A steric antagonism of actin polymerization by a salmonella virulence protein

Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.     

Control by TSH of protein turnover in thyroid subcellular fractions.

The action of TSH on protein turnover in various subcellular fractions has been investigated in dog thyroid slices incubated in vitro. The results suggest a general inhibition by TSH of protein catabolism. Using double labeline (3/ and 14C) of the proteins, an increase of the disappearance of some labeled material from the microsomal fraction in the presence of TSH has been observed. The protein nature of this material has been established by testing its susceptibility to hydrolysis by trypsin. The fact that the microsomal pellet had to be treated by triton X 100 before hydrolysis by trypsin could occur, suggests that the material is probably enclosed in, or protected by membrane vesicles. Its high molecular weight and its ability to be immunoprecipitated by an antithyroglobulin serum suggest that the microsomal protein, the disappearance of which is stimulated by TSH, is thyroglobulin or one of its subunits. It is suggested that our results reflect the acceleration by TSH of the vectorial transfer of thyroglobulin through the membranes of the endoplasmic reticulum to the colloid space.     

Uncoupling actin filament fragmentation by cofilin from increased subunit turnover

The actin depolymerizing factor (ADF)/cofilin family of proteins interact with actin monomers and filaments in a pH-sensitive manner. When ADF/cofilin binds F-actin it induces a change in the helical twist and fragmentation; it also accelerates the dissociation of subunits from the pointed ends of filaments, thereby increasing treadmilling or depolymerization. Using site-directed mutagenesis we characterized the two actin-binding sites on human cofilin. One target site was chosen because we previously showed that the villin head piece competes with ADF for binding to F-actin. Limited sequence homology between ADF/cofilin and the part of the villin headpiece essential for actin binding suggested an actin-binding site on cofilin involving a structural loop at the opposite end of the molecule to the alpha-helix already implicated in actin binding. Binding through the alpha-helix is primarily to monomeric actin, whereas the loop region is specifically involved in filament association. We have characterized the actin binding properties of each site independently of the other. Mutation of a single lysine residue in the loop region abolishes binding to filaments, but not to monomers. Using the mutation analogous to the phosphorylated form of cofilin (S3D), we show that filament binding is inhibited at physiological ionic strength but not under low salt conditions. At low ionic strength, this mutant induces both the twist change and fragmentation characteristic of wild-type cofilin, but does not activate subunit dissociation. The results suggest a two-site binding to filaments, initiated by association through the loop site, followed by interaction with the adjacent subunit through the "helix" site at the opposite end of the molecule. Together, these interactions induce twist and fragmentation of filaments, but the twist change itself is not responsible for the enhanced rate of actin subunit release from filaments.     

RhoJ modulates melanoma invasion by altering actin cytoskeletal dynamics

Rho family GTPases regulate diverse processes in human melanoma ranging from tumor formation to metastasis and chemoresistance. In this study, a combination of in vitro and in vivo approaches was utilized to determine whether RHOJ, a CDC42 homologue that regulates melanoma chemoresistance, also controls melanoma migration. Depletion or overexpression of RHOJ altered cellular morphology, implicating a role for RHOJ in modulating actin cytoskeletal dynamics. RHOJ depletion inhibited melanoma cell migration and invasion in vitro and melanoma tumor growth and lymphatic spread in mice. Molecular studies revealed that RHOJ alters actin cytoskeletal dynamics by inducing the phosphorylation of LIMK, cofilin, and p41‐ARC (ARP2/3 complex subunit) in a PAK1‐dependent manner in vitro and in tumor xenografts. Taken together, these observations identify RHOJ as a melanoma linchpin determinant that regulates both actin cytoskeletal dynamics and chemoresistance by activating PAK1.     

Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein

Podosomes, small actin-based adhesion structures, differ from focal adhesions in two aspects: their core structure and their ability to organize into large patterns in osteoclasts. To address the mechanisms underlying these features, we imaged live preosteoclasts expressing green fluorescent protein-actin during their differentiation. We observe that podosomes always form inside or close to podosome groups, which are surrounded by an actin cloud. Fluorescence recovery after photobleaching shows that actin turns over in individual podosomes in contrast to cortactin, suggesting a continuous actin polymerization in the podosome core. The observation of podosome assemblies during osteoclast differentiation reveals that they evolve from simple clusters into rings that expand by the continuous formation of new podosomes at their outer ridge and inhibition of podosome formation inside the rings. This self-organization of podosomes into dynamic rings is the mechanism that drives podosomes at the periphery of the cell in large circular patterns. We also show that an additional step of differentiation, requiring microtubule integrity, stabilizes the podosome circles at the cell periphery to form the characteristic podosome belt pattern of mature osteoclasts. These results therefore provide a mechanism for the patterning of podosomes in osteoclasts and reveal a turnover of actin inside the podosome.     

