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.     

Heat shock gene expression and cytoskeletal alterations in mouse neuroblastoma cells

The cytoskeleton of neuroblastoma cells, clone Neuro 2A, is altered by two stress conditions: heat shock and arsenite treatment. Microtubules are reorganized, intermediate filaments are aggregated around the nucleus, and the number of stress fibers is reduced. Since both stress modalities induce similar cytoskeletal alterations, no thermic denaturation of one or more cytoskeletal components can be involved in this process. Heat shock proteins are induced both by heat and by arsenite. However, cells treated with arsenite synthesize hsp28 which is not detected in heat-treated cells. Synthesis of all hsps is prevented by addition of actinomycin D or cycloheximide. Under these conditions no alterations are observed in the organization of microtubules and intermediate filaments during heat or arsenite treatment. However, these drugs are not able to prevent the rapid loss of stress fibers. A re-formation of the cytoskeleton during the recovery period proceeds within 3 h and is also found to occur in the presence of a protein synthesis inhibitor. These data suggest that reorganization of microtubules and intermediate filaments during a stress treatment requires the synthesis of a new protein(s), probably hsp(s).     

Interaction of frog virus-3 with the cytoskeleton. I. Altered organization of microtubules, intermediate filaments, and microfilaments

The progressive cytoskeletal alterations of frog virus 3-infected baby hamster kidney (BHK) and fathead minnow (FHM) cells were studied by immunofluorescence and electron microscopy. The virus assembly sites, which contain viral genomes and viral proteins, were detected in the cytoplasm at 4 h (FHM) or 6 h (BHK) and mature virions appeared 2 h later. When infected cells were treated with Triton X-100, the assembly sites were found in association with the cytoskeleton. In infected cells, the number of microtubules progressively decreased but a few microtubules traversing in the vicinity of the assembly sites remained intact. Early in infection, the intermediate filaments retracted from the cell periphery, delimited the forming assembly sites, and remained there throughout infection. We suggest that intermediate filaments are involved in the formation of assembly sites. In addition, the filaments either by themselves or in conjunction with microtubules may anchor the assembly sites near the nucleus. The microfilament bundles (stress fibers) disappeared with the formation of assembly sites, and late in infection many projections containing microfilaments and virus particles appeared at the cell surface. The observation suggests a role for microfilaments in virus release. Taken together, these results provide the first example of a virus-infected cell in which all three cytoskeletal filaments show profound organizational changes and suggest an active participation of the host cytoskeleton in viral functions.     

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.     

Translation and the cytoskeleton: a mechanism for targeted protein synthesis

This review describes the critical evidence that in eukaryotic cells polyribosomes, mRNAs and components of the protein synthetic machinery are associated with the cytoskeleton. The role of microtubules, intermediate filaments and microfilaments are discussed; at present most evidence suggests that polyribosomes interact with the actin filaments. The use of non-ionic detergent/deoxycholate treatment in the isolation of cytoskeletal-bound polysomes is described and the conclusion reached that at low salt concentrations this leads to mixed preparations of polysomes derived from both the cytoskeleton and the endoplasmic reticulum. At present the best approach for isolation of cytoskeletal-bound polysomes appears to involve extraction with salt concentrations greater than 130 mM after an initial non-ionic detergent treatment. Such polysomes appear to be enriched in certain mRNAs and thus it is suggested that they are involved in translation of a unique set of proteins. The evidence for mRNA localisation is presented and the role of the cytoskeleton in transport and localisation of RNA discussed. Recent data on the role of the 3 untranslated region in the targeting of mRNAs both to particular regions of the cell and for translation on cytoskeletal-bound polysomes is described. The hypothesis is developed that the association of polysomes with the cytoskeleton is the basis of a mechanism for the targeting of mRNAs and the compartmentalization of protein synthesis.Abbreviations CBP cytoskeletal-bound polysomes - FP free polysomes - MBP membrane-bound polysomes - ER endoplasmic reticulum     

Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro

Summary The extracellular matrix, particularly basement membranes, plays an important role in angiogenesis (blood vessel formation). Previous work has demonstrated that a basement membranelike substrate (Matrigel) induces human umbilical vein endothelial cells to rapidly form vessel-like tubes (Kubota, et al., 1988; Grant et al., 1989b); however, the precise mechanism of tube formation is unclear. Using this in vitro model, we have investigated morphologic changes occurring during tube formation and the cytoskeletal and protein synthesis requirements of this process. Electron microscopy showed that endothelial cells attach to the Matrigel surface, align, and form cylindrical structures that contain a lumen and polarized cytoplasmic organelles. The cytoskeleton is reorganized into bundles of actin filaments oriented along the axis of the tubes and is located at the periphery of the cells. The addition of colchicine or cytochalasin D blocked tube formation, indicating that both microfilaments and microtubules are involved in this process. Cycloheximide blocked tube formation by 100%, indicating that the process also required protein synthesis. In particular, collagen synthesis seems to be required for tube formation because cis-hydroxyproline inhibited tube formation, whereas either the presence of ascorbic acid or the addition of exogenous collagen IV to the Matrigel increased tube formation. Our results indicate that endothelial cell attachment to Matrigel induces the reorganization of the cytoskeleton and elicits the synthesis of specific proteins required for the differentiated phenotype of the cells.     

Cell signaling pathways to alphaB-crystallin following stresses of the cytoskeleton

Small heat shock proteins (sHSPs) act as chaperone, but also in protecting the different cytoskeletal components. Recent results suggest that αB-crystallin, a member of sHSPs family, might regulate actin filament dynamics, stabilize them in a phosphorylation dependent manner, and protect the integrity of intermediate filaments (IF) against extracellular stress. We demonstrate that vinblastin and cytochalasin D, which respectively disorganize microtubules and actin microfilaments, trigger the activation of the p38/MAPKAP2 kinase pathway and lead to the specific αB-crystallin phosphorylation at serine 59. Upstream of p38, we found that RhoK, PKC and PKA are selectively involved in the activation of p38 and phosphorylation of αB-crystallin, depending on the cytoskeletal network disorganized. Moreover, we demonstrate that chronic perturbations of IF network result in the same activation of p38 MAPK and αB-crystallin phosphorylation, as with severe disorganization of other cytoskeletal networks. Finally, we also show that Ser 59 phosphorylated αB-crystallin colocalizes with cytoskeletal components. Thus, disturbance of cytoskeleton leads by converging signaling pathways to the phosphorylation of αB-crystallin, which probably acts as a protective effector of the cytoskeleton.     

Organisation and regulation of the cytoskeleton in plant programmed cell death

Programmed cell death (PCD) involves precise integration of cellular responses to extracellular and intracellular signals during both stress and development. In recent years much progress in our understanding of the components involved in PCD in plants has been made. Signalling to PCD results in major reorganisation of cellular components. The plant cytoskeleton is known to play a major role in cellular organisation, and reorganization and alterations in its dynamics is a well known consequence of signalling. There are considerable data that the plant cytoskeleton is reorganised in response to PCD, with remodelling of both microtubules and microfilaments taking place. In the majority of cases, the microtubule network depolymerises, whereas remodelling of microfilaments can follow two scenarios, either being depolymerised and then forming stable foci, or forming distinct bundles and then depolymerising. Evidence is accumulating that demonstrate that these cytoskeletal alterations are not just a consequence of signals mediating PCD, but that they also may have an active role in the initiation and regulation of PCD. Here we review key data from higher plant model systems on the roles of the actin filaments and microtubules during PCD and discuss proteins potentially implicated in regulating these alterations.     

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501- 520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.     

Cytoskeletal networks and the regulation of cardiac contractility: microtubules, hypertrophy, and cardiac dysfunction

The cytoskeleton as classically defined for eukaryotic cells consists of three systems of protein filaments: the microtubules, the intermediate filaments, and the microfilaments. In mature striated muscle such as the heart of the adult mammal, these three types of cytoskeletal filaments are superimposed spatially on the myofilaments, a specialized system of contractile protein filaments. Each of these systems of protein filaments has the potential to respond in an adaptive or maladaptive manner during load-induced hypertrophic cardiac growth. However, the extent to which such hypertrophy is compensatory is also critically dependent on the type of hemodynamic overload that serves as the hypertrophic stimulus. Thus cardiac hypertrophy is not intrinsically maladaptive; rather, it is the nature of the inducing load rather than hypertrophy itself that is responsible, through effects on structural and/or regulatory proteins, for the frequent deterioration of initially compensatory hypertrophy into the congestive heart failure state. As one example reviewed here of this load specificity of maladaptation, increased microtubule network density is a persistent feature of severely pressure-overloaded, hypertrophied, and failing myocardium that imposes a primarily viscous load on active myofilaments during contraction.     

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.     

Phytohormones and Cytoskeleton

Various aspects of the interaction between phytohormones and principal components of the cytoskeleton are considered. Four levels of this interaction are discussed: (1) the spatial organization of microtubules and microfilaments; (2) the synthesis and modification of cytoskeletal proteins; (3) the cytoskeleton involvement in processes, which are under hormonal control; and (4) the cytoskeleton involvement in phytohormone functioning.     

