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M D Welch

Publications and source records attributed to M D Welch.

At least 19 recordsLinked to original sources
PubMed2001

Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility.

The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding. Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation. These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L. monocytogenes. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility.

Actin-Related Protein 2↗
PubMed2001

Cytoskeleton: actin and endocytosis--no longer the weakest link.

The actin cytoskeleton has long been believed to play a role in endocytosis, but its actual function in this process has been unclear. Now, three proteins that promote actin nucleation have been found to provide a link between the actin cytoskeleton and the endocytic machinery.

Actin-Related Protein 2↗
PubMed2001

Reconstitution of human Arp2/3 complex reveals critical roles of individual subunits in complex structure and activity.

The Arp2/3 complex is a seven-protein assembly that is critical for actin nucleation and branching in cells. Here we report the reconstitution of active human Arp2/3 complex after expression of all seven subunits in insect cells. Expression of partial complexes revealed that a heterodimer of the p34 and p20 subunits constitutes a critical structural core of the complex, whereas the remaining subunits are peripherally located. Arp3 is crucial for nucleation, consistent with it being a structural component of the nucleation site. p41, p21, and p16 contribute differently to nucleation and stimulation by ActA and WASP, whereas p34/p20 bind actin filaments and likely function in actin branching. This study reveals that the nucleating and organizing functions of Arp2/3 complex subunits are separable, indicating that these activities may be differentially regulated in cells.

Actin Cytoskeleton↗
PubMed2001

Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility.

The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged-to-alanine mutations in the NH2-terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin-binding sequence of ActA spans 40 amino acids. We also identified a 'motility rate and cloud-to-tail transition' region in which nine contiguous mutations spanning amino acids 165-260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to 'skid' or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin-based motility of L. monocytogenes.

Actins↗
PubMed2000

Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility.

The Listeria monocytogenes ActA protein induces actin-based motility by enhancing the actin nucleating activity of the host Arp2/3 complex. Using systematic truncation analysis, we identified a 136-residue NH(2)-terminal fragment that was fully active in stimulating nucleation in vitro. Further deletion analysis demonstrated that this fragment contains three regions, which are important for nucleation and share functional and/or limited sequence similarity with host WASP family proteins: an acidic stretch, an actin monomer-binding region, and a cofilin homology sequence. To determine the contribution of each region to actin-based motility, we compared the biochemical activities of ActA derivatives with the phenotypes of corresponding mutant bacteria in cells. The acidic stretch functions to increase the efficiency of actin nucleation, the rate and frequency of motility, and the effectiveness of cell-cell spread. The monomer-binding region is required for actin nucleation in vitro, but not for actin polymerization or motility in infected cells, suggesting that redundant mechanisms may exist to recruit monomer in host cytosol. The cofilin homology sequence is critical for stimulating actin nucleation with the Arp2/3 complex in vitro, and is essential for actin polymerization and motility in cells. These data demonstrate that each region contributes to actin-based motility, and that the cofilin homology sequence plays a principal role in activation of the Arp2/3 complex, and is an essential determinant of L. monocytogenes pathogenesis.

Actin-Related Protein 2↗
PubMed1999

The Wiskott-Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex.

Actin polymerization at the cell cortex is thought to provide the driving force for aspects of cell-shape change and locomotion. To coordinate cellular movements, the initiation of actin polymerization is tightly regulated, both spatially and temporally. The Wiskott-Aldrich syndrome protein (WASP), encoded by the gene that is mutated in the immunodeficiency disorder Wiskott-Aldrich syndrome [1], has been implicated in the control of actin polymerization in cells [2] [3] [4] [5]. The Arp2/3 complex, an actin-nucleating factor that consists of seven polypeptide subunits [6] [7] [8], was recently shown to physically interact with WASP [9]. We sought to determine whether WASP is a cellular activator of the Arp2/3 complex and found that WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro. Moreover, WASP-coated microspheres polymerized actin, formed actin tails and exhibited actin-based motility in cell extracts, similar to those behaviors displayed by the pathogenic bacterium Listeria monocytogenes. In extracts depleted of the Arp2/3 complex, WASP-coated microspheres and L. monocytogenes were non-motile and exhibited only residual actin polymerization. These results demonstrate that WASP is sufficient to direct actin-based motility in cell extracts and that this function is mediated by the Arp2/3 complex. WASP interacts with diverse signaling proteins and may therefore function to couple signal transduction pathways to Arp2/3-complex activation and actin polymerization.

