Vinculin activation by talin through helical bundle conversion

Vinculin is a conserved component and an essential regulator of both cell-cell (cadherin-mediated) and cell-matrix (integrin-talin-mediated focal adhesions) junctions, and it anchors these adhesion complexes to the actin cytoskeleton by binding to talin in integrin complexes or to alpha-actinin in cadherin junctions. In its resting state, vinculin is held in a closed conformation through interactions between its head (Vh) and tail (Vt) domains. The binding of vinculin to focal adhesions requires its association with talin. Here we report the crystal structures of human vinculin in its inactive and talin-activated states. Talin binding induces marked conformational changes in Vh, creating a novel helical bundle structure, and this alteration actively displaces Vt from Vh. These results, as well as the ability of alpha-actinin to also bind to Vh and displace Vt from pre-existing Vh-Vt complexes, support a model whereby Vh functions as a domain that undergoes marked structural changes that allow vinculin to direct cytoskeletal assembly in focal adhesions and adherens junctions. Notably, talin's effects on Vh structure establish helical bundle conversion as a signalling mechanism by which proteins direct cellular responses.     

Sequence and domain structure of talin

Talin is a high-molecular-weight cytoskeletal protein concentrated at regions of cell-substratum contact and, in lymphocytes, at cell-cell contacts. Integrin receptors are involved in the attachment of adherent cells to extracellular matrices and of lymphocytes to other cells. In these situations, talin codistributes with concentrations of integrins in the cell surface membrane. Furthermore, in vitro binding studies suggest that integrins bind to talin, although with low affinity. Talin also binds with high affinity to vinculin, another cytoskeletal protein concentrated at points of cell adhesion. Finally, talin is a substrate for the Ca2(+)-activated protease, calpain II, which is also concentrated at points of cell-substratum contact. To learn more about the structure of talin and its involvement in transmembrane connections between extracellular adhesions and the cytoskeleton, we have cloned and sequenced murine talin. We describe a model for the structure of talin based on this sequence and other data. Homologies between talin and other proteins define a novel family of submembranous cytoskeleton-associated proteins all apparently involved in connections to the plasma membrane.     

Interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage

Many observations suggest the presence of transmembrane linkages between the cytoskeleton and the extracellular matrix. In fibroblasts both light and electron microscopic observations reveal a co-alignment between actin filaments at the cell surface and extracellular fibronectin. These associations are seen at sites of cell matrix interaction, frequently along stress fibres and sometimes where these bundles of microfilaments terminate at adhesion plaques (focal contacts). Non-morphological evidence also indicates a functional linkage between the cytoskeleton and extracellular matrix. Addition of fibronectin to transformed cells induces flattening of the cells and a reorganization of the actin cytoskeleton, with the concomitant appearance of arrays of stress fibres. Conversely, disruption of the actin cytoskeleton by treatment with cytochalasin B leads to release of fibronectin from the cell surface. As yet, there is no detailed knowledge of the molecules involved in this transmembrane linkage, although several proteins have been suggested as candidates in the chain of attachment between bundles of actin filaments and the cytoplasmic face of the plasma membrane: these include vinculin, alpha-actinin and talin, each one having been identified at regions where bundles of actin filaments interact with the plasma membrane and underlying cell-surface fibronectin. Recently, the cell-substrate attachment (CSAT) antigen has been identified as a plasma membrane receptor for fibronectin, raising the possibility that this glycoprotein complex may serve as a bridge between fibronectin and one or more of the underlying cytoskeletal components mentioned. Here we have investigated the interaction of the purified CSAT antigen with these cytoskeletal components, and we demonstrate an interaction specifically between the CSAT antigen and talin.     

Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain

Microtubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 A resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly.     

Structural basis for vinculin activation at sites of cell adhesion

Vinculin is a highly conserved intracellular protein with a crucial role in the maintenance and regulation of cell adhesion and migration. In the cytosol, vinculin adopts a default autoinhibited conformation. On recruitment to cell-cell and cell-matrix adherens-type junctions, vinculin becomes activated and mediates various protein-protein interactions that regulate the links between F-actin and the cadherin and integrin families of cell-adhesion molecules. Here we describe the crystal structure of the full-length vinculin molecule (1,066 amino acids), which shows a five-domain autoinhibited conformation in which the carboxy-terminal tail domain is held pincer-like by the vinculin head, and ligand binding is regulated both sterically and allosterically. We show that conformational changes in the head, tail and proline-rich domains are linked structurally and thermodynamically, and propose a combinatorial pathway to activation that ensures that vinculin is activated only at sites of cell adhesion when two or more of its binding partners are brought into apposition.     

