Sox2 has yet to be determined. Sox2 can act synergistically with Oct3/4 in vitro to activate Oct-Sox enhancers, which regulate the expression of pluripotent stem cellspecific genes, including Nanog, Oct3/4 and Sox2 itself. These findings suggest that Sox2 is required by ES cells for its Oct-Sox enhancer activity. Using inducible Sox2-null mouse ES cells, we show that Sox2 is dispensable for the activation of these Oct-Sox enhancers. In contrast, we demonstrate that Sox2 is necessary for regulating multiple transcription factors that affect Oct3/4 expression and that the forced expression of Oct3/4 rescues the pluripotency of Sox2-null ES cells. These results indicate that the essential function of Sox2 is to stabilize ES cells in a pluripotent state by maintaining the requisite level of Oct3/4 expression.

The DNA damage response triggered by bacterial cytolethal distending toxins (CDTs) is associated with activation of the actin-regulating protein RhoA and phosphorylation of the downstream-regulated mitogen-activated protein kinase (MAPK) p38, which promotes the survival of intoxicated (i.e. cells exposed to a bacterial toxin) cells. To identify the effectors of this CDT-induced survival response, we screened a library of 4492 Saccharomyces cerevisiae mutants that carry deletions in nonessential genes for reduced growth following inducible expression of CdtB. We identified 78 genes whose deletion confers hypersensitivity to toxin. Bioinformatics analysis revealed that DNA repair and endocytosis were the two most overrepresented signaling pathways. Among the human orthologs present in our data set, FEN1 and TSG101 regulate DNA repair and endocytosis, respectively, and also share common interacting partners with RhoA. We further demonstrate that FEN1, but not TSG101, regulates cell sur...

The human oropharyngeal muscles have a unique anatomy with diverse and intricate functions. To investigate if this specialization is also reflected in the cytoarchitecture of muscle fibers, intermediate filament proteins and the dystrophin-associated protein complex have been analyzed in two human palate muscles, musculus uvula (UV) and musculus palatopharyngeus (PP), with immunohistochenmical and morphological techniques. Human limb muscles were used as reference. The findings show that the soft palate muscle fibers have a cytoskeletal architecture that differs from the limb muscles. While all limb muscles showed immunoreaction for a panel of antibodies directed against different domains of cytoskeletal proteins desmin and dystrophin, a subpopulation of palate muscle fibers lacked or had a faint immunoreaction for desmin (UV 11.7% and PP 9.8%) and the C-terminal of the dystrophin molecule (UV 4.2% and PP 6.4%). The vast majority of these fibers expressed slow contractile protein myosin heavy chain I. Furthermore, an unusual staining pattern was also observed in these fibers for b-dystroglycan, caveolin-3 and neuronal nitric oxide synthase nNOS, which are all membrane-linking proteins associated with the dystrophin C-terminus. While the immunoreaction for nNOS was generally weak or absent, b-dystroglycan and caveolin-3 showed a stronger immunostaining. The absence or a low expression of cytoskeletal proteins otherwise considered ubiquitous and important for integration and contraction of muscle cells indicate a unique cytoarchitecture designed to meet the intricate demands of the upper airway muscles. It can be concluded that a subgroup of muscle fibers in the human soft palate appears to have special biomechanical properties, and their unique cytoarchitecture must be taken into account while assessing function and pathology in oropharyngeal muscles.

Evidence from a variety of sources suggests that pericytes have contractile properties and may therefore function in the regulation of capillary blood flow. However, it has been suggested that contractility is not a ubiquitous function of pericytes, and that pericytes surrounding true capillaries apparently lack the machinery for contraction. The present study used a variety of techniques to investigate the expression of contractile proteins in the pericytes of the CNS. The results of immunocytochemistry on cryosections of brain and retina, retinal whole-mounts and immunoblotting of isolated brain capillaries indicate strong expression of the smooth muscle isoform of actin (α-SM actin) in a significant number of mid-capillary pericytes. Immunogold labelling at the ultrastructural level showed that α-SM actin expression in capillaries was exclusive to pericytes, and endothelial cells were negative. Compared to α-SM actin, non-muscle myosin was present in lower concentrations. By contrast, smooth muscle myosin isoforms, were absent. Pericytes were strongly positive for the intermediate filament protein vimentin, but lacked desmin which was consistently found in vascular smooth muscle cells. These results add support for a contractile role in pericytes of the CNS microvasculature, similar to that of vascular smooth muscle cells.

