Dynamics of F-actin and F-actin complexes

Carlson, Francis D.; Fraser, Allan B.

doi:10.1016/0022-2836(74)90518-x

Cited by 73 publications

(20 citation statements)

References 14 publications

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“…2 mg/ml (exceeding those in the gelled extracts) was incapable of gelation. This is consistent with the properties of purified macrophage actin (58), muscle actin (6), and Acanthamoeba actin (38) which do not gel at low concentrations (ca. 2 mg/ml) in the absence of actin-binding proteins.…”

Section: Molecular Basis Of Gelationsupporting

confidence: 89%

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

Condeelis¹,

Taylor²

1977

The Journal of Cell Biology

195

153

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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,

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Section: Molecular Basis Of Gelationsupporting

confidence: 89%

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

Condeelis¹,

Taylor²

1977

The Journal of Cell Biology

195

153

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“…Swelling may break crosslinks in the cytoplasm, resulting in a looser structure with decreased viscosity. This effect may be similar to the sol-to-gel transformation of actin/heavy meromyosin mixtures, where larger and more rapid intensity fluctuations are measured in the presence of ATP (18). Piddington and Sattelle (5) reported that dilution of the During periods of time indicated by the solid bars 500 mM K+ and only 12 mM Cl-were present.…”

Section: Resultssupporting

confidence: 57%

Effect of increased potassium concentrations on particle motion within a neurosecretory structure.

Englert¹,

Edwards²

1977

Proc. Natl. Acad. Sci. U.S.A.

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Intensity fluctuation spectroscopy was used to detect particle motion within the pericardial organ of the crab Carcinus while it was perfused with media with increased potassium concentrations. Replacement of some or all of the sodium ions of the normal medium with potassium ions caused a graded increase in motion. Evidence is presented which indicates that this effect is not a consequence of membrane depolarization or calcium ion influx, but is due to the flux of potassium chloride and water into the cytoplasm of the cells. The observation of Shaw and Newby has been confirmed and extended by Piddington and Sattelle (5). They found, however, that omission of calcium ions from the medium had no effect on the potassium response (6). Despite this, they nevertheless attributed the scattering change to calcium entry.We have investigated this phenomenon to determine whether it has any relevance to the mechanism of neurotransmitter release. The pericardial organ of the crab Carcinus was used. This is a specialized neurosecretory structure, the cortex of which is packed tightly with neurosecretory endings containing a variety of granules and vesicles (7). It usually has a bluish color when viewed under a dissecting lamp, probably due to light scattering by the granular inclusions of the nerve endings (8). METHODSSpecimens of Carcinus were obtained from the Marine Biological Laboratory at Woods Hole and kept in a tank of artificial sea water at about 15'C. The pericardial organ was dissected from an animal in artificial sea water and transferred to a microscope slide to which it was fastened with Vaseline. The microscope slide formed the bottom of a perfusion chamber which could be covered and sealed with a cover slip. This chamber was then mounted on the stage of a specially modified Zeiss WL microscope (Fig. 1).This microscope permits observation of preparations with the incandescent illuminator of the microscope as well as with laser light. The beam of a 4-mW He/Ne laser (Coherent Radiation model 80) passes through a lens and is reflected from a half-silvered mirror and focused onto the back focal plane of the microscope condenser (numerical aperture = 1.4). The beam then passes through the preparation at an angle determined by the lateral displacement of the beam off the optical axis of the microscope (9). This angle can be adjusted between 0°and almost 90°by means of the micrometer drive on the lens-mirror assembly.An iris diaphragm in the image plane of the microscope can limit the field to an area 27 gam in diameter (using a 40X objective). This diaphragm and the image of the preparation can be observed through an ocular or, by removing a mirror from the system, the light scattered from the preparation can be passed through a pair of lenses that bring the back focal plane of the microscope objective (40X, numerical aperture = 0.65) to focus on an aperture in front of the cathode of a photomultiplier tube. The position of this aperture in the back focal plane determines the angle at which light is collected...

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“…stroyed by very small shear forces (2 1). Muscle actin combined with heavy meromyosin (22) or alpha-actinin (21) will form a solid gel. Similarly, pure macrophage actin does not form a solid gel unless combined with high molecular weight actin-binding protein (1 7).…”

Section: Contemporary Biochemical Stud1 Esmentioning

confidence: 99%

Cytoskeletal functions of cytoplasmic contractile proteins

Pollard

1976

J Supramol Struct

104

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This is a review of the evidence that the cytoplasmic contractile proteins function as a cytoskeletal system in the cytoplasmic matrix. Biochemical experiments show that cycoplasmic actin filaments can form a solid gel under conditions likely to exist in living cells. The actin filaments are associated with other proteins which amy stabilize the gel and which are involved with motile force generation like myosin. Ultrastructural studies show that actin filaments are difficult to preserve, but that under stabilizing conditions networks of actin filaments are found throughtout the cytoplasmic matrix.

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Dynamics of F-actin and F-actin complexes

Cited by 73 publications

References 14 publications

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

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

Effect of increased potassium concentrations on particle motion within a neurosecretory structure.

Cytoskeletal functions of cytoplasmic contractile proteins

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