Rapid signal transduction in living cells is a unique feature of mechanotransduction

It is widely postulated that mechanotransduction is initiated at the local force-membrane interface by inducing local conformational changes of proteins, similar to soluble ligand-induced signal transduction. However, all published reports are limited in time scale to address this fundamental issue. Using a FRET-based cytosolic Src reporter in a living cell, we quantified changes of Src activities as a local stress via activated integrins was applied. The stress induced rapid (<0.3 s) activation of Src at remote cytoplasmic sites, which depends on the cytoskeletal prestress. In contrast, there was no Src activation within 12 s of soluble epidermal growth factor (EGF) stimulation. A 1.8-Pa stress over a focal adhesion activated Src to the same extent as 0.4 ng/ml EGF at long times (minutes), and the energy levels for mechanical stimulation and chemical stimulation were comparable. The effect of both stress and EGF was less than additive. Nanometer-scale cytoskeletal deformation analyses revealed that the strong activation sites of Src by stress colocalized with large deformation sites of microtubules, suggesting that microtubules are essential structures for transmitting stresses to activate cytoplasmic proteins. These results demonstrate that rapid signal transduction via the prestressed cytoskeleton is a unique feature of mechanotransduction.     

Caldesmon-dependent switching between capillary endothelial cell growth and apoptosis through modulation of cell shape and contractility

Caldesmon (CaD), a protein component of the actomyosin filament apparatus, modulates cell shape and cytoskeletal structure when overexpressed. When capillary endothelial cells were infected with an adenoviral vector encoding GFP-CaD under Tet-Off control, progressive inhibition of contractility, loss of actin stress fibers, disassembly of focal adhesions, and cell retraction resulted. This was accompanied by a cell shape (rounding)-dependent increase in apoptosis and concomitant inhibition of cell cycle progression. Cell growth also was inhibited in low expressor cells in which cell tension was suppressed independently of significant changes in cell shape, cytoskeletal structure, or focal adhesions. Thus, changes in both cytoskeletal structure and contractility appear to be central to the mechanism by which extracellular matrix-dependent changes in capillary cell shape influence growth and apoptosis during angiogenesis, and hence the cytoskeleton may represent a potential target for anti-angiogenesis therapy.     

Mechanical Remodeling of the Endothelial Surface and Actin Cytoskeleton Induced by Fluid Flow

Objective : The mechanism by which cultured endothelial cells respond to shear stress is controversial. The cell surface and cytoskeleton are involved, but their roles are undefined. In this study, previously unknown changes in the surface detail and actin cytoskeleton of bovine aortic endothelial cells were identified. Methods : Actin filament content and filament number in resting and flow-oriented cells were determined by biochemical assays. The three-dimensional organization of the actin cytoskeleton in cells was defined in the confocal microscope and in the electron microscope after rapid-freezing, freeze-drying, and metal coating of detergent-permeabilized cells. Results : Endothelial cells have smooth apical membranes in situ. However, cultured cells exhibit surface microvilli which increase the apical surface area, exposing the ruffled surface to forces from fluid flow and potentially enhancing cell interactions with blood-borne white cells. Stereoscopic micrographs show that stress fibers are integrated into a complex distributed cytoplasmic structural actin network (DCSA). This lattice is formed by actin filaments that frequently cross and connect to each other, stress fibers, and microfilaments and microtubules. The cytoskeletons of cells cultured in static media lack apparent order when compared to cytoskeletons from cells which have been exposed to 24 hours of laminar flow. Conclusions : The DCSA physically connects the apical and basal cell membranes and fills the volume between nucleus and membrane, providing a mechanism for transmitting mechanical forces across cells and a signaling pathway from membrane to nucleus. Stress fibers increase the mechanical modulus of the DCSA, although this increase is probably unnecessary to withstand the increase in shear stress caused by blood flow in vivo. This implies that actin rearrangements are not required for mechanical integrity, but serve an alternate function.     

Src-dependent repression of ARF6 is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts

Bone digestion occurs when osteoclasts adhere onto bone surfaces and polarize to form acidic, hydrolase-rich resorption lacunae. For this process, they condense their actin-rich podosomes in tight belts to establish sealing zones, which segregate their basal membranes from those facing resorption lacunae. This polarization process remains poorly understood. Here, we combined quantitative proteomics and gene silencing to identify new substrates of the Src tyrosine kinase, a key regulator of osteoclast function. We now report that a depletion of the ARF GTPase-activating protein GIT2, which localizes to sealing zones upon Src phosphorylation, or a lack of GTP hydrolysis on ARF6 impairs sealing zone formation and polarized membrane traffic. Surprisingly, the Rho guanine nucleotide exchange factors α and β PIX, which usually coordinate ARF and Rho signaling, were found to be dispensable. We conclude that the Src-dependent localization of GIT2 is essential for down-regulating ARF6 activity at sealing zones, and thus for maintaining osteoclast polarity.     

