Reconstitution of Arp2/3-Nucleated Actin Assembly with CP, V-1 and CARMIL.

Actin polymerization is often associated with membrane proteins containing capping-protein-interacting (CPI) motifs, such as CARMIL, CD2AP, and WASHCAP/Fam21. CPI motifs bind directly to actin capping protein (CP), and this interaction weakens the binding of CP to barbed ends of actin filaments, lessening the ability of CP to functionally cap those ends. The protein V-1 / myotrophin binds to the F-actin binding site on CP and sterically blocks CP from binding barbed ends. CPI-motif proteins also weaken the binding between V-1 and CP, which decreases the inhibitory effects of V-1, thereby freeing CP to cap barbed ends. Here, we address the question of whether CPI-motif proteins on a surface analogous to a membrane lead to net activation or inhibition of actin assembly nucleated by Arp2/3 complex. Using reconstitution with purified components, we discovered that CARMIL at the surface promotes and enhances actin assembly, countering the inhibitory effects of V-1 and thus activating CP. The reconstitution involves the presence of an Arp2/3 activator on the surface, along with Arp2/3 complex, V-1, CP, profilin and actin monomers in solution, recreating key features of cell physiology.

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein.

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Förster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. Interaction with CPI-motif peptides induced conformations within CP that bring the cap and stalk closer, while interaction with V-1 moves them away from one another. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wild-type (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein.

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Förster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. CPI-motif peptide association induced conformational changes within CP that propagate in one direction, while V-1 association induced conformational changes in the opposite direction. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wildtype (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI-motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

Septin and actin contributions to endothelial cell-cell junctions and monolayer integrity.

Septins in endothelial cells (ECs) have important roles supporting the integrity of the endothelial monolayer. Cell-cell junctions in EC monolayers are highly dynamic, with continuous retractions and protrusions. Depletion of septins in ECs leads to disruption of cell-cell junctions, which are composed of VE-cadherin and other junctional proteins. In EC monolayers, septins are concentrated at the plasma membrane at sites of cell-cell contact, in curved- and scallop-shaped patterns. These membrane-associated septin accumulations are located in regions of positive membrane curvature, and those regions are often associated with and immediately adjacent to actin-rich protrusions with negative membrane curvature. EC septins associate directly with plasma membrane lipids, based on findings with site-specific mutations of septins in ECs, which is consistent with biochemical and cell biological studies in other systems. Loss of septins leads to disruption of the EC monolayer, and gaps form between cells. The number and breadth of cell-cell contacts and junctions decreases, and the number and frequency of retractions, ruffles, and protrusions at cell edges also decreases. In addition, loss of septins leads to decreased amounts of F-actin at the cortical membrane, along with increased amounts of F-actin in stress fibers of the cytoplasm. Endothelial monolayer disruption from loss of septins is also associated with decreased transendothelial electric resistance (TEER) and increased levels of transendothelial migration (TEM) by immune and cancer cells, owing to the gaps in the monolayer. A current working model is that assembly of septin filaments at regions of positive membrane curvature contributes to a mechanical footing or base for actin-based protrusive forces generated at adjoining regions of the membrane. Specific molecular interactions between the septin and actin components of the cytoskeleton may also be important contributors. Regulators of actin assembly may promote and support the assembly of septin filaments at the membrane, as part of a molecular feedback loop between the assembly of septin and actin filaments.

Comparative Analysis of CPI-Motif Regulation of Biochemical Functions of Actin Capping Protein.

The heterodimeric actin capping protein (CP) is regulated by a set of proteins that contain CP-interacting (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families (a) to bind to CP, (b) to allosterically inhibit barbed-end capping by CP, and (c) to allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino acid residues that are conserved but are not part of the strictly defined consensus, as it was originally identified in comparisons of sequences of CPI motifs across all protein families [Hernandez-Valladares, M., et al. (2010) Structural characterization of a capping protein interaction motif defines a family of actin filament regulators. Nat. Struct. Mol. Biol. 17, 497-503; Bruck, S., et al. (2006) Identification of a Novel Inhibitory Actin-capping Protein Binding Motif in CD2-associated Protein. J. Biol. Chem. 281, 19196-19203]. These biochemical differences may be important for conserved distinct functions of CPI-motif protein families in cells with respect to the regulation of CP activity and actin assembly near membranes.