Identification of talin as a major cytoplasmic protein implicated in platelet activation

During platelet activation there is a major reorganization in the platelet cytoskeleton that accompanies a rapid change in platelet shape. Many of the events associated with activation are attributed to a rise in calcium concentration within the platelet cytoplasm. One direct consequence of the elevated calcium is the activation of a calcium-dependent protease that cleaves a major platelet protein of relative molecular mass (Mr) approximately 235,000 (235K) to 200K. This protein, P235, has been purified and reported to interact with actin, but the significance of the proteolytic cleavage is unknown. Talin, a cytoskeletal protein in smooth muscle and fibroblasts, binds vinculin and, together with vinculin, is localized in fibroblasts at sites of actin-membrane attachment. Talin and P235 have similar purification procedures, sedimentation coefficients and Stokes' radii (ref. 6 and Molony et al., unpublished observations). Of particular significance, talin is readily cleaved by proteases from approximately 215K to a fragment of approximately 190K. Given these similarities we have investigated the possible relationship between these proteins. Here we demonstrate that platelet P235 is recognized by anti-talin antibody and that it binds vinculin. Both proteins are cleaved in vitro by the calcium-activated protease to yield similar fragments. We conclude that P235 corresponds to the platelet form of talin.     

Novel form of growth cone motility involving site-directed actin filament assembly.

Regulation of cytoskeletal structure and motility by extracellular signals is essential for all directed forms of cell movement and underlies the developmental process of axonal guidance in neuronal growth cones. Interaction with polycationic microbeads can trigger morphogenic changes in neurons and muscle cells normally associated with formation of pre- and postsynaptic specializations. Furthermore, when various types of microscopic particles are applied to the lamellar surface of a neuronal growth cone or motile cell they often exhibit retrograde movement at rates of 1-6 microns min-1 (refs 3-6). There is strong evidence that this form of particle movement results from translocation of membrane proteins associated with cortical F-actin networks, not from bulk retrograde lipid flow and may be a mechanism behind processes such as cell locomotion, growth cone migration and capping of cell-surface antigens. Here we report a new form of motility stimulated by polycationic bead interactions with the growth-cone membrane surface. Bead binding rapidly induces intracellular actin filament assembly, coincident with a production of force sufficient to drive bead movements. These extracellular bead movements resemble intracellular movements of bacterial parasites known to redirect host cell F-actin assembly for propulsion. Our results suggest that site-directed actin filament assembly may be a widespread cellular mechanism for generating force at membrane-cytoskeletal interfaces.     

Nucleolar proteome dynamics

The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.     

细胞骨架在植物细胞周期进程中的动态变化及其调控?

细胞周期,即细胞生长与分裂的周期,是生命得以世代繁衍而生生不息的基础.真核细胞有丝分裂周期进程调控的分子机制高度保守.其间,微管和微丝骨架进行有规律的动态变化,顺次组成各种细胞生长和分裂装置,主动参与细胞周期进程的调节.然而,高等植物细胞周期不同时相分别有着与动物细胞不完全相同的、独特的细胞骨架列阵.而这些列阵的产生和维持直接依赖于众多细胞骨架结合蛋白以及上游信号分子的调控.本文重点综述了植物细胞周期进程中微管和微丝骨架的动态变化规律以及参与植物细胞骨架动态和有丝分裂装置组装调控的细胞骨架结合蛋白的最新研究进展,同时对细胞骨架在植物细胞周期进程中研究进行总结和展望.     

Type I gamma phosphatidylinositol phosphate kinase targets and regulates focal adhesions

The ability of cells to form cell contacts, adhere to the extracellular matrix, change morphology, and migrate is essential for development, wound healing, metastasis, cell survival and the immune response. These events depend on the binding of integrin to the extracellular matrix, and assembly of focal adhesions, which are complexes comprising scaffolding and signalling proteins organized by adhesion to the extracellular matrix. Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) regulates interactions between these proteins, including the interaction of vinculin with actin and talin. The binding of talin to beta-integrin is strengthened by PtdIns(4,5)P(2), suggesting that the basis of focal adhesion assembly is regulated by this lipid mediator. Here we show that the type I phosphatidylinositol phosphate kinase isoform-gamma 661 (PIPKI gamma 661), an enzyme that makes PtdIns(4,5)P(2), is targeted to focal adhesions by an association with talin. PIPKI gamma 661 is tyrosine phosphorylated by focal adhesion associated kinase signalling, increasing both the activity of phosphatidylinositol phosphate kinase and its association with talin. This defines a mechanism for spatial generation of PtdIns(4,5)P(2) at focal adhesions.     

