An epidermal plakin that integrates actin and microtubule networks at cellular junctions

Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.     

Actin and Microtubules in Cell Motility: Which One is in Control?

The cytoskeleton is composed of three distinct elements: actin microfilaments, microtubules and intermediate filaments. The actin cytoskeleton is thought to provide protrusive and contractile forces, and microtubules to form a polarized network allowing organelle and protein movement throughout the cell. Intermediate filaments are generally considered the most rigid component, responsible for the maintenance of the overall cell shape. Cytoskeletal elements must be coordinately regulated for the cell to fulfill complex cellular functions, as diverse as cell migration, cell adhesion and cell division. Coordination between cytoskeletal elements is achieved by signaling pathways, involving common regulators such as the Rho guanosine-5'-triphosphatases (GTPases). Furthermore, evidence is now accumulating that cytoskeletal elements participate in regulating each other. As a consequence, although their functions seem well defined, they are in fact overlapping, with actin playing a role in membrane trafficking and microtubules being involved in the control of protrusive and contractile forces. This cytoskeletal crosstalk is both direct and mediated by signaling molecules. Cell motility is a well-studied example where the interplay between actin and microtubules appears bidirectional. This leads us to wonder which, if any, cytoskeletal element leads the way.     

Qualitative and quantitative differences of cytoskeleton proteins in embryos produced in vitro, in vivo, and by somatic nuclear transfer

The cytoskeleton, consisting of complex and dynamic systems of structural filaments, intermediate filaments and microtubules, is not only a structural element but also contributes to many cellular processes such as functional compartments, transportation, mitosis, secretion, formation of cell extensions, and intercellular communication. Suggestions in rat 2-cell embryos that abnormal distributions of cytoskeletal proteins occurred following the initiations of developmental arrest and our former studies showing reduced intercellular contact zones in cloned bovine embryos prompted us to conduct comparative studies on 8-cell stage bovine embryos from nuclear transfer (NT), in vitro, and in vivo production. Immunohistochemistry and Laser-Scanning-Microscopy facilitated detection of cytoskeleton proteins--alpha-tubulin, F-actin, beta-catenin, and the cell adhesion protein cadherin; image and cluster analysis were subsequently used to study the distribution pattern of the proteins, whereas Western blot was carried out for their qualitative and quantitative analysis. The maximum fluorescence intensity of stained alpha-tubulin was observed in the cloned and the in vitro embryos. A significant higher intensity of staining for F-actin was observed in the in vivo and in vitro embryos. In contrast, Western blot revealed no differences of actin, tubulin, and catenin between the three tested groups whereas a lower abundance of cadherin proteins in the cloned embryos was visible. The distribution of actin filaments in cloned embryos was more centric or one-sided and not peripheral whereas the stained spots of catenin were smaller in comparison to in vivo or in vitro produced embryos. These differences recorded in the distribution patterns may be associated with cell physiological processes related to an influenced actin-catenin-cadherin system. In conclusion, reduced intercellular contacts coupled with abnormal distribution of cytoskeletal proteins seem to play an important role in the developmental arrest encountered normally at the 8-cell stage in bovine cloned embryos.     

Distribution of microtubules and other cytoskeletal filaments during myotube elongation as revealed by fluorescence microscopy

Summary Distribution of microtubules and other cytoskeletal filaments in growing skeletal muscle cells (myotubes) was studied in vitro by fluorescence microscopy using fluorescin-labeled antibodies and phalloidin, a specific antiactin drug. In the distal elongating tips of myotubes, microtubules were the major cytoskeletal elements; actin and intermediate filaments were much less abundant. On the other hand, colcemidand nocodozole-treatments caused disruption of microtubules and also prompt retraction of growth tips to form myosacs, a type of deformed myotube. Actin filaments remained unaffected during the retraction. The difference in the distribution of the 3 cytoskeletal filaments in the region of growth tips was most remarkable in the case of those myotubes in the process of recovery from myosacs. In an early phase of recovery, the cellular processes extending from myosacs were enriched with both microtubules and intermediate filaments, but not with actin filaments. Later, when the processes became further developed, intermediate filaments were scarce at the extreme ends. Fluorescein-labeled actin introduced by a micro-injection method was minimally incorporated into filaments in the cellular processes. We conclude that microtubules make up the cytoskeletal element which is most responsible for elongation or spreading of growth tips of myotubes in vitro.     