Store-operated calcium entry (SOCE) is the predominant Ca2+ entry mechanism in nonexcitable cells and controls a variety of physiological and pathological processes. Although significant progress has been made in identifying the components required for SOCE, the molecular mechanisms underlying it are elusive. The present study provides evidence for a direct involvement of kinase suppressor of Ras 2 (KSR2) in SOCE. Using lymphocytes and fibroblasts from ksr2−/− mice and shKSR2-depleted cells, we find that KSR2 is critical for the elevation of cytosolic Ca2+ concentration. Specifically, our results show that although it is dispensable for Ca2+-store depletion, KSR2 is required for optimal calcium entry. We observe that KSR2 deficiency affects stromal interaction molecule 1 (STIM1)/ORAI1 puncta formation, which is correlated with cytoskeleton disorganization. Of interest, we find that KSR2-associated calcineurin is crucial for SOCE. Blocking calcineurin activity impairs STIM1/ORAI1 pun...

Bleb formation has been correlated with nonmuscle myosin II (NM-II) activity. Whether three isoforms of NM-II (NM-IIA, -IIB, and -IIC) have the same or differential roles in bleb formation is not well understood. Here, we report that ectopically expressed GFP-tagged NM-II isoforms exhibit different types of membrane protrusions such as multiple blebs, lamellipodia, combination of both or absence of any such protrusions in MCF-7 cells. Quantification suggests that 50% of NM-IIA-GFP, 29% of NM-IIB-GFP and 19% of NM-IIC1-GFP expressing MCF-7 cells show multiple blebs formation compared with 36% of GFP alone expressing cells. Interestingly, NM-IIB has almost 50% lesser rate of dissociation from actin filament compared with NM-IIA and -IIC1 as determined by FRET analysis both at cell and bleb cortices. When we induce bleb formation by disruption of the cortex, we find that all three NM-II-GFP isoforms can re-appear and form filaments to a different degree in the growing bleb. NM-IIB-GFP ...

In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production.

Synchrony between mechanically separated biological systems is well known. We posed the question: can cells induce synchronous behavior in neighboring cells which are mechanically separated and which cannot communicate via chemical or electrical mechanisms. Caco-2 cell cultures were divided into three groups. "Inducer" cells were exposed to H 2 O 2 . "Detector" cells were placed in separate containers near the inducer cells but were not exposed to H 2 O 2 . Control cells were exposed to fresh media and were kept in a distant laboratory area. Samples were measured for total protein concentration, NFκB activation and structural changes, 10, 30 and 60 min after exposure respectively. Exposing inducer cells to H 2 O 2 resulted in a significant reduction in total protein content (−50%), an increase in nuclear NFκB activation (+38%), and structural damage (56%) compared to controls. There was a similar reduction in total protein content (−48%), increase in the nuclear fraction of NFκB (+ 35%) and structural damage (25%) in detector cells. These findings provide evidence in support of a non-chemical, non-electrical communication. This signaling system possibly plays a role in synchronous, stimulus-appropriate cell responses to noxious stimuli and may explain a number of cellular behaviors that are hard to explain based only on conventional cell signaling systems.

Myoclonic epilepsy and ragged-red fibers (MERRF) syndrome is a rare disorder characterized by myoclonus, muscle weakness, cerebellar ataxia, heart conduction block, and dementia. It has been documented that 80-90% of the patients with MERRF syndrome are caused by the A8344G mutation in the tRNA Lys gene of mitochondrial DNA (mtDNA). We and other investigators have reported that the mtDNA mutation results in not only inefficient generation of adenosine triphosphate but also increased production of reactive oxygen species (ROS) in cultured cells harboring A8344G mutation of mtDNA. In addition, we found an imbalance in the gene expression of antioxidant enzymes in the skin fibroblasts of MERRF patients. The mRNA, protein, and enzyme activity levels of manganese-superoxide dismutase were increased, but those of Cu,Zn-SOD, catalase, and glutathione peroxidase did not show significant changes. Recently, we showed that the excess ROS could damage voltage-dependent anion channel, prohibitin, Lon protease, and aconitase in the MERRF cells. Moreover, there was a dramatic increase in the gene expression and activity of matrix metalloproteinase 1, which may contribute to the cytoskeleton remodeling involved in the weakness and atrophy of muscle commonly seen in MERRF patients. Taken together, we suggest that mtDNA mutation-elicited oxidative stress, oxidative dam-age, and altered gene expression are involved in the pathogenesis and progression of MERRF syndrome.