An interaction between vinculin and talin

In cultured fibroblasts, microfilament bundles terminate at adhesion plaques (focal contacts), the specialized regions where the cells adhere most tightly to the underlying substrate. Vinculin is a protein concentrated in adhesion plaques and has been suggested as a possible link between the ends of the bundles of actin filaments and the plasma membrane. If vinculin is one protein in a chain of attachment between the bundles of microfilaments and the plasma membrane, it is important to identify other components which interact with vinculin. We have recently discovered a new protein in adhesion plaques which we refer to as talin. Here we show that talin binds to vinculin, which suggests that talin may be involved with vinculin in the attachment of microfilament bundles to the plasma membrane at the adhesion plaques.     

Surface mechanics mediate pattern formation in the developing retina

Pattern formation of biological structures involves organizing different types of cells into a spatial configuration. In this study, we investigate the physical basis of biological patterning of the Drosophila retina in vivo. We demonstrate that E- and N-cadherins mediate apical adhesion between retina epithelial cells. Differential expression of N-cadherin within a sub-group of retinal cells (cone cells) causes them to form an overall shape that minimizes their surface contact with surrounding cells. The cells within this group, in both normal and experimentally manipulated conditions, pack together in the same way as soap bubbles do. The shaping of the cone cell group and packing of its components precisely imitate the physical tendency for surfaces to be minimized. Thus, simple patterned expression of N-cadherin results in a complex spatial pattern of cells owing to cellular surface mechanics.     

Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly

The atomic structure of tubulin in a polymerized, straight protofilament is clearly distinct from that in a curved conformation bound to a cellular depolymerizer. The nucleotide contents are identical, and in both cases the conformation of the GTP-containing, intra-dimer interface is indistinguishable from the GDP-containing, inter-dimer contact. Here we present two structures corresponding to the start and end points in the microtubule polymerization and hydrolysis cycles that illustrate the consequences of nucleotide state on longitudinal and lateral assembly. In the absence of depolymerizers, GDP-bound tubulin shows distinctive intra-dimer and inter-dimer interactions and thus distinguishes the GTP and GDP interfaces. A cold-stable tubulin polymer with the non-hydrolysable GTP analogue GMPCPP, containing semi-conserved lateral interactions, supports a model in which the straightening of longitudinal interfaces happens sequentially, starting with a conformational change after GTP binding that straightens the dimer enough for the formation of lateral contacts into a non-tubular intermediate. Closure into a microtubule does not require GTP hydrolysis.     

Nanoscale architecture of integrin-based cell adhesions

Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signalling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a?<200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a ～40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin's polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.     

Synergistic response to oncogenic mutations defines gene class critical to cancer phenotype

Understanding the molecular underpinnings of cancer is of critical importance to the development of targeted intervention strategies. Identification of such targets, however, is notoriously difficult and unpredictable. Malignant cell transformation requires the cooperation of a few oncogenic mutations that cause substantial reorganization of many cell features and induce complex changes in gene expression patterns. Genes critical to this multifaceted cellular phenotype have therefore only been identified after signalling pathway analysis or on an ad hoc basis. Our observations that cell transformation by cooperating oncogenic lesions depends on synergistic modulation of downstream signalling circuitry suggest that malignant transformation is a highly cooperative process, involving synergy at multiple levels of regulation, including gene expression. Here we show that a large proportion of genes controlled synergistically by loss-of-function p53 and Ras activation are critical to the malignant state of murine and human colon cells. Notably, 14 out of 24 'cooperation response genes' were found to contribute to tumour formation in gene perturbation experiments. In contrast, only 1 in 14 perturbations of the genes responding in a non-synergistic manner had a similar effect. Synergistic control of gene expression by oncogenic mutations thus emerges as an underlying key to malignancy, and provides an attractive rationale for identifying intervention targets in gene networks downstream of oncogenic gain- and loss-of-function mutations.     

