Study of the influence of actin-binding proteins using linear analyses of cell deformability

The actin cytoskeleton plays a key role in the deformability of the cell and in mechanosensing. Here we analyze the contributions of three major actin cross-linking proteins, myosin II, a-actinin and filamin, to cell deformability, by using micropipette aspiration of Dictyostelium cells. We examine the applicability of three simple mechanical models: for small deformation, linear viscoelasticity and drop of liquid with a tense cortex; and for large deformation, a Newtonian viscous fluid. For these models, we have derived linearized equations and we provide a novel, straightforward methodology to analyze the experiments. This methodology allowed us to differentiate the effects of the cross-linking proteins in the different regimes of deformation. Our results confirm some previous observations and suggest important relations between the molecular characteristics of the actin-binding proteins and the cell behavior: the effect of myosin is explained in terms of the relation between the lifetime of the bond to actin and the resistive force; the presence of a-actinin obstructs the deformation of the cytoskeleton, presumably mainly due to the higher molecular stiffness and to the lower dissociation rate constants; and filamin contributes critically to the global connectivity of the network, possibly by rapidly turning over crosslinks during the remodeling of the cytoskeletal network, thanks to the higher rate constants, flexibility and larger size. The results suggest a sophisticated relationship between the expression levels of actinbinding proteins, deformability and mechanosensing.

Physical mechanism of the compressive response of F-actin networks : significance of crosslinker unbinding events

A model of crosslinker unbinding is implemented in a highly coarsegrained granular model of F-actin cytoskeleton. We employ this specific granular model to study the mechanisms of the compressive responses of F-actin networks. It is found that the compressive response of F-actin cytoskeleton has dependency on the strain rate. The evolution of deformation energy in the network indicates that crosslinker unbinding events can induce the remodelling of F-actin cytoskeleton in response to external loadings. The internal stress in F-actin cytoskeleton can efficiently dissipate with the help of crosslinker unbinding, which could lead to the spontaneous relaxation of living cells.

A Superfamily of Actin-Binding Proteins at the Actin-Membrane Nexus of Higher Plants

Complex animals use a wide variety of adaptor proteins to produce specialized sites of interaction between actin and membranes. Plants do not have these protein families, yet actin-membrane interactions within plant cells are critical for the positioning of subcellular compartments, for coordinating intercellular communication, and for membrane deformation [1]. Novel factors are therefore likely to provide interfaces at actin-membrane contacts in plants, but their identity has remained obscure. Here we identify the plantspecific Networked (NET) superfamily of actin-binding proteins, members of which localize to the actin cytoskeleton and specify different membrane compartments. The founding member of the NET superfamily, NET1A, is anchored at the plasma membrane and predominates at cell junctions, the plasmodesmata. NET1A binds directly to actin filaments via a novel actin-binding domain that defines a superfamily of thirteen Arabidopsis proteins divided into four distinct phylogenetic clades. Members of other clades identify interactions at the tonoplast, nuclear membrane, and pollen tube plasma membrane, emphasizing the role of this superfamily in mediating actin-membrane interactions.

How HIV takes advantage of the cytoskeleton in entry and replication

The host cell cytoskeleton plays a key role in the life cycle of viral pathogens whose propagation depends on mandatory intracellular steps. Accordingly, also the human immunodeficiency virus type 1 (HIV-1) has evolved strategies to exploit and modulate in particular the actin cytoskeleton for its purposes. This review will recapitulate recent findings on how HIV-1 hijacks the cytoskeleton to facilitate entry into, transport within and egress from host cells as well as to commandeer communication of infected with uninfected bystander cells.

