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The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. Our cells contain a network of filaments made up of a protein called actin. Just like the skeleton that supports our body, the actin ‘cytoskeleton’ gives a cell its shape and strength. Actin filaments are also critical for many other processes including enabling cells to move and divide. The assembly of actin filaments must be properly controlled so that they are formed at the right time and place within the cell. A complex of proteins known as the WAVE Regulatory Complex (WRC) promotes the assembly of actin filaments. The complex contains a region called the VCA, which is able to bind to and activate another protein to make the new actin filaments. The WRC regulates filament assembly by controlling the availability of the VCA in a way that is similar to opening and closing a safe box. When new actin filaments are not needed, the safe box is closed and the VCA is not available. However, when cells need to make new actin filaments, the WRC is opened to release the VCA region so that it is able to bind to the filament-producing protein. Previous studies have shown that a protein called Rac1 acts as a key to open the WRC and trigger actin filament assembly. But it remains unclear how this works. A major obstacle to studying this process is that Rac1 and the WRC only weakly interact with each other, which makes it difficult to capture the interaction under experimental conditions. To overcome this obstacle, Chen et al. tethered a Rac1 molecule to the WRC in order to make the interaction more stable. A technique called cryo-electron microscopy was used to study the three-dimensional shape of this Rac1-WRC complex. Unexpectedly, Rac1 was attached to a different part of the WRC than the site predicted by previous studies. Further experiments showed that Rac1 needs to bind to both of these sites at the same time in order to open the WRC and release VCA, similar to using two keys to open one safe box for increased security. Some cancers, neurological disorders and other diseases can be caused by defects in WRC and Rac1 activity. Therefore, these findings could lead to new ways to treat these conditions in human patients.

Expression of receptor for hyaluronan-mediated motility (RHAMM), a breast cancer susceptibility gene, is tightly controlled in normal tissues but elevated in many tumors, contributing to tumorigenesis and metastases. However, how the expression of RHAMM is regulated remains elusive. Statins, inhibitors of mevalonate metabolic pathway widely used for hypercholesterolemia, have been found to also have antitumor effects, but little is known of the specific targets and mechanisms. Moreover, Hippo signaling pathway plays crucial roles in organ size control and cancer development, yet its downstream transcriptional targets remain obscure. Here we show that RHAMM expression is regulated by mevalonate and Hippo pathways converging onto Yes-associated protein (YAP)/TEAD, which binds RHAMM promoter at specific sites and controls its transcription and consequently breast cancer cell migration and invasion (BCCMI); and that simvastatin inhibits BCCMI via targeting YAP-mediated RHAMM transcription. Required for ERK phosphorylation and BCCMI, YAP-activated RHAMM transcription is dependent on mevalonate and sensitive to simvastatin, which modulate RHAMM transcription by modulating YAP phosphorylation and nuclear-cytoplasmic localization. Further, modulation by mevalonate/simvastatin of YAP-activated RHAMM transcription requires geranylgeranylation, Rho GTPase activation, and actin cytoskeleton rearrangement, but is largely independent of MST and LATS kinase activity. These findings from in vitro and in vivo investigations link mevalonate and Hippo pathways with RHAMM as a downstream effector, a YAP-transcription and simvastatin-inhibition target, and a cancer metastasis mediator; uncover a mechanism regulating RHAMM expression and cancer metastases; and reveal a mode whereby simvastatin exerts anticancer effects; providing potential targets for cancer therapeutic agents.

Vasodilator‐stimulated phosphoprotein (VASP) is a key regulator of dynamic actin structures like filopodia and lamellipodia, but its precise function in their formation is controversial. Using in vitro TIRF microscopy, we show for the first time that both human and Dictyostelium VASP are directly involved in accelerating filament elongation by delivering monomeric actin to the growing barbed end. In solution, DdVASP markedly accelerated actin filament elongation in a concentration‐dependent manner but was inhibited by low concentrations of capping protein (CP). In striking contrast, VASP clustered on functionalized beads switched to processive filament elongation that became insensitive even to very high concentrations of CP. Supplemented with the in vivo analysis of VASP mutants and an EM structure of the protein, we propose a mechanism by which membrane‐associated VASP oligomers use their WH2 domains to effect both the tethering of actin filaments and their processive elongation in sites of active actin assembly.