Direct observation of proteolytic cleavage at the S2 site upon forced unfolding of the Notch negative regulatory region

The conserved Notch signaling pathway plays crucial roles in developing and self-renewing tissues. Notch is activated upon ligand-induced conformation change of the Notch negative regulatory region (NRR) unmasking a key proteolytic site (S2) and facilitating downstream events. Thus far, the molecular mechanism of this signal activation is not defined. However, strong indirect evidence favors a model whereby transendocytosis of the Notch extracellular domain, in tight association with ligand into the ligand-bearing cell, exerts a force on the NRR to drive the required structure change. Here, we demonstrate that force applied to the human Notch2 NRR can indeed expose the S2 site and, crucially, allow cleavage by the metalloprotease TACE (TNF-alpha-converting enzyme). Molecular insight into this process is achieved using atomic force microscopy and molecular dynamics simulations on the human Notch2 NRR. The data show near-sequential unfolding of its constituent LNR (Lin12-Notch repeat) and HD (heterodimerization) domains, at forces similar to those observed for other protein domains with a load-bearing role. Exposure of the S2 site is the first force “barrier” on the unfolding pathway, occurring prior to unfolding of any domain, and achieved via removal of the LNRA∶B linker region from the HD domain. Metal ions increase the resistance of the Notch2 NRR to forced unfolding, their removal clearly facilitating unfolding at lower forces. The results provide direct demonstration of force-mediated exposure and cleavage of the Notch S2 site and thus firmly establish the feasibility of a mechanotransduction mechanism for ligand-induced Notch activation.     

Spatial organization of the extracellular matrix regulates cell-cell junction positioning

The organization of cells into epithelium depends on cell interaction with both the extracellular matrix (ECM) and adjacent cells. The role of cell-cell adhesion in the regulation of epithelial topology is well-described. ECM is better known to promote cell migration and provide a structural scaffold for cell anchoring, but its contribution to multicellular morphogenesis is less well-understood. We developed a minimal model system to investigate how ECM affects the spatial organization of intercellular junctions. Fibronectin micropatterns were used to constrain the location of cell-ECM adhesion. We found that ECM affects the degree of stability of intercellular junction positioning and the magnitude of intra- and intercellular forces. Intercellular junctions were permanently displaced, and experienced large perpendicular tensional forces as long as they were positioned close to ECM. They remained stable solely in regions deprived of ECM, where they were submitted to lower tensional forces. The heterogeneity of the spatial organization of ECM induced anisotropic distribution of mechanical constraints in cells, which seemed to adapt their position to minimize both intra- and intercellular forces. These results uncover a morphogenetic role for ECM in the mechanical regulation of cells and intercellular junction positioning.     

Implications of Mechanical Stretch on Wound Repair of Gastric Smooth Muscle Cells In Vitro

Gastric smooth muscle cells continually receive repetitive physical stretching by food storage, peristalsis and fasting contraction; therefore mechanical stretch can not be disregarded in gastric events. The aim of this study was to clarify the effects of mechanical stretch on wound repair using a rabbit gastric smooth muscle cell sheet. Mechanical stretch was imposed on adherent cells on a flexible membrane in order to increase elongation by an average of 5% and 10%, respectively, at 5 cycles per minute after artificial wounding. Adherent cells not subjected to mechanical stretch served as controls. The restoration process was monitored by measuring wound size for 48 h. Proliferation was assessed by BrdU staining and the influence on the cytoskeletal system was assessed by actin staining. The speed of restoration was highest in controls and lowest in the 10% stretch groups. Proliferation was almost equal to that of controls in the stretch groups. Under the condition of mechanical stretch, stress fibers appeared weakened and the direction of fibers was not consistent but random. In conclusion, mechanical stretch inhibited the migration of gastric smooth muscle cells, leading to cytoskeletal dysfunction. It is suggested that physical stretch, such as mechanical stretch, might be an important factor in the process of gastric wound repair.     

