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R D Mullins

Publications and source records attributed to R D Mullins.

At least 19 recordsLinked to original sources
PubMed2001

Calcium spiking activity and baseline calcium levels in ROS 17/2.8 cells exposed to extremely low frequency electromagnetic fields (ELF EMF).

PURPOSE: To determine whether extremely low frequency electromagnetic fields can alter average free cytosolic calcium ion concentrations [Ca2+]i and transient increases in [Ca2+]i in populations of ROS 17/2.8 cells. MATERIALS AND METHODS: Cells loaded with the calcium-selective luminescent photoprotein, aequorin, were placed in the bottom of a sample chamber, which was inserted into the gap of a previously described air gap reactor system where they were exposed either to sinusoidal magnetic fields at a variety of frequencies and flux densities or to sham conditions. Real-time recordings of photon counts due to aequorin luminescence were obtained and data were analysed with the use of probit plots. RESULTS: Probit plots of data obtained from cells exposed to the various magnetic fields were virtually superimposable over the data obtained for the same cultures during pre- and post-exposure sham or no-field periods. CONCLUSION: These experiments provided no evidence for any effects of ELF EMF, either positive or negative, on either average [Ca2+]i or on transient increases in [Ca2+]i.

Aequorin↗
PubMed2001

Actin dynamics.

Explore the source record for details and available documents.

Actin Depolymerizing Factors↗
PubMed2000

Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex.

The protein N-WASP [a homolog to the Wiskott-Aldrich syndrome protein (WASP)] regulates actin polymerization by stimulating the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. N-WASP is tightly regulated by multiple signals: Only costimulation by Cdc42 and phosphatidylinositol (4,5)-bisphosphate (PIP2) yields potent polymerization. We found that regulation requires N-WASP's constitutively active output domain (VCA) and two regulatory domains: a Cdc42-binding domain and a previously undescribed PIP(2)-binding domain. In the absence of stimuli, the regulatory modules together hold the VCA-Arp2/3 complex in an inactive "closed" conformation. In this state, both the Cdc42- and PIP2-binding sites are masked. Binding of either input destabilizes the closed state and enhances binding of the other input. This cooperative activation mechanism shows how combinations of simple binding domains can be used to integrate and amplify coincident signals.

Actin Cytoskeleton↗
PubMed2000

Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks.

BACKGROUND: Cellular movements are powered by the assembly and disassembly of actin filaments. Actin dynamics are controlled by Arp2/3 complex, the Wiskott-Aldrich syndrome protein (WASp) and the related Scar protein, capping protein, profilin, and the actin-depolymerizing factor (ADF, also known as cofilin). Recently, using an assay that both reveals the kinetics of overall reactions and allows visualization of actin filaments, we showed how these proteins co-operate in the assembly of branched actin filament networks. Here, we investigated how they work together to disassemble the networks. RESULTS: Actin filament branches formed by polymerization of ATP-actin in the presence of activated Arp2/3 complex were found to be metastable, dissociating from the mother filament with a half time of 500 seconds. The ADF/cofilin protein actophorin reduced the half time for both dissociation of gamma-phosphate from ADP-Pi-actin filaments and debranching to 30 seconds. Branches were stabilized by phalloidin, which inhibits phosphate dissociation from ADP-Pi-filaments, and by BeF3, which forms a stable complex with ADP and actin. Arp2/3 complex capped pointed ends of ATP-actin filaments with higher affinity (Kd approximately 40 nM) than those of ADP-actin filaments (Kd approximately 1 microM), explaining why phosphate dissociation from ADP-Pi-filaments liberates branches. Capping protein prevented annealing of short filaments after debranching and, with profilin, allowed filaments to depolymerize at the pointed ends. CONCLUSIONS: The low affinity of Arp2/3 complex for the pointed ends of ADP-actin makes actin filament branches transient. By accelerating phosphate dissociation, ADF/cofilin promotes debranching. Barbed-end capping proteins and profilin allow dissociated branches to depolymerize from their free pointed ends.

Acanthamoeba↗
PubMed2000

Activation of the Arp2/3 complex by the Listeria acta protein. Acta binds two actin monomers and three subunits of the Arp2/3 complex.

ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent actin nucleation, but its interactions with cellular components of the nucleation machinery are not well understood. Here we show that two domains of ActA (residues 85-104 and 121-138) with sequence similarity to WASP homology 2 domains bind two actin monomers with submicromolar affinity. ActA binds Arp2/3 with a K(d) of 0.6 microm and competes for binding with the WASP family proteins N-WASP and Scar1. By chemical cross-linking, ActA, N-WASP, and Scar1 contact the same three subunits of the Arp2/3 complex, p40, Arp2, and Arp3. Interestingly, profilin competes with ActA for binding of Arp2/3, but actophorin (cofilin) does not. The minimal Arp2/3-binding site of ActA (residues 144-170) is C-terminal to both actin-binding sites and shares sequence homology with Arp2/3-binding regions of WASP family proteins. The maximal activity at saturating concentrations of ActA is identical to the most active domains of the WASP family proteins. We propose that ActA and endogenous WASP family proteins promote Arp2/3-dependent nucleation by similar mechanisms and require simultaneous binding of Arp2 and Arp3.

Actin-Related Protein 2↗
PubMed2000

beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2.

Recently, a requirement for beta-arrestin-mediated endocytosis in the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by several G protein-coupled receptors (GPCRs) has been proposed. However, the importance of this requirement for function of ERK1/2 is unknown. We report that agonists of Galphaq-coupled proteinase-activated receptor 2 (PAR2) stimulate formation of a multiprotein signaling complex, as detected by gel filtration, immunoprecipitation and immunofluorescence. The complex, which contains internalized receptor, beta-arrestin, raf-1, and activated ERK, is required for ERK1/2 activation. However, ERK1/2 activity is retained in the cytosol and neither translocates to the nucleus nor causes proliferation. In contrast, a mutant PAR2 (PAR2deltaST363/6A), which is unable to interact with beta-arrestin and, thus, does not desensitize or internalize, activates ERK1/2 by a distinct pathway, and fails to promote both complex formation and cytosolic retention of the activated ERK1/2. Whereas wild-type PAR2 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(deltaST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, beta-arrestin, raf-1, and activated ERK1/2 might ensure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists.

Animals↗
PubMed2000

How WASP-family proteins and the Arp2/3 complex convert intracellular signals into cytoskeletal structures.

In most cells, the structure of the actin cytoskeleton is regulated by Rho-family G proteins. Recent work has outlined a highly conserved signaling pathway from G protein activation to actin assembly. The key downstream components are WASP family proteins - adaptor molecules that bind multiple signaling and cytoskeletal proteins - and the Arp2/3 complex - a multi-functional protein complex that nucleates and crosslinks actin filaments.

Actin Cytoskeleton↗
PubMed2000

Molecular mechanisms controlling actin filament dynamics in nonmuscle cells.

We review how motile cells regulate actin filament assembly at their leading edge. Activation of cell surface receptors generates signals (including activated Rho family GTPases) that converge on integrating proteins of the WASp family (WASp, N-WASP, and Scar/WAVE). WASP family proteins stimulate Arp2/3 complex to nucleate actin filaments, which grow at a fixed 70 degrees angle from the side of pre-existing actin filaments. These filaments push the membrane forward as they grow at their barbed ends. Arp2/3 complex is incorporated into the network, and new filaments are capped rapidly, so that activated Arp2/3 complex must be supplied continuously to keep the network growing. Hydrolysis of ATP bound to polymerized actin followed by phosphate dissociation marks older filaments for depolymerization by ADF/cofilins. Profilin catalyzes exchange of ADP for ATP, recycling actin back to a pool of unpolymerized monomers bound to profilin and thymosin-beta 4 that is poised for rapid elongation of new barbed ends.

Actin Cytoskeleton↗
PubMed2000

Mechanisms underlying spontaneous calcium spiking in aequorin-loaded ROS 17/2.8 cells.

In earlier studies, McLeod and coworkers reported the detection of spontaneous calcium spiking in ROS 17/2.8 cells which they suggested was derived from individual cells progressing through mitosis or the cell cycle. They also indicated that the degree of spiking could be modulated by exposure of the cells to time-varying extremely low frequency electric fields. Given the implications of such observations for our understanding of the effects of electromagnetic fields on biological systems, it appeared important for mechanistic reasons to understand the basis of this spiking. In this study, we were able to confirm that spontaneous calcium spiking activity could be detected in ROS 17/2.8 cells and that this appeared to emanate from individual cells. We found this spiking to be completely dependent on extracellular calcium ions and to be independent of the inositol 1,4,5-trisphosphate-sensitive intracellular calcium store. This spiking is not reduced by treatments which slow down or block the passage of cells through the cell cycle. Further, we found that spiking was only detectable in the most highly aequorin-loaded subpopulation of cells whose growth rate is reduced and whose morphological appearance is abnormal. In conjunction with what is known about calcium spiking in other, nonexcitable mammalian cells in culture, the data presented strongly argue that the spontaneous calcium spiking observed in ROS 17/2.8 cells is unrelated to normal events of the cell cycle and most likely result from the damaging effects of excessive loading with aequorin.