Role of RhoA/ROCK-dependent actin contractility in the induction of tenascin-C by cyclic tensile strain

In chick embryo fibroblasts, the mRNA for extracellular matrix protein tenascin-C is induced 2-fold by cyclic strain (10%, 0.3 Hz, 6 h). This response is attenuated by inhibiting Rho-dependent kinase (ROCK). The RhoA/ROCK signaling pathway is primarily involved in actin dynamics. Here, we demonstrate its crucial importance in regulating tenascin-C expression. Cyclic strain stimulated RhoA activation and induced fibroblast contraction. Chemical activators of RhoA synergistically enhanced the effects of cyclic strain on cell contractility. Interestingly, tenascin-C mRNA levels perfectly matched the extent of RhoA/ROCK-mediated actin contraction. First, RhoA activation by thrombin, lysophosphatidic acid, or colchicine induced tenascin-C mRNA to a similar extent as strain. Second, RhoA activating drugs in combination with cyclic strain caused a super-induction (4- to 5-fold) of tenascin-C mRNA, which was again suppressed by ROCK inhibition. Third, disruption of the actin cytoskeleton with latrunculin A abolished induction of tenascin-C mRNA by chemical RhoA activators in combination with cyclic strain. Lastly, we found that myosin II activity is required for tenascin-C induction by cyclic strain. We conclude that RhoA/ROCK-controlled actin contractility has a mechanosensory function in fibroblasts that correlates directly with tenascin-C gene expression. Previous RhoA/ROCK activation, either by chemical or mechanical signals, might render fibroblasts more sensitive to external tensile stress, e.g., during wound healing.

The interplay between neuronal activity and actin dynamics mimic the setting of an LTD synaptic tag

Persistent forms of plasticity, such as long-term depression (LTD), are dependent on the interplay between activity-dependent synaptic tags and the capture of plasticity-related proteins. We propose that the synaptic tag represents a structural alteration that turns synapses permissive to change. We found that modulation of actin dynamics has different roles in the induction and maintenance of LTD. Inhibition of either actin depolymerisation or polymerization blocks LTD induction whereas only the inhibition of actin depolymerisation blocks LTD maintenance. Interestingly, we found that actin depolymerisation and CaMKII activation are involved in LTD synaptic-tagging and capture. Moreover, inhibition of actin polymerisation mimics the setting of a synaptic tag, in an activity-dependent manner, allowing the expression of LTD in non-stimulated synapses. Suspending synaptic activation also restricts the time window of synaptic capture, which can be restored by inhibiting actin polymerization. Our results support our hypothesis that modulation of the actin cytoskeleton provides an input-specific signal for synaptic protein capture.

The Functions and Regulation of Ornithine Decarboxylase

Ornithine decarboxylase (ODC) regulates the synthesis of polyamines which are involved in many cellular functions e.g. proliferation and differentiation. Due to its critical role, ODC is a tightly regulated enzyme by antizymes and antizyme inhibitors. If the regulation fails, the activity of ODC increases and may lead to malignant transformation of a cell. Increased ODC activity is found in many common cancers, including colon, prostate, and breast cancer. In a transformed cell, dynamics of the actin cytoskeleton is disturbed. A small G-protein, RhoA regulates organization of the cytoskeleton, and its overactivity increases malignant potential of the cell. The present results indicate that covalent attachment of polyamines by transglutaminase is a physiological means of regulating the activity of RhoA. The translocation of RhoA to the plasma membrane, where it exerts its activity is dependent on the presence of catalytically active ODC. As the overactivity of ODC and RhoA are implicated in cell transformation, the results provide a mechanistic explanation of the interrelationship between the polyamine metabolism and the reorganization of the actin cytoskeleton occurring in cancer cells. ODC and polyamines have also an important role in the function of central nervous system. They participate in the regulation of brain morphogenesis in embryos. In adult nervous tissue, polyamines regulate K+ and glutamate channels. K+ inward rectifying channels control membrane potentials and NMDA-type glutamate receptors (NMDAR) regulate synaptic plasticity. High ODC activity and polyamine levels are considered important in the development of ischemic brain damage and they are implicated in the pathogenesis of Alzheimer s disease (AD). A homolog of ODC was cloned from a human brain cDNA library, and several alternatively spliced variants were detected in human brain and testis. The novel protein was nevertheless devoid of ODC catalytic activity. It was subsequently found to be a novel inductor of ODC activity and polyamine synthesis, called antizyme inhibitor 2 (AZIN2). The accumulation of AZIN2 in vesicle-like formations along the axons and beneath the plasma membrane of neurons as well as in steroid hormone producing Leydig cells and luteal cells of the gonads implies that AZIN2 plays a role in secretion and vesicle trafficking. An accumulation of AZIN2 was detected also in specimens of AD brains. This increased expression of AZIN2 was specific for AD and was not found in brains with other neurodegenerative diseases including CADASIL or dementia with Lewy bodies.

