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T D Pollard

Publications and source records attributed to T D Pollard.

At least 73 records · Page 4Linked to original sources
PubMed1997

Identification of a second myosin-II in Schizosaccharomyces pombe: Myp2p is conditionally required for cytokinesis.

As in many eukaryotic cells, fission yeast cytokinesis depends on the assembly of an actin ring. We cloned myp2(+), a myosin-II in Schizosaccharomyces pombe, conditionally required for cytokinesis. myp2(+), the second myosin-II identified in S. pombe, does not completely overlap in function with myo2(+). The catalytic domain of Myp2p is highly homologous to known myosin-IIs, and phylogenetic analysis places Myp2p in the myosin-II family. The Myp2p sequence contains well-conserved ATP- and actin-binding motifs, as well as two IQ motifs. However, the tail sequence is unusual, since it is predicted to form two long coiled-coils separated by a stretch of sequence containing 19 prolines. Disruption of myp2(+) is not lethal but under nutrient limiting conditions cells lacking myp2(+) function are multiseptated, elongated, and branched, indicative of a defect in cytokinesis. The presence of salt enhances these morphological defects. Additionally, Deltamyp2 cells are cold sensitive in high salt, failing to form colonies at 17 degrees C. Thus, myp2(+) is required under conditions of stress, possibly linking extracellular growth conditions to efficient cytokinesis and cell growth. GFP-Myp2p localizes to a ring in the middle of late mitotic cells, consistent with a role in cytokinesis. Additionally, we constructed double mutants of Deltamyp2 with temperature-sensitive mutant strains defective in cytokinesis. We observed synthetic lethal interactions between Deltamyp2 and three alleles of cdc11ts, as well as more modest synthetic interactions with cdc14ts and cdc16ts, implicating myp2(+) function for efficient cytokinesis under normal conditions.

Actins↗
PubMed1996

Structural requirements and thermodynamics of the interaction of proline peptides with profilin.

The binding to poly(L-proline) is used for the affinity purification of profilins, but little is known about the structural and thermodynamic aspects of the interaction. We used changes in the intrinsic fluorescence of profilin, CD spectroscopy, and isothermal titration calorimetry to assess how the size and composition of synthetic proline-rich peptides influence binding to Acanthamoeba and human profilins. Although a 6 residue type II poly(L-proline) helix can span the binding site, highest affinity binding is achieved by proline oligomers > or = 10 residues. Binding is stereospecific since (D-proline)11 does not bind. In 75 mM KCI the dissociation equilibrium constant for poly(L-proline) is about 10 microM proline decamer units for amoeba profilin and 20-30 microM for human profilin. Consistent with a significant hydrophobic component of the interaction, delta Cp is negative and higher salt concentrations enhance the affinity. No protons dissociate or bind during the interaction. Binding of poly(L-proline) is favored both entropically and enthalpically. Substitution of glycine in proline undecamers reduces affinity by about 1 kcal mol-1 for each substitution due to increased rotational freedom of the free peptides. Substitution of alanine has a similar effect. Disorder in the free peptides imparts an unfavorable entropic cost for immobilizing the substituted peptides on the binding site on profilin.

Acanthamoeba↗
PubMed1996

Kinetics and thermodynamics of phalloidin binding to actin filaments from three divergent species.