Aequorin↗
PubMed1999

Rho-family GTPases require the Arp2/3 complex to stimulate actin polymerization in Acanthamoeba extracts.

BACKGROUND: Actin filaments polymerize in vivo primarily from their fast-growing barbed ends. In cells and extracts, GTPgammaS and Rho-family GTPases, including Cdc42, stimulate barbed-end actin polymerization; however, the mechanism responsible for the initiation of polymerization is unknown. There are three formal possibilities for how free barbed ends may be generated in response to cellular signals: uncapping of existing filaments; severing of existing filaments; or de novo nucleation. The Arp2/3 complex localizes to regions of dynamic actin polymerization, including the leading edges of motile cells and motile actin patches in yeast, and in vitro it nucleates the formation of actin filaments with free barbed ends. Here, we investigated actin polymerization in soluble extracts of Acanthamoeba. RESULTS: Addition of actin filaments with free barbed ends to Acanthamoeba extracts is sufficient to induce polymerization of endogenous actin. Addition of activated Cdc42 or activation of Rho-family GTPases in these extracts by the non-hydrolyzable GTP analog GTPgammaS stimulated barbed-end polymerization, whereas immunodepletion of Arp2 or sequestration of Arp2 using solution-binding antibodies blocked Rho-family GTPase-induced actin polymerization. CONCLUSIONS: For this system, we conclude that the accessibility of free barbed ends regulates actin polymerization, that Rho-family GTPases stimulate polymerization catalytically by de novo nucleation of free barbed ends and that the primary nucleation factor in this pathway is the Arp2/3 complex.

Acanthamoeba↗
PubMed1999

Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex.

The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex was not known. We report that full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament-a dendritic nucleation mechanism. The Arp2/3 complex with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex.

Acanthamoeba↗
PubMed1999

Structure and function of the Arp2/3 complex.

The Arp2/3 complex is a ubiquitous and essential component of the actin cytoskeleton in eukaryotic cells. It nucleates actin filaments, caps their pointed ends and cross-links them into orthogonal networks. In amoeba, vertebrates and fungi, the complex consists of actin-related proteins Arp2 and Arp3 and individual copies of five novel polypeptides. The Arps are thought to mediate pointed-end capping and nucleation. Chemical cross-linking implicates three subunits in binding the complex to the side of another actin filament.

Actin-Related Protein 2↗
PubMed1998

The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments.

The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 microM. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 +/- 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

Acanthamoeba↗
PubMed1998

Arp2/3 complex from Acanthamoeba binds profilin and cross-links actin filaments.

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 microM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of alpha-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.

Acanthamoeba↗
PubMed1997

Structure, subunit topology, and actin-binding activity of the Arp2/3 complex from Acanthamoeba.

The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actin-related proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of approximately 13 x 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 microM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actin-rich regions of Acanthamoeba.

Acanthamoeba↗
PubMed1993

Design and characterization of a system for exposure of cultured cells to extremely low frequency electric and magnetic fields over a wide range of field strengths.

A system is described that is capable of producing extremely low frequency (ELF) magnetic fields for relatively short-term exposure of cultured mammalian cells. The system utilizes a ferromagnetic core to contain and direct the magnetic field of a 1,000 turn solenoidal coil and can produce a range of flux densities and induced electric fields much higher than those produced by Helmholtz coils. The system can generate magnetic fields from the microtesla (microT) range up to 0.14 T with induced electric field strengths on the order of 1.0 V/m. The induced electric field can be accurately varied by changing the sample chamber configuration without changing the exposure magnetic field. This gives the system the ability to separate the bioeffects of magnetic and induced electric fields. In the frequency range of 4-100 Hz and magnetic flux density range of 0.005-0.14 T, the maximum total harmonic distortion of the induced electric field is typically less than 1.0%. The temperature of the samples is held constant to within 0.4 degrees C by constant perfusion of warmed culture medium through the sample chamber.

Cells, Cultured↗