Characterization of small GTPase Cdc42 from the ectomycorrhizal fungus Suillus bovinus and Agrobacterium tumefaciens-mediated transformation of fungi

Ectomycorrhizal formation between the host tree, Pinus sylvestris and fungal symbiont, Suillus bovinus was investigated at the molecular level by isolating genes regulating the organization of the actin cytoskeleton in the fungal partner S. bovinus. An Agrobacterium tumefaciens mediated transformation (ATMT) system was developed for the ectomycorrhizal fungi in order to assign specific functions to the cloned molecules. The developed ATMT system was also used to transform a plant pathogenic fungus, Helminthosporium turcicum, to hygromycin B resistance. Small GTPases Cdc42 and Rac1, the regulators of actin cytoskeleton in eukaryotes were isolated from S. bovinus. Sbcdc42 and Sbrac1, are both expressed in vegetative and in the symbiotic hyphae of S. bovinus . Using IIF microscopy, Cdc42 and actin were co-localized at the tips of vegetative hyphae and were visualized in association with the plasma membrane in swollen cells typical to the symbiotic hyphae. These results suggest that the small GTPases Cdc42 may play a significant role in the polarized growth of S. bovinus hyphae and regulate fungal morphogenesis during ectomycorrhiza formation through reorganization of the actin cytoskeleton. The functional equality of Cdc42 was tested in yeast complementation experiments using a Saccharomyces cerevisiae temperature sensitive mutant, cdc42-1ts. The genomic clone of CDC42 was isolated from S. bovinus genomic DNA via specific primers for Cdc42. The analogous S. cerevisiae cdc42 mutations, dominant active G12V and dominant negative D118A, were generated in the Sbcdc42 gene by in-vitro mutagenesis. The ectomycorrhizal fungi, S. bovinus, P. involutus and H. cylindroporum were transformed using ATMT and phleomycin as a selectable marker. PCR screeing suggested that the T-DNA was inserted in all the three fungal genomes but the fate of integration could not be proved by Southern blot analysis. An alternative Agrobacterium strain, AGL-1 and selection marker, hygromycin was used to transform our model fungus S. bovinus. PCR and Southern analysis suggested an improved efficiency of transformation. All the transformed fungal colonies selected for hygromycin gave positives in PCR and the Southerns showed multiple or single copy T-DNA integrations into the S. bovinus genome. Using the same Agrobacterium strain and the selectable marker, a maize pathogen, H. turcicum was also subjected to ATMT. The H. turcicum transformation data suggested the single copy T-DNA integrations into the genome of the screened transformants that further confirms wider applicability of the ATMT. The plasmids carrying the wild-type (pHGCDC42) and the mutated Sbcdc42 alleles (pHGGV; pHGDA) under Agaricus bisporus gpd promoter were constructed in an A. tumefaciens vector. ATMT was used to transform S. bovinus with the plasmids carrying the wild-type and mutated Sbcdc42 alleles. The isolation of Sbcdc42 and Sbrac1 genes and some other functionally related genes from ectomycorrhizal fungus, S. bovinus will form the basis of future work to resolve the signalling pathway leading to ectomycorrhizal symbiosis. The development of ATMT system will be a valuable tool in analysing the exact function of signalling pathway components in ectomycorrhizal symbiosis or in plant pathogenic interactions. The transformation frequency and broad applicability along with the simplicity of T-DNA integration make Agrobacterium a valuable, new and a powerfull tool for targeted and insertional mutagenesis in these plant associated fungi. The developed ATMT systems should therefore make it possible to generate large number of transformants with tagged genes which could then be screened for their specific roles in symbiosis and pathogenecity, respectively.

The bacterial cytoskeleton modulates motility, type 3 secretion, and colonization in Salmonella.

Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase RcsC.

