Trypanin is a cytoskeletal linker protein and is required for cell motility in African trypanosomes

The cytoskeleton of eukaryotic cells is comprised of a complex network of distinct but interconnected filament systems that function in cell division, cell motility, and subcellular trafficking of proteins and organelles. A gap in our understanding of this dynamic network is the identification of proteins that connect subsets of cytoskeletal structures. We previously discovered a family of cytoskeleton-associated proteins that includes GAS11, a candidate human tumor suppressor upregulated in growth-arrested cells, and trypanin, a component of the flagellar cytoskeleton of African trypanosomes. Although these proteins are intimately associated with the cytoskeleton, their function has yet to be determined. Here we use double-stranded RNA interference to block trypanin expression in Trypanosoma brucei, and demonstrate that this protein is required for directional cell motility. Trypanin(minus sign) mutants have an active flagellum, but are unable to coordinate flagellar beat. As a consequence, they spin and tumble uncontrollably, occasionally moving backward. Immunofluorescence experiments demonstrate that trypanin is located along the flagellum/flagellum attachment zone and electron microscopic analysis revealed that cytoskeletal connections between the flagellar apparatus and subpellicular cytoskeleton are destabilized in trypanin(minus sign) mutants. These results indicate that trypanin functions as a cytoskeletal linker protein and offer insights into the mechanisms of flagellum-based cell motility.     

Crystal structure of vinculin in complex with vinculin binding site 50 (VBS50), the integrin binding site 2 (IBS2) of talin

The cytoskeletal protein talin activates integrin receptors by binding of its FERM domain to the cytoplasmic tail of β‐integrin. Talin also couples integrins to the actin cytoskeleton, largely by binding to and activating the cytoskeletal protein vinculin, which binds to F‐actin through the agency of its five‐helix bundle tail (Vt) domain. Talin activates vinculin by means of buried amphipathic α‐helices coined vinculin binding sites (VBSs) that reside within numerous four‐ and five‐helix bundle domains that comprise the central talin rod, which are released from their buried locales by means of mechanical tension on the integrin:talin complex. In turn, these VBSs bind to the N‐terminal seven‐helix bundle (Vh1) domain of vinculin, creating an entirely new helix bundle that severs its head‐tail interactions. Interestingly, talin harbors a second integrin binding site coined IBS2 that consists of two five‐helix bundle domains that also contain a VBS (VBS50). Here we report the crystal structure of VBS50 in complex with vinculin at 2.3 Å resolution and show that intramolecular interactions of VBS50 within IBS2 are much more extensive versus its interactions with vinculin. Indeed, the IBS2‐vinculin interaction only occurs at physiological temperature and the affinity of VBS50 for vinculin is about 30 times less than other VBSs. The data support a model where integrin binding destabilizes IBS2 to allow it to bind to vinculin.     

Intermolecular versus intramolecular interactions of the vinculin binding site 33 of talin

The cytoskeletal proteins talin and vinculin are localized at cell‐matrix junctions and are key regulators of cell signaling, adhesion, and migration. Talin couples integrins via its FERM domain to F‐actin and is an important regulator of integrin activation and clustering. The 220 kDa talin rod domain comprises several four‐ and five‐helix bundles that harbor amphipathic α‐helical vinculin binding sites (VBSs). In its inactive state, the hydrophobic VBS residues involved in binding to vinculin are buried within these helix bundles, and the mechanical force emanating from bound integrin receptors is thought necessary for their release and binding to vinculin. The crystal structure of a four‐helix bundle of talin that harbors one of these VBSs, coined VBS33, was recently determined. Here we report the crystal structure of VBS33 in complex with vinculin at 2 Å resolution. Notably, comparison of the apo and vinculin bound structures shows that intermolecular interactions of the VBS33 α‐helix with vinculin are more extensive than the intramolecular interactions of the VBS33 within the talin four‐helix bundle.     

c-Abl-mediated phosphorylation of WAVE3 is required for lamellipodia formation and cell migration

