Involvement of the conserved adaptor protein Alix in actin cytoskeleton assembly

The conserved adaptor protein Alix, also called AIP1 or Hp95, promotes flattening and alignment of cultured mammalian fibroblasts; however, the mechanism by which Alix regulates fibroblast morphology is not understood. Here we demonstrate that Alix in WI38 cells, which require Alix expression for maintaining typical fibroblast morphology, associates with filamentous actin (F-actin) and F-actin-based structures lamellipodia and stress fibers. Reducing Alix expression by small interfering RNA (siRNA) decreases F-actin content and inhibits stress fiber assembly. In cell-free systems, Alix directly interacts with F-actin at both the N-terminal Bro1 domain and the C-terminal proline-rich domain. In Alix immunoprecipitates from WI38 cell lysates, actin is the most abundant partner protein of Alix. In addition, the N-terminal half of the middle region of Alix binds cortactin, an activator of the ARP2/3 complex-mediated initiation of actin polymerization. Alix is required for lamellipodial localization of cortactin. The C-terminal half of the middle region of Alix interacts with alpha-actinin, a key factor that bundles F-actin in stress fibers. Alix knockdown decreases the amount of alpha-actinin that associates with F-actin. These findings establish crucial involvement of Alix in actin cytoskeleton assembly.     

Cdc42-dependent actin polymerization during compensatory endocytosis in Xenopus eggs

The actin filament (F-actin) cytoskeleton associates dynamically with the plasma membrane and is thus ideally positioned to participate in endocytosis. Indeed, a wealth of genetic and biochemical evidence has confirmed that actin interacts with components of the endocytic machinery, although its precise function in endocytosis remains unclear. Here, we use 4D microscopy to visualize the contribution of actin during compensatory endocytosis in Xenopus laevis eggs. We show that the actin cytoskeleton maintains exocytosing cortical granules as discrete invaginated compartments, such that when actin is disrupted, they collapse into the plasma membrane. Invaginated, exocytosing cortical granule compartments are directly retrieved from the plasma membrane by F-actin coats that assemble on their surface. These dynamic F-actin coats seem to drive closure of the exocytic fusion pores and ultimately compress the cortical granule compartments. Active Cdc42 and N-WASP are recruited to exocytosing cortical granule membranes before F-actin coat assembly and coats assemble by Cdc42-dependent, de novo actin polymerization. Thus, F-actin may power fusion pore resealing and function in two novel endocytic capacities: the maintenance of invaginated compartments and the processing of endosomes.     

Mechanics and multiple-particle tracking microheterogeneity of alpha-actinin-cross-linked actin filament networks

Cell morphology is controlled by the actin cytoskeleton organization and mechanical properties, which are regulated by the available contents in actin and actin regulatory proteins. Using rheometry and the recently developed multiple-particle tracking method, we compare the mechanical properties and microheterogeneity of actin filament networks containing the F-actin cross-linking protein alpha-actinin. The elasticity of F-actin/alpha-actinin networks increases with actin concentration more rapidly for a fixed molar ratio of actin to alpha-actinin than in the absence of alpha-actinin, for networks of fixed alpha-actinin concentration and of fixed actin concentration, but more slowly than theoretically predicted for a homogeneous cross-linked semiflexible polymer network. These rheological measurements are complemented by multiple-particle tracking of fluorescent microspheres imbedded in the networks. The distribution of the mean squared displacements of these microspheres becomes progressively more asymmetric and wider for increasing concentration in alpha-actinin and, to a lesser extent, for increasing actin concentration, which suggests that F-actin networks become progressively heterogeneous for increasing protein content. This may explain the slower-than-predicted rise in elasticity of F-actin/alpha-actinin networks. Together these in vitro results suggest that actin and alpha-actinin provides the cell with an unsuspected range of regulatory pathways to modulate its cytoskeleton's micromechanics and local organization in vivo.     

