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T D Pollard

Publications and source records attributed to T D Pollard.

At least 19 recordsLinked to original sources
PubMed1992

Nucleotide exchange, structure, and mechanical properties of filaments assembled from ATP-actin and ADP-actin.

Actin monomers with bound ATP, ADP, or fluorescent analogues of these nucleotides exchange the nucleotide on a second time scale, whereas filaments assembled from each of these species exchange their nucleotide with the solution at least 1,000 times slower than monomers. Filaments assembled from either ATP-actin or ADP-actin are indistinguishable by electron microscopy after negative staining. The dynamic elasticity and viscosity of filaments assembled from ATP-actin or ADP-actin or mixtures of these two species are the same over a wide range of frequencies. These observations do not support a recent suggestion (Janmey, P. A., Hvidt, S., Oster, G. F., Lamb, J., Stossel, T. P., and Hartwig, J. H. (1990) Nature 347, 95-99) that ATP hydrolysis within actin filaments stiffens the polymer and alters both their structure and affinity for nucleotides. The difference in observations between these two studies may be related to time-dependent changes in ADP-actin prepared in slightly different ways.

Actin Cytoskeleton↗
PubMed1992

Cytokinesis.

The actomyosin contractile-ring mechanism remains the paradigm for cytokinesis after 20 years of experimental testing. Recent evidence suggests that Ca2+ triggers the contraction and that cell-cycle kinases regulate the timing of cytokinesis. New work is required to identify the signals from the mitotic spindle that specify the position of the furrow.

Actomyosin↗
PubMed1992

Myosin-I moves actin filaments on a phospholipid substrate: implications for membrane targeting.

Acanthamoeba myosin-I bound to substrates of nitrocellulose or planar lipid membranes on glass moved actin filaments at an average velocity of 0.2 micron/s. This movement required ATP and phosphorylation of the myosin-I heavy chain. We prepared planar lipid membranes on a glass support by passive fusion of lipid vesicles (Brian, A. A., and H. M. McConnell. 1984. Proc. Natl. Acad. Sci. USA. 81:6159-6163) composed of phosphatidylcholine and containing 0-40% phosphatidylserine. The mass of lipid that bound to the glass was the same for membranes of 2 and 20% phosphatidylserine in phosphatidylcholine and was sufficient to form a single bilayer. Myosin-I moved actin filaments on planar membranes of 5-40% but not 0-2% phosphatidylserine. At the low concentrations of phosphatidylserine, actin filaments tended to detach suggesting that less myosin-I was bound. We used the cooperative activation of Acanthamoeba myosin-I ATPase by low concentrations of actin to assess the association of phospholipids with myosin-I. Under conditions where activity depends on the binding of actin to the tail of myosin-I (Albanesi, J. P., H. Fujisaki, and E. D. Korn. 1985. J. Biol. Chem. 260:11174-11179), phospholipid vesicles with 5-40% phosphatidylserine inhibited ATPase activity. The motility and ATPase results demonstrate a specific interaction of the tail of myosin-I with physiological concentrations of phosphatidylserine. This interaction is sufficient to support motility and may provide a mechanism to target myosin-I to biological membranes.

Acanthamoeba↗
PubMed1992

Localization and specificity of the phospholipid and actin binding sites on the tail of Acanthamoeba myosin IC.

We used bacterially expressed beta-galactosidase fusion proteins to localize the phospholipid binding domain of Acanthamoeba myosin IC to the region between amino acids 701 and 888 in the NH2-terminal half of the tail. Using a novel immobilized ligand lipid binding assay, we determined that myosin I can bind to several different acidic phospholipids, and that binding requires a minimum of 5 mol% acidic phospholipid in a neutral lipid background. The presence of di- and triglycerides and sterols in the lipid bilayer do not contribute to the affinity of myosin I for membranes. We confirm that the ATP-insensitive actin binding site is contained in the COOH-terminal 30 kD of the tail as previously shown for Acanthamoeba myosin IA. We conclude that the association of the myosin IC tail with acidic phospholipid head groups supplies much of the energy for binding myosin I to biological membranes, but probably not specificity for targeting myosin I isoforms to different cellular locations.