Ena/VASP proteins are implicated in a variety of fundamental cellular processes including axon guidance and cell migration. In vitro , they enhance elongation of actin filaments, but at rates differing in nearly an order of magnitude according to species, raising questions about the molecular determinants of rate control. Chimeras from fast and slow elongating VASP proteins were generated and their ability to promote actin polymerization and to bind G‐actin was assessed. By in vitro TIRF microscopy as well as thermodynamic and kinetic analyses, we show that the velocity of VASP‐mediated filament elongation depends on G‐actin recruitment by the WASP homology 2 motif. Comparison of the experimentally observed elongation rates with a quantitative mathematical model moreover revealed that Ena/VASP‐mediated filament elongation displays a saturation dependence on the actin monomer concentration, implying that Ena/VASP proteins, independent of species, are fully saturated with actin in vivo and generally act as potent filament elongators. Moreover, our data showed that spontaneous addition of monomers does not occur during processive VASP‐mediated filament elongation on surfaces, suggesting that most filament formation in cells is actively controlled. Ena/VASP proteins have important functions in actin‐dependent processes. A model for the actin elongation activity of Ena/VASP based on the affinity and saturation state of WH2‐domain‐mediated actin monomer binding is presented.

Actin polymerization drives cell movement and provides cells with structural integrity. Intracellular environments contain high concentrations of solutes, including organic compounds, macromolecules, and proteins. Macromolecular crowding has been shown to affect actin filament stability and bulk polymerization kinetics. However, the molecular mechanisms behind how crowding influences individual actin filament assembly are not well understood. In this study, we investigated how crowding modulates filament assembly kinetics using total internal reflection fluorescence (TIRF) microscopy imaging and pyrene fluorescence assays. The elongation rates of individual actin filaments analyzed from TIRF imaging depended on the type of crowding agent (polyethylene glycol, bovine serum albumin, and sucrose) as well as their concentrations. Further, we utilized all-atom molecular dynamics (MD) simulations to evaluate the effects of crowding molecules on the diffusion of actin monomers during filament assembly. Taken together, our data suggest that solution crowding can regulate actin assembly kinetics at the molecular level.

Autophagy of the cortical ER in budding yeast was unexpectedly found to require End3, a component of the endocytic machinery that promotes the assembly of actin at endocytic pits on the plasma membrane. The cortical ER transiently interacts with invaginating endocytic pits through a linkage consisting of VAP proteins, oxysterol binding proteins and type I myosins. These proteins are required for actin assembly and for autophagy of the ER. Assembly of actin at these contact sites may direct the movement of ER away from the cortex towards sites of autophagosome assembly.

Several biological macromolecules, such as proteins, nucleic acids, and polysaccharides, occupy about 30% of the space in cells, resulting in a crowded macromolecule environment. The crowding effect within cells exerts an impact on the functions of biological components, the assembly behavior of biomacromolecules, and the thermodynamics and kinetics of metabolic reactions. Cell-like structures provide confined and independent compartments for studying the working mechanisms of cells, which can be used to study the physiological functions arising from the crowding effect of macromolecules in cells. This article mainly summarizes the progress of research on the macromolecular crowding effects in cell-like structures. It includes the effects of this crowding on actin assembly behavior, tubulin aggregation behavior, and gene expression. The challenges and future trends in this field are presented at the end of the paper.

