeelis¹,

Taylor²

1977

195

153

“…A drop of each sample was applied to a Formvar carbon-coated grid and treated according to the technique described by Moore et al (26).…”

Section: Negative Stainingmentioning

confidence: 99%

“…is interesting that both spindle-and spread-shaped myosin filaments are also observed in negatively stained preparations of amoeba cytoplasm (26). 2 The physiological implications are being investigated.…”

Section: L Taylor Slitdie$ On Polarization Optical Properties Of mentioning

confidence: 99%

Quantitative studies on the polarization optical properties of striated muscle. I. Birefringence changes of rabbit psoas muscle in the transition from rigor to relaxed state.

Dl¹

1976

The changes in birefringence in the rigor to relax transition of single tritonextracted rabbit psoas muscle fibers have been investigated with quantitative polarized light techniques. The total birefringence of rest length fibers in rigor was (1.46 -4-0.08) x 10 -s and increased to (1.67 • 0.05) • 10 -s after Mg-ATP relaxation. Pyrophosphate relaxation increased the total birefringence only slightly, whereas subsequent Mg-ATP relaxation elicited the maximum increase in birefringence. Changes in lattice spacing did not account for the total increase in birefringence during relaxation. Moreover, the increase in total birefringence was attributable to increases in intrinsic birefringence as well as form birefringence. No change in birefringence was exhibited upon exposure to a relaxation solution after myosin extraction.Synthetic myosin filaments were prepared and treated with relaxation and rigor solutions. The negatively stained filaments treated with a rigor solution had gross irregular projections at either end, while the filaments treated with a relaxing solution were more spindle shaped. The results are compatible with the view that the subfragment-2 moieties of myosin angle away from the myosin aggregates (light meromyosin) to permit the attachment of the subfragment-I moieties to actin.It is commonly accepted that the force generation mechanism in vertebrate striated muscle involves the angular movements of the subfragment-I (S-I) moieties upon attachment to the actin filaments (16,15,9,27). However, a simple mechanical interaction between the S-I moieties and actin is not possible since the center-to-center spacing between actin and myosin varies at different sarcomere lengths (18). At rest length (ca. 2.70 /~m) in rabbit psoas muscle, the surface-to-surface spacing between actin and myosin is greater than the largest lengths attributed to the S-I moieties of myosin (16,23,25).H. E. Huxley postulated that the subfragment-2 (S-2) moieties of myosin might function as hinges between the light meromyosin (LMM) backbones and the S-I moieties of myosin. Therefore the movements of S-2 moieties away from the long axis of the myosin backbones would permit the attachment of the S-I moieties at the same orienta-

“…The negative staining procedure was modified from previously published methods (23,33,7). Aliquots of the Taylor, D. L., and R. Zeh.…”

Section: Negative Staining Of Cytoplasmic Extractsmentioning

confidence: 99%

“…11 b). Many regions exhibited both actin and thicker filaments which appeared similar to myosin aggregates (33,23,7) (Fig. 12 a), as well as areas suggestive of a transformation from a less filamentous to a more filamentous state of actin (Fig.…”

mentioning

confidence: 99%

The contractile basis of ameboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts.

Taylor¹,

Rhodes²,

Hammond³

1976

The role of calcium and magnesium-ATP on the structure and contractility in motile extracts of Amoeba proteus and plasmalemma-ectoplasm "ghosts" of Chaos carolinensis has been investigated by correlating light and electron microscope observations with turbidity and birefringence measurements. The extract is nonmotile and contains very few F-actin filaments and myosin aggregates when prepared in the presence of both low calcium ion and ATP concentrations at an ionic strength of I = 0.05, pH 6.8. The addition of 1.0 mM magnesium chloride, 1.0 mM ATP, in the presence of a low calcium ion concentration (relaxation solution) induced the formation of some fibrous bundles of actin without contracting, whereas the addition of a micromolar concentration of calcium in addition to 1.0 mM magnesium-ATP (contraction solution) (Taylor, D. L., J. S. Condeelis, P. L. Moore, and R. D. Allen. 1973. J. Cell Biol. 59:378-394) initiated the formation of large arrays of F-actin filaments followed by contractions. Furthermore, plasmalemma-ectoplasm ghosts prepared in the relaxation solution exhibited very few straight F-actin filaments and myosin aggregates. In contrast, plasmalemma-ectoplasm ghosts treated with the contraction solution contained many straight F-actin filaments and myosin aggregates. The increase in the structure of ameba cytoplasm at the endoplasm-ectoplasm interface can be explained by a combination of the transformation of actin from a less filamentous to a more structured filamentous state possibly involving the cross-linking of actin to form fibrillar arrays (see above-mentioned reference) followed by contractions of the actin and myosin along an undetermined distance of the endoplasm and/or ectoplasm.Experimental motile model systems have played a significant role in understanding the structural and chemical dynamics of cell movements. Simard-Duquesne and Couillard (31) elicited visible contractions in glycerinated models of Amoeba proteus upon the addition of magnesium and ATP. Cytoplasmic contractions and streaming have been identified both in cytoplasm removed from the plasmalemma (l, 12, 33) and in fractionated cytoplasm (39,46,27). In addition, it has been shown that cytoplasm isolated from single specimens of Chaos carolinensis contracted upon the addition of at least 7.0 • l0 -7 M free calcium ions in the presence of an endogenous magnesium-ATP