Acanthamoeba↗
PubMed1992

Phosphorylation of myosin-II regulatory light chain by cyclin-p34cdc2: a mechanism for the timing of cytokinesis.

To understand how cytokinesis is regulated during mitosis, we tested cyclin-p34cdc2 for myosin-II kinase activity, and investigated the mitotic-specific phosphorylation of myosin-II in lysates of Xenopus eggs. Purified cyclin-p34cdc2 phosphorylated the regulatory light chain of cytoplasmic and smooth muscle myosin-II in vitro on serine-1 or serine-2 and threonine-9, sites known to inhibit the actin-activated myosin ATPase activity of smooth muscle and nonmuscle myosin (Nishikawa, M., J. R. Sellers, R. S. Adelstein, and H. Hidaka. 1984. J. Biol. Chem. 259:8808-8814; Bengur, A. R., A. E. Robinson, E. Appella, and J. R. Sellers. 1987. J. Biol. Chem. 262:7613-7617; Ikebe, M., and S. Reardon. 1990. Biochemistry. 29:2713-2720). Serine-1 or -2 of the regulatory light chain of Xenopus cytoplasmic myosin-II was also phosphorylated in Xenopus egg lysates stabilized in metaphase, but not in interphase. Inhibition of myosin-II by cyclin-p34cdc2 during prophase and metaphase could delay cytokinesis until chromosome segregation is initiated and thus determine the timing of cytokinesis relative to earlier events in mitosis.

Amino Acid Sequence↗
PubMed1992

The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells.

We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.

Actins↗
PubMed1992

The localization of myosin I and myosin II in Acanthamoeba by fluorescence microscopy.

We used several fixation protocols and a panel of monoclonal antibodies to re-examine the localization of myosin I and myosin II in Acanthamoeba. Two monoclonal antibodies that bind to the head of myosin II stain a range of particles in the cytoplasm. The smallest and most numerous cytoplasmic particles are about the same size and intensity as myosin II minifilaments and are distributed throughout the endoplasm. The largest particles stain like myosin II thick filaments and are concentrated in the cleavage furrow of dividing cells and in the tail of locomoting cells. Five different monoclonal antibodies that bind to the myosin II tail also stain cytoplasmic particles but with a limited range of intensity. None of the myosin II monoclonal antibodies stains the contractile vacuole or plasma membrane. Two monoclonal antibodies to myosin I gave punctate cytoplasmic staining that did not correspond clearly to any of the phase-dense particles in the cytoplasm. In many, but not all, locomoting cells, the myosin I staining was concentrated at the leading edge. Both myosin I antibodies stained a single cytoplasmic vacuole of variable size that was presumed to be the contractile vacuole. The antibody that binds myosin IA but not myosin IB stained novel intercellular contacts and the antibody that binds both myosin IA and myosin IB stained the plasma membrane, especially the tips of filopodia.

Acanthamoeba↗
PubMed1991

Evidence for a functional link between profilin and CAP in the yeast S. cerevisiae.

CAP is a component of the S. cerevisiae adenylyl cyclase complex. The N-terminal domain is required for cellular RAS responsiveness. Loss of the C-terminal domain is associated with morphological and nutritional defects. Here we report that cap- cells bud randomly and are defective in actin distribution. The morphological and nutritional defects associated with loss of the CAP C-terminal domain are suppressed by over-expression of PFY, the gene encoding profilin, an actin- and polyphosphoinositide-binding protein. The phenotype of cells lacking PFY resembles that of cells lacking the CAP C-terminal domain. Study of mutated yeast profilins and profilins from Acanthamoeba suggests that the ability of profilin to suppress cap- cells is dependent upon a property other than, or in addition to, its ability to bind actin. This property may be its ability to bind polyphosphoinositides. We propose that CAP and profilin provide a link between growth signals and remodeling of the cellular cytoskeleton.