Actin-Related Protein 2↗
PubMed1999

The world according to Arp: regulation of actin nucleation by the Arp2/3 complex.

The coordination of cell shape change and locomotion requires that actin polymerization at the cell cortex be tightly controlled in response to both intracellular and extracellular cues. The Arp2/3 complex - an actin filament nucleating and organizing factor - appears to be a central player in the cellular control of actin assembly. Recently, a molecular pathway leading from key signalling molecules to actin filament nucleation by the Arp2/3 complex has been discovered. In this pathway, the GTPase Cdc42 acts in concert with WASP family proteins to activate the Arp2/3 complex. These findings have led to a more complete picture of the mechanism of actin filament generation and organization during cell motility.

Actin-Related Protein 2↗
PubMed1999

Spatial control of actin polymerization during neutrophil chemotaxis.

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.

Actin-Related Protein 2↗
PubMed1998

Visualization and molecular analysis of actin assembly in living cells.

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM-kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP-CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

Actin Cytoskeleton↗
PubMed1998

Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation.

Actin filament assembly at the cell surface of the pathogenic bacterium Listeria monocytogenes requires the bacterial ActA surface protein and the host cell Arp2/3 complex. Purified Arp2/3 complex accelerated the nucleation of actin polymerization in vitro, but pure ActA had no effect. However, when combined, the Arp2/3 complex and ActA synergistically stimulated the nucleation of actin filaments. This mechanism of activating the host Arp2/3 complex at the L. monocytogenes surface may be similar to the strategy used by cells to control Arp2/3 complex activity and hence the spatial and temporal distribution of actin polymerization.

Actin-Related Protein 2↗
PubMed1997

The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly.

The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

Actin Cytoskeleton↗
PubMed1997

Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes.

The pathogenic bacterium Listeria monocytogenes is capable of directed movement within the cytoplasm of infected host cells. Propulsion is thought to be driven by actin polymerization at the bacterial cell surface, and moving bacteria leave in their wake a tail of actin filaments. Determining the mechanism by which L. monocytogenes polymerizes actin may aid the understanding of how actin polymerization is controlled in the cell. Actin assembly by L. monocytogenes requires the bacterial surface protein ActA and protein components present in host cell cytoplasm. We have purified an eight-polypeptide complex that possesses the properties of the host-cell actin polymerization factor. The pure complex is sufficient to initiate ActA-dependent actin polymerization at the surface of L. monocytogenes, and is required to mediate actin tail formation and motility. Two subunits of this protein complex are actin-related proteins (ARPs) belonging to the Arp2 and Arp3 subfamilies. The Arp3 subunit localizes to the surface of stationary bacteria and the tails of motile bacteria in tissue culture cells infected with L. monocytogenes; this is consistent with a role for the complex in promoting actin assembly in vivo. The activity and subunit composition of the Arp2/3 complex suggests that it forms a template that nucleates actin polymerization.

Actin-Related Protein 2↗
PubMed1997

Actin dynamics in vivo.

Actin dynamics in lamellipodia are driven by continuous cycles of actin polymerization, retrograde flow, and depolymerization. In the past year, advances have been made in identifying signaling pathways that regulate actin-filament uncapping and polymerization, in determining the role of myosin motor proteins in retrograde flow, and in evaluating the role of severing proteins in actin depolymerization. Both Listeria monocytogenes and Saccharomyces cerevisiae have emerged as powerful model organisms for studying actin dynamics in cells.

Actins↗
PubMed1994

The yeast actin cytoskeleton.

Budding and fission yeast present significant advantages for studies of the actin cytoskeleton. The application of classical and molecular genetic techniques provides a facile route for the analysis of structure/function relationships, for the isolation of novel proteins involved in cytoskeletal function, and for deciphering the signals that regulate actin assembly in vivo. This review focuses on the budding yeast Saccharomyces cerevisiae and also identifies some recent advances from studies on the fission yeast Schizosaccharomyces pombe, for which studies on the actin cytoskeleton are still in their infancy.