The current understanding of glioma biology reveals targets for anti-invasive therapy which include manipulations of extracellular matrix and receptors, growth factors and cytokines, proteases, cytoskeletal components, oncogenes and tumor suppressor genes. A better understanding of the complex regulation and the signalling molecules involved in glioma invasion is still needed in order to design new and effective treatment modalities towards invasive tumor cells. Representative and valid in vitro experimental systems and animal models of gliomas are necessary for the characterization of the invasive phenotype and further development of anti-invasive therapy. In the future, it will probably be important to move from comparative genomic modelling through protein characterization based on advanced proteomic techniques to analyse tissue samples, where the aim for gliomas should be to compare invaded and non-invaded tissue. This will hopefully render promising new therapeutic targets for ...

Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is able to specifically bind and dephosphorylate activated PDGF and insulin receptors, modulating the onset of mitogenic process. LMW-PTP is present in two distinct intracellular locations. While the cytosolic LMW-PTP pool interacts directly with activated insulin or PDGF receptors, the cytoskeletonassociated LMW-PTP is tyrosine phosphorylated upon PDGF stimulation and is involved in cytoskeleton rearrangement acting on p190Rho-GAP. We investigated the differential role of LMW-PTP in PDGF-or insulininduced mitogenesis and cytoskeleton rearrangement.

Moritella viscosa is considered the main aetiological agent of 'winter ulcer' disease in farmed salmonid fish. To further understand the pathogenesis of this disease, M. viscosa interaction with fish cells was studied using a Chinook salmon embryo cell line (CHSE-214). As winter ulcer appears exclusively at temperatures below 7-8 C, we attempted to identify if this connection is explained by temperature regulated bacterial virulence. Therefore, infection studies were performed at a temperature range from 4 to 15 C. At all temperatures, M. viscosa caused CHSE cells to retract and round up, lose their attachment abilities and finally disintegrate. The bacterium adhered to CHSE cells and caused changes to the cytoskeleton, however, it did not invade the cells. Increased adherence was demonstrated at 4 C compared to adherence at higher temperatures. Extracellular proteins exerted rapid pore formation and lysis of CHSE cells at a temperature range from 4 to 22 C. Furthermore, only small differences were found comparing extracellular proteomes of M. viscosa from 4 and 15 C. We propose that the pathogenic mechanisms exerted by M. viscosa on CHSE cells are disruption of the cytoskeleton which affects cell rigidity and structure, followed by pore formation and lysis caused by secreted products from the bacterium. These processes can also occur at temperatures above those experienced from winter ulcer outbreaks. However, the adhesion mechanisms appear to be temperature regulated and may contribute to temperature dependent disease outbreaks.

Villin, a 95-kD cytoskeletal protein selectively expressed in the microvilli of some absorptive cells was localized immunohistochemically in the oviduct and the seminiferous excretory ducts of the mouse. Villin was found in the proximal part of the oviduct, comprising the preampulla, ampulla, and part of the isthmus. Distal to the isthmus the oviductal cells lining the junctura and the intrauterine colliculus tubaris were devoid of villin. No villin could be detected in the uterine cells. Ductuli efferentes, connecting the rete testis with the epididymis were the only portion of the male seminiferous ductal system expressing villin. The cells lining the epididymis and the vas deferens were devoid of villin. These data show that villin is selectively expressed in male and female reproductive systems and that it is limited to anatomically defined proximal portions of the reproductive ducts.

Protease-activated receptor 2 (PAR2) has been implicated in the pathogenesis of airway inflammation. We report that epithelial PAR2 stimulation with trypsin (0.05-1 U/ml) or an agonist peptide (SLIGKV-NH 2 , 1-100 μM) for 0.5-3 h dose-and time-dependently enhanced neutrophil adhesion to alveolar type II epithelial cells (A549 cells) and that this stimulation also induced the formation of epithelial actin filaments. Both responses in neutrophil adhesion and epithelial actin reorganization were reduced by a Rho inhibitor, mevastatin and by a Rho-associated kinase (ROCK) inhibitor, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide). Neutrophil adherence was also inhibited by an inhibitor of actin polymerization, cytochalasin D and a tyrosine kinase inhibitor, genistein. Further, the PAR2-mediated tyrosine phosphorylation of focal adhesion kinase (FAK), a major cytoskeleton protein, was detected, and this response was inhibited by mevastatin or Y-27632. These results suggest that PAR2 stimulation of alveolar epithelial cells enhances neutrophil adhesion presumably at least in part through Rho/ROCK signal-mediated actin cytoskeleton reorganization associated with the tyrosine phosphorylation of FAK.