Mst1 Kinase Regulates the Actin-Bundling Protein L-Plastin To Promote T Cell Migration.

Exploring the mechanisms controlling lymphocyte trafficking is essential for understanding the function of the immune system and the pathophysiology of immunodeficiencies. The mammalian Ste20-like kinase 1 (Mst1) has been identified as a critical signaling mediator of T cell migration, and loss of Mst1 results in immunodeficiency disease. Although Mst1 is known to support T cell migration through induction of cell polarization and lamellipodial formation, the downstream effectors of Mst1 are incompletely defined. Mice deficient for the actin-bundling protein L-plastin (LPL) have phenotypes similar to mice lacking Mst1, including decreased T cell polarization, lamellipodial formation, and cell migration. We therefore asked whether LPL functions downstream of Mst1. The regulatory N-terminal domain of LPL contains a consensus Mst1 phosphorylation site at Thr(89) We found that Mst1 can phosphorylate LPL in vitro and that Mst1 can interact with LPL in cells. Removal of the Mst1 phosphorylation site by mutating Thr(89) to Ala impaired localization of LPL to the actin-rich lamellipodia of T cells. Expression of the T89A LPL mutant failed to restore migration of LPL-deficient T cells in vitro. Furthermore, expression of T89A LPL in LPL-deficient hematopoietic cells, using bone marrow chimeras, failed to rescue the phenotype of decreased thymic egress. These results identify LPL as a key effector of Mst1 and establish a novel mechanism linking a signaling intermediate to an actin-binding protein critical to T cell migration.

Role of N-WASP in Endothelial Monolayer Formation and Integrity.

Endothelial cells (ECs) form a monolayer that serves as a barrier between the blood and the underlying tissue. ECs tightly regulate their cell-cell junctions, controlling the passage of soluble materials and immune cells across the monolayer barrier. We studied the role of N-WASP, a key regulator of Arp2/3 complex and actin assembly, in EC monolayers. We report that N-WASP regulates endothelial monolayer integrity by affecting the organization of cell junctions. Depletion of N-WASP resulted in an increase in transendothelial electrical resistance, a measure of monolayer integrity. N-WASP depletion increased the width of cell-cell junctions and altered the organization of F-actin and VE-cadherin at junctions. N-WASP was not present at cell-cell junctions in monolayers under resting conditions, but it was recruited following treatment with sphingosine-1-phosphate. Taken together, our results reveal a novel role for N-WASP in remodeling EC junctions, which is critical for monolayer integrity and function.

Role of cortactin homolog HS1 in transendothelial migration of natural killer cells.

Natural Killer (NK) cells perform many functions that depend on actin assembly, including adhesion, chemotaxis, lytic synapse assembly and cytolysis. HS1, the hematopoietic homolog of cortactin, binds to Arp2/3 complex and promotes actin assembly by helping to form and stabilize actin filament branches. We investigated the role of HS1 in transendothelial migration (TEM) by NK cells. Depletion of HS1 led to a decrease in the efficiency of TEM by NK cells, as measured by transwell assays with endothelial cell monolayers on porous filters. Transwell assays involve chemotaxis of NK cells across the filter, so to examine TEM more specifically, we imaged live-cell preparations and antibody-stained fixed preparations, with and without the chemoattractant SDF-1α. We found small to moderate effects of HS1 depletion on TEM, including whether the NK cells migrated via the transcellular or paracellular route. Expression of HS1 mutants indicated that phosphorylation of HS1 tyrosines at positions 222, 378 and 397 was required for rescue in the transwell assay, but HS1 mutations affecting interaction with Arp2/3 complex or SH3-domain ligands had no effect. The GEF Vav1, a ligand of HS1 phosphotyrosine, influenced NK cell transendothelial migration. HS1 and Vav1 also affected the speed of NK cells migrating across the surface of the endothelium. We conclude that HS1 has a role in transendothelial migration of NK cells and that HS1 tyrosine phosphorylation may signal through Vav1.

Endothelial monolayers and transendothelial migration depend on mechanical properties of the substrate.