Focal contacts and the cytoskeleton

Cultured cells attach to the substratum by means of specialized domains of cell surface, called focal contacts. The inner side of the cell membrane is associated in these structures with cytoskeletal elements, while the outer side is connected with extracellular matrix. The present review describes both light and electron microscopic methods of studying the focal contacts and ultrastructure of adhesion plaque, that is the cytoskeletal domain of focal contact. The proteins of adhesion plaque and focal contact membranes are also characterized. The processes of the formation of focal contacts and their association with the bundles of actin microfilaments in normal cultured fibroblasts are described in detail. Association of focal contacts with other cytoskeletal elements microtubules and intermediate filaments is discussed. The neoplastic transformation induced changes of focal contact system and cytoskeletal structures associated with contact sites are described.     

Dissection of keratin dynamics: different contributions of the actin and microtubule systems

It has only recently been recognized that intermediate filaments (IFs) and their assembly intermediates are highly motile cytoskeletal components with cell-type- and isotype-specific characteristics. To elucidate the cell-type-independent contribution of actin filaments and microtubules to these motile properties, fluorescent epithelial IF keratin polypeptides were introduced into non-epithelial, adrenal cortex-derived SW13 cells. Time-lapse fluorescence microscopy of stably transfected SW13 cell lines synthesizing fluorescent human keratin 8 and 18 chimeras HK8-CFP and HK18-YFP revealed extended filament networks that are entirely composed of transgene products and exhibit the same dynamic features as keratin systems in epithelial cells. Detailed analyses identified two distinct types of keratin motility: (I) Slow (approximately 0.23 microm/min), inward-directed, continuous transport of keratin filament precursor particles from the plasma membrane towards the cell interior, which is most pronounced in lamellipodia. (II) Fast (approximately 17 microm/min), bidirectional and intermittent transport of keratin particles in axonal-type cell processes. Disruption of actin filaments inhibited type I motility while type II motility remained. Conversely, microtubule disruption inhibited transport mode II while mode I continued. Combining the two treatments resulted in a complete block of keratin motility. We therefore conclude that keratin motility relies both on intact actin filaments and microtubules and is not dependent on epithelium-specific cellular factors.     

Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3-E1 osteoblasts does not require intact microfilaments or microtubules.

Cultured osteoblasts express three major types of cytoskeleton: actin microfilaments, microtubules, and intermediate filaments. The cytoskeletal network is thought to play an important role in the transmission and conversion of a mechanical stimulus into a biochemical response. To examine a role for the three different cytoskeletal networks in fluid shear stress-induced signaling in osteoblasts, we individually disrupted actin microfilaments, micro-tubules, and intermediate filaments in MC3T3-E1 osteoblasts with multiple pharmacological agents. We subjected these cells to 90 min of laminar fluid shear stress (10 dyn/cm(2)) and compared the PGE(2) and PGI(2) release and induction of cyclooxygenase-2 protein to control cells with intact cytoskeletons. Disruption of actin microfilaments, microtubules, or intermediate filaments in MC3T3-E1 cells did not prevent a significant fluid shear stress-induced release of PGE(2) or PGI(2). Furthermore, disruption of actin microfilaments or microtubules did not prevent a significant fluid shear stress-induced increase in cyclooxygenase-2 protein levels. Disruption of intermediate filaments with acrylamide did prevent the fluid shear stress-induced increase in cyclooxygenase-2 but also prevented a PGE(2)-induced increase in cyclooxygenase-2. Thus none of the three major cytoskeletal networks are required for fluid shear stress-induced prostaglandin release. Furthermore, although neither actin microfilaments nor microtubules are required for fluid shear stress-induced increase in cyclooxygenase-2 levels, the role of intermediate filaments in regulation of cyclooxygenase-2 expression is less clear.     

Rho-associated coiled-coil kinase (ROCK) signaling and disease

Abstract

The small Rho GTPase family of proteins, encompassing the three major G-protein classes Rho, Rac and cell division control protein 42, are key mitogenic signaling molecules that regulate multiple cancer-associated cellular phenotypes including cell proliferation and motility. These proteins are known for their role in the regulation of actin cytoskeletal dynamics, which is achieved through modulating the activity of their downstream effector molecules. The Rho-associated coiled-coil kinase 1 and 2 (ROCK1 and ROCK2) proteins were the first discovered Rho effectors that were primarily established as players in RhoA-mediated stress fiber formation and focal adhesion assembly. It has since been discovered that the ROCK kinases actively phosphorylate a large cohort of actin-binding proteins and intermediate filament proteins to modulate their functions. It is well established that global cellular morphology, as modulated by the three cytoskeletal networks: actin filaments, intermediate filaments and microtubules, is regulated by a variety of accessory proteins whose activities are dependent on their phosphorylation by the Rho-kinases. As a consequence, they regulate many key cellular functions associated with malignancy, including cell proliferation, motility and viability. In this current review, we focus on the role of the ROCK-signaling pathways in disease including cancer.     