We compared the kinetics and thermodynamics of rhodamine phalloidin binding to actin purified from rabbit skeletal muscle, Acanthamoeba castellanii, and Saccharomyces cerevisiae in 50 mM KCl, 1 mM MgCl2, and pH 7.0 buffer at 22 degrees C. Filaments of S. cerevisiae actin bind rhodamine phalloidin more weakly than Acanthamoeba and rabbit skeletal muscle actin filaments due to a more rapid dissociation rate in spite of a significantly faster association rate constant. The higher dissociation rate constant and lower binding affinity of rhodamine phalloidin for S. cerevisiae actin filaments provide a quantitative explanation for the inefficient staining of yeast actin filaments, compared with that of rabbit skeletal muscle actin filaments [Kron et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4466-4470]. The temperature dependence of the rate constants was interpreted according to transition state theory. There is a small enthalpic difference (delta H++) between the ground states and the transition state. Consequently, the free energy of activation (delta G++) for association and dissociation of rhodamine phalloidin is dominated by entropic changes (delta S++). At equilibrium, rhodamine phalloidin binding generates a positive entropy change (delta S0). The rates of rhodamine phalloidin binding are independent of the pH, ionic strength, and filament length. Rhodamine covalently bound decreases the association rate and affinity of phalloidin for actin. The association rate constant is low for both phalloidin and rhodamine phalloidin because the filaments must undergo conformational changes (i.e. "breathe") to expose the phalloidin binding site [De La Cruz, E. M., & Pollard, T. D. (1994) Biochemistry 33, 14387-14392]. Raising the solvent microviscosity, but not the macroviscosity, dampens these conformational fluctuations, and phalloidin binding kinetics are inhibited. Yeast actin filaments bind rhodamine phalloidin more rapidly, suggesting that perhaps they are more flexible and can breathe more easily than rabbit or Acanthamoeba actin filaments.

Acanthamoeba↗
PubMed1996

Characterization of actin and poly-L-proline binding sites of Acanthamoeba profilin with monoclonal antibodies and by mutagenesis.

We characterized several deletion and substitution mutations of Acanthamoeba profilin and nine monoclonal antibodies to Acanthamoeba profilin. The results provide two independent lines of evidence about the binding sites for actin and poly-L-proline on the profilin molecule. This new evidence is consistent with the main conclusions about these binding sites from previous structural and mutagenic studies. Mutagenesis also revealed that the native structure of profilin is very sensitive to substitutions and deletions at the C terminus. For example, profilin with a deletion of the eight C-terminal residues has many of the physical properties of a molten globule, yet remarkably still binds to actin. This instability may account for the lack of function of similar mutants in yeast.

Acanthamoeba↗
PubMed1996

Actin' like actin?

The most biologically significant property of actin is its ability to self-associate and form two-stranded polymeric microfilaments. In living cells, these micro filaments form the actin cytoskeleton, essential for maintenance of the shape, passive mechanical properties and active motility of eukaryotic cells. Recently discovered actin-related proteins (ARPs) appear to share a common ancestor with conventional actin. At present, six classes of ARPs have been discovered, three of which have representatives in diverse species across eukaryotic phyla and may share functional characteristics with conventional actin. The three most ubiquitous ARPs are predicted to share a common core structure with actin and contain all the residues required for ATP binding. Surface residues involved in protein protein interactions, however, have diverged. Models of these proteins based on the atomic structure of actin provide some clues about how ARPs interact with each other, with conventional actin and with conventional actin-binding proteins.

Journal Article↗
PubMed1996

Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase.

Acanthamoeba myosin-IA and myosin-IB are single-headed molecular motors that may play an important role in membrane-based motility. To better define the types of motility that myosin-IA and myosin IB can support, we determined the rate constants for key steps on the myosin-I ATPase pathway using fluorescence stopped-flow, cold-chase, and rapid-quench techniques. We determined the rate constants for ATP binding, ATP hydrolysis, actomyosin-I dissociation, phosphate release, and ADP release. We also determined equilibrium constants for myosin-I binding to actin filaments, ADP binding to actomyosin-I, and ATP hydrolysis. These rate constants define an ATPase mechanism in which (a) ATP rapidly dissociates actomyosin-I, (b) the predominant steady-state intermediates are in a rapid equilibrium between actin-bound and free states, (c) phosphate release is rate limiting and regulated by heavy-chain phosphorylation, and (d) ADP release is fast. Thus, during steady-state ATP hydrolysis, myosin-I is weakly bound to the actin filament like skeletal muscle myosin-II and unlike the microtubule-based motor kinesin. Therefore, for myosin-I to support processive motility or cortical contraction, multiple myosin-I molecules must be specifically localized to a small region on a membrane or in the actin-rich cell cortex. This conclusion has important implications for the regulation of myosin-I via localization through the unique myosin-I tails. This is the first complete transient kinetic characterization of a member of the myosin superfamily, other than myosin-II, providing the opportunity to obtain insights about the evolution of all myosin isoforms.