Hyperphosphorylated forms of the microtubule-associated protein (MAP) tau accumulate in Alzheimer's disease and related tauopathies and are thought to have an important role in neurodegeneration. However, the mechanisms through which phosphorylated tau induces neurodegeneration have remained elusive. Here, we show that tau-induced neurodegeneration is associated with accumulation of filamentous actin (F-actin) and the formation of actin-rich rods in Drosophila and mouse models of tauopathy. Importantly, modulating F-actin levels genetically leads to dramatic modification of tau-induced neurodegeneration. The ability of tau to interact with F-actin in vivo and in vitro provides a molecular mechanism for the observed phenotypes. Finally, we show that the Alzheimer's disease-linked human β-amyloid protein (Aβ) synergistically enhances the ability of wild-type tau to promote alterations in the actin cytoskeleton and neurodegeneration. These findings raise the possibility that a direct interaction between tau and actin may be a critical mediator of tau-induced neurotoxicity in Alzheimer's disease and related disorders.

The αIIbβ3 integrin receives signals in agonist-activated platelets, resulting in its conversion to an active conformation that binds fibrinogen, thereby mediating platelet aggregation. Fibrinogen binding to αIIbβ3 subsequently induces a cascade of intracellular signalling events. The molecular mechanisms of this bi-directional αIIbβ3-mediated signalling are unknown but may involve the binding of proteins to the integrin cytoplasmic domains. We reported previously the sequence of a novel 22-kDa, EF-handcontaining, protein termed CIB (calcium-and integrin-binding protein) that interacts specifically with the αIIb cytoplasmic domain in the yeast two-hybrid system. Further analysis of numerous tissues and cell lines indicated that CIB mRNA and protein are widely expressed. In addition, isothermal titration Abbreviations : CIB, calcium-and integrin-binding protein ; CaM, calmodulin ; ITC, isothermal titration calorimetry ; mAb, monoclonal antibody. ). calorimetry indicated that CIB binds to an αIIb cytoplasmicdomain peptide in a Ca# + -dependent manner, with moderate affinity (K d , 700 nM) and 1 : 1 stoichiometry. In aggregated platelets, endogenous CIB and αIIbβ3 translocate to the Triton X-100-insoluble cytoskeleton in a parallel manner, demonstrating that the cellular localization of CIB is regulated, potentially by αIIbβ3. Thus CIB may contribute to integrin-related functions by mechanisms involving Ca# + -modulated binding to the αIIb cytoplasmic domain and changes in intracellular distribution.

Polar transport of the phytohormone auxin mediates various processes in plant growth and development, such as apical dominance, tropisms, vascular patterning and axis formation. This view is based largely on the effects of polar auxin transport inhibitors. These compounds disrupt auxin efflux from the cell but their mode of action is unknown. It is thought that polar auxin flux is caused by the asymmetric distribution of efflux carriers acting at the plasma membrane. The polar localization of efflux carrier candidate PIN1 supports this model. Here we show that the seemingly static localization of PIN1 results from rapid actin-dependent cycling between the plasma membrane and endosomal compartments. Auxin transport inhibitors block PIN1 cycling and inhibit trafficking of membrane proteins that are unrelated to auxin transport. Our data suggest that PIN1 cycling is of central importance for auxin transport and that auxin transport inhibitors affect efflux by generally interfering with...