An innovative fixative for cytoskeletal components allows high resolution in colocalization studies using immunofluorescence techniques

Using a new fixation solution, CytoSkelFix, it is now possible to obtain superior fixation and thus resolution of cytoskeletal components using immunofluorescence and fluorescence microscopy. This fixative combines rapid cell penetration and cellular crosslinking of proteins such that both preservation and resolution of cellular proteins can be detected. The cytoskeleton has proven very difficult to preserve, partly because of the lability of one of the filament systems (microtubules), and one single fixative is incapable of properly preserving microtubules, microfilaments, and intermediate filaments for localization in the same cell. Further, the motor proteins associated with the cytoskeletal elements are even more difficult to preserve, particularly simultaneously with the fiber system with which they associate. We present evidence that CytoSkelFix is a superior preservative and would be useful in fixation for all types of immunofluorescence colocalization studies where superior preservation is required.     

Isolation and characterization of cytoskeletons from cotton fiber cytoplasts

Summary Over the last 25 yr, success in characterizing the individual protein components of animal cytoskeletons was possible, in part, due to technical advances in the isolation and purification of anucleate cytoskeletons from animal cells. As a step towards characterizing protein components of the plant cytoskeleton, we have isolated cytoskeletons from cytoplasts (anucleate protoplasts) prepared from cotton fiber cells grown in ovule culture. Cytoplasts isolated into a hypertonic, Ca²⁺-free medium at pH 6.8 retained internal structures after extraction with the detergent, Triton X-100. These structures were shown to include microtubule and microfilament arrays by immunofluorescence and electron microscopy. Actin and tubulin were the only abundant proteins in these preparations, suggesting that microfilaments and microtubules were the major cytoskeleta elements in the isolated cytoskeletons. The absence of additional, relatively abundant proteins suggests that (a) other cytoskeletal arrays potentially present in fiber cells (e.g., intermediate filaments) were either lost during detergent extraction or were minor components of the fiber cell cytoskeleton; and (b) high ratios of individual cytoskeletal-associated proteins relative to actin and tubulin were not required to maintain microtubules and microfilaments in organized structures.     

The Association of Tau-Like Proteins with Vimentin Filaments in Cultured Cells

There is increasing evidence that the different polymers that constitute the cytoskeleton are interconnected to form a three-dimensional network. The macromolecular interaction patterns that stabilize this network and its intrinsic dynamics are the basis for numerous cellular processes. Within this context,in vitrostudies have pointed to the existence of specific associations between microtubules, microfilaments, and intermediate filaments. It has also been postulated that microtubule-associated proteins (MAPs) are directly involved in mediating these interactions. The interactions of tau with vimentin filaments, and its relationships with other filaments of the cytoskeletal network, were analyzed in SW-13 adenocarcinoma cells, through an integrated approach that included biochemical and immunological studies. This cell line has the advantage of presenting a wild-type clone (vim+) and a mutant clone (vim−) which is deficient in vimentin expression. We analyzed the cellular roles of tau, focusing on its interactions with vimentin filaments, within the context of its functional aspects in the organization of the cytoskeletal network. Cosedimentation experiments of microtubular protein with vimentin in cell extracts enriched in intermediate filaments, combined with studies on the direct interaction of tau with nitrocellulose-bound vimentin and analysis of tau binding to vimentin immobilized in single-strand DNA affinity columns, indicate that tau interacts with the vimentin network. These studies were confirmed by a quantitative analysis of the immunofluorescence patterns of cytoskeleton-associated tubulin, tau, and vimentin using flow cytometry. In this regard, a decrease in the levels of tau associated to the cytoskeletal network in the vim− cell mutant compared with the wild-type clones was observed. However, immunofluorescence data on SW-13 cells suggest that the absence of a structured network of vimentin in the mutant vim− cells does not affect the cytoplasmic organization formed by microtubules and actin filaments, when compared with the wild-type vim+ cells. These studies suggest that tau associates with vimentin filaments and that these interactions may play a structural role in cells containing these filaments.     