Actins↗
PubMed1994

A nuclear protein with sequence similarity to proteins implicated in human acute leukemias is important for cellular morphogenesis and actin cytoskeletal function in Saccharomyces cerevisiae.

The cellular functions of the product of the Saccharomyces cerevisiae ANC1 (actin non-complementing) gene were investigated. ANC1 was previously identified in a screen for mutations that enhance the defect caused by a mutation in the actin gene. Here, we show that anc1-1 and anc1 delta 1::HIS3 (gene deletion) mutants exhibit a novel combination of defects in the organization of the actin cytoskeleton and the localization of Spa2p, a protein implicated in polarity development and cytokinesis. Morphological abnormalities exhibited by anc1 mutants include failure to form a mating projection in response to alpha-factor and development of swollen or elongated cell shapes during proliferation. These morphological aberrations correlate with cytoskeletal defects that were also observed. These phenotypes demonstrate that Anc1p is important for actin function and for the functions of other proteins involved in morphogenesis. In further support of these roles for Anc1p, the anc1 delta 1::HIS3 mutation was found to be synthetically lethal in combination with a null mutation in SLA1, a gene that is important for membrane cytoskeleton function. Surprisingly, Anc1p was found to be a nuclear protein and to have sequence similarity to the human proteins ENL and AF-9. These human proteins are implicated in the development of a subset of acute lymphoblastic leukemias, acute myeloid leukemias, and lymphomas. Our findings suggest that changes in the functions or organization of actin filaments might contribute to the establishment of the neoplastic state for these leukemias and lymphomas.

Actins↗
PubMed1994

Strength training effects on rearfoot motion in running.

The investigation examined isokinetic (IK) and nonisokinetic (NIK) strength training programs for the inversion (INV) and eversion (EV) muscles on pronation during running. Seventy-seven volunteers were videotaped running on a treadmill at 3.8 m.s-1 and total pronation (delta beta PRO) was computed. Eighteen heel-strike runners with the largest values of delta beta PRO (X = 16.7 degrees) were selected as subjects. During the pre- and posttests, isokinetic muscle strength at 20 and 180 degrees.s-1 was determined for the concentric (CON) and eccentric (ECC) actions of the INV and EV muscle groups. The subjects also were videotaped running on a treadmill (3.8 m.s-1). The IK training group performed three sets of eight CON and ECC repetitions at 20, 90, and 180 degrees.s-1 for both muscle groups; and the NIK subjects did exercises commonly used in ankle rehabilitation. Each group trained three times weekly for 8 wk. The IK group showed significantly (P < or = 0.05) CON and ECC strength increases for all INV test conditions and three of the four EV conditions (20 degrees.s-1 CON and ECC, and 180 degrees.s-1 CON). They also demonstrated significant decreases in the rearfoot (2.2 degrees) and pronation/supination (2.9 degrees) angles at heel strike and in delta beta PRO (-2.2 degrees).l The NIK group exhibited no change in rearfoot motion and only increased INV strength at the 180 degrees.s-1 ECC test condition. The findings suggest that pronation can be decreased by an isokinetic strength training program for the INV and EV muscles.

Adolescent↗
PubMed1993

Screens for extragenic mutations that fail to complement act1 alleles identify genes that are important for actin function in Saccharomyces cerevisiae.

Null mutations in SAC6 and ABP1, genes that encode actin-binding proteins, failed to complement the temperature-sensitive phenotype caused by a mutation in the ACT1 gene. To identify novel genes whose protein products interact with actin, mutations that fail to complement act1-1 or act1-4, two temperature-sensitive alleles of ACT1, were isolated. A total of 14 extragenic noncomplementing mutations and 12 new alleles of ACT1 were identified in two independent screens. The 14 extragenic noncomplementing mutations represent alleles of at least four different genes, ANC1, ANC2, ANC3 and ANC4 (Actin NonComplementing). Mutations in the ANC1 gene were shown to cause osmosensitivity and defects in actin organization; phenotypes that are similar to those caused by act1 mutations. We conclude that the ANC1 gene product plays an important role in actin cytoskeletal function. The 12 new alleles of ACT1 will be useful for further elucidation of the functions of actin in yeast.

Actins↗