Structure of Cdc42 in complex with the GTPase-binding domain of the 'Wiskott-Aldrich syndrome' protein.

The Rho-family GTP-hydrolysing proteins (GTPases), Cdc42, Rac and Rho, act as molecular switches in signalling pathways that regulate cytoskeletal architecture, gene expression and progression of the cell cycle. Cdc42 and Rac transmit many signals through GTP-dependent binding to effector proteins containing a Cdc42/Rac-interactive-binding (CRIB) motif. One such effector, the Wiskott-Aldrich syndrome protein (WASP), is postulated to link activation of Cdc42 directly to the rearrangement of actin. Human mutations in WASP cause severe defects in haematopoletic cell function, leading to clinical symptoms of thrombocytopenia, immunodeficiency and eczema. Here we report the solution structure of a complex between activated Cdc42 and a minimal GTPase-binding domain (GBD) from WASP. An extended amino-terminal GBD peptide that includes the CRIB motif contacts the switch I, beta2 and alpha5 regions of Cdc42. A carboxy-terminal beta-hairpin and alpha-helix pack against switch II. The Phe-X-His-X2-His portion of the CRIB motif and the alpha-helix appear to mediate sensitivity to the nucleotide switch through contacts to residues 36-40 of Cdc42. Discrimination between the Rho-family members is likely to be governed by GBD contacts to the switch I and alpha5 regions of the GTPases. Structural and biochemical data suggest that GBD-sequence divergence outside the CRIB motif may reflect additional regulatory interactions with functional domains that are specific to individual effectors.     

Induction of formation of presynaptic terminals in neuroblastoma cells by synapsin IIb

The synapsins are a family of closely related phosphoproteins (termed synapsins Ia, Ib, IIa and IIb) associated with synaptic vesicles and implicated in the short-term regulation of neurotransmitter release from nerve endings. During development, expression of the synapsins correlates temporally with synapse formation, but there has been no direct evidence that they are involved in synaptogenesis. Here we report that overexpression of synapsin IIb in the neuroblastoma x glioma hybrid clonal cell line NG108-15 leads, during cell differentiation, to marked increases in the number of neuritic varicosities and in the numbers of small clear vesicles and large dense core vesicles per varicosity, as well as to the appearance of synapse-like cell-cell contacts. Thus, synapsin IIb may be involved in the regulation of synapse formation and, as a result, in long-term neuronal signalling.     

Heterogeneity of membrane phospholipid mobility in endothelial cells depends on cell substrate

Cellular growth control and differentiation have been shown to be dependent on both cell-cell and cell-substrate contacts. Interactions of cells with extracellular material are critical events during embryonic development and maintenance of tissue function. Plasma membrane receptors have been described for components of the extracellular matrix such as fibronectin, laminin and various collagen types. Transmembrane signalling has been shown to be influenced by the lateral mobilities of the plasma membrane constituents. The interaction of cells with their extracellular matrix could thus have a significant effect on the mobility properties of the plasma membrane components. Here we have studied the dynamic properties of fluorescent membrane phospholipids in bovine endothelial cells using fluorescence recovery after photo-bleaching measurements. At this molecular level we find that the phospholipid lateral diffusion coefficient is dependent on the substrate upon which cells are allowed to adhere (collagen, fibronectin or a natural basement membrane) and on the topography of the cell (basal versus apical plasma membrane).     

Self-organizing optic-cup morphogenesis in three-dimensional culture

Balanced organogenesis requires the orchestration of multiple cellular interactions to create the collective cell behaviours that progressively shape developing tissues. It is currently unclear how individual, localized parts are able to coordinate with each other to develop a whole organ shape. Here we report the dynamic, autonomous formation of the optic cup (retinal primordium) structure from a three-dimensional culture of mouse embryonic stem cell aggregates. Embryonic-stem-cell-derived retinal epithelium spontaneously formed hemispherical epithelial vesicles that became patterned along their proximal-distal axis. Whereas the proximal portion differentiated into mechanically rigid pigment epithelium, the flexible distal portion progressively folded inward to form a shape reminiscent of the embryonic optic cup, exhibited interkinetic nuclear migration and generated stratified neural retinal tissue, as seen in vivo. We demonstrate that optic-cup morphogenesis in this simple cell culture depends on an intrinsic self-organizing program involving stepwise and domain-specific regulation of local epithelial properties.