Acanthamoeba↗
PubMed1996

The chemical mechanism of myosin-I: implications for actin-based motility and the evolution of the myosin family of motor proteins.

The Acanthamoeba myosin-IA and myosin-IB molecular motors bind to membranes, so they may produce the force to move organelles and membranes along actin filaments. We have determined the rate constants for the actin-activated myosin-I ATPase by pre-steady state kinetic analysis. ATP binds rapidly to myosin-I and dissociates the enzyme from actin filaments at a rate > 500 s-1. Myosin-I hydrolyzes ATP to ADP and inorganic phosphate (Pi) at 20-50 s-1. Phosphate dissociation is the rate limiting step in the ATPase cycle, 0.01 s-1 for myosin-I alone and at 10 s-1 when myosin-I is bound to actin filaments. ADP dissociation is rapid. Phosphorylation controls the ATPase cycle by increasing the rate of phosphate release from myosin-I bound to actin. At steady state the major species are myosin-ATP and myosin-ADP-Pi, which rapidly bind to and dissociate from actin filaments. During the ATPase cycle myosin-I binds so weakly to actin filaments that it cannot support processive movement like kinesin, unless several motors cluster together on a membrane or actin filament. These properties of the enzyme emphasize the importance of characterizing mechanisms that promote the self-association of myosin-I isoforms at specific binding sites in cells.

Acanthamoeba↗
PubMed1995

Phosphorylation on threonine-18 of the regulatory light chain dissociates the ATPase and motor properties of smooth muscle myosin II.

We cloned the full-length cDNA for the cytoplasmic myosin II regulatory light chain (RLC) from a stage 1-2 Xenopus oocyte library. The Xenopus RLC is 94% identical to the chicken smooth muscle myosin RLC. All of the protein kinase C and myosin light chain kinase phosphorylation sites are conserved. Using trifluoperazine [Trybus, K. M., Waller, G. S., & Chatman, T. A. (1994) J. Cell Biol. 124, 963-969], we removed the RLC of smooth muscle myosin and replaced it with recombinant Xenopus RLCs. The wild-type Xenopus RLC substitutes for the gizzard RLC in actin-activated ATPase and in vitro motility assays. We made alanine substitutions of the two residues phosphorylated by myosin light chain kinase, Ser-19 and Thr-18. All of the myosin hybrids, regardless of their mutations or phosphorylation, have similar K+EDTA ATPase activities. As expected, the T18A, S19A hybrid had no actin-activated ATPase, whereas the T18A hybrid phosphorylated on Ser-19 had an actin-activated ATPase similar to that of wild-type hybrids phosphorylated only on Ser-19. The actin-activated ATPase of myosin phosphorylated only on Thr-18 is approximately 15-fold lower than that of myosin phosphorylated on Ser-19. Phosphorylation of either Ser-19 or Thr-18 permits the formation of filaments. Remarkably, in the gliding filament assay, myosin phosphorylated only on Thr-18 moves actin filaments at velocities similar to myosin phosphorylated on Ser-19 or both Thr-18 and Ser-19.

Actins↗
PubMed1995

Fluorescent erythrocyte ghosts as standards for quantitative flow cytometry.

We report here a quick and inexpensive method for preparing standards of known fluorochrome content for calibration and quantitation of flow cytometry fluorescence signals. Erythrocyte ghosts prepared by hypotonic lysis are filled with solutions containing fluorescently labeled dextran. Standards prepared by this technique have a narrow range of fluorescence and a linear response of fluorescence to fluorochrome content up to 2 x 10(6) fluorochrome molecules/cell. The volume of ghost standard particles is roughly 70 femtoliters (fl)/cell. The fluorescence of ghost standards is nearly identical to that of commercially available microbead standards of similar fluorochrome content. Ghost standards have stable fluorescence for at least 3 weeks at 4 degrees C. These standards can be made with any fluorochrome or combination of fluorochromes over a wide concentration range.

Erythrocyte Membrane↗
PubMed1995

Nucleotide-free actin: stabilization by sucrose and nucleotide binding kinetics.

