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Thomas D Pollard

Publications and source records attributed to Thomas D Pollard.

14 recordsLinked to original sources
PubMed2003

The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin.

Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline.

Actin Cytoskeleton↗
PubMed2003

The cytoskeleton, cellular motility and the reductionist agenda.

Eukaryotic cells depend on cytoskeletal polymers and molecular motors to establish their asymmetrical shapes, to transport intracellular constituents and to drive their motility. Cell biologists are using diverse experimental approaches to understand the molecular basis of cellular movements and to explain why defects in the component proteins cause disease. Much of the molecular machinery for motility evolved in early eukaryotes, so a limited set of general principles can explain the motility of most cells.

Actins↗
PubMed2003

Cellular motility driven by assembly and disassembly of actin filaments.

Motile cells extend a leading edge by assembling a branched network of actin filaments that produces physical force as the polymers grow beneath the plasma membrane. A core set of proteins including actin, Arp2/3 complex, profilin, capping protein, and ADF/cofilin can reconstitute the process in vitro, and mathematical models of the constituent reactions predict the rate of motion. Signaling pathways converging on WASp/Scar proteins regulate the activity of Arp2/3 complex, which mediates the initiation of new filaments as branches on preexisting filaments. After a brief spurt of growth, capping protein terminates the elongation of the filaments. After filaments have aged by hydrolysis of their bound ATP and dissociation of the gamma phosphate, ADF/cofilin proteins promote debranching and depolymerization. Profilin catalyzes the exchange of ADP for ATP, refilling the pool of ATP-actin monomers bound to profilin, ready for elongation.

Actin Depolymerizing Factors↗
PubMed2003

EPLIN regulates actin dynamics by cross-linking and stabilizing filaments.

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.

Actin Cytoskeleton↗
PubMed2003

Dynamic localization of myosin-I to endocytic structures in Acanthamoeba.

We used fluorescence microscopy of live Acanthamoeba to follow the time course of the concentration of myosin-I next to the plasma membrane at sites of macropinocytosis and phagocytosis. We marked myosin-I with a fluorescently labeled monoclonal antibody (Cy3-M1.7) introduced into the cytoplasm by syringe loading. M1.7 binds myosin-IA and -IC without affecting their activities, but does not bind myosin-IB. Cy3-M1.7 concentrates at two different macropinocytic structures: large circular membrane ruffles that fuse to create macropinosomes, and smaller endocytic structures that occur at the end of stalk-like pseudopodia. These dynamic structures enclose macropinosomes every 30-60 s. Cy3-M1.7 accumulates rapidly as these endocytic structures form and dissipate rapidly after they internalize. Double labeling fixed cells with Cy3-M1.7 and polyclonal antibodies specific for myosin-IA, -IB, or -IC revealed that all three myosin-I isoforms associate with macropinocytic structures, but individual structures vary in their myosin-I isoform composition. Myosin-I and actin also concentrate transiently at sites where amoebae ingest yeast or the pseudopodia of neighboring cells (heterophagy) by the process of phagocytosis. Within 3 min of yeast attachment to the amoeba, myosin-I concentrates around the phagocytic cup, yeast are internalized, and myosin-I de-localizes. Despite known differences in the regulation of macropinocytosis and phagocytosis, the morphology, protein composition, and dynamics of phagocytosis and macropinocytosis are similar, indicating that they share common structural properties and contractile mechanisms.

Acanthamoeba↗
PubMed2003

A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex.

Members of the Wiskott-Aldrich syndrome protein (WASP) family link Rho GTPase signaling pathways to the cytoskeleton through a multiprotein assembly called Arp2/3 complex. The C-terminal VCA regions (verprolin-homology, central hydrophobic, and acidic regions) of WASP and its relatives stimulate Arp2/3 complex to nucleate actin filament branches. Here we show by differential line broadening in NMR spectra that the C (central) and A (acidic) segments of VCA domains from WASP, N-WASP and Scar bind Arp2/3 complex. The C regions of these proteins have a conserved sequence motif consisting of hydrophobic residues and an arginine residue. Point mutations in this conserved sequence motif suggest that it forms an amphipathic helix that is required in biochemical assays for activation of Arp2/3 complex. Key residues in this motif are buried through contacts with the GTPase binding domain in the autoinhibited structure of WASP and N-WASP, indicating that sequestration of these residues is an important aspect of autoinhibition.