Covering common ground: F-actin-dependent transport of plant viral protein inclusions reveals a novel mechanism for movement utilized by unrelated viral proteins

Plant viruses are composed of diverse genomes (e.g., RNA or DNA) encoding proteins that vary widely in sequence. It is becoming clear, however, that some apparently unrelated viral proteins have similar functions. The P6 protein encoded by Cauliflower mosaic virus (CaMV) and the 126-kDa protein encoded by Tobacco mosaic virus (TMV) are examples of this convergence in protein function. Although having no apparent sequence similarity, both proteins are pathogenicity determinants during infection, are components of novel intracellular cytoplasmic inclusions and suppress RNA silencing. Here we review our recent results demonstrating an additional novel convergent activity between these proteins: both proteins traffic along the actin cytoskeleton (microfilaments). We also discuss results showing a unique property of the P6 protein: a non-mobile strong association with microtubules. Lastly, we discuss the potential mechanism by which the P6 and 126-kDa proteins traffic along microfilaments. We provide new results suggesting that actin filament polymerization-driven movement does not support 126-kDa protein transport, thus leading to a focus on myosins as the driving force for this movement.Key words: actin polymerization, cytoskeleton, cauliflower mosaic virus, microfilaments, microtubules, myosin, tobacco mosaic virus, virus movement, intracellular transport     

Caldesmon is necessary for maintaining the actin and intermediate filaments in cultured bladder smooth muscle cells

Caldesmon (CaD), a component of microfilaments in all cells and thin filaments in smooth muscle cells, is known to bind to actin, tropomyosin, calmodulin, and myosin and to inhibit actin-activated ATP hydrolysis by smooth muscle myosin. Thus, it is believed to regulate smooth muscle contraction, cell motility and the cytoskeletal structure. Using bladder smooth muscle cell cultures and RNA interference (RNAi) technique, we show that the organization of actin into microfilaments in the cytoskeleton is diminished by siRNA-mediated CaD silencing. CaD silencing significantly decreased the amount of polymerized actin (F-actin), but the expression of actin was not altered. Additionally, we find that CaD is associated with 10 nm intermediate-sized filaments (IF) and in vitro binding assay reveals that it binds to vimentin and desmin proteins. Assembly of vimentin and desmin into IF is also affected by CaD silencing, although their expression is not significantly altered when CaD is silenced. Electronmicroscopic analyses of the siRNA-treated cells showed the presence of myosin filaments and a few surrounding actin filaments, but the distribution of microfilament bundles was sparse. Interestingly, the decrease in CaD expression had no effect on tubulin expression and distribution of microtubules in these cells. These results demonstrate that CaD is necessary for the maintenance of actin microfilaments and intermediate-sized filaments in the cytoskeletal structure. This finding raises the possibility that the cytoskeletal structure in smooth muscle is affected when CaD expression is altered, as in smooth muscle de-differentiation and hypertrophy seen in certain pathological conditions.     

Modulation of milk protein synthesis through alteration of the cytoskeleton in mouse mammary epithelial cells cultured on a reconstituted basement membrane

Recent studies indicate that the cytoskeleton may be involved in modulating tissue-specific gene expression in mammalian cells. We have studied the role of the cytoskeleton in regulating milk protein synthesis and secretion by primary mouse mammary epithelial cells cultured on a reconstituted basement membrane that promotes differentiation. After 8 days in culture, cells were treated with cytochalasin D (CD) (0.5-1 micrograms/ml) to alter actin filaments or acrylamide (Ac) (5 mM) to alter intermediate filaments (cytokeratins). CD inhibited synthesis of most proteins in a concentration-dependent manner, with beta-casein being the first affected. In contrast, Ac increased protein synthesis and secretion by 17-31% after a 12 hr treatment. Polyacrylamide gel electrophoresis of total secreted proteins indicates that synthetic rates of most proteins were increased equally by Ac treatment. This increase is apparently controlled at the level of translation, because control and Ac-treated cells contained the same amount of poly-A+ RNA, and neither CD nor Ac altered mRNA levels for beta-casein. There was also no indication that either CD or Ac can induce the expression of milk proteins in quiescent cells cultured on a plastic substratum. In conjunction with the biochemical studies, changes in cytoskeletal morphology caused by the drug treatments were analyzed by immunofluorescence microscopy. As has been observed in other cell types, low concentrations of CD caused cells to round up by disrupting actin filaments. Ac treatment slightly decreased the intensity of actin staining, but no changes in microfilament organization were observed. Ac-treated cells showed slight disorganization of the cytokeratin filaments, with some peripheral interfibrillar bundling, but the cytokeratin network did not collapse and no retraction of cell extensions or breakdown of cell-cell contacts was observed. These results confirm previous reports that the actin cytoskeleton may play a role in regulating tissue-specific protein synthesis. How Ac stimulates protein synthesis is unknown, but it is unlikely that this effect is directly mediated through intermediate filaments.