We prepared nucleotide-free actin in buffer containing 48% (w/v) sucrose. Sucrose inhibits the irreversible denaturation of actin that follows nucleotide dissociation [Kasai et al. (1965) Biochim. Biophys. Acta 94, 494-503]. Our conditions removed nucleotide from approximately 80% of the actin. Stabilization of nucleotide-free actin depends on the sucrose concentration. The CD ellipticity (x 10(3) deg cm2 dmol-1) at 222 nm of nucleotide-free actin in 48% sucrose is -3.54. The ellipticity of denatured nucleotide-free actin in dilute buffer is -2.01 and that of native actin is -4.19. In 48% sucrose nucleotide-free actin has 1.12 and native actin has 0.5 solvent-exposed thiol residues. The conformation of native actin is recovered when ATP and Mg2+ are added. Our ability to generate stable nucleotide-free actin permitted us to study the kinetics of nucleotide binding to actin. The observed rate constant of the reaction is linearly dependent on the concentration of epsilon ATP, a fluorescent analog of ATP. The inverse of the association rate constant is proportional to the viscosity of the solvent with an intercept near the origin as expected for a diffusion-limited reaction. The second-order association rate constant for Mg(2+)-ATP and Ca(2+)-ATP binding to nucleotide-free actin in water at 22 degrees C is 5 x 10(6) M-1 s-1. The Smoluchowski collision rate constant for actin and ATP is calculated to be 6.5 x 10(9) M-1 s-1, which makes the "orientation factor" 7.7 x 10(-4). From the ratio of the dissociation and association rate constants, we calculate dissociation equilibrium constants of 1.2 x 10(-9) M for Mg(2+)-ATP-actin, 4.4 x 10(-9) M for Mg(2+)-epsilon ATP-actin, and 1.2 x 10(-10) M for Ca(2+)-ATP-actin.

Actins↗
PubMed1995

Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba.

We cloned and sequenced the two actin-related proteins (Arps) present in the profilin-binding complex of Acanthamoeba (Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994, J. Cell Biol. 127:107-115). The sequence of Arp2 is more similar to other Arp2s than to actin, while the sequence of Arp3 is more similar to other Arp3s than to actin. Phylogenetic analysis of all known Arps demonstrates that most group into three major families, which are likely to be shared across all eukaryotic phyla. Together with conventional actins, the Arps form a larger family distinct from structurally related ATPases such as Hsp70's and sugar kinases. Atomic models of the Arps based on their sequences and the structure of actin provide some clues about function. Both Arps have atoms appropriately placed to bind ATP and divalent cation. Arp2, but not Arp3, has a conserved profilin-binding site. Neither Arp has the residues required to copolymerize with actin, but an Arp heterodimer present in the profilin-binding complex might serve as a pointed end nucleus for actin polymerization. Both Acanthamoeba Arps are soluble in cell homogenates, and both are concentrated in the cortex of Acanthamoeba. The cellular concentrations are 1.9 microM Arp2 and 5.1 microM Arp3, substoichiometric to actin (200 microM) but comparable to many actin-binding proteins.

Acanthamoeba↗
PubMed1994

Transient kinetic analysis of rhodamine phalloidin binding to actin filaments.

We have characterized the binding of rhodamine phalloidin to actin filaments and actin filaments saturated with either myosin subfragment-1 or tropomyosin in 50 mM KCl, 1 mM MgCl2 buffer at pH 7.0. Direct transient kinetic measurements of rhodamine phalloidin binding to actin filaments indicate an association rate constant of 2.8 x 10(4) M-1 s-1 and a dissociation rate constant of 4.8 x 10(-4) s-1. The ratio of the rate constants yields a dissociation equilibrium constant of 17 nM. From equilibrium measurements, the apparent affinity of rhodamine phalloidin for actin filaments is 116 nM. The difference between the affinities determined by equilibrium and kinetic experiments is attributed to the depolymerization of filaments at low actin concentrations in the equilibrium samples. The binding stoichiometry is one rhodamine phalloidin molecule per actin subunit. When myosin subfragment-1 and tropomyosin are bound to actin filaments, the rate constants for rhodamine phalloidin binding are the same as for actin alone and in agreement with the binding affinities measured in equilibrium experiments. Presumably these proteins stabilize the filaments. Neither substitution of CaCl2 for MgCl2 nor the inclusion of 20 mM phosphate altered the rate or equilibrium constants.