Rapid re-organization of the actin cytoskeleton supports T-cell trafficking towards immune sites and interaction with antigen presenting cells (APCs). F-actin rearrangement enables T-cell trafficking by stabilizing adhesion to vascular endothelial cells and promoting transendothelial migration. T-cell/APC immune synapse (IS) maturation also relies upon f-actin-anchored LFA-1:ICAM-1 ligation. Therefore, efficient T-cell responses require tight regulation of f-actin dynamics. In this review, we summarize how the actin-bundling protein L-plastin (LPL) regulates T-cell activation and migration. LPL enhances f-actin polymerization and also directly binds to the β2 chain of the integrin LFA-1 to support intercellular adhesion and IS formation in human and murine T cells. LPL- deficient T cells migrate slowly in response to chemo-attractants such as CXCL12, CCL19, and poorly polarize towards ICAM-1. Loss of LPL impairs thymic egress and intranodal motility. LPL is also required for T-cell IS maturation with APCs, and therefore for efficient cytokine production and proliferation. LPL -/- mice are less susceptible to T-cell mediated pathologies, such as allograft rejection and experimental autoimmune encephalomyelitis (EAE). LPL activity is regulated by its N-terminal “headpiece”, which contains serine and threonine phosphorylation and calcium- and calmodulin-binding sites. LPL phosphorylation is required for lamellipodia formation during adhesion and migration, and also for LFA-1 clustering during IS formation. However, the precise molecular interactions by which LPL supports T-cell functional responses remain unclear. Future studies elucidating LPL-mediated regulation of T-cell migration and/or activation may illuminate pathways for therapeutic targeting in T-cell-mediated diseases.

Background Polarity is necessary for epithelial cells to perform distinct functions at their apical and basal surfaces. Oral epithelial cell-derived ameloblasts at secretory stage (SABs) synthesize large amounts of enamel matrix proteins (EMPs), largely amelogenins. EMPs are unidirectionally secreted into the enamel space through their apical cytoplasmic protrusions, or Tomes’ processes (TPs), to guide the enamel formation. Little is known about the transcriptional regulation underlying the establishment of cell polarity and unidirectional secretion of SABs. Results The higher-order chromatin architecture of eukaryotic genome plays important roles in cell- and stage-specific transcriptional programming. A genome organizer, special AT-rich sequence-binding protein 1 (SATB1), was discovered to be significantly upregulated in ameloblasts compared to oral epithelial cells using a whole-transcript microarray analysis. The Satb1 −/− mice possessed deformed ameloblasts and a thin layer of hypomineralized and non-prismatic enamel. Remarkably, Satb1 −/− ameloblasts at the secretory stage lost many morphological characteristics found at the apical surface of wild-type ( wt) SABs, including the loss of Tomes’ processes, defective inter-ameloblastic adhesion, and filamentous actin architecture. As expected, the secretory function of Satb1 −/− SABs was compromised as amelogenins were largely retained in cells. We found the expression of epidermal growth factor receptor pathway substrate 8 ( Eps8 ), a known regulator for actin filament assembly and small intestinal epithelial cytoplasmic protrusion formation, to be SATB1 dependent. In contrast to wt SABs, EPS8 could not be detected at the apical surface of Satb1 −/− SABs. Eps8 expression was greatly reduced in small intestinal epithelial cells in Satb1 −/− mice as well, displaying defective intestinal microvilli. Conclusions Our data show that SATB1 is essential for establishing secretory ameloblast cell polarity and for EMP secretion. In line with the deformed apical architecture, amelogenin transport to the apical secretory front and secretion into enamel space were impeded in Satb1 −/− SABs resulting in a massive cytoplasmic accumulation of amelogenins and a thin layer of hypomineralized enamel. Our studies strongly suggest that SATB1-dependent Eps8 expression plays a critical role in cytoplasmic protrusion formation in both SABs and in small intestines. This study demonstrates the role of SATB1 in the regulation of amelogenesis and the potential application of SATB1 in ameloblast/enamel regeneration.

Microbial pathogens use a variety of mechanisms to disrupt the actin cytoskeleton during infection. Vibrio parahaemolyticus (V. para) is a Gram-negative bacterium that causes gastroenteritis, and new pandemic strains are emerging throughout the world. Analysis of the V. para genome revealed a type III secretion system effector, VopL, encoding three Wiskott-Aldrich homology 2 domains that are interspersed with three proline-rich motifs. Infection of HeLa cells with V. para induces the formation of long actin fibers in a VopL-dependent manner. Transfection of VopL promotes the assembly of actin stress fibers. In vitro, recombinant VopL potently induces assembly of actin filaments that grow at their barbed ends, independent of eukaryotic factors. Vibrio VopL is predicted to be a bacterial virulence factor that disrupts actin homeostasis during an enteric infection of the host.