Actins↗
PubMed1991

Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation.

Epidermal growth factor and platelet-derived growth factor can stimulate the production of the second messenger inositol trisphosphate in responsive cells, but the biochemical pathway for these signaling events has been uncertain because the reactions have not been reconstituted with purified molecules in vitro. A reconstitution is described that requires not only the growth factor, its receptor with tyrosine kinase activity, and the soluble phospholipase C-gamma 1, but also the small soluble actin-binding protein profilin. Profilin binds to the substrate phosphatidylinositol 4,5-bisphosphate and inhibits its hydrolysis by unphosphorylated phospholipase C-gamma 1. Phosphorylation of phospholipase C-gamma 1 by the epidermal growth factor receptor tyrosine kinase overcomes the inhibitory effect of profilin and results in an effective activation of phospholipase C-gamma 1.

Animals↗
PubMed1991

Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments.

We used electron microscopy to measure the effects of cytochalasins, phalloidin, and pH on the rates of elongation at the barbed and pointed ends of actin filaments. In the case of the cytochalasins, we compared the effects on ATP- and ADP-actin monomers. Micromolar concentrations of either cytochalasin B (CB) or cytochalasin D (CD) inhibit elongation at both ends of the filament, about 95% at the barbed end and 50% at the pointed end, so that the two ends contribute about equally to the rate of growth. Half-maximal inhibition of elongation at the barbed end is at 0.1 microM CB and 0.02 microM CD for ATP-actin and at 0.1 microM CD for ADP-actin. At the pointed end, CD inhibits elongation by ATP-actin and ADP-actin about equally. At high (2 microM) concentrations, the cytochalasins reduce the association and dissociation rate constants in parallel for both ADP- and ATP-actin, so their effects on the critical concentrations are minimal. These observations confirm and extend those of Bonder and Mooseker [Bonder, E. M., & Mooseker, M. S. (1986) J. Cell Biol. 102, 282-288]. The dependence of the elongation rate on the concentration of both cytochalasin and actin can be explained quantitatively by a mechanism that includes the effects of cytochalasin binding to actin monomers [Godette, D. W., & Frieden, C. (1986) J. Biol. Chem. 261, 5974-5980] and a partial cap of the barbed end of the filament by the complex of ADP-actin and cytochalasin.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗
PubMed1991

Analysis of cDNA clones for Acanthamoeba profilin-I and profilin-II shows end to end homology with vertebrate profilins and a small family of profilin genes.

We have cloned and sequenced full length cDNAs for Acanthamoeba profilin-I and profilin-II. The genes and the encoded proteins are nearly identical except for the region between bp 121 and 210 where 35% of the nucleotides and 47% of amino acids differ. Most of these substitutions are conservative, although three of them are responsible for the differences in the isoelectric points of the isoforms [Kaiser et al., Cell Biol., 102:221-226, 1986]. The DNA sequence revealed six corrections in the previously published protein sequence of profilin-I [Ampe et al., J. Biol. Chem. 260:834-840, 1985] and for the first time resolved the ambiguities at the five positions where profilin-IA and -IB differ. The DNA sequence of profilin-II also allowed us to make two corrections in the protein sequence [Ampe et al., FEBS Lett. 228:17-21, 1988a]. Probes prepared from the cDNAs revealed 1 profilin-IA gene, one strongly cross-hybridizing profilin-I gene and one strongly reacting profilin-II gene on Southern blots of Acanthamoeba DNA. Weaker reactions with other genomic DNA fragments leave open the possibility of one additional gene each for profilin-I and profilin-II. Four different profilin RNAs were resolved on Northern blots. It possible to align the sequences of the three Acanthamoeba profilins with the sequences of nine other profilins from five different phyla. There are only two invariant residues in these profilin sequences, but many pairwise identities and conservative substitutions that indicate considerable divergence of this family of proteins from its ancestral precursor.

