Using the atomic force microscope to locally probe the cell membrane, we observed the formation of multiple tethers (thin nanotubes, each requiring a similar pulling force) as reproducible features within force profiles recorded on individual cells. Forces obtained with Chinese hamster ovary cells, a malignant human brain tumor cell line, and human endothelial cells (EA hy926) were found to be 28 +/- 10 pN, 29 +/- 9 pN, and 29 +/- 10 pN, respectively, independent of the nature of attachment to the cantilever. The rather large variation of the tether pulling forces measured at several locations on individual cells points to the existence of heterogeneity in the membrane properties of a morphologically homogeneous cell. Measurement of the summary lengths of the simultaneously extracted tethers provides a measure of the size of the available membrane reservoir through which co-existing tethers are associated. As expected, partial disruption of the actin cytoskeleton and removal of the hyaluronan backbone of the glycocalyx were observed to result in a marked decrease (30-50%) in the magnitude and a significant sharpening of the force distribution indicating reduced heterogeneity of membrane properties. Taken together, our results demonstrate the ability of the plasma membrane to locally produce multiple interdependent tethers-a process that could play an important role in the mechanical association of cells with their environment.

Protein-directed intracellular transport has not been observed in bacteria despite the existence of dynamic protein localization and a complex cytoskeleton. However, protein trafficking has clear potential uses for important cellular processes such as growth, development, chromosome segregation, and motility. Conflicting models have been proposed to explain Myxococcus xanthus motility on solid surfaces, some favoring secretion engines at the rear of cells and others evoking an unknown class of molecular motors distributed along the cell body. Through a combination of fluorescence imaging, force microscopy, and genetic manipulation, we show that membrane-bound cytoplasmic complexes consisting of motor and regulatory proteins are directionally transported down the axis of a cell at constant velocity. This intracellular motion is transmitted to the exterior of the cell and converted to traction forces on the substrate. Thus, this study demonstrates the existence of a conserved class of processive intracellular motors in bacteria and shows how these motors have been adapted to produce cell motility.murein cluster B | proton motive force

We study intact and bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. We find strong evidence of power-law stress-stiffening in the E. coli cell wall, with an exponent of 1.22 ± 0.12, such that the wall is significantly stiffer in intact cells (E = 23 ± 8 MPa and 49 ± 20 MPa in the axial and circumferential directions) than in unpressurized sacculi. These measurements also indicate that the turgor pressure in living cells E. coli is 29 ± 3 kPa.Many cellular-scale processes in biology, such as cell growth, division and motility, necessarily involve mechanical interactions. Recent theoretical work in bacteria has led to a number of physically-realistic models of bacterial cells [1][2][3]. However, in many instances, precise, direct measurements of the mechanical properties of cellular components in live cells are lacking.The cell envelope in most bacteria is made of one or two layers of membrane and a rigid cell wall consisting of a network of peptidoglycan (PG) polymers. These two materials serve different cellular functions. The semipermeable plasma membrane maintains a chemical separation between the cell interior and the surrounding medium. The large concentration of solutes in the cytoplasm generates an osmotic pressure, termed turgor pressure, that pushes the plasma membrane against the cell wall. The cell wall, on the other hand, defines the cell shape and constrains the volume under turgor.The magnitude of the turgor pressure under physiological conditions has been estimated using several