Actin-Related Protein 2↗
PubMed2002

Intracellular localization and dynamics of myosin-II and myosin-IC in live Acanthamoeba by transient transfection of EGFP fusion proteins.

We developed a reliable method for transient transfection of Acanthamoeba using Superfect (Qiagen) and a vector with the Acanthamoeba ubiquitin promoter and enhanced green fluorescent protein (EGFP) as the reporter gene. The transfection efficiency was 3% for profilin-I-EGFP and EGFP-myosin-II tail, and less than 0.5% for larger constructs such as full length myosin-II or myosin-IC. Profilin-I-EGFP was distributed throughout the cytoplasm as observed previously with rhodamine-labeled profilin, while EGFP alone accumulated in the nucleus. EGFP fused to full length myosin-II or to the C-terminal 256 residues of the myosin-II tail concentrated in fluorescent spots similar to thick filaments and minifilaments identified previously in fixed cells with fluorescent antibodies. Thick filaments were located in the dorsal cytoplasm and along the lateral margins of the back half of the cell. Thick filaments formed behind the leading edge and moved continuously towards the rear of the cell, where they disassembled. If phosphorylation of the myosin-II heavy chain was prevented by mutation of all three phosphorylated serines to alanine, thick filaments of unphosphorylated myosin-II accumulated around vesicles of various sizes. EGFP-myosin-IC was spread throughout the cytoplasm but concentrated transiently around contractile vacuoles and macropinocytosis cups providing that the construct included both the head and a tail with the SH3 domain.

Acanthamoeba↗
PubMed2002

Xenopus actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends.

Xenopus actin-interacting protein 1 (XAip1) is thought to promote fragmentation of actin filaments by cofilin. To examine the mechanism of XAip1, we measured polymer lengths by fluorescence microscopy and the concentration of filament ends with an elongation assay. Cofilin creates ends by severing actin filaments. XAip1 alone does not sever actin filaments or prevent annealing/redistribution of mechanically severed filaments and has no effect on the concentration of ends available for subunit addition. In the presence of XAip1, the apparent filament fragmentation by cofilin is enhanced, but XAip1 reduces rather than increases the concentration of ends capable of adding subunits. Electron microscopy with gold-labeled antibodies showed that a low concentration of XAip1 bound preferentially to one end of the filament. A high concentration of XAip1 bound along the length of the filament. In the presence of gelsolin-actin to cap filament barbed ends, XAip1 does not enhance cofilin activity. We conclude that XAip1 caps the barbed end of filaments severed by cofilin. This capping blocks annealing and depolymerization and allows more extensive severing by cofilin.

Actin Depolymerizing Factors↗
PubMed2002

Hydrolysis of ATP by polymerized actin depends on the bound divalent cation but not profilin.

Growing evidence suggests that the nucleotide bound to actin filaments serves as a timer to control actin filament turnover during cell motility (Pollard, T. D., Blanchoin, L., and Mullins, R. D. (2000) Annu. Rev. Biophys. Biomol. Struct. 29, 545-576). We re-examined the hydrolysis of ATP by polymerized actin using mechanical quenched-flow methods to improve temporal resolution. The rate constant for ATP hydrolysis by polymerized Mg actin is 0.3 s(-1), 3-fold faster than that measured manually. The ATP hydrolysis rate is similar when Mg ATP actin elongates either the pointed end or the barbed end of filaments. Polymerized Ca actin hydrolyzes ATP at 0.05 s(-1). Mg ATP actin saturated with profilin can elongate barbed ends at >60 s(-1), 2 orders of magnitude faster than ATP hydrolysis (0.3 s(-1)). Given that profilin binds to a surface on actin that is buried in the Holmes model of the actin filament, we expect that profilin will block subunit addition at the barbed end of a filament. Profilin must move from this site at rates much faster than it dissociates from monomers (4 s(-1)). ATP hydrolysis is not required for this movement.

Actins↗
PubMed2002

Structure and function of the Arp2/3 complex.

The Arp2/3 complex, a 220 kDa macromolecular assembly comprising two actin-related proteins and five other subunits, plays a key role in cellular motility by initiating the polymerization of new actin filaments. Crystal and cryo-electron microscopic structures of the Arp2/3 complex and branch junctions have clarified extensive biochemical data on the mechanism of this process.

Actin-Related Protein 2↗