Oncogenes induce a vimentin filament collapse mediated by HDAC6 that is linked to cell stiffness

Oncogenes deregulate fundamental cellular functions, which can lead to development of tumors, tumor-cell invasion, and metastasis. As the mechanical properties of cells govern cell motility, we hypothesized that oncogenes promote cell invasion by inducing cytoskeletal changes that increase cellular stiffness. We show that the oncogenes simian virus 40 large T antigen, c-Myc, and cyclin E induce spatial reorganization of the vimentin intermediate filament network in cells. At the cellular level, this reorganization manifests as increased width of vimentin fibers and the collapse of the vimentin network. At nanoscale resolution, the organization of vimentin fibers in these oncogene-expressing cells was more entangled, with increased width of the fibers compared with control cells. Expression of these oncogenes also resulted in up-regulation of the tubulin deacetylase histone deacetylase 6 (HDAC6) and altered spatial distribution of acetylated microtubules. This oncogene expression also induced increases in cellular stiffness and promoted the invasive capacity of the cells. Importantly, HDAC6 was required and sufficient for the structural collapse of the vimentin filament network, and was required for increased cellular stiffness of the oncogene-expressing cells. Taken together, these data are consistent with the possibility that oncogenes can induce cellular stiffness via an HDAC6-induced reorganization of the vimentin intermediate filament network.Oncogenes induce major changes in cell behavior that can result in the development of tumors and tumor-cell metastases. Although expression of oncogenes promotes the ability of cells to invade the surrounding environment and to form metastases (1–3), the mechanisms behind this remain to be further elucidated. Many tumors show increased tissue stiffness, which can at least in part be explained by an increase in the stiffness of the extracellular microenvironment in tumors (4). Nonetheless, this increase in stiffness might also be due to an increase in the intrinsic stiffness of the cytoplasm of transformed cells. As the mechanical properties of cells govern their motile behavior, we hypothesized that oncogenes promote cell invasion via the control of cellular stiffness.The cell cytoskeleton is composed of actin filaments, microtubules, and intermediate filaments (IFs), and it is believed to govern the mechanical properties of cells. In particular, the actin filaments have been shown to control cell mechanics (5, 6). In addition, there are a number of observations that indicate that vimentin IFs also contribute to the mechanical properties of cells. Vimentin IFs are a major cytoskeletal component in motile mesenchymal cells and metastatic tumors of epithelial origin. Epithelial-to-mesenchymal transition (EMT) is a diagnostic marker of migration and invasion of cancer cells, and it is characterized by expression of vimentin. Vimentin IFs functionally control the cell shape changes that occur during EMT and are strongly associated with cell invasion and poor tumor prognosis (7–10). This suggests that vimentin IFs have roles in the mechanical and motile properties of cells. In vitro, the mechanical properties of IFs are clearly distinct from the actin microfilament and microtubule systems (11). Under slight deformation, IFs provide compliance to cells. However, under great stress and deformation, IFs provide cells with mechanical strength and stiffness (12, 13). Vimentin fills the entire cytoplasm, and fibroblasts from mice that lack functional vimentin are significantly more pliant than normal fibroblasts (14, 15). Taken together, these observations suggest that the vimentin IF network can contribute to the mechanical strength and stiffness of cells.In addition to the mechanical properties of individual filaments, the mechanical properties of the IF network depend equally on the geometry and bonds that link individual filaments together in a network (13). Hence, not only the presence, but also the organization of the vimentin IF network will have major effects on the mechanical properties of cells. Therefore, we set out to determine whether, and if so how, oncogenes can change the spatial organization of the vimentin IF network, cellular stiffness, and cell invasion.Simian virus 40 large T antigen (SV40T), c-Myc, and cyclin E oncogenes are all intimately linked to development and progression of tumors. The SV40T antigen induces cell transformation and promotes tumor formation in various cell systems and animal models through its ability to initiate DNA replication (16). Many cancers show defective c-Myc activity, and aberrant expression of c-Myc induces major changes in gene expression, which result in enhanced cell proliferation (17). Overexpression of cyclin E, which results in accelerated G1 progression and chromosomal instability, is seen in many different types of tumors (18).Many studies have shown that increased activity of histone deacetylase 6 (HDAC6) is intimately linked to tumor formation and the invasive capacity of tumor cells. HDAC6 is up-regulated in many tumor types, and is required for optimal tumor growth; it is also used as a marker for patient prognosis (19). In addition, the Ras oncogene requires a functional HDAC6 to transform cells, as defined through anchorage-independent cell proliferation (20). Also, whereas increased expression of HDAC6 increases the invasive phenotype of cells (21), inhibition of HDAC6 inhibits cell migration (22, 23). Together, this suggests that HDAC6 is required for the cellular changes that lead to cell transformation and tumor-cell invasion. Consequently, HDAC6 is a key target for the development of anticancer drugs (24).In the present study we analyzed how oncogenes change the organization of the vimentin IF network and how this is linked to changes in cellular stiffness and invasion. Our data indicate that oncogenes cause an HDAC6-mediated reorganization of the vimentin IF network in cells, which is accompanied by increased cellular stiffness and cell invasion.     