Actin Cytoskeleton↗
PubMed1994

Vaccinia virus expresses a novel profilin with a higher affinity for polyphosphoinositides than actin.

We expressed in Escherichia coli the vaccinia virus gene for a protein similar to vertebrate profilins, purified the recombinant viral profilin, and characterized its interactions with actin and polyphosphoinositides. Compared with cellular profilins, this viral profilin has a low affinity (Kd > or = 35 microM) for human platelet actin monomers, a weak effect on the exchange of the nucleotide bound to the actin, and no detectable affinity for poly(L-proline). Vaccinia profilin binds to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate in micelles and large unilamellar vesicles, but not to phosphatidylserine or phosphatidylcholine. Kinetic analysis by surface plasmon resonance showed that both vaccinia and amoeba profilins bind slowly to polyphosphoinositides, with association rate constants in the range of (1-4) x 10(4) M-1 s-1. The higher affinity of vaccinia profilin for polyphosphoinositides (Kd = 0.2-8.5 microM) than for actin or poly(L-proline) and the concentration of vaccinia profilin expressed in infected HeLa cells (approximately 20 microM) suggest that vaccinia profilin binds preferentially to PIP and PIP2 in vivo. Consequently, vaccinia profilin is more likely to influence phosphoinositide metabolism than actin assembly. Expression of 7-105 microM vaccinia profilin in a Saccharomyces cerevisiae profilin null mutant did not rescue the null phenotype, so that the affinity of vaccinia profilin for phosphoinositides alone is insufficient for normal profilin function in yeast.

Actins↗
PubMed1994

X-ray structures of isoforms of the actin-binding protein profilin that differ in their affinity for phosphatidylinositol phosphates.

We determined the structures of Acanthamoeba profilin I and profilin II by x-ray crystallography at resolutions of 2.0 and 2.8 A, respectively. The polypeptide folds and the actin-binding surfaces of the amoeba profilins are very similar to those of bovine and human profilins. The electrostatic potential surfaces of the two Acanthamoeba isoforms differ. Two areas of high positive potential on the surface of profilin II are candidate binding sites for phosphatidylinositol phosphates. The proximity of these sites to the actin binding site provides an explanation for the competition between actin and lipids for binding profilin.

Acanthamoeba↗
PubMed1994

Purification, characterization and crystallization of human platelet profilin expressed in Escherichia coli.

Human platelet profilin was expressed in Escherichia coli using a T7 based expression vector. The recombinant material is similar to authentic human platelet profilin based on the measured Kd for rabbit skeletal muscle actin. Crystals of the recombinant material were obtained from both PEG 8000 and (NH4)2SO4. These crystals are isomorphous and belong to the monoclinic space group C2, a = 75.0, b = 32.0, c = 62.5, beta = 123 degrees. These crystals contain one molecule in the asymmetric unit and diffract to at least 2.0 A.

Actins↗
PubMed1994

Wild-type but not mutant APC associates with the microtubule cytoskeleton.

The adenomatous polyposis coli protein (APC) is mutated in familial adenomatous polyposis patients as well as in sporadic colorectal tumors. In an attempt to further understand the function of APC, the subcellular localization of APC was examined. Wild-type and mutant forms of APC were expressed in mammalian cells and protein detected by immunofluorescence using monoclonal and polyclonal antibodies. Staining of wildtype APC protein revealed a filamentous network which extended throughout the cytoplasm and colocalized with microtubules. In striking contrast, mutant APC protein gave a diffuse cytoplasmic staining pattern. Treatment with a microtubule depolymerizing agent, nocodazole, caused APC as well as tubulin to become diffusely cytoplasmic. In addition, immunoperoxidase staining of transfected APC protein followed by transmission electron microscopy revealed staining of microtubules. These results suggest that wild-type but not mutant APC protein may be associated with the microtubule cytoskeleton.

3T3 Cells↗