Mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance are the major players in the protection of bacterial cells against hypoosmotic shock. Although a great deal is known about structure and function of these channels, much less is known about how membrane lipids may influence their mechanosensitivity and function. In this study, we use liposome coreconstitution to examine the effects of different types of lipids on MscS and MscL mechanosensitivity simultaneously using the patch-clamp technique and confocal microscopy. Fluorescence lifetime imaging (FLIM)-FRET microscopy demonstrated that coreconstitution of MscS and MscL led to clustering of these channels causing a significant increase in the MscS activation threshold. Furthermore, the MscL/MscS threshold ratio dramatically decreased in thinner compared with thicker bilayers and upon addition of cholesterol, known to affect the bilayer thickness, stiffness and pressure profile. In contrast, application of micromolar concentrations of lysophosphatidylcholine (LPC) led to an increase of the MscL/MscS threshold ratio. These data suggest that differences in hydrophobic mismatch and bilayer stiffness, change in transbilayer pressure profile, and close proximity of MscL and MscS affect the structural dynamics of both channels to a different extent. Our findings may have far-reaching implications for other types of ion channels and membrane proteins that, like MscL and MscS, may coexist in multiple molecular complexes and, consequently, have their activation characteristics significantly affected by changes in the lipid environment and their proximity to each other.

The accumulation of misfolded and unfolded proteins in the endoplasmic reticulum (ER) induces ER stress, activating the unfolded protein response (UPR). One of the effectors of the UPR is XBP1, a critical transcriptional factor for genes responsible for cell survival. ER stress is also known to play a vital role in mediating ischemic reperfusion damage in the brain. In this study, we investigated the role of XBP1 in rat primary hippocampal neurons subjected to oxygen and glucose deprivation followed by reoxygenation (OGD/R) stress, an in vitro model of ischemia/reperfusion (I/R) injury. Primary neurons subjected to OGD had increased levels of spliced XBP1 (XBP1s) mRNA. Interestingly, the level of XBP1s decreased during the initial reoxygenation stress period. The combination of OGD and the subsequent 20-h reoxygenation stress period significantly increased the apoptotic death of primary cells. Overexpression of XBP1s suppressed cell death induced by OGD/R stress. These results suggest that suppression of XBP1 activation accelerates neuronal cell death after I/R and that activation of the XBP1 pathway may provide a therapeutic approach for the treatment of cerebral I/R injury.

Both in vivo and in vitro studies have shown that neurosteroids promote learning and memory by modulating synaptic functions in the hippocampus. However, we do not know to what degree endogenously synthesized neurosteroids contribute to the hippocampal synaptic functions. Cytochrome P450scc is the enzyme that converts cholesterol to pregnenolone (PREG), which is required for the biosynthesis of all other neurosteroids. To investigate the physiological roles of endogenous neurosteroids in synaptic functions, we electrophysiologically examined the effects of aminoglutethimide (AG), a selective inhibitor of P450scc, on the synaptic transmission and plasticity in the dentate gyrus of rat hippocampal slices. The application of AG (100 μM) decreased the slope of the field excitatory postsynaptic potentials (fEPSPs) in granule cells by 20-30% in 20 min through the modulation of postsynaptic AMPA receptors, while it did not affect the presynaptic properties, including the paired-pulse ratio and the probability of glutamate release from presynaptic terminals. The AG-induced depression was nearly completely rescued by exogenously applied 500 nM PREG or by 1 nM dehydroepiandrosterone sulfate (DHEAS), one of the neurosteroids synthesized from PREG, suggesting that the AG-induced depression was caused by the loss of DHEAS. AG also reduced NMDA receptor activity, and suppressed high-frequency stimulation (HFS)-induced long-term potentiation (LTP). These findings provide novel evidence that the endogenous neurosteroids locally synthesized in the brain are required to maintain the normal excitatory synaptic transmission and plasticity in the dentate gyrus of the rat hippocampus.

Neurosteroids pregnenolone-sulfate (PREGS) and dehydroepiandrosterone (DHEA) have been shown to enhance neurogenesis in the hippocampal dentate gyrus (DG) of adult rodents. In Alzheimer's disease (AD) brain, the levels of these neurosteroids are known to be altered compared to age-matched non-demented controls. The aim of this study was to examine the effects of PREGS and DHEA on the hippocampal neurogenesis in 8-month-old male APPswe/PS1dE9 transgenic (APP/PS1) mice that show amyloid plaques and impaired spatial cognitive performance. In the DG of APP/PS1 mice the proliferation of progenitor cells was increased, while the neurite growth and survival of newborn neuronal cells were markedly impaired. Treatment with PREGS or DHEA rescued perfectly the hypoplastic neurite of newborn neurons in APP/PS1 mice, while neither of them affected the over-proliferation of progenitor cells. Notably, the administration of PREGS, but not DHEA, to APP/PS1 mice could protect the survival and maturation of newborn neuronal cells, which was accompanied by the improvement of spatial cognitive performance. The results indicate that treatment of AD like brains of APP/PS1 mice with PREGS might protect the hippocampal neurogenesis, leading to the improved spatial cognitive performance.

Intracellular and extracellular mechanical forces affect the structure and dynamics of the actin cytoskeleton. However, the underlying molecular and biophysical mechanisms, including how mechanical forces are sensed, are largely unknown. Actin-depolymerizing factor/cofilin proteins are actin-modulating proteins that are ubiquitously distributed in eukaryotes, and they are the most likely candidate as proteins to drive stress fiber disassembly in response to changes in tension in the fiber. In this study, we propose a novel hypothesis that tension in an actin filament prevents the filament from being severed by cofilin. To test this, we placed single actin filaments under tension using optical tweezers. When a fiber was tensed, it was severed after the application of cofilin with a significantly larger delay in comparison with control filaments suspended in solution. The binding rate of cofilin to an actin bundle decreased when the bundle was tensed. These results suggest that tension in an actin filament reduces the cofilin binding, resulting in a decrease in its effective severing activity.