Background/purpose: The knowledge how surfactants and hydrolyzed proteins influence the elastic properties of living epidermal keratinocytes is sparse. We demonstrate that the stiffness of cells measured by atomic force microscope (AFM) can be correlated with viability test. Methods and materials: The effects of sodium lauryl sulphate (SLS) and hydrolyzed collagen (HK) of molecular weight 9 kDa were examined with respect to human keratinocytes viability and elasticity. MTT assay was applied to determine the survival fraction of keratinocytes treated with SLS and HK solutions of various molar ratios. The AFM measurements of the keratinocytes stiffness were carried out immediately after the exposure of cells to the SLS and HK, respectively. Results: The increase of the SLS concentration resulted in the decrease of cells proliferation and this effect was inhibited by addition of HK. The strongest inhibition was observed for the SLS:HK molar ratio equals to 2:1. AFM study shows decrease in the cell stiffness for cells treated with SLS. Fluorescence microscopy reveals remodeling of actin filaments of SLS-treated cells. SLS:HK mixture treatment results in mechanical stiffness close to untreated cells. Conclusion: These results provide possible correlations between mechanical properties and viability of keratinocytes when the chemical stress occurs.

We study the shapes of human red blood cells using continuum mechanics. In particular, we model the crenated, echinocytic shapes and show how they may arise from a competition between the bending energy of the plasma membrane and the stretching/shear elastic energies of ...

Aim To examine, at a global scale, patterns in the direction in which climbing plants twine. We tested three hypotheses: (1) that twining direction is determined randomly; (2) that twining direction is determined by apices following the apparent movement of the sun across the sky; and (3) that twining direction is determined by the Coriolis effect. Location Seventeen sites spanning nine countries, both hemispheres and 65 ° of latitude. Methods Twining direction was recorded for the first c. 100 stems encountered along transects through natural vegetation at each site. Results Ninety-two per cent of the 1485 twining stems we recorded grew in righthanded helices, i.e. they twined in an anticlockwise direction. This is significantly (P < 0.001) different from random. The proportion of stems twining right-handedly (anticlockwise) was independent of both latitude (P = 0.33) and hemisphere (P = 0.63). These data are inconsistent with the idea that twining direction is determined by either the relative passage of the sun through the celestial sphere or by the Coriolis effect. Thus, we reject all three of our hypotheses. Main conclusions The predominance of right-handed helical growth in climbing plants cannot be explained by hypotheses attempting to link plant growth behaviour and global location. One alternative hypothesis for our findings is that the widespread phenomenon of anticlockwise twining arises as a function of microtubule orientation operating at a subcellular level.

Collagen remodelling by fibroblasts has a crucial role in organizing tissue structures that are essential to motility during wound repair, development and regulation of cell growth. However, the mechanism of collagen fibre movement in threedimensional (3D) matrices is not understood. Here, we show that fibroblast lamellipodia extend along held collagen fibres, bind, and retract them in a 'hand-over-hand' cycle, involving α2β1 integrin. Wild-type fibroblasts move collagen fibres three to four times farther per cycle than fibroblasts lacking myosin II-B (myosin II-B −/−). Similarly, myosin II-B −/− fibroblasts contract 3D collagen gels threefold less than controls. On twodimensional (2D) substrates, however, rates of collagen bead and cell movement are not affected by loss of myosin II-B. Green fluorescent protein (GFP)-tagged myosin II-B, but not II-A, restores normal function in knockout cells and localizes to cell processes, whereas myosin II-A is more centrally located. Additionally, GFP-myosin II-B moves out to the periphery and back during handover hand fibre movement, whereas on 2D collagen, myosin II-B is more centrally distributed. Thus, we suggest that cyclic myosin II-B assembly and contraction in lamellipodia power 3D fibre movements. Remodelling of extracellular matrix proteins is critical to the organization of tissues and organs; therefore, it is important to understand the cellular and molecular steps in single collagen fibre movement. Surprisingly, cell motility on glass and other 2D substrates is not necessarily relevant to the movement of fibres in 3D matrices. There is limited overlap between the proteins localized to 3D matrix contacts and those at 2D adhesions 1,2. Furthermore, the morphology of fibroblasts in a 3D collagen matrix bears little resemblance to their morphology on a 2D collagen-coated surface 3,4. Thus, there is a fundamentally different mode of motility on 3D fibres compared with 2D surfaces 5. Carefully orchestrated movements of actin and myosin underlie most cellular motile processes 6-8. This motile machinery forms the cortical cytoskeleton. Upon binding extracellular matrix proteins, integrin receptors initiate a complex signalling cascade culminating in the

Background: Estrogen is an established enhancer of breast cancer development, but less is known on its effect on local progression or metastasis. We studied the effect of estrogen receptor recruitment on actin cytoskeleton remodeling and breast cancer cell movement and invasion. Moreover, we characterized the signaling steps through which these actions are enacted.