Endothelial cells (ECs) line the microvasculature and constitute a barrier between the vessel lumen and surrounding tissues. ECs inform circulating immune cells of the health and integrity of surrounding tissues, recruiting them in response to pathogens and tissue damage. ECs play an active role in the transmigration of immune cells across the vessel wall. We have discovered important differences in the properties of ECs on soft hydrogel substrates of varying stiffness, in comparison to glass. Primary ECs from several human sources were tested; all formed monolayers normally on soft substrates. EC monolayers formed more mature cell-cell junctions on soft substrates, relative to glass, based on increased recruitment of vinculin and F-actin. EC monolayers supported transendothelial migration (TEM) on soft substrates. Immune cells, including peripheral blood lymphocytes (PBLs) and natural killer cells, showed decreasing numbers of paracellular (between-cell) transmigration events with decreasing substrate stiffness, while the number of transcellular (through-cell) events increased for PBLs. Melanoma cancer cells showed increased transmigration with decreased stiffness. Our findings demonstrate that endothelial monolayers respond to the mechanical properties of their surroundings, which can regulate the integrity and function of the monolayer independently from inflammatory signals. Soft hydrogel substrates are a more appropriate and physiological model for tissue environments than hard substrates, with important implications for the experimental analysis of TEM.

Endothelial cells use dynamic actin to facilitate lymphocyte transendothelial migration and maintain the monolayer barrier.

The vascular endothelium is a highly dynamic structure, and the integrity of its barrier function is tightly regulated. Normally impenetrable to cells, the endothelium actively assists lymphocytes to exit the bloodstream during inflammation. The actin cytoskeleton of the endothelial cell (EC) is known to facilitate transmigration, but the cellular and molecular mechanisms are not well understood. Here we report that actin assembly in the EC, induced by Arp2/3 complex under control of WAVE2, is important for several steps in the process of transmigration. To begin transmigration, ECs deploy actin-based membrane protrusions that create a cup-shaped docking structure for the lymphocyte. We found that docking structure formation involves the localization and activation of Arp2/3 complex by WAVE2. The next step in transmigration is creation of a migratory pore, and we found that endothelial WAVE2 is needed for lymphocytes to follow a transcellular route through an EC. Later, ECs use actin-based protrusions to close the gap behind the lymphocyte, which we discovered is also driven by WAVE2. Finally, we found that ECs in resting endothelial monolayers use lamellipodial protrusions dependent on WAVE2 to form and maintain contacts and junctions between cells.

Roles for actin assembly in endocytosis.

Endocytosis includes a number of processes by which cells internalize segments of their plasma membrane, enclosing a wide variety of material from outside the cell. Endocytosis can contribute to uptake of nutrients, regulation of signaling molecules, control of osmotic pressure, and function of synapses. The actin cytoskeleton plays an essential role in several of these processes. Actin assembly can create protrusions that encompass extracellular materials. Actin can also support the processes of invagination of a membrane segment into the cytoplasm, elongation of the invagination, scission of the new vesicle from the plasma membrane, and movement of the vesicle away from the membrane. We briefly discuss various types of endocytosis, including phagocytosis, macropinocytosis, and clathrin-independent endocytosis. We focus mainly on new findings on the relative importance of actin in clathrin-mediated endocytosis (CME) in yeast versus mammalian cells.

Actin dynamics and endocytosis in yeast and mammals.

Tight regulation of the actin cytoskeleton is critical for many cell functions, including various forms of cellular uptake. Clathrin-mediated endocytosis (CME) is one of the main methods of uptake in many cell types. An intact and properly regulated actin cytoskeleton is required for CME in Saccharomyces cerevisiae. Yeast CME requires the proper regulation of actin polymerization, filament cross-linking, and filament disassembly. Recent studies also point to a role for F-BAR and BAR-domain containing proteins in linking the processes of generating and sensing plasma membrane curvature with those regulating the actin cytoskeleton. Many of these same proteins are conserved in mammalian CME. However, until recently the requirement for actin in mammalian CME was less clear. Several recent studies in mammalian cells provide new support for an actin requirement in the invagination and late stages of CME. This review focuses on the regulation of the actin cytoskeleton during CME in yeast and the emerging evidence for a role for actin during mammalian CME.

Dynamin2 GTPase and cortactin remodel actin filaments.