Reorganization of endothelial cells cytoskeleton during formation of functional monolayer in vitro

Endothelium lining the inner surface of vessels regulates permeability of vascular wall by providing exchange between blood circulation in vessels and tissue fluid and therefore performs a barrier function. Endothelial cells (ECs) in culture are able to maintain the barrier function peculiar to cells of vascular endothelium in vivo. The endothelial monolayer in vitro is a unique model system that allows studying interaction of cytoskeletal and adhesive structures of endotheliocytes from the earliest stages of its formation. In the present work, we described and quantitatively characterized the changes of EC cytoskeleton from the moment of spreading of endotheliocytes on glass and the formation of the first contacts between neighbor cells until formation of a functional confluent monolayer. The main type of intermediate filaments of ECs are vimentin filaments. At different stages of endothelial monolayer formation, disposition of vimentin filaments and their amount do not change essentially, they occupy more than 80% of the cell area. Actin filaments system of endotheliocytes is represented by cortical actin at the cell periphery and by bundles of actin stress fibers organized in parallel. With formation of contacts between cells in native endothelial cells, the number of actin filaments rises and thickness of their bundles increases. With formation of endothelial monolayer, there are also changes in the microtubules system—their number increases at the cell edge. At all stages of EC monolayer formation, the number of microtubules in the region of the already formed intercellular contacts exceeds the number of microtubules in the free lamella region of the cell.     

Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella.

Salmonella causes severe gastroenteritis in humans, entering non-phagocytic cells to initiate intracellular replication. Bacterial engulfment occurs by macropinocytosis, which is dependent upon nucleation of host cell actin polymerization and condensation ('bundling') of actin filaments into cables. This is stimulated by contact-induced delivery of an array of bacterial effector proteins, including the four Sips (Salmonella invasion proteins). Here we show in vitro that SipC bundles actin filaments independently of host cell components, a previously unknown pathogen activity. Bundling is directed by the SipC N-terminal domain, while additionally the C-terminal domain nucleates actin polymerization, an activity so far known only in eukaryotic proteins. The ability of SipC to cause actin condensation and cytoskeletal rearrangements was confirmed in vivo by microinjection into cultured cells, although as SipC associates with lipid bilayers it is possible that these activities are normally directed from the host cell membrane. The data suggest a novel mechanism by which a pathogen directly modulates the cytoskeletal architecture of mammalian target cells.     

Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement

Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.     

Cooperative control of Akt phosphorylation, bcl-2 expression, and apoptosis by cytoskeletal microfilaments and microtubules in capillary endothelial cells

Capillary endothelial cells can be switched between growth and apoptosis by modulating their shape with the use of micropatterned adhesive islands. The present study was carried out to examine whether cytoskeletal filaments contribute to this response. Disruption of microfilaments or microtubules with the use of cytochalasin D or nocodazole, respectively, led to levels of apoptosis in capillary cells equivalent to that previously demonstrated by inducing cell rounding with the use of micropatterned culture surfaces containing small (<20 microm in diameter) circular adhesive islands coated with fibronectin. Simultaneous disruption of microfilaments and microtubules led to more pronounced cell rounding and to enhanced levels of apoptosis approaching that observed during anoikis in fully detached (suspended) cells, indicating that these two cytoskeletal filament systems can cooperate to promote cell survival. Western blot analysis revealed that the protein kinase Akt, which is known to be critical for control of cell survival became dephosphorylated during cell rounding induced by disruption of the cytoskeleton, and that this was accompanied by a decrease in bcl-2 expression as well as a subsequent increase in caspase activation. This ability of the cytoskeleton to control capillary endothelial cell survival may be important for understanding the relationship among extracellular matrix turnover, cell shape changes, and apoptosis during angiogenesis inhibition.     

Control of actin turnover by a salmonella invasion protein

Salmonella force their way into nonphagocytic host intestinal cells to initiate infection. Uptake is triggered by delivery into the target cell of bacterial effector proteins that stimulate cytoskeletal rearrangements and membrane ruffling. The Salmonella invasion protein A (SipA) effector is an actin binding protein that enhances uptake efficiency by promoting actin polymerization. SipA-bound actin filaments (F-actin) are also resistant to artificial disassembly in vitro. Using biochemical assays of actin dynamics and actin-based motility models, we demonstrate that SipA directly arrests cellular mechanisms of actin turnover. SipA inhibits ADF/cofilin-directed depolymerization both by preventing binding of ADF and cofilin and by displacing them from F-actin. SipA also protects F-actin from gelsolin-directed severing and reanneals gelsolin-severed F-actin fragments. These data suggest that SipA focuses host cytoskeletal reorganization by locally inhibiting both ADF/cofilin- and gelsolin-directed actin disassembly, while simultaneously stimulating pathogen-induced actin polymerization.     