An abnormal increase in intestinal paracellular permeability may be an important pathogenic factor in various intestinal diseases. The intracellular factors and pro-cesses that regulate and cause alteration of intestinal paracellular permeability are not well understood. The ...

After the definitive ban on tin-based antifouling substances, new organic compounds have recently been introduced in antifouling paint formulations, as either principal or booster biocides. In most cases, previous risk assessment of these biocides has been inadequate so that their possible effects on aquatic ecosystems is a matter of great concern. We studied the effects of two new organic biocides often associated in paint formulations, Sea-Nine 211 TM (4,5 dichloro-2-n-octyl-4-isothiazoline-3-one) and chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile), on haemocytes of the compound ascidian Botryllus schlosseri exposed for 60 min to various concentrations (from 0.1 to 10 M) of the xenobiotics. This species had previously proved to be a good bioindicator of organotin compounds. Both compounds, at concentrations of 1 and 10 M, altered the morphology of phagocytes, and these changes were closely related to disrupting effects on cytoskeletal components. At the same concentrations, phagocytosis, which requires cytoskeletal modifications for pseudopod formation, was severely hindered. Both compounds were able to induce apoptosis of Botryllus blood cells, probably as a consequence of severe oxidative stress related to the reported decrease of intracellular reduced glutathione (GSH) content. In the case of Sea-Nine 211 TM , a substantial increase in intracellular Ca 2+ and a negative effect on Ca 2+-ATPase activity may also be involved in the activation of the cell death machinery. Cytochrome-c-oxidase was also significantly inhibited by the two biocides, indicating perturbation of the mitochondrial respiratory chain. Isodynamic mixtures of Sea-Nine 211 TM and chlorothalonil were used to evaluate the occurrence of interactions between the two compounds. Results suggest the combined action of partial additivity when cell-spreading and cytochrome-c-oxidase activity were considered, and were indicative of antagonism in the case of the GSH depletion. On the whole, our results indicate that short-term in vitro exposure of haemocytes to high concentrations of Sea-Nine 211 TM and chlorothalonil provokes a marked reduction in haemocyte functionality, higher than or comparable to that of TBT. These assays of acute toxicity stress the immunosuppressive potential of these compounds, which, although counterbalanced by their short half-life in the marine environment, can lead to biocoenosis dismantling through rapid bioaccumulation by filter-feeding non-target benthic organisms.

disorders . It is characterized by specific morpho-2-deoxy-D-Ribose (dRib), the most reducing sugar, logical and biochemical alterations, including cytoinduces apoptosis in normal human fibroblasts, as plasmic shrinkage, condensation and margination of judged by cytoplasmic shrinkage, chromatin condenchromatin, DNA fragmentation and appearence of sation, DNA fragmentation and mitochondrial depoapoptotic bodies; all the above distinguish this type of larization. This effect is independent from culture concell death from necrosis . Apoptosis can be trigditions, such as cell density and the presence or abgered by various stimuli, such as glucocorticoids, oxidasence of serum in the culture milieu, suggesting that tive stress, ionizing irradiation, cytostatic drugs, with-dRib-induced apoptosis is cell cycle-independent.

rescuing collapsed replication forks. In contrast, uncontrolled HR can lead to chromosome translocations, loss of heterozygosity, and deletion of repetitive sequences. Controlled HR is particularly important for the preservation of repetitive sequences of the ribosomal gene (rDNA) cluster. Here we show that recombinational repair of a DSB in rDNA in Saccharomyces cerevisiae involves the transient relocalization of the lesion to associate with the recombination machinery at an extranucleolar site. The nucleolar exclusion of Rad52 recombination foci entails Mre11 and Smc5-Smc6 complexes and depends on Rad52 SUMO (small ubiquitin-related modifier) modification. Remarkably, mutations that abrogate these activities result in the formation of Rad52 foci within the nucleolus and cause rDNA hyperrecombination and the excision of extrachromosomal rDNA circles. Our study also suggests a key role of sumoylation for nucleolar dynamics, perhaps in the compartmentalization of nuclear activities.