The large GTPase dynamin, best known for its activities that remodel membranes during endocytosis, also regulates F-actin-rich structures, including podosomes, phagocytic cups, actin comet tails, subcortical ruffles, and stress fibers. The mechanisms by which dynamin regulates actin filaments are not known, but an emerging view is that dynamin influences F-actin via its interactions with proteins that interact directly or indirectly with actin filaments. We show here that dynamin2 GTPase activity remodels actin filaments in vitro via a mechanism that depends on the binding partner and F-actin-binding protein, cortactin. Tightly associated actin filaments cross-linked by dynamin2 and cortactin became loosely associated after GTP addition when viewed by transmission electron microscopy. Actin filaments were dynamically unraveled and fragmented after GTP addition when viewed in real time using total internal reflection fluorescence microscopy. Cortactin stimulated the intrinsic GTPase activity of dynamin2 and maintained a stable link between actin filaments and dynamin2, even in the presence of GTP. Filaments remodeled by dynamin2 GTPase in vitro exhibit enhanced sensitivity to severing by the actin depolymerizing factor, cofilin, suggesting that GTPase-dependent remodeling influences the interactions of actin regulatory proteins and F-actin. The global organization of the actomyosin cytoskeleton was perturbed in U2-OS cells depleted of dynamin2, implicating dynamin2 in remodeling actin filaments that comprise supramolecular F-actin arrays in vivo. We conclude that dynamin2 GTPase remodels actin filaments and plays a role in orchestrating the global actomyosin cytoskeleton.

Characterization of power induced heating and damage in fiber optic probes for near-field scanning optical microscopy.

Tip-induced sample heating in near-field scanning optical microscopy (NSOM) is studied for fiber optic probes fabricated using the chemical etching technique. To characterize sample heating from etched NSOM probes, the spectra of a thermochromic polymer sample are measured as a function of probe output power, as was previously reported for pulled NSOM probes. The results reveal that sample heating increases rapidly to approximately 55-60 degrees C as output powers reach approximately 50 nW. At higher output powers, the sample heating remains approximately constant up to the maximum power studied of approximately 450 nW. The sample heating profiles measured for etched NSOM probes are consistent with those previously measured for NSOM probes fabricated using the pulling method. At high powers, both pulled and etched NSOM probes fail as the aluminum coating is damaged. For probes fabricated in our laboratory we find failure occurring at input powers of 3.4+/-1.7 and 20.7+/-6.9 mW for pulled and etched probes, respectively. The larger half-cone angle for etched probes ( approximately 15 degrees for etched and approximately 6 degrees for pulled probes) enables more light delivery and also apparently leads to a different failure mechanism. For pulled NSOM probes, high resolution images of NSOM probes as power is increased reveal the development of stress fractures in the coating at a taper diameter of approximately 6 microm. These stress fractures, arising from the differential heating expansion of the dielectric and the metal coating, eventually lead to coating removal and probe failure. For etched tips, the absence of clear stress fractures and the pooled morphology of the damaged aluminum coating following failure suggest that thermal damage may cause coating failure, although other mechanisms cannot be ruled out.

The role of nuclear envelope calcium in modifying nuclear pore complex structure.

Some of the most important trafficking processes in cells involve transport across the nuclear envelope. Whether it is the import of transcription factors or the export of RNA, the only known portal across the double lipid bilayer that forms the nuclear envelope are the macromolecular pores known as nuclear pore complexes (NPCs). Understanding how signals influence the conformation of the NPC is important for testing models of, and perhaps modifying, transport across the nuclear envelope. Here we summarize high-resolution atomic force microscopy studies of NPC structure following manipulation of nuclear envelope calcium stores of nuclei from Xenopus laevis oocytes. The results show that the release of calcium from these stores through the specific activation of inositol 1,4,5-trisphosphate receptors leads to changes in NPC structure observable from both sides of the nuclear envelope. The diameter of the NPC is also sensitive to these calcium stores and increases upon calcium release. Western blot analysis reveals the presence of ryanodine receptors in the nuclear envelope of X. laevis oocytes, although in low abundance. Activation of these calcium channels also leads to the displacement of the central mass and changes in NPC diameter. This change in structure may involve a displacement of the cytoplasmic and nuclear rings of the NPC towards each other, leading to the apparent emergence of the central mass from both sides of the NPC. The changes in conformation and diameter of the NPC may alter cargo access and binding to phenylalanine-glycine repeats lining the pore, thus altering transport.

Activation of ryanodine receptors in the nuclear envelope alters the conformation of the nuclear pore complex.