Strain hardening of actin filament networks. Regulation by the dynamic cross-linking protein alpha-actinin

Mechanical stresses applied to the plasma membrane of an adherent cell induces strain hardening of the cytoskeleton, i.e. the elasticity of the cytoskeleton increases with its deformation. Strain hardening is thought to mediate the transduction of mechanical signals across the plasma membrane through the cytoskeleton. Here, we describe the strain dependence of a model system consisting of actin filaments (F-actin), a major component of the cytoskeleton, and the F-actin cross-linking protein alpha-actinin, which localizes along contractile stress fibers and at focal adhesions. We show that the amplitude and rate of shear deformations regulate the resilience of F-actin networks. At low temperatures, for which the lifetime of binding of alpha-actinin to F-actin is long, F-actin/alpha-actinin networks exhibit strong strain hardening at short time scales and soften at long time scales. For F-actin networks in the absence of alpha-actinin or for F-actin/alpha-actinin networks at high temperatures, strain hardening appears only at very short time scales. We propose a model of strain hardening for F-actin networks, based on both the intrinsic rigidity of F-actin and dynamic topological constraints formed by the cross-linkers located at filaments entanglements. This model offers an explanation for the origin of strain hardening observed when shear stresses are applied against the cellular membrane.     

Enaptin, a giant actin-binding protein, is an element of the nuclear membrane and the actin cytoskeleton

Enaptin belongs to a family of recently identified giant proteins that associate with the F-actin cytoskeleton as well as the nuclear membrane. It is composed of an N-terminal alpha-actinin type actin-binding domain (ABD) followed by a long coiled coil rod and a transmembrane domain at the C-terminus. The ABD binds to F-actin in vivo and in vitro and leads to bundle formation. The human Enaptin gene spreads over 515 kb and gives rise to several splicing isoforms (Nesprin-1, Myne-1, Syne-1, CPG2). The longest assembled cDNA encompasses 27,669 bp and predicts a 1014 kDa protein. Antibodies against the ABD of Enaptin localise the protein at F-actin-rich structures throughout the cell and in focal contacts as well as at the nuclear envelope. In COS7 cells, the protein is also present within the nuclear compartment. With the discovery of the actin-binding properties of Enaptin and the highly homologous Nuance, we define a family of proteins that integrate the cytoskeleton with the nucleoskeleton.     

Localization of FMRP-associated mRNA granules and requirement of microtubules for activity-dependent trafficking in hippocampal neurons

Fragile X syndrome is caused by the absence of the fragile X mental-retardation protein (FMRP), an mRNA-binding protein, which may play important roles in the regulation of dendritic mRNA localization and/or synaptic protein synthesis. We have recently applied high-resolution fluorescence imaging methods to document the presence, motility and activity-dependent regulation of FMRP granule trafficking in dendrites and spines of cultured hippocampal neurons. In this study, we show that FMRP granules distribute to F-actin-rich compartments, including filopodia, spines and growth cones during the staged development of hippocampal neurons in culture. Fragile X mental-retardation protein granules were shown to colocalize with ribosomes, ribosomal RNA and MAP1B mRNA, a known FMRP target, which encodes a protein important for microtubule and actin stabilization. The levels of FMRP within dendrites were reduced by disruption of microtubule dynamics, but not by disruption of F-actin. Direct measurements of FMRP transport kinetics using fluorescence recovery after photobleaching in living neurons showed that microtubules were required to induce the mGluR-dependent translocation into dendrites. This study provides further characterization of the composition and regulated trafficking of FMRP granules in dendrites of hippocampal neurons.     

A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.     

Comparison of the effects on secretion in chromaffin and PC12 cells of Rab3 family members and mutants. Evidence that inhibitory effects are independent of direct interaction with Rabphilin3.