The activity of the Wiskott-Aldrich syndrome-related WAVE3 protein is critical for the regulation of the Arp2/3-dependent cytoskeleton organization downstream of Rac-GTPase. The Ableson (Abl) non-receptor tyrosine kinase is also involved in the remolding of actin cytoskeleton in response to extracellular stimuli. Here we show that platelet-derived growth factor stimulation of cultured cells results in WAVE3-Abl interaction and localization to the cell periphery. WAVE3-Abl interaction promotes the tyrosine phosphorylation of WAVE3 by Abl, and STI-571, a specific inhibitor of Abl kinase activity, abrogates the Abl-mediated phosphorylation of WAVE3. We have also shown that Abl targets and phosphorylates four tyrosine residues in WAVE3 and that the Abl-dependent phosphorylation of WAVE3 is critical for the stimulation of lamellipodia formation and cell migration. Our results show that the activation of WAVE3 to promote actin remodeling is enhanced by the c-Abl-mediated tyrosine phosphorylation of WAVE3.     

Stimulation of fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42

Cell adhesion to extracellular matrix is an important physiological stimulus for organization of the actin-based cytoskeleton. Adhesion to the matrix glycoprotein thrombospondin-1 (TSP-1) triggers the sustained formation of F-actin microspikes that contain the actin-bundling protein fascin. These structures are also implicated in cell migration, which may be an important function of TSP-1 in tissue remodelling and wound repair. To further understand the function of fascin microspikes, we examined whether their assembly is regulated by Rho family GTPases. We report that expression of constitutively active mutants of Rac or Cdc42 triggered localization of fascin to lamellipodia, filopodia, and cell edges in fibroblasts or myoblasts. Biochemical assays demonstrated prolonged activation of Rac and Cdc42 in C2C12 cells adherent to TSP-1 and activation of the downstream kinase p21-activated kinase (PAK). Expression of dominant-negative Rac or Cdc42 in C2C12 myoblasts blocked spreading and formation of fascin spikes on TSP-1. Spreading and spike assembly were also blocked by pharmacological inhibition of F-actin turnover. Shear-loading of monospecific anti-fascin immunoglobulins, which block the binding of fascin to actin into cytoplasm, strongly inhibited spreading, actin cytoskeletal organization and migration on TSP-1 and also affected the motility of cells on fibronectin. We conclude that fascin is a critical component downstream of Rac and Cdc42 that is needed for actin cytoskeletal organization and cell migration responses to thrombospondin-1.     

Neurotrophin regulation of beta-actin mRNA and protein localization within growth cones.

Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of beta-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that beta-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of beta-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA. Localization of beta-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.     

A novel human protein of the maternal centriole is required for the final stages of cytokinesis and entry into S phase

Centrosomes nucleate microtubules and contribute to mitotic spindle organization and function. They also participate in cytokinesis and cell cycle progression in ways that are poorly understood. Here we describe a novel human protein called centriolin that localizes to the maternal centriole and functions in both cytokinesis and cell cycle progression. Centriolin silencing induces cytokinesis failure by a novel mechanism whereby cells remain interconnected by long intercellular bridges. Most cells continue to cycle, reenter mitosis, and form multicellular syncytia. Some ultimately divide or undergo apoptosis specifically during the protracted period of cytokinesis. At later times, viable cells arrest in G1/G0. The cytokinesis activity is localized to a centriolin domain that shares homology with Nud1p and Cdc11p, budding and fission yeast proteins that anchor regulatory pathways involved in progression through the late stages of mitosis. The Nud1p-like domain of centriolin binds Bub2p, another component of the budding yeast pathway. We conclude that centriolin is required for a late stage of vertebrate cytokinesis, perhaps the final cell cleavage event, and plays a role in progression into S phase.     