B cells encounter both soluble Ag (sAg) and membrane-associated Ag (mAg) in the secondary lymphoid tissue, yet how the physical form of Ag modulates B cell activation remains unclear. This study compares actin reorganization and its role in BCR signalosome formation in mAg-and sAg-stimulated B cells. Both mAg and sAg induce F-actin accumulation and actin polymerization at BCR microclusters and at the outer rim of BCR central clusters, but the kinetics and magnitude of F-actin accumulation in mAgstimulated B cells are greater than those in sAg-stimulated B cells. Accordingly, the actin regulatory factors, cofilin and gelsolin, are recruited to BCR clusters in both mAg-and sAg-stimulated B cells but with different kinetics and patterns of cellular redistribution. Inhibition of actin reorganization by stabilizing F-actin inhibits BCR clustering and tyrosine phosphorylation induced by both forms of Ag. Depolymerization of F-actin leads to unpolarized microclustering of BCRs and tyrosine phosphorylation in BCR microclusters without mAg and sAg, but with much slower kinetics than those induced by Ag. Therefore, actin reorganization, mediated via both polymerization and depolymerization, is required for the formation of BCR signalosomes in response to both mAg and sAg.

Cochlear outer hair cells (OHCs) rapidly change their length and stiffness when their membrane potential is altered. Prestin, the motor protein for this electromotility, is present along the OHC lateral plasma membrane where there is a high density of intra-membrane protein particles (IMPs). However, it is not known to what extent prestin contributes to this unusual dense population of proteins and overall organization of the membrane to generate the unique electromechanical response of OHCs. We investigated the relationship of prestin with the IMPs, the underlying cortical cytoskeletal lattice, and electromotility in prestin-deficient mice. Using freezefracture, we observed a reduction in density and size of the IMPs that correlates with the reduction and absence of prestin in the heterozygous and homozygous mice, respectively. We also observed a reduction or absence of electromotilityrelated charge density, axial stiffness, and piezoelectric properties of the OHC. A comparison of the charge density with the number of IMPs suggests that prestin forms tetramers in the wild type but is likely to form lower number oligomers in the prestin-deficient OHCs from the heterozygous mice. Interestingly, the characteristic actin-based cortical cytoskeletal lattice that underlies the membrane is absent in the prestin-null OHCs, suggesting that prestin is also required for recruiting or maintaining the cortical cytoskeletal lattice. These results suggest that the majority of the IMPs are indeed prestin and that electrically evoked length and stiffness changes are interrelated and dependent on both prestin and on the cortical actin cytoskeletal lattice of the OHC lateral membrane. Published 2009 Wiley-Liss, Inc.

Anyone who is skilled in the art of physical therapy knows that the mechanical properties, behavior and movement of our bodies are as important for human health as chemicals and genes. However, only recently have scientists and physicians begun to appreciate the key role that mechanical forces play in biological control at the molecular and cellular levels. This article provides a brief overview of a lecture presented at the 1 st International Fascia Research Congress that convened at Harvard Medical School in Boston, MA on October 4, 2007. (see figure 1) In this lecture, I described what we have learned over the past thirty years as a result of our research focused on the molecular mechanisms by which cells sense mechanical forces and convert them into changes in intracellular biochemistry and gene expression-a process called "mechanotransduction". This work has revealed that molecules, cells, tissues, organs, and our entire bodies use "tensegrity" architecture to mechanically stabilize their shape, and to seamlessly integrate structure and function at all size scales. Through use of this tension-dependent building system, mechanical forces applied at the macroscale produce changes in biochemistry and gene expression within individual living cells. This structurebased system provides a mechanistic basis to explain how application of physical therapies might influence cell and tissue physiology.

It has always been a desire of mankind to conquest Space. A major step in realizing this dream was the completion of the International Space Station (ISS). Living there for several months confirmed early observations of short-term spaceflights that a loss of gravity affects the health of astronauts. Space medicine tries to understand the mechanism of microgravity-induced health problems and to conceive potent countermeasures. There are four different aspects which make space medicine appealing: i) finding better strategies for adapting astronauts to weightlessness; ii) identification of microgravity-induced diseases (e.g. osteoporosis, muscle atrophy, cardiac problems and others); iii) defining new therapies to conquer these diseases which will benefit astronauts as well as people on Earth in the end; and iv) on top of that, unveiling the mechanisms of weightlessness-dependent molecular and cellular changes is a requirement for improving space medicine. In mammalian cells, microgravity induces apoptosis and alters the cytoskeleton and affects signal transduction pathways, cell differentiation, growth, proliferation, migration and adhesion.