The Rab class of low molecular weight GTPases has been implicated in the regulation of vesicular trafficking between membrane compartments in eukaryotic cells. The Rab3 family consisting of four highly homologous isoforms is associated with secretory granules and synaptic vesicles. Many different types of experiments indicate that Rab3a is a negative regulator of exocytosis and that its GTP-bound form interacts with Rabphilin3, a possible effector. Overexpression of Rabphilin3 in chromaffin cells enhances secretion. We have investigated the expression, localization, and effects on secretion of the various members of the Rab3 family in bovine chromaffin and PC12 cells. We found that Rab3a, Rab3b, Rab3c, and Rab3d are expressed to varying degrees in PC12 cells and in a fraction enriched in chromaffin granule membranes from the adrenal medulla. Immunocytochemistry revealed that all members of the family when overexpressed in PC12 cells localize to secretory granules. Binding constants for the interaction of the GTP-bound forms of Rab3a, Rab3b, Rab3c, and Rab3d with Rabphilin3 were comparable (Kd = 10-20 nM). Overexpression of each of the four members of the Rab3 family inhibited secretion. Mutations in Rab3a were identified that strongly impaired the ability of the GTP-bound form to interact with Rabphilin3. The mutated proteins inhibited secretion similarly to wild type Rab3a. Although Rab3a and Rabphilin3 are located on the same secretory granule or secretory vesicle and interact both in vitro and in situ, it is concluded that the inhibition of secretion by overexpression of Rab3a is unrelated to its ability to interact with Rabphilin3.     

Synaptopodin, a molecule involved in the formation of the dendritic spine apparatus, is a dual actin/alpha-actinin binding protein

Synaptopodin (SYNPO) is a cytoskeletal protein that is preferentially located in mature dendritic spines, where it accumulates in the spine neck and closely associates with the spine apparatus. Formation of the spine apparatus critically depends on SYNPO. To further determine its molecular action, we screened for cellular binding partners. Using the yeast two-hybrid system and biochemical assays, SYNPO was found to associate with both F-actin and alpha-actinin. Ectopic expression of SYNPO in neuronal and non-neuronal cells induced actin aggregates, thus confirming a cytoplasmic interaction with the actin cytoskeleton. Whereas F-actin association is mediated by a central SYNPO motif, binding to alpha-actinin requires the C-terminal domain. Notably, the alpha-actinin binding domain is also essential for dendritic targeting and postsynaptic accumulation of SYNPO in primary neurons. Taken together, our data suggest that dendritic spine accumulation of SYNPO critically depends on its interaction with postsynaptic alpha-actinin and that SYNPO may regulate spine morphology, motility and function via its distinct modes of association with the actin cytoskeleton.     

Interaction of fluorescently-labeled contractile proteins with the cytoskeleton in cell models

To determine if a living cell is necessary for the incorporation of actin, alpha-actinin, and tropomyosin into the cytoskeleton, we have exposed cell models to fluorescently labeled contractile proteins. In this in vitro system, lissamine rhodamine-labeled actin bound to attachment plaques, ruffles, cleavage furrows and stress fibers, and the binding could not be blocked by prior exposure to unlabeled actin. Fluorescently labeled alpha-actinin also bound to ruffles, attachment plaques, cleavage furrows, and stress fibers. The periodicity of fluorescent alpha-actinin along stress fibers was wider in gerbil fibroma cells than it was in PtK2 cells. The fluorescent alpha-actinin binding in cell models could not be blocked by the prior addition of unlabeled alpha-actinin suggesting that alpha-actinin was binding to itself. While there was only slight binding of fluorescent tropomyosin to the cytoskeleton of interphase cells, there was stronger binding in furrow regions of models of dividing cells. The binding of fluorescently labeled tropomyosin could be blocked by prior exposure of the cell models to unlabeled tropomyosin. If unlabeled actin was permitted to polymerize in the stress fibers in cell models, fluorescently labeled tropomyosin stained the fibers. In contrast to the labeled contractile proteins, fluorescently labeled ovalbumin and BSA did not stain any elements of the cytoskeleton. Our results are discussed in terms of the structure and assembly of stress fibers and cleavage furrows.     