Nuclear pore complexes (NPCs) are supramolecular protein pores that traverse the nuclear envelope and form the only known direct route of transport between the cytoplasmic and nuclear spaces. Detailed studies have identified both active and passive mechanisms of transport through the NPC and structural studies have revealed its three-dimensional architecture. Under certain conditions, structural studies have found evidence for a mass in the central pore of the NPC whose identity remains unclear. Some studies suggest this mass represents cargo caught in transit, while others suggest it is an integral component of the NPC, the position of which is sensitive to sample conditions. Regardless of its identity, previous studies have shown that the central mass location within the NPC pore is influenced by the presence of calcium in the cisternal spaces of the nuclear membrane. Specific depletion of these calcium stores through inositol 1,4,5-trisphosphate (IP(3)) receptor activation leads to the apparent displacement of the central mass towards both the cytoplasmic and nucleoplasmic sides of the NPC. Whether the central mass is cargo or a NPC component, these observations may offer interesting insights linking transport and calcium signaling pathways. Here, we show that ryanodine (Ry) receptors are also present in the nuclear envelope of Xenopus laevis oocytes, and their specific activation can affect the conformational state of the NPC. Although previously undetected, Western blot analysis of isolated oocyte nuclei reveals the presence of Ry receptors in the nuclear envelope, albeit in low abundance. Extensive atomic force microscopy (AFM) studies at the single pore level of isolated, fixed nuclei reveal changes in the NPC conformational state following treatments that stimulate Ry receptor activity. At resting calcium levels ( approximately 200 nM Ca(2+)), the central mass within the lumen of the NPC is recessed 5.3 nm below the cytoplasmic rim of the NPC. Following treatment with 10 nM ryanodine, the central mass displaces towards the cytoplasmic face occupying a new position only 2.9 nm below the cytoplasmic rim. Interestingly, at high ryanodine concentrations (20 microM), which are reported to deactivate Ry receptors, the central mass is observed to return to the recessed position, 5.4 nm below the cytoplasmic rim. Treatments with caffeine also lead to large changes in the NPC conformation, confirming the link to specific activation of Ry receptors. These observations are consistent with a new mechanism of NPC regulation in which specific activation of Ry receptors located in the nuclear envelope can modulate cisternal calcium levels, leading to changes in the NPC conformation. Together with previous studies, it now appears that both IP(3) and Ry receptors are present in the nuclear envelope of Xenopus oocytes and are capable, through activation, of indirectly influencing the conformational state of the NPC.

Nuclear side conformational changes in the nuclear pore complex following calcium release from the nuclear membrane.

Changes in nuclear pore complex (NPC) structure are studied following treatments modifying the cisternal calcium levels located between the two lipid bilayers that together form the nuclear envelope. Since the NPC forms the only known passageway across the nuclear envelope, it plays a central role in nucleocytoplasmic transport. Understanding the origin of conformational changes that may affect this trafficking or modify cargo interactions with the NPC is, therefore, necessary to completely understand the function of these complex molecules. In previous studies on the cytoplasmic side of the nuclear envelope, a central mass was observed in the pore of the NPC and its location was shown to be sensitive to the cisternal calcium levels. Here we report atomic force microscopy (AFM) measurements on the nuclear side of the envelope, which also reveal a cisternal calcium dependence in the conformational state of the NPC. These measurements, made at the single nuclear pore level, reveal a displacement of the central mass towards the nuclear side of the membrane following treatments with adenophostin A, a specific agonist of calcium channels (inositol 1,4,5-trisphosphate (IP(3)) receptors) located in the nuclear envelope. We further demonstrate that these conformational changes are observed in nuclear pores lacking the basket structure while samples prepared in the presence of protease inhibitors retain baskets and block AFM measurements of the channel. While these measurements are unable to distinguish whether the central mass is cargo or an integral component of the NPC, its dose-dependent displacement with cisternal calcium levels does suggest links to transport or to changes in cargo interactions with the NPC. Taken together with previous measurements done on the cytoplasmic side of the nuclear envelope, these studies argue against a piston-like displacement of the central mass and instead suggest a more complicated mechanism. One possibility involves a concerted collapse of the NPC rings towards one another following cisternal calcium release, thus leading to the apparent emergence of the central mass from each side of the NPC.