Temporal regulation of salmonella virulence effector function by proteasome-dependent protein degradation

Salmonella enterica invasion of host cells requires the reversible activation of the Rho-family GTPases Cdc42 and Rac1 by the bacterially encoded GEF SopE and the GAP SptP, which exert their function at different times during infection and are delivered into host cells by a type III secretion system. We found that SopE and SptP are delivered in equivalent amounts early during infection. However, SopE is rapidly degraded through a proteosome-mediated pathway, while SptP exhibits much slower degradation kinetics. The half-lives of these effector proteins are determined by their secretion and translocation domains. Chimeric protein analysis indicated that delivery of SptP into host cells by the SopE secretion and translocation domain drastically shortened its half-life. Conversely, delivery of SopE by the SptP secretion and translocation signals significantly increased its half-life, resulting in persistent actin cytoskeleton rearrangements. This regulatory mechanism constitutes a remarkable example of a pathogen's adaptation to modulate cellular functions.     

Analysis of turnover dynamics of the submembranous actin cortex

The cell cortex is a thin network of actin, myosin motors, and associated proteins that underlies the plasma membrane in most eukaryotic cells. It enables cells to resist extracellular stresses, perform mechanical work, and change shape. Cortical structural and mechanical properties depend strongly on the relative turnover rates of its constituents, but quantitative data on these rates remain elusive. Using photobleaching experiments, we analyzed the dynamics of three classes of proteins within the cortex of living cells: a scaffold protein (actin), a cross-linker (α-actinin), and a motor (myosin). We found that two filament subpopulations with very different turnover rates composed the actin cortex: one with fast turnover dynamics and polymerization resulting from addition of monomers to free barbed ends, and one with slow turnover dynamics with polymerization resulting from formin-mediated filament growth. Our data suggest that filaments in the second subpopulation are on average longer than those in the first and that cofilin-mediated severing of formin-capped filaments contributes to replenishing the filament subpopulation with free barbed ends. Furthermore, α-actinin and myosin minifilaments turned over significantly faster than F-actin. Surprisingly, only one-fourth of α-actinin dimers were bound to two actin filaments. Taken together, our results provide a quantitative characterization of essential mechanisms under­lying actin cortex homeostasis.     

The role of actin turnover in retrograde actin network flow in neuronal growth cones

The balance of actin filament polymerization and depolymerization maintains a steady state network treadmill in neuronal growth cones essential for motility and guidance. Here we have investigated the connection between depolymerization and treadmilling dynamics. We show that polymerization-competent barbed ends are concentrated at the leading edge and depolymerization is distributed throughout the peripheral domain. We found a high-to-low G-actin gradient between peripheral and central domains. Inhibiting turnover with jasplakinolide collapsed this gradient and lowered leading edge barbed end density. Ultrastructural analysis showed dramatic reduction of leading edge actin filament density and filament accumulation in central regions. Live cell imaging revealed that the leading edge retracted even as retrograde actin flow rate decreased exponentially. Inhibition of myosin II activity before jasplakinolide treatment lowered baseline retrograde flow rates and prevented leading edge retraction. Myosin II activity preferentially affected filopodial bundle disassembly distinct from the global effects of jasplakinolide on network turnover. We propose that growth cone retraction following turnover inhibition resulted from the persistence of myosin II contractility even as leading edge assembly rates decreased. The buildup of actin filaments in central regions combined with monomer depletion and reduced polymerization from barbed ends suggests a mechanism for the observed exponential decay in actin retrograde flow. Our results show that growth cone motility is critically dependent on continuous disassembly of the peripheral actin network.     

Cortical actin turnover during cytokinesis requires myosin II

The involvement of myosin II in cytokinesis has been demonstrated with microinjection, genetic, and pharmacological approaches; however, the exact role of myosin II in cell division remains poorly understood. To address this question, we treated dividing normal rat kidney (NRK) cells with blebbistatin, a potent inhibitor of the nonmuscle myosin II ATPase. Blebbistatin caused a strong inhibition of cytokinesis but no detectable effect on the equatorial localization of actin or myosin. However, whereas these filaments dissociated from the equator in control cells during late cytokinesis, they persisted in blebbistatin-treated cells over an extended period of time. The accumulation of equatorial actin was caused by the inhibition of actin filament turnover, as suggested by a 2-fold increase in recovery half-time after fluorescence photobleaching. Local release of blebbistatin at the equator caused localized accumulation of equatorial actin and inhibition of cytokinesis, consistent with the function of myosin II along the furrow. However, treatment of the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a second, global role. Our observations indicate that myosin II ATPase is not required for the assembly of equatorial cortex during cytokinesis but is essential for its subsequent turnover and remodeling.     