Cleavage of focal adhesion kinase by different proteases during SRC-regulated transformation and apoptosis. Distinct roles for calpain and caspases

Integrin-associated focal adhesion complexes provide the main adhesive links between the cellular actin cytoskeleton and the surrounding extracellular matrix. In vitro, cells utilize a complex temporal and spatially regulated mechanism of focal adhesion assembly and disassembly required for cell migration. Recent studies indicate that members of both calpain and caspase protease families can promote limited proteolytic cleavage of several components of focal adhesions leading to disassembly of these complexes. Such mechanisms that influence cell adhesion may be deregulated under pathological conditions characterized by increased cell motility, such as tumor invasion. v-Src-induced oncogenic transformation is associated with loss of focal adhesion structures and transition to a less adherent, more motile phenotype, while inactivating temperature-sensitive v-Src in serum-deprived transformed cells leads to detachment and apoptosis. In this report, we demonstrate that v-Src-induced disassembly of focal adhesions is accompanied by calpain-dependent proteolysis of focal adhesion kinase. Furthermore, inhibitors of calpain repress v-Src-induced focal adhesion disruption, loss of substrate adhesion, and cell migration. In contrast, focal adhesion loss during detachment and apoptosis induced after switching off temperature-sensitive v-Src in serum-deprived transformed cells is accompanied by caspase-mediated proteolysis of focal adhesion kinase. Thus, calpain and caspase differentially regulate focal adhesion turnover during Src-regulated cell transformation, motility, and apoptosis.     

Monitoring the Cytoskeletal EGF Response in Live Gastric Carcinoma Cells

Altered cell motility is considered to be a key factor in determining tumor invasion and metastasis. Epidermal growth factor (EGF) signaling has been implicated in this process by affecting cytoskeletal organization and dynamics in multiple ways. To sort the temporal and spatial regulation of EGF-dependent cytoskeletal re-organization in relation to a cell’s motile behavior time-lapse microscopy was performed on EGF-responsive gastric carcinoma-derived MKN1 cells co-expressing different fluorescently labeled cytoskeletal filaments and focal adhesion components in various combinations. The experiments showed that EGF almost instantaneously induces a considerable increase in membrane ruffling and lamellipodial activity that can be inhibited by Cetuximab EGF receptor antibodies and is not elicited in non-responsive gastric carcinoma Hs746T cells. The transient cell extensions are rich in actin but lack microtubules and keratin intermediate filaments. We show that this EGF-induced increase in membrane motility can be measured by a simple image processing routine. Microtubule plus-ends subsequently invade growing cell extensions, which start to accumulate focal complexes at the lamellipodium-lamellum junction. Such paxillin-positive complexes mature into focal adhesions by tyrosine phosphorylation and recruitment of zyxin. These adhesions then serve as nucleation sites for keratin filaments which are used to enlarge the neighboring peripheral keratin network. Focal adhesions are either disassembled or give rise to stable zyxin-rich fibrillar adhesions which disassemble in the presence of EGF to support formation of new focal adhesion sites in the cell periphery. Taken together the results serve as a basis for modeling the early cytoskeletal EGF response as a tightly coordinated and step-wise process which is relevant for the prediction of the effectiveness of anti-EGF receptor-based tumor therapy.     

Procedures for the biochemical enrichment and proteomic analysis of the cytoskeletome

The cell cytoskeleton is composed of microtubules, intermediate filaments, and actin that provide a rigid support structure important for cell shape. However, it is also a dynamic signaling scaffold that receives and transmits complex mechanosensing stimuli that regulate normal physiological and aberrant pathophysiological processes. Studying cytoskeletal functions in the cytoskeleton’s native state is inherently difficult due to its rigid and insoluble nature. This has severely limited detailed proteomic analyses of the complex protein networks that regulate the cytoskeleton. Here, we describe a purification method that enriches for the cytoskeleton and its associated proteins in their native state that is also compatible with current mass spectrometry-based protein detection methods. This method can be used for biochemical, fluorescence, and large-scale proteomic analyses of numerous cell types. Using this approach, 2346 proteins were identified in the cytoskeletal fraction of purified mouse embryonic fibroblasts, of which 635 proteins were either known cytoskeleton proteins or cytoskeleton-interacting proteins. Functional annotation and network analyses using the Ingenuity Knowledge Database of the cytoskeletome revealed important nodes of interconnectivity surrounding well-established regulators of the actin cytoskeleton and focal adhesion complexes. This improved cytoskeleton purification method will aid our understanding of how the cytoskeleton controls normal and diseased cell functions.     

Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies

The intracellular polymerization of cytoskeletal proteins into their supramolecular assemblies raises many questions regarding the regulatory patterns that control this process. Binding experiments using the ELISA solid phase system, together with protein assembly assays and electron microscopical studies provided clues on the protein-protein associations in the polymerization of tubulin and actin networks. In vitro reconstitution experiments of these cytoskeletal filaments using purified tau, tubulin, and actin proteins were carried out. Tau protein association with tubulin immobilized in a solid phase support system was inhibited by actin monomer, and a higher inhibition was attained in the presence of preassembled actin filaments. Conversely, tubulin and assembled microtubules strongly inhibited tau interaction with actin in the solid phase system. Actin filaments decreased the extent of in vitro tau-induced tubulin assembly. Studies on the morphological aspects of microtubules and actin filaments coexisting in vitro, revealed the association between both cytoskeletal filaments, and in some cases, the presence of fine filamentous structures bridging these polymers. Immunogold studies showed the association of tau along polymerized microtubules and actin filaments, even though a preferential localization of labeled tau with microtubules was revealed. The studies provide further evidence for the involvement of tau protein in modulating the interactions of microtubules and actin polymers in the organization of the cytsokeletal network.     

Cytoskeletal network in colon cancer: from genes to clinical application

Colorectal cancer arises from well-defined sequential steps characterised by distinct genetic events. Abnormalities in the expression and functional activity of cell adhesion molecules are implicated in the development and progression of the majority of colorectal cancers. Intercellular (e.g. E-cadherin/catenin complex) and cell-matrix (e.g. integrins) adhesion molecules are more than just cementing substances but regulate cell polarity, differentiation, proliferation, migration and invasion. Many of these cellular events are mediated through their intimate association with the actin cytoskeletal network. A dynamic actin cytoskeleton characterises normal epithelial cells and polymerisation and depolymerisation of actin filaments enables cell shape to change during migration and mitosis. In colorectal cancer, cells lose actin cytoskeletal organisation and normal cell adhesion when they become invasive. Future investigations should allow the unravelling of new cytoskeletal network functions in tumour biology and may lead to the development of novel therapeutic strategies based on the manipulation of its associated molecules.     

Mechanics of microtubules

Microtubules are rigid cytoskeletal filaments, and their mechanics affect cell morphology and cellular processes. For instance, microtubules for the support structures for extended morphologies, such as axons and cilia. Further, microtubules act as tension rods to pull apart chromosomes during cellular division. Unlike other cytoskeletal filaments (e.g., actin) that work as large networks, microtubules work individually or in small groups, so their individual mechanical properties are quite important to their cellular function. In this review, we explore the past work on the mechanics of individual microtubules, which have been studied for over a quarter of a century. We also present some prospective on future endeavors to determine the molecular mechanisms that control microtubule rigidity.     

Dendrite-like process formation and cytoskeletal remodeling regulated by delta-catenin expression

Actin- and microtubule-mediated changes in cell shape are essential for many cellular activities. However, the molecular mechanisms underlying the interplay between the two are complex and remain obscure. Here we show that the expression of delta-catenin (or NPRAP/Neurojungin), a member of p120(ctn) subfamily of armadillo proteins can induce the branching of dendrite-like processes in 3T3 cells and enhance dendritic morphogenesis in primary hippocampal neurons. This induction of branching phenotype involves initially the disruption of filamentous actin, and requires the growth of microtubules. The carboxyl-terminal truncation mutant of delta-catenin can cluster and redistribute the full-length protein, and dominantly inhibit its branching effect. delta-Catenin forms protein complexes and can bind directly to actin in vitro. The carboxyl-terminal truncation of delta-catenin does not interfere with its actin-binding capability; therefore the actin interaction alone is not sufficient for the induction of dendrite-like processes. When delta-catenin-transformed cells establish elaborate dendrite-like branches, the main cellular processes become stabilized and resist the disruption of both actin filaments and microtubules, as determined by fluorescent light microscopy and time-lapse recording analyses. We suggest that delta-catenin can effect a biphasic cytoskeletal remodeling event which differentially regulates actin and microtubules and promotes cellular morphogenesis.