The effects of mechanical force applied to the integrin clusters at focal contacts were examined in cultured human umbilical vein endothelial cells. When a fibronectin-coated glass bead was attached to the apical cell surface, focal contacts formed beneath the bead that became linked to focal contacts at the basal cell membrane by actin stress fibers in 5 minutes. Integrin dynamics at the basal focal contacts were monitored in live cells in response to a localized mechanical stimulus generated by displacing the glass bead. Traction force transmitted to the basal focal contacts through the stress fibers was monitored by measuring the deformation of the polyacrylamide gel substratum. The force declined in a few seconds, probably owing to decreases in the elastic modulus of the stress fibers. This transient mechanical stimulus caused the dephosphorylation of paxillin and disassembly of integrin clusters at the basal cell membrane in 20 minutes. The disassembly was mediated mainly by clathrin-dependent endocytosis of integrins. The integrin internalization was inhibited in Ca(2+)- and K(+)-free solution, and by phenylarsine oxide, a phosphatase inhibitor. These results suggest that a transient mechanical stimulus applied to focal contacts induces Ca(2+)-dependent dephosphorylation of some proteins, including paxillin, and facilitates clathrin-dependent endocytosis of integrins.

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma.

Endothelial cells (ECs) release ATP in response to shear stress, a fluid mechanical force generated by flowing blood but, although its release has a crucial role in controlling a variety of vascular functions by activating purinergic receptors, the mechanism of ATP release has never been established. To analyze the dynamics of ATP release, we developed a novel chemiluminescence imaging method by using cell-surface-attached firefly luciferase and a CCD camera. Upon stimulation of shear stress, cultured human pulmonary artery ECs simultaneously released ATP in two different manners, a highly concentrated, localized manner and a less concentrated, diffuse manner. The localized ATP release occurred at caveolin-1-rich regions of the cell membrane, and was blocked by caveolin-1 knockdown with siRNA and the depletion of plasma membrane cholesterol with methyl-β-cyclodexrin, indicating involvement of caveolae in localized ATP release. Ca(2+) imaging with Fluo-4 combined with ATP imaging revealed that shear stress evoked an increase in intracellular Ca(2+) concentration and the subsequent Ca(2+) wave that originated from the same sites as the localized ATP release. These findings suggest that localized ATP release at caveolae triggers shear-stress-dependent Ca(2+) signaling in ECs.

The delivery of Met-tRNA(i) to the 40S ribosomal subunit is thought to occur by way of a ternary complex (TC) comprising eIF2, GTP and Met-tRNA(i). We have generated from purified human proteins a stable multifactor complex (MFC) comprising eIF1, eIF2, eIF3 and eIF5, similar to the MFC reported in yeast and plants. A human MFC free of the ribosome also is detected in HeLa cells and rabbit reticulocytes, indicating that it exists in vivo. In vitro, the MFC-GTP binds Met-tRNA(i) and delivers the tRNA to the ribosome at the same rate as the TC. However, MFC-GDP shows a greatly reduced affinity to Met-tRNA(i) compared to that for eIF2-GDP, suggesting that MFC components may play a role in the release of eIF2-GDP from the ribosome following AUG recognition. Since an MFC-Met-tRNA(i) complex is detected in cell lysates, it may be responsible for Met-tRNA(i)-40S ribosome binding in vivo, possibly together with the TC. However, the MFC protein components also bind individually to 40S ribosomes, creating the possibility that Met-tRNA(i) might bind directly to such 40S-factor complexes. Thus, three distinct pathways for Met-tRNA(i) delivery to the 40S ribosomal subunit are identified, but which one predominates in vivo remains to be elucidated.

Disrupted-In-Schizophrenia 1 (DISC1) is a promising candidate gene for susceptibility to psychiatric disorders, including schizophrenia. DISC1 appears to be involved in neurogenesis, neuronal migration, axon/dendrite formation, and synapse formation; during these processes, DISC1 acts as a scaffold protein by interacting with various partners. However, the lack of DISC1 knockout mice and a well-characterized antibody to DISC1 has made it difficult to determine the exact role of DISC1 in vivo. In this study, we generated mice lacking exons 2 and 3 of the DISC1 gene and prepared specific antibodies to the N- and C-termini of DISC1. The DISC1 mutant mice are viable and fertile, and no gross phenotypes, such as disorganization of the brain's cytoarchitecture, were observed. Western blot analysis revealed that the DISC1-specific antibodies recognize a protein with an apparent molecular mass of approximately 100 kDa in brain extracts from wild-type mice but not in brain extracts from DISC1 mutant mice. Immunochemical studies demonstrated that DISC1 is mainly localized to the vicinity of the Golgi apparatus in hippocampal neurons and astrocytes. A deficiency of full-length DISC1 induced a threshold shift in the induction of long-term potentiation (LTP) in the dentate gyrus. The DISC1 mutant mice displayed abnormal emotional behavior as assessed by the elevated-plus maze and cliff avoidance tests, thereby suggesting that a deficiency of full-length DISC1 may result in lower anxiety and/or higher impulsivity. Based on these results, we suggest that full-length DISC1-deficient mice and DISC1-specific antibodies are powerful tools for dissecting the pathophysiological functions of DISC1.