Fungal colonisation and fluconazole therapy in acute liver disease

Abstract: Background/Aims: Fungal infection, particulary with Candida spp., has been identified as an important cause of morbidity and mortality in patients with acute liver failure. Fungal colonisation of superficial mucosal sites usually precedes invasive infection. We investigated colonisation patterns in patients with acute liver disease receiving fluconazole therapy in order to investigate the possibility of emergence of fluconazole-resistant C. albicans or other species. Methods: During a 6-month study period, we studied all patients referred to our unit with acute liver disease by twice-weekly sampling and mycological analysis of specimens from superficial mucosal and other sites as appropriate. Patients were treated with prophylactic antimicrobials including 100 mg fluconazole daily in accordance with our usual protocol. Results: Twenty-two patients with acute liver disease were studied, eight of whom underwent transplantation. Eighteen patients were colonised by fungi at presentation, and six developed secondary colonisation during fluconazole therapy. Four of these patients (all transplanted) became colonised by resistant species; one of these was Aspergillus fumigatus, which led to death. There were no other invasive fungal infections identified during the study period, and no fluconazole-resistant C. albicans were identified. Conclusions: Resistance to fluconazole is unlikely to develop in C. albicans during short-term fluconazole prophylaxis in acute liver disease, and in this study we did not find evidence of invasive disease from other Candida spp. during fluconazole therapy. However, in patients at particularly high risk, other strategies are required to prevent infection with Aspergillus spp.     

A contact-activated kinase signals Candida albicans invasive growth and biofilm development

For mammalian cells, contact-dependent regulatory controls are crucially important for controlling cellular proliferation and preventing diseases such as cancer. Candida albicans, an opportunistic fungal pathogen that normally resides within a mammalian host, also exhibits contact-dependent cellular behaviors such as invasive hyphal growth and biofilm development. Results reported here demonstrate that, in C. albicans, physical contact results in activation of the mitogen-activated protein kinase Mkc1p. This kinase is part of the fungal cell integrity pathway, a signal transduction pathway known to be activated by cell wall stress. It is demonstrated here that Mkc1p is required for invasive hyphal growth and normal biofilm development. Therefore, Mkc1p signaling contributes to contact-dependent regulation. Because responding to contact appropriately allows coordinated cellular behavior in a metazoan, commensal C. albicans cells behave like a part of the host, using contact-activated signaling to regulate fungal behavior.     

Dandruff-associated Malassezia genomes reveal convergent and divergent virulence traits shared with plant and human fungal pathogens

Fungi in the genus Malassezia are ubiquitous skin residents of humans and other warm-blooded animals. Malassezia are involved in disorders including dandruff and seborrheic dermatitis, which together affect >50% of humans. Despite the importance of Malassezia in common skin diseases, remarkably little is known at the molecular level. We describe the genome, secretory proteome, and expression of selected genes of Malassezia globosa. Further, we report a comparative survey of the genome and secretory proteome of Malassezia restricta, a close relative implicated in similar skin disorders. Adaptation to the skin environment and associated pathogenicity may be due to unique metabolic limitations and capabilities. For example, the lipid dependence of M. globosa can be explained by the apparent absence of a fatty acid synthase gene. The inability to synthesize fatty acids may be complemented by the presence of multiple secreted lipases to aid in harvesting host lipids. In addition, an abundance of genes encoding secreted hydrolases (e.g., lipases, phospholipases, aspartyl proteases, and acid sphingomyelinases) was found in the M. globosa genome. In contrast, the phylogenetically closely related plant pathogen Ustilago maydis encodes a different arsenal of extracellular hydrolases with more copies of glycosyl hydrolase genes. M. globosa shares a similar arsenal of extracellular hydrolases with the phylogenetically distant human pathogen, Candida albicans, which occupies a similar niche, indicating the importance of host-specific adaptation. The M. globosa genome sequence also revealed the presence of mating-type genes, providing an indication that Malassezia may be capable of sex.     

高病死率真菌肺炎的预防与管理

深部真菌感染是院内感染的重要病原。在传染病院里,真菌肺炎是重症肝炎病人高病死率的重要诱因。但又缺乏早期特异性诊断方法。为此,我们对影响真菌肺炎的高危因素进行早期预测,以求能及时采取预防措施。