Ten commercially available denture adhesives, nine soluble formulations (six creams, three powders) and one insoluble product (pad), were analyzed regarding the cytotoxicity profile in direct and indirect assays using L929 fibroblast cells. In the direct assay, fibroblasts were seeded over the surface of a thick adhesive gel (5%, creams; 2.5%, powders and pad). In the indirect assay, cells were cultured in the presence of adhesive extracts prepared in static and dynamic conditions (0.5-2%, creams; 0.25-1%, powders and pad). Cell toxicity was assessed for cell viability/proliferation (MTT assay) and cell morphology (observation of the F-actin cytoskeleton organization by confocal laser scanning microscopy). Direct contact of the L929 fibroblasts with the thick adhesive gels caused no, or only a slight, decrease in cell viability/proliferation. The adhesive extracts (especially those prepared in dynamic conditions) caused significantly higher growth inhibition of fibroblasts and, in addition, caused doseand time-dependent effects, throughout the 6-72 h exposure time. Also, dose-dependent effects on cell morphology, with evident disruption of the F-actin cytoskeleton organization, were seen in the presence of most adhesives. In conclusion, the adhesives possessed different degrees of cytotoxicity, but similar dose-and time-dependent biological profiles.

The Rho family of small GTPases (RhoA, Rac1 and Cdc42) controls signal-transduction pathways that influence many aspects of cell behaviour, including cytoskeletal dynamics 1-3 . At the leading edge, Rac1 and Cdc42 promote cell motility through the formation of lamellipodia and filopodia, respectively. On the contrary, RhoA promotes the formation of contractile actin−myosin-containing stress fibres in the cell body and at the rear 1,2,4 . Here, we identify synaptopodin, an actinassociated protein, as a novel regulator of RhoA signalling and cell migration in kidney podocytes. We show that synaptopodin induces stress fibres by competitive blocking of Smurf1mediated ubiquitination of RhoA, thereby preventing the targeting of RhoA for proteasomal degradation. Gene silencing of synaptopodin in kidney podocytes causes the loss of stress fibres and the formation of aberrant non-polarized filopodia and impairment of cell migration. Together, these data show that synaptopodin is essential for the integrity of the podocyte actin cytoskeleton and for the regulation of podocyte cell migration.

The haploid gametophyte stage of the moss life cycle is amenable to genetic and biochemical studies. Many species can be cultured on simple defined media, where growth is rapid, making them ideal material for metabolic studies. Developmental responses to hormones and to environmental inputs can be studied both at the level of individual cells and in multicellular tissues. The protonemal stage of gametophyte development comprises cell filaments that extend by the serial division of their apical cells, allowing the investigation of the generation and modification of cell polarity and the role of the cytoskeleton in these processes. Molecular techniques including gene inactivation by targeted gene replacement or by RNA interference, together with the nearly completed sequencing of the Physcomitrella patens genome, open the way for detailed study of the functions of genes involved in both development and metabolism. 497 Annu. Rev. Plant. Biol. 2006.57:497-520. Downloaded from arjournals.annualreviews.org by David Cove on 07/07/06. For personal use only.

Cells of clone NB41A3 of the C-1300 mouse neuroblastoma were grown to a critical density at which many of the cells flatten, assume a variety of shapes and sizes and some sprout processes resembling neurites. We have studied the distribution of actin and tubulin in these cells using fluorescence microscopy and antibodies against actin or tubulin under these conditions. Actin-containing structures are variably arranged and predominantly associated with motile areas of\the cell periphery including the growth cone. Microtubules appear to run radially from the perinuclear area towards the cell periphery. When neurites are present, microtubules converge into them and run to the growth cone but rarely contact its edge.

▪   Many pathogens actively exploit the actin cytoskeleton during infection. This exploitation may take place during entry into mammalian cells after engagement of a receptor and/or as series of signaling events culminating in the engulfment of the microorganism. Although actin rearrangements are a common feature of most internalization events (e.g. entry of Listeria, Salmonella, Shigella, Yersinia, Neisseria, and Bartonella), bacterial and other cellular factors involved in entry are specific to each bacterium. Another step during which pathogens harness the actin cytoskeleton takes place in the cytosol, within which some bacteria (Listeria, Shigella, Rickettsia) or viruses (vaccinia virus) are able to move. Movement is coupled to a polarized actin polymerization process, with the formation of characteristic actin tails. Increasing attention has focused on this phenomenon due to its striking similarity to cellular events occurring at the leading edge of locomoting cells. Thus patho...