Characterizing the mechanics of cultured cell monolayers.

One-cell-thick monolayers are the simplest tissues in multicellular organisms, yet they fulfill critical roles in development and normal physiology. In early development, embryonic morphogenesis results largely from monolayer rearrangement and deformation due to internally generated forces. Later, monolayers act as physical barriers separating the internal environment from the exterior and must withstand externally applied forces. Though resisting and generating mechanical forces is an essential part of monolayer function, simple experimental methods to characterize monolayer mechanical properties are lacking. Here, we describe a system for tensile testing of freely suspended cultured monolayers that enables the examination of their mechanical behavior at multi-, uni-, and subcellular scales. Using this system, we provide measurements of monolayer elasticity and show that this is two orders of magnitude larger than the elasticity of their isolated cellular components. Monolayers could withstand more than a doubling in length before failing through rupture of intercellular junctions. Measurement of stress at fracture enabled a first estimation of the average force needed to separate cells within truly mature monolayers, approximately ninefold larger than measured in pairs of isolated cells. As in single cells, monolayer mechanical properties were strongly dependent on the integrity of the actin cytoskeleton, myosin, and intercellular adhesions interfacing adjacent cells. High magnification imaging revealed that keratin filaments became progressively stretched during extension, suggesting they participate in monolayer mechanics. This multiscale study of monolayer response to deformation enabled by our device provides the first quantitative investigation of the link between monolayer biology and mechanics.

Cooperation of Mtmr8 with PI3K Regulates Actin Filament Modeling and Muscle Development in Zebrafish

Background: It has been shown that mutations in at least four myotubularin family genes (MTM1, MTMR1, 2 and 13) are causative for human neuromuscular disorders. However, the pathway and regulative mechanism remain unknown. Methodology/Principal Findings: Here, we reported a new role for Mtmr8 in neuromuscular development of zebrafish. Firstly, we cloned and characterized zebrafish Mtmr8, and revealed the expression pattern predominantly in the eye field and somites during early somitogenesis. Using morpholino knockdown, then, we observed that loss-of-function of Mtmr8 led to defects in somitogenesis. Subsequently, the possible underlying mechanism and signal pathway were examined. We first checked the Akt phosphorylation, and observed an increase of Akt phosphorylation in the morphant embryos. Furthermore, we studied the PH/G domain function within Mtmr8. Although the PH/G domain deletion by itself did not result in embryonic defect, addition of PI3K inhibitor LY294002 did give a defective phenotype in the PH/G deletion morphants, indicating that the PH/G domain was essential for Mtmr8's function. Moreover, we investigated the cooperation of Mtmr8 with PI3K in actin filament modeling and muscle development, and found that both Mtmr8-MO1 and Mtmr8-MO2+LY294002 led to the disorganization of the actin cytoskeleton. In addition, we revealed a possible participation of Mtmr8 in the Hedgehog pathway, and cell transplantation experiments showed that Mtmr8 worked in a non-cell autonomous manner in actin modeling. Conclusion/Significance: The above data indicate that a conserved functional cooperation of Mtmr8 with PI3K regulates actin filament modeling and muscle development in zebrafish, and reveal a possible participation of Mtmr8 in the Hedgehog pathway. Therefore, this work provides a new clue to study the physiological function of MTM family members.

Burkholderia cenocepacia disrupts host cell actin cytoskeleton by inactivating Rac and Cdc42

Burkholderia cenocepacia, a member of the Burkholderia cepacia complex, is an opportunistic pathogen that causes devastating infections in patients with cystic fibrosis. The ability of B. cenocepacia to survive within host cells could contribute significantly to its virulence in immunocompromised patients. In this study, we explored the mechanisms that enable B. cenocepacia to survive inside macrophages. We found that B. cenocepacia disrupts the actin cytoskeleton of infected macrophages, drastically altering their morphology. Submembranous actin undergoes depolymerization, leading to cell retraction. The bacteria perturb actin architecture by inactivating Rho family GTPases, particularly Rac1 and Cdc42. GTPase inactivation follows internalization of viable B. cenocepacia and compromises phagocyte function: macropinocytosis and phagocytosis are markedly inhibited, likely impairing the microbicidal and antigen-presenting capability of infected macrophages. The type VI secretion system is essential for the bacteria to elicit these changes. This is the first report demonstrating inactivation of Rho family GTPases by a member of the B. cepacia complex.

