Membrane interactions with the actin cytoskeleton (original) (raw)
Related papers
The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly
Journal of Cell Biology, 1993
Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma mem-9 The Rockefeller University Press, over-end with the actin beads in a plastic tube for 1 h at 4~ Beads and sample were returned to the column and the unbound fraction (run-throngh) was collected. The beads were washed with I0 ml of I% 0(3, 0.5 ~M phalloidin, CB and the ponticulin-containing fraction was eluted with 5 mi of 2 M NaCI, 2 mM EGTA, I% OG, 0.5 ~M phalloidin, CB. The beads were re.equilibrated, reincubated with the run-through fraction, and eluted as above once or twice more. (With freshly preFared F-actin beads, two incubations and NaCl elutions were sufficient to remove all ponticulin from the run-through; older beads sometimes required a third round.) The NaCl eluates were pooled and concentrated to ~2 ml in a Centricon-10 (Amicon). The concentrated NaCI eluates were dialyzed against I% OG, CB at 4~ for at least 24 h.
Journal of Cell Biology, 1987
F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gpl7) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gpl7 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gpl7 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gpl7 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gpl7 9 The Rockefeller University Press, 0021-9525/
2000
In novel, low-speed sedimentation assays, highly purified, sonicated Dictyostelium discoideum plasma membrane fragments bind to F-actin beads (fluoresce in-labeled F-actin on antifluorescein IgG-Sephacryl S-1000 beads) . Binding was found to be (a) specific, since beads containing bound fluorescein-labeled ovalbumin or beads without bound fluorescein-labeled protein do not bind membranes, (b) saturable at -0.6 ug of membrane protein per microgram of bead-bound F-actin, (c) rapid with a b,, of 4-20 min, and (d) apparently of reasonable affinity since the off rate is too slow to be measured by present techniques. Using low-speed sedimentation assays, we found that sonicated plasma membrane fragments, after extraction with chaotropes, still bind F-actin beads. Heat-denatured membranes, proteolyzed membranes, and D. discoideum lipid vesicles did not bind F-actin beads. These results indicate that integral membrane proteins are responsible for the binding between sonicated membrane fragments and F-actin on beads . This finding agrees with the previous observation that integral proteins mediate interactions between D. discoideum plasma membranes and F-actin in solution (Luna, E. J ., V. M. Fowler, J . Swanson, D. Branton, and D. L. Taylor, 1981, J. Cell Biol., 88 :396-409). We conclude that low-speed sedimentation assays using F-actin beads are a reliable method for monitoring the associations between F-actin and membranes . Since these assays are relatively quantitative and require only micrograms of membranes and F-actin, they are a significant improvement over other existing techniques for exploring the biochemical details of F-actin-membrane interactions .
2000
Dictyosteliumdiscoideum plasma membranes isolatedby each of three procedures bind F-actin.The interactions between these membranes and actin are examined by a novel application of fallingball viscometry .Treating the membranes as multivalent actin-binding particlesanalogous to divalent actin-gelation factors,we observe large increases in viscosity (actincross-linking)when membranes depleted of actinand myosin are incubated with rabbit skeletalmuscle F-actin.Pre-extractionof peripheral membrane proteins with chaotropes or the
How actin binds and assembles onto plasma membranes from Dictyostelium discoideum
Journal of Cell Biology, 1988
We have shown previously . 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, Samples with or without 350 ktg/ml plasma membranes and with or without 5 ~tM phalloidin were prepared in 20 I.tl of polymerization buffer with 0.02 % Tween 20. Then, either ~25I-labeled EF actin or ~25I-labeled untreated actin
Diacylglycerol-stimulated formation of actin nucleation sites at plasma membranes
Science, 1992
lation in normal embryos to that in embryos that were UV-irradiated as zygotes to block subcortical rotation. The IP3 mass at the 256-cell stage was not significantly different in UV-irradiated (100.2 ± 10.8 fmol per embryo) and control embryos (103.6 ± 9.0 fmol per embryo) (16). Subcortical rotation is therefore not a prerequisite for IP3 accumulation in the cleavage stage embryo, and our data do not support the suggestion that zygotic UV irradiation should globally enhance PI cycle activity during mesoderm induction .
Binding and assembly of actin filaments by plasma membranes from dictyostelium discoideum
Journal of Cell Biology, 1986
The binding of native, 125I-Bolton-Hunterlabeled 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 ~g/mg of membrane protein. The respective concentrations of actin at half-saturation were 8, 12, and 18 ~g/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.
Ponticulin is an atypical membrane protein
Journal of Cell Biology, 1994
We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at •8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no u-helical membrane-spanning domains are apparent, several hydrophobic and/or sided/3-strands, each long enough to traverse the membrane, are predicted. Al-