Acanthamoeba↗
PubMed1991

Functional implications of the unusual amino acid sequence of the regulatory light chain of Acanthamoeba castellanii myosin-II.

We have determined by protein chemistry methods the amino acid sequence of light chain 2 from Acanthamoeba castellanii myosin-II (ALC2). This is the first reported sequence for any protozoan myosin light chain. ALC2 consists of 154 amino acid residues, including a single residue of His and two residues each of Pro and Tyr, and lacks Cys and Trp. The N-terminus is blocked, and if an N-terminal acetyl group is assumed. ALC2 has a calculated molecular weight of 17,657. ALC2 is an acidic protein, with a calculated net charge of -7 at pH 7. The sequence of ALC2 is most similar to those of the calmodulins (identity approximately 35%), followed by myosin regulatory light chains. ALC2 appears to lack the potential N-terminal phosphorylation site and single Ca(2+)-binding site in region I which are characteristic of most myosin regulatory light chains. Instead, ALC2, unlike any other myosin light chain characterized to date, may have a functional Ca(2+)-binding site only in region II, suggesting a novel role of ALC2 in the Ca2+ regulation of the activity of Acanthamoeba myosin-II.

Acanthamoeba↗
PubMed1991

Mechanism of the interaction of human platelet profilin with actin.

We have reexamined the interaction of purified platelet profilin with actin and present evidence that simple sequestration of actin monomers in a 1:1 complex with profilin cannot explain many of the effects of profilin on actin assembly. Three different methods to assess binding of profilin to actin show that the complex with platelet actin has a dissociation constant in the range of 1 to 5 microM. The value for muscle actin is similar. When bound to actin, profilin increases the rate constant for dissociation of ATP from actin by 1,000-fold and also increases the rate of dissociation of Ca2+ bound to actin. Kinetic simulation showed that the profilin exchanges between actin monomers on a subsecond time scale that allows it to catalyze nucleotide exchange. On the other hand, polymerization assays give disparate results that are inconsistent with the binding assays and each other: profilin has different effects on elongation at the two ends of actin filaments; profilin inhibits the elongation of platelet actin much more strongly than muscle actin; and simple formation of 1:1 complexes of actin with profilin cannot account for the strong inhibition of spontaneous polymerization. We suggest that the in vitro effects on actin polymerization may be explained by a complex mechanism that includes weak capping of filament ends and catalytic poisoning of nucleation. Although platelets contain only 1 profilin for every 5-10 actin molecules, these complex reactions may allow substoichiometric profilin to have an important influence on actin assembly. We also confirm the observation of I. Lassing and U. Lindberg (1985. Nature [Lond.] 318:472-474) that polyphosphoinositides inhibit the effects of profilin on actin polymerization, so lipid metabolism must also be taken into account when considering the functions of profilin in a cell.

Actins↗
PubMed1991

The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model.