C3G is required for c-Abl-induced filopodia and its overexpression promotes filopodia formation

The Rap1 guanine nucleotide exchange factor, C3G (also known as Rap1GEF-1) is involved in signaling from growth factors, cytokines and integrins and plays a role in cell adhesion and migration, but the mechanism by which C3G regulates various cellular functions is poorly understood. We, therefore, investigated the ability of C3G to affect actin cytoskeleton-dependent morphological changes in cells. Using RNA interference, we provide evidence that C3G is required for c-Abl-induced filopodia during cell spreading on fibronectin. C3G expression induces actin cytoskeletal reorganization and promotes filopodia formation independent of its catalytic activity. It showed enrichment at filopodia tips characteristic of molecules involved in filopodia dynamics. C3G-induced filopodia were not inhibited by dominant negative mutants of Rho, Rac and Cdc42, but required Abl catalytic activity. Coexpression of N-Wasp-Crib inhibited C3G induced as well as c-Abl-induced filopodia and wiskostatin, a pharmacological inhibitor of N-Wasp attenuates C3G-induced filopodia. Cellular C3G interacts with c-Abl and C3G expression results in enhanced localization of endogenous c-Abl in the cytoplasm. We suggest that C3G and c-Abl function in an interdependent manner, in linking external signals to remodeling the cytoskeleton to induce filopodia.     

Dynasore, a dynamin inhibitor, suppresses lamellipodia formation and cancer cell invasion by destabilizing actin filaments

Dynamic remodeling of actin filaments are bases for a variety of cellular events including cell motility and cancer invasion, and the regulation of actin dynamics implies dynamin, well characterized endocytotic protein. Here we report that dynasore, a inhibitor of dynamin GTPase, potently destabilizes F-actin in vitro, and it severely inhibits the formation of pseudopodia and cancer cell invasion, both of which are supported by active F-actin formation. Dynasore rapidly disrupted F-actin formed in brain cytosol in vitro, and the dynasore’s effect on F-actin was indirect. Dynasore significantly suppressed serum-induced lamellipodia formation in U2OS cell. Dynasore also destabilized F-actin in resting cells, which caused the retraction of the plasma membrane. A certain amount of dynamin 2 in U2OS cells localized along F-actin, and co-localized with cortactin, a physiological binding partner of dynamin and F-actin. However, these associations of dynamin were partially disrupted by dynasore treatment. Furthermore, invasion activity of H1080 cell, a lung cancer cell line, was suppressed by approximately 40% with dynasore treatment. These results strongly suggest that dynasore potently destabilizes F-actin, and the effect implies dynamin. Dynasore or its derivative would be suitable candidates as potent anti-cancer drugs.     

JSAP1 is required for the cell adhesion and spreading of mouse embryonic fibroblasts

The roles of JSAP1 and JIP1 in cell adhesion and spreading were examined using mouse embryonic fibroblasts (MEFs) deficient in JIP1 (JIP1-KO), JSAP1 (JSAP1-KO), and in both JIP1 and JSAP1 (double-KO), and by using their wild type. After being plated on fibronectin-coated culture plates, wild type MEFs rapidly adhered and differentiated to typical longitudinal fibroblasts in 4 h. JSAP1-KO MEFs showed a similar sequence of adhesion and cell spreading, but their adhesion was weak, and cell spreading sequence proceeded in a delayed manner compared with the wild type. In spreading JSAP1-KO MEFs, adhesion-triggered actin cytoskeleton reorganization and FAK activation proceeded at a slower pace than in wild type MEFs. The cellular properties of double-KO MEFs and JIP1-KO MEFs were similar to those of JSAP1-KO MEFs and wild type MEFs, respectively. These results suggest that JSAP1 plays a role in adhesion and cell spreading by regulating the rapid reorganization of the actin cytoskeleton.     

Correlation between the interaction of the vinculin tail domain with lipid membranes,its phosphorylation and cell mechanical behaviour

Cell adhesion and cell–cell contacts are a pre‐requisite for proper metabolism, protein synthesis and cell survival. Integrins are the transmembrane receptors that link the extracellular matrix via the FAC (focal adhesion complex) with the cytoskeleton. Vinculin is a pivotal FAC protein that has not only been implicated in regulating FAC formation and transmitting mechanical forces, but also in associating with membranous lipids in biological systems.     

Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination

Early steps in myelination in the central nervous system (CNS) include a specialized and extreme form of cell spreading in which oligodendrocytes extend large lamellae that spiral around axons to form myelin. Recent studies have demonstrated that laminin-2 (LN-2; alpha2beta1gamma1) stimulates oligodendrocytes to extend elaborate membrane sheets in vitro (cell spreading), mediated by integrin alpha6beta1. Although a congenital LN-2 deficiency in humans is associated with CNS white matter changes, LN-2-deficient (dy/dy) mice have shown abnormalities primarily within the peripheral nervous system. Here, we demonstrate a critical role for LN-2 in CNS myelination by showing that dy/dy mice have quantitative and morphologic defects in CNS myelin. We have defined the molecular pathway through which LN-2 signals oligodendrocyte cell spreading by demonstrating requirements for phosphoinositide 3-kinase activity and integrin-linked kinase (ILK). Interaction of oligodendrocytes with LN-2 stimulates ILK activity. A dominant negative ILK inhibits LN-2-induced myelinlike membrane formation. A critical component of the myelination signaling cascade includes LN-2 and integrin signals through ILK.     

Lava lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization

Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, we have conducted a biochemical screen for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. We now show that a novel MMAP, Lava Lamp (Lva), is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Our functional analysis shows that cellularization is dramatically inhibited upon injecting anti-Lva antibodies (IgG and Fab) into embryos. In addition, we show that brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. We suggest that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. Our results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion.     

Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts

Nuclear inheritance is highly ordered, ensuring stringent, unbiased partitioning of chromosomes before cell division. In plants, however, little is known about the analogous cellular processes that might ensure unbiased inheritance of non-nuclear organelles, either in meristematic cell divisions or those induced during the acquisition of totipotency. We have investigated organelle redistribution and inheritance mechanisms during cell division in cultured tobacco mesophyll protoplasts. Quantitative analysis of organelle repositioning observed by autofluorescence of chloroplasts or green fluorescent protein (GFP), targeted to mitochondria or endoplasmic reticulum (ER), demonstrated that these organelles redistribute in an ordered manner before division. Treating protoplasts with cytoskeleton-disrupting drugs showed that redistribution depended on actin filaments (AFs), but not on microtubules (MTs), and furthermore, that an intact actin cytoskeleton was required to achieve unbiased organelle inheritance. Labelling the actin cytoskeleton with a novel GFP-fusion protein revealed a highly dynamic actin network, with local reorganisation of this network itself, appearing to contribute substantially to repositioning of chloroplasts and mitochondria. Our observations show that each organelle exploits a different strategy of redistribution to ensure unbiased partitioning. We conclude that inheritance of chloroplasts, mitochondria and ER in totipotent plant cells is an ordered process, requiring complex interactions with the actin cytoskeleton.     

Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis

Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G₀-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthelic integrin ligand (RGD-containing peptide), cell spreading, nuclear extention, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12–15 h and hence, most of G₁, in order to enter S phase. After this restriction point was passed, normally ‘anchorage-dependent’ endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25–1000 ng ml⁻¹): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml⁻¹) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed when cytoskeletal stiffness was measured directly in living cells using magnetic twisting cytometry. These results emphasize the importance of matrix-dependent changes in cell and nuclear shape as well as higher order structural interactions between different cytoskeletal filament systems for control of capillary cell growth during angiogenesis.     

The Arabidopsis SPIKE1 gene is required for normal cell shape control and tissue development

Regulated growth and cell shape control are fundamentally important to the function of plant cells, tissues, and organs. The signal transduction cascades that control localized growth and cell shape, however, are not known. To better understand the relationship between cytoskeletal organization, organelle positioning, and regulated vesicle transport, we conducted a forward genetic screen to identify genes that regulate cytoskeletal organization in plants. Because of the distinct requirements for microtubules and actin filaments during leaf trichome development, a trichome-based morphology screen is an efficient approach to identify genes that affect cytoplasmic organization. The seedling lethal spike1 mutant was identified based on trichome, cotyledon, and leaf-shape defects. The predicted SPIKE1 protein shares amino acid identity with a large family of adapter proteins present in humans, flies, and worms that integrate extracellular signals with cytoskeletal reorganization. Both the trichome phenotype and immunolocalization data suggest that SPIKE1 also is involved in cytoskeletal reorganization. The assembly of laterally clustered foci of microtubules and polarized growth are early events in cotyledon development, and both processes are misregulated in spike1 epidermal cells.