Binding and assembly of actin filaments by plasma membranes from Dictyostelium discoideum.

The binding of native, 125I-Bolton-Hunter-labeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. In the presence of gelsolin, the amount of actin bound at saturation to three different membrane preparations was 80, 120, and 200 micrograms/mg of membrane protein. The respective concentrations of actin at half-saturation were 8, 12, and 18 micrograms/ml. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape. In kinetic experiments, actin added as monomers bound to membranes at a rate of 0.6 microgram ml-1 min-1, while pre-polymerized actin bound at a rate of 3.0 micrograms ml-1 min-1. Even in the absence of phalloidin, actin bound to membranes at concentrations well below the normal critical concentration. This membrane-bound actin stained with rhodamine-phalloidin and was cross-linked by m-maleimidobenzoyl succinimide ester, a bifunctional cross-linker, into multimers with the same pattern observed for cross-linked F-actin. We conclude that D. discoideum plasma membranes bind actin specifically and saturably and that these membranes organize actin into filaments below the normal critical concentration for polymerization. This interaction probably occurs between multiple binding sites on the membrane and the side of the actin filament, and may be related to the clustering of membrane proteins.

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How actin binds and assembles onto plasma membranes from Dictyostelium discoideum.

The Journal of Cell Biology ◽

10.1083/jcb.107.1.201 ◽

1988 ◽

Vol 107 (1) ◽

pp. 201-209 ◽

Cited By ~ 26

Author(s):

M A Schwartz ◽

E J Luna

Keyword(s):

Dictyostelium Discoideum ◽

Actin Polymerization ◽

Plasma Membranes ◽

Critical Concentration ◽

Membrane Surface ◽

Tight Binding ◽

Actin Binding ◽

Positive Cooperativity ◽

Tightly Coupled ◽

Membrane Bound

We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross-linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell.

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Rac GTPases regulate the morphology and deformability of the erythrocyte cytoskeleton

Blood ◽

10.1182/blood-2006-03-005942 ◽

2006 ◽

Vol 108 (12) ◽

pp. 3637-3645 ◽

Cited By ~ 69

Author(s):

Theodosia A. Kalfa ◽

Suvarnamala Pushkaran ◽

Narla Mohandas ◽

John H. Hartwig ◽

Velia M. Fowler ◽

...

Keyword(s):

Microcytic Anemia ◽

Cytoskeletal Proteins ◽

Rhodamine Phalloidin ◽

Capping Protein ◽

Cytoskeleton Organization ◽

Rac Gtpases ◽

Actin Capping Protein ◽

Cellular Deformability ◽

Actin Capping ◽

Erythrocyte Cytoskeleton

Abstract Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. Rac1 and Rac2 GTPases regulate actin structures and have multiple overlapping as well as distinct roles in hematopoietic cells; therefore, we studied their role in red blood cells (RBCs). Conditional gene targeting with a loxP-flanked Rac1 gene allowed Crerecombinase–induced deletion of Rac1 on a Rac2 null genetic background. The Rac1–/–;Rac2–/– mice developed microcytic anemia with a hemoglobin drop of about 20% and significant anisocytosis and poikilocytosis. Reticulocytes increased more than 2-fold. Rac1–/–;Rac2–/– RBCs stained with rhodamine-phalloidin demonstrated F-actin meshwork gaps and aggregates under confocal microscopy. Transmission electron microscopy of the cytoskeleton demonstrated junctional aggregates and pronounced irregularity of the hexagonal spectrin scaffold. Ektacytometry confirmed that these cytoskeletal changes in Rac1–/–;Rac2–/– erythrocytes were associated with significantly decreased cellular deformability. The composition of the cytoskeletal proteins was altered with an increased actin-to-spectrin ratio and increased phosphorylation (Ser724) of adducin, an F-actin capping protein. Actin and phosphorylated adducin of Rac1–/–;Rac2–/– erythrocytes were more easily extractable by Triton X-100, indicating weaker association to the cytoskeleton. Thus, deficiency of Rac1 and Rac2 GTPases in mice alters actin assembly in RBCs and causes microcytic anemia with reticulocytosis, implicating Rac GTPases as dynamic regulators of the erythrocyte cytoskeleton organization.

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Structural insights into the regulation of actin capping protein by twinfilin C-terminal tail

Journal of Molecular Biology ◽

10.1016/j.jmb.2021.166891 ◽

2021 ◽

pp. 166891

Author(s):

Shuichi Takeda ◽

Ryotaro Koike ◽

Ikuko Fujiwara ◽

Akihiro Narita ◽

Makoto Miyata ◽

...

Keyword(s):

Capping Protein ◽

Actin Capping Protein ◽

Actin Capping ◽

Structural Insights

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Ca2+-dependent actin-binding phosphoprotein in Physarum polycephalum. Subunit b is a DNase I-binding and F-actin capping protein.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42976-0 ◽

1984 ◽

Vol 259 (8) ◽

pp. 5208-5213

Author(s):

H Maruta ◽

G Isenberg

Keyword(s):

Physarum Polycephalum ◽

Dnase I ◽

Actin Binding ◽

Capping Protein ◽

Actin Capping Protein ◽

Actin Capping ◽

Subunit B

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CapZ integrates several signaling pathways in response to mechanical stiffness

The Journal of General Physiology ◽

10.1085/jgp.201812199 ◽

2019 ◽

Vol 151 (5) ◽

pp. 660-669 ◽

Cited By ~ 3

Author(s):

Christopher Solís ◽

Brenda Russell

Keyword(s):

Mechanical Load ◽

Phosphorylation Site ◽

Tight Binding ◽

Neonatal Rat ◽

Actin Binding ◽

Mechanical Stimuli ◽

Muscle Adaptation ◽

Capping Protein ◽

Actin Capping Protein ◽

Actin Capping

Muscle adaptation is a response to physiological demand elicited by changes in mechanical load, hormones, or metabolic stress. Cytoskeletal remodeling processes in many cell types are thought to be primarily regulated by thin filament formation due to actin-binding accessory proteins, such as the actin-capping protein. Here, we hypothesize that in muscle, the actin-capping protein (named CapZ) integrates signaling by a variety of pathways, including phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) binding, to regulate muscle fiber growth in response to mechanical load. To test this hypothesis, we assess mechanotransduction signaling that regulates muscle growth using neonatal rat ventricular myocytes cultured on substrates with the stiffness of the healthy myocardium (10 kPa), fibrotic myocardium (100 kPa), or glass. We investigate how PIP2 signaling affects CapZ using the PIP2 sequestering agent neomycin and the effect of PKC-mediated CapZ phosphorylation using the PKC-activating drug phorbol 12-myristate 13-acetate (PMA). Molecular simulations suggest that close interactions between PIP2 and the β-tentacle of CapZ are modified by phosphorylation at T267. Fluorescence recovery after photobleaching (FRAP) demonstrates that the kinetic binding constant of CapZ to sarcomeric thin filaments in living muscle cells increases with stiffness or PMA treatment but is diminished by PIP2 reduction. Furthermore, CapZ with a deletion of the β-tentacle that lacks the phosphorylation site T267 shows increased FRAP kinetics with lack of sensitivity to PMA treatment or PIP2 reduction. Förster resonance energy transfer (FRET) probes the molecular interactions between PIP2 and CapZ, which are decreased by PIP2 availability or by the β-tentacle truncation. These data suggest that CapZ is bound to actin tightly in the idle, locked state, with little phosphorylation or PIP2 binding. However, this tight binding is loosened in growth states triggered by mechanical stimuli such as substrate stiffness, which may have relevance to fibrotic heart disease.

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