Three-dimensional (3-D) helical reconstructions computed from electron micrographs of negatively stained dispersed F-actin filaments invariably revealed two uninterrupted columns of mass forming the "backbone" of the double-helical filament. The contact between neighboring subunits along the thus defined two long-pitch helical strands was spatially conserved and of high mass density, while the intersubunit contact between them was of lower mass density and varied among reconstructions. In contrast, phalloidinstabilized F-actin filaments displayed higher and spatially more conserved mass density between the two long-pitch helical strands, suggesting that this bicyclic hepta-peptide toxin strengthens the intersubunit contact between the two strands. Consistent with this distinct intersubunit bonding pattern, the two long-pitch helical strands of unstabilized filaments were sometimes observed separated from each other over a distance of two to six subunits, suggesting that the intrastrand intersubunit contact is also physically stronger than the interstrand contact. The resolution of the filament reconstructions, extending to 2.5 nm axially and radially, enabled us to reproducibly "cut out" the F-actin subunit which measured 5.5 nm axially by 6.0 nm tangentially by 3.2 nm radially. The subunit is distinctly polar with a massive "base" pointing towards the "barbed" end of the filament, and a slender "tip" defining its "pointed" end (i.e., relative to the "arrowhead" pattern revealed after stoichiometric decoration of the filaments with myosin subfragment 1). Concavities running approximately parallel to the filament axis both on the inner and outer face of the subunit define a distinct cleft separating the subunit into two domains of similar size: an inner domain confined to radii less than or equal to 2.5-nm forms the uninterrupted backbone of the two long-pitch helical strands, and an outer domain placed at radii of 2-5-nm protrudes radially and thus predominantly contributes to the outer part of the massive base. Quantitative evaluation of successive crossover spacings along individual F-actin filaments revealed the deviations from the mean repeat to be compensatory, i.e., short crossovers frequently followed long ones and vice versa. The variable crossover spacings and diameter of the F-actin filament together with the local unraveling of the two long-pitch helical strands are explained in terms of varying amounts of compensatory "lateral slipping" of the two strands past each other roughly perpendicular to the filament axis. This intrinsic disorder of the actin filament may enable the actin moiety to play a more active role in actin-myosin-based force generation than merely act as a rigid passive cable as has hitherto been assumed.

Actins↗
PubMed1991

Characterization of actin filament severing by actophorin from Acanthamoeba castellanii.

Actophorin is an abundant 15-kD actinbinding protein from Acanthamoeba that is thought to form a nonpolymerizable complex with actin monomers and also to reduce the viscosity of polymerized actin by severing filaments (Cooper et al., 1986. J. Biol. Chem. 261:477-485). Homologous proteins have been identified in sea urchin, chicken, and mammalian tissues. Chemical crosslinking produces a 1:1 covalent complex of actin and actophorin. Actophorin and profilin compete for crosslinking to actin monomers. The influence of actophorin on the steady-state actin polymer concentration gave a Kd of 0.2 microM for the complex of actophorin with actin monomers. Several new lines of evidence, including assays for actin filament ends by elongation rate and depolymerization rate, show that actophorin severs actin filaments both at steady state and during spontaneous polymerization. This is confirmed by direct observation in the light microscope and by showing that the effects of actophorin on the low shear viscosity of polymerized actin cannot be explained by monomer sequestration. The severing activity of actophorin is strongly inhibited by stoichiometric concentrations of phalloidin or millimolar concentrations of inorganic phosphate.

Acanthamoeba↗
PubMed1991

The actin filament severing protein actophorin promotes the formation of rigid bundles of actin filaments crosslinked with alpha-actinin.

The actin filament severing protein, Acanthamoeba actophorin, decreases the viscosity of actin filaments, but increases the stiffness and viscosity of mixtures of actin filaments and the crosslinking protein alpha-actinin. The explanation of this paradox is that in the presence of both the severing protein and crosslinker the actin filaments aggregate into an interlocking meshwork of bundles large enough to be visualized by light microscopy. The size of these bundles depends on the size of the containing vessel. The actin filaments in these bundles are tightly packed in some areas while in others they are more disperse. The bundles form a continuous reticulum that fills the container, since the filaments from a particular bundle may interdigitate with filaments from other bundles at points where they intersect. The same phenomena are seen when rabbit muscle aldolase rather than alpha-actinin is used as the crosslinker. We propose that actophorin promotes bundling by shortening the actin filaments enough to allow them to rotate into positions favorable for lateral interactions with each other via alpha-actinin. The network of bundles is more rigid and less thixotropic than the corresponding network of single actin filaments linked by alpha-actinin. One explanation may be that alpha-actinin (or aldolase) normally in rapid equilibria with actin filaments may become trapped between the filaments increasing the effective concentration of the crosslinker.

Acanthamoeba↗
PubMed1991

Myosin-I.

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Animals↗