Cytoplasmic gamma actin as a Z-disc protein

To investigate the precise localization of cytoplasmic gamma actin in skeletal muscle and the relationship to dystrophin molecules, we designed an antibody against the N-terminal peptide of cytoplasmic gamma actin. Western blot analysis using SDS-PAGE and isoelectric focusing (IEF) gel revealed that the antibody reacted only with the actin isoforms having gamma motility, confirming that the antibody is specific to the cytoplasmic (nonmuscle) gamma actin. Immunohistochemical analysis of the skeletal muscle of the adult mouse revealed a dot-like staining pattern of the antibody in transverse sections and a striated staining pattern in longitudinal sections. The double immunostaining technique revealed the colocalization of cytoplasmic gamma actin with alpha-actinin, implying the localization of the actin on the Z-disc. Contrary to previous findings (1), we did not detect the colocalization of cytochrome oxidase, a mitochondria marker, with this actin.     

Control of actin filament length and turnover by actin depolymerizing factor (ADF/cofilin) in the presence of capping proteins and ARP2/3 complex.

The effect of Arabidopsis thaliana ADF1 and human ADF on the number of filaments in F-actin solutions has been examined using a seeded polymerization assay. ADF did not sever filaments in a catalytic fashion, but decreased the steady-state length distribution of actin filaments in correlation with its effect on actin dynamics. The increase in filament number was modest as compared with the large increase in filament turnover. ADF did not decrease the length of filaments shorter than 1 micrometer. ADF promoted the rapid turnover of gelsolin-capped filaments in a manner dependent on the number of pointed ends. To explain these results, we propose that, as a consequence of the cooperative binding of ADF to F-actin, two populations of energetically different filaments coexist in solution pending a flux of subunits from one to the other. The ADF-decorated filaments depolymerize rapidly from their pointed ends, while undecorated filaments polymerize. ADF also promotes rapid turnover of gelsolin-capped filaments in the presence of the pointed end capper Arp2/3 complex. It is shown that the Arp2/3 complex steadily generates new barbed ends in solutions of gelsolin-capped filaments, which represents an important aspect of its function in actin-based motility.     

The PDZ-LIM protein RIL modulates actin stress fiber turnover and enhances the association of alpha-actinin with F-actin

ALP, CLP-36 and RIL form the ALP subfamily of PDZ-LIM proteins. ALP has been implicated in sarcomere function in muscle cells in association with alpha-actinin. The closely related CLP-36 is predominantly expressed in nonmuscle cells, where it localizes to actin stress fibers also in association with alpha-actinin. Here we have studied the expression and functions of RIL originally identified as a gene downregulated in H-ras-transformed cells. RIL was mostly expressed in nonmuscle epithelial cells with a pattern distinct from that of CLP-36. RIL protein was found to localize to actin stress fibers in nonmuscle cells similarly to CLP-36. However, RIL expression led to partially abnormal actin filaments showing thick irregular stress fibers not seen with CLP-36. Furthermore, live cell imaging demonstrated altered stress fiber dynamics with rapid formation of new fibers and frequent collapse of thick irregular fibers in EGFP-RIL-expressing cells. These effects may be mediated through the association of RIL with alpha-actinin, as RIL was found to associate with alpha-actinin via its PDZ domain, and RIL enhanced the ability of alpha-actinin to cosediment with actin filaments. These results implicate the RIL PDZ-LIM protein as a regulator of actin stress fiber turnover.     

The measurement of protein turnover by density labelling

A method for measuring the rate of protein degradation in plant tissue is described. The method uses density labelling to avoid difficulties associated with compartmentation and recycling of amino acids. Although the technique cannot be readily adapted to measure the rate of degradation of single proteins, it avoids difficulties of interpretation due to enzyme activation or inactivation. Values for the half-life of Lemna minor protein obtained by this method are compared with values obtained by a number of other methods. To obtain satisfactory results it was necessary to improve the method of isopycnic centrifugation in CsCl gradients. A considerable improvement was achieved by using KBr gradients, and the advantages of using KBr rather than CsCl for the separation of density-labelled protein are discussed.     

Regulation of protein turnover by heat shock proteins

Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin–proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system.     

Quantitative analysis of actin turnover in Listeria comet tails: evidence for catastrophic filament turnover

Rapid assembly and disassembly (turnover) of actin filaments in cytoplasm drives cell motility and shape remodeling. While many biochemical processes that facilitate filament turnover are understood in isolation, it remains unclear how they work together to promote filament turnover in cells. Here, we studied cellular mechanisms of actin filament turnover by combining quantitative microscopy with mathematical modeling. Using live cell imaging, we found that actin polymer mass decay in Listeria comet tails is very well fit by a simple exponential. By analyzing candidate filament turnover pathways using stochastic modeling, we found that exponential polymer mass decay is consistent with either slow treadmilling, slow Arp2/3-dissociation, or catastrophic bursts of disassembly, but is inconsistent with acceleration of filament turnover by severing. Imaging of single filaments in Xenopus egg extract provided evidence that disassembly by bursting dominates isolated filament turnover in a cytoplasmic context. Taken together, our results point to a pathway where filaments grow transiently from barbed ends, rapidly terminate growth to enter a long-lived stable state, and then undergo a catastrophic burst of disassembly. By keeping filament lengths largely constant over time, such catastrophic filament turnover may enable cellular actin assemblies to maintain their mechanical integrity as they are turning over.