Interaction of pollen-specific actin-depolymerizing factor with actin

We have examined the interaction of recombinant lily pollen ADF, LIADF1, with actin and found that whilst it bound both G- and F-actin, it had a much smaller effect on the polymerization and depolymerization rate constants than the maize vegetative ADF, ZmADF3. An antiserum specific to pollen ADF, antipADF, was raised and used to localize pollen ADF in daffodil - a plant in which massive reorganizations of the actin cytoskeleton have been seen to occur as pollen enters and exits dormancy. We show, for the first time, an ADF decorating F-actin in cells that did not result from artificial increase in ADF concentration. In dehydrated pollen this ADF:actin array is replaced by actin:ADF rodlets and aggregates of actin, which presumably act as a storage form of actin during dormancy. In germinated pollen ADF has no specific localization, except when an adhesion is made at the tip where actin and ADF now co-localize. These activities of pollen ADF are discussed with reference to the activities of ZmADF3 and other members of the ADF/cofilin group of proteins.

Inhibition of cell migration and invasion mediated by the TAT-RasGAP317-326 peptide requires the DLC1 tumor suppressor.

TAT-RasGAP317-326, a peptide corresponding to the 317-326 sequence of p120 RasGAP coupled with a cell-permeable TAT-derived peptide, sensitizes the death response of various tumor cells to several anticancer treatments. We now report that this peptide is also able to increase cell adherence, prevent cell migration and inhibit matrix invasion. This is accompanied by a marked modification of the actin cytoskeleton and focal adhesion redistribution. Interestingly, integrins and the small Rho GTP-binding protein, which are well-characterized proteins modulating actin fibers, adhesion and migration, do not appear to be required for the pro-adhesive properties of TAT-RasGAP317-326. In contrast, deleted in liver cancer-1, a tumor suppressor protein, the expression of which is often deregulated in cancer cells, was found to be required for TAT-RasGAP317-326 to promote cell adherence and inhibit migration. These results show that TAT-RasGAP317-326, besides its ability to favor tumor cell death, hampers cell migration and invasion.

Cloning of actin genes from a genomic library of the newt, Notophthalmus viridescens

The regenerating urodele limb is a useful model system in which to study, in vivo, the controls of cell proliferation and differentiation. Techniques are available which enable one to experimentally manipulate mitogenic influences upon the blastema, as well the morphogenesis of the regenerating 11mb. Although classical regeneration studies have generated a wealth of knowledge concerning tissue interactions, little 1s known about the process at the level of gene expression. The aim of this project was to clone potentially developmentally regulated genes from a newt genomic library for use in future studies of gene expression during limb regeneration. We decided to clone the cytoskeletal actin gene for the following reasons: 1. its expression reflects the proliferative and differentiatlve states of cells in other systems 2. the high copy number of cytoplasmic actin pseudogenes in other vertebrates and the high degree of evolutionary sequence conservation among actin genes increased the chance of cloning one of the newt cytoplasmic actin genes. 3. Preliminary experiments indicated that a newt actin could probably be identified using an available chick ~-actln gene for a molecular probe. Two independent recombinant phage clones, containing actin homologous inserts, were isolated from a newt genomic library by hybridization with the chick actin probe. Restriction mapping identified actin homologous sequences within the newt DNA inserts which were subcloned into the plasmid pTZ19R. The recombinant plasmids were transformed into the Escherichia coli strain, DHsa. Detailed restriction maps were produced of the 5.7Kb and 3.1Kb newt DNA inserts in the plasmids, designated pTNAl and pTNA2. The short «1.3 Kb) length of the actin homologous sequence in pTNA2 indicated that it was possibly a reverse transcript pseudogene. Problems associated with molecular cloning of DNA sequences from N. viridescens are discussed with respect to the large genome size and abundant highly repetitive DNA sequences.