Regulation of TGF-beta1/MAPK-mediated PAI-1 gene expression by the actin cytoskeleton in human mesangial cells

The importance of transforming growth factor-beta1 (TGF-beta1) in plasminogen activator inhibitor-1 (PAI-1) gene expression has been established, but the precise intracellular mechanisms are not fully understood. Our hypothesis is that the actin cytoskeleton is involved in TGF-beta1/MAPK-mediated PAI-1 expression in human mesangial cells. Examination of the distributions of actin filaments (F-actin), alpha-actinin, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by immunofluorescence and immunoprecipitation revealed that ERK and JNK associate with alpha-actinin along F-actin and that TGF-beta1 stimulation promote the dissociation of ERK and JNK with F-actin. Disassembly of the actin cytoskeleton inhibited phosphorylation of ERK and JNK and modulated PAI-1 expression and promoter activity under both basal and TGF-beta1-stimulated conditions. Stabilizing actin prevented dephosphorylation of ERK and JNK. ERK and JNK inhibitors and overexpressed dominant negative mutants antagonized the ability of TGF-beta1 to increase PAI-1 expression and promoter activity. Disassembly of F-actin also inhibited AP-1 DNA binding activity as determined by electrophoretic mobility shift assay using AP-1 consensus oligonucleotides derived from human PAI-1 promoter. F-actin stabilization prevented loss of AP-1 DNA binding activity. Therefore, changes in actin cytoskeleton modulate the ability of TGF-beta1 to stimulate PAI-1 expression through a mechanism dependent on the activation of MAPK/AP-1 pathways.     

Adhesion of L1210 cells to sulfonated styrene copolymer surfaces: imaging of F-actin AND alpha-actinin

The static adhesion of living L1210 cells to sulfonated copolymer surfaces of different sulfonic group content and the actin cytoskeleton organization in the adhering cells were studied. The strength of the cell-substratum interaction was estimated by determining the relative number of cells remaining adherent despite experiencing a shearing force equal to 1.25 x 10(-11) N caused by the laminar flow of the medium. The cell-substratum interaction took place in a medium with or without serum. The distribution of F-actin and alpha-actinin in the adhering cells was determined in sequences of fluorescent images of cell optical slices with the use of a computer method of cell image analysis. It was shown that the surface sulfonic groups affect not only the rate and strength of cell-substratum adhesion but also the F-actin and alpha-actinin distribution (in the cell regions near the substratum surface) in cells adhering in the medium containing serum. These proteins, concentrated in the tips of microvilli, were observed as dots. The distinctness (discernibleness) and sizes of these dots depend on the surface content of sulfonic groups. F-actin is located at the periphery of the cells in cells adhering in the medium without serum and alpha-actinin is concentrated in small dots at the periphery and in the central part of the cells.     

Identification of a novel protein complex containing annexin A4, rabphilin and synaptotagmin

Rabphilin is a synaptic vesicle-associated protein proposed to play a role in regulating neurotransmitter release. Here we report the isolation and identification of a novel protein complex containing rabphilin, annexin A4 and synaptotagmin 1. We show that the rabphilin C2B domain interacts directly with the N-terminus of annexin A4 and mediates the co-complexing of these two proteins in PC12 cells. Analyzing the cellular localisation of these co-complexing proteins we find that annexin A4 is located on synaptic membranes and co-localises with rabphilin at the plasma membrane in PC12 cells. Given that rabphilin and synaptotagmin are synaptic vesicle proteins involved in neurotransmitter release, the identification of this complex suggests that annexin A4 may play a role in synaptic exocytosis.     

The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alpha-actinin

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L- selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L- selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha- actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L- selectin cytoplasmic domain. In contrast, a deletion mutant of L- selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L- selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L- selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton.     

Real-time measurement of F-actin remodelling during exocytosis using Lifeact-EGFP transgenic animals

F-actin remodelling is essential for a wide variety of cell processes. It is important in exocytosis, where F-actin coats fusing exocytic granules. The purpose of these F-actin coats is unknown. They may be important in stabilizing the fused granules, they may play a contractile role and promote expulsion of granule content and finally may be important in endocytosis. To elucidate these functions of F-actin remodelling requires a reliable method to visualize F-actin dynamics in living cells. The recent development of Lifeact-EGFP transgenic animals offers such an opportunity. Here, we studied the characteristics of exocytosis in pancreatic acinar cells obtained from the Lifeact-EGFP transgenic mice. We show that the time-course of agonist-evoked exocytic events and the kinetics of each single exocytic event are the same for wild type and Lifeact-EGFP transgenic animals. We conclude that Lifeact-EGFP animals are a good model to study of exocytosis and reveal that F-actin coating is dependent on the de novo synthesis of F-actin and that development of actin polymerization occurs simultaneously in all regions of the granule. Our insights using the Lifeact-EGFP mice demonstrate that F-actin coating occurs after granule fusion and is a granule-wide event.     

Adrenal paraneurone contractile proteins and stimulus-secretion coupling

Actin, myosin, and alpha-actinin have been isolated from adrenal chromaffin cells and characterized. Their physicochemical properties have been studied and their cell localization revealed by biochemical, immunocytochemical, and ultrastructural techniques. Alpha-actinin is a component of chromaffin granule membranes and some of the cell actin copurifies with these secretory granules. Myosin is not detected in the granules but is present mainly in the cytosol. Trifluoperazine, a calmodulin antagonist, blocks stimulation-induced hormone release from chromaffin cells at a step distal from Ca2+ entry. High affinity calmodulin binding sites have also been found in chromaffin granule membranes. Furthermore, microinjection of calmodulin antibodies into chromaffin cells blocks hormone output in response to stimulation. In view of the above findings, the possible roles of contractile proteins and calmodulin in chromaffin cell functions is discussed.     

The adaptor protein Bam32 regulates Rac1 activation and actin remodeling through a phosphorylation-dependent mechanism

The B cell adaptor molecule of 32 kDa (Bam32) is an adaptor that links the B cell antigen receptor (BCR) to ERK and JNK activation and ultimately to mitogenesis. After BCR cross-linking, Bam32 is recruited to the plasma membrane and accumulates within F-actin-rich membrane ruffles. Bam32 contains one Src homology 2 and one pleckstrin homology domain and is phosphorylated at a single site, tyrosine 139. To define the function of Bam32 in membrane-proximal signaling events, we established human B cell lines overexpressing wild-type or mutant Bam32 proteins. The basal level of F-actin increased in cells expressing wild-type or myristoylated Bam32 but decreased in cells expressing either an Src homology-2 or Tyr-139 Bam32 mutant. Overexpression of wild-type Bam32 also affected BCR-induced actin remodeling, which was visualized as increases in F-actin-rich membrane ruffles. In contrast, Bam32 mutants largely blocked the BCR-induced increase in cellular F-actin. The positive and negative effects of Bam32 variants on F-actin levels were closely mirrored by their effects on the activation of the GTPase Rac1, which is known to regulate actin remodeling in lymphocytes. Bam32-deficient DT40 B cells showed decreased Rac1 activation and a failure of Rac1 to co-localize with the BCR, whereas cells overexpressing Bam32 had increased constitutive Rac1 activation. These results suggest that Bam32 regulates the cytoskeleton through Rac1. Bam32 variants also affected downstream signaling to JNK in a manner similar to that of Rac1, suggesting that the effect of Bam32 on JNK activation may be at least partially mediated through Rac1. Our results demonstrate a novel phosphorylation-dependent function of Bam32 in regulating Rac1 activation and actin remodeling.     

Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras.

GTP activates the interaction between the synaptic vesicle proteins rabphilin and rab3. This raises the question of whether rabphilin is a resident vesicle protein that recruits rab3 in a stage-dependent fashion, or if it is instead an effector protein recruited by rab3. We now show that rabphilin, like rab3, dissociates from synaptic vesicles after exocytosis in a manner requiring both Ca2+ and membrane fusion. Rabphilin interacts with GTP-rab3 via a N-terminal domain comprising a novel Zn2+(-)finger motif, and this interaction is essential for rabphilin binding to synaptic vesicles. Thus, in the same way that ras recruits raf to the plasma membrane, rab3 reversibly recruits rabphilin to synaptic vesicles in a stage-dependent manner. These results reveal an unexpected similarity between the molecular mechanisms by which small G protein function in recruiting effector proteins to membranes during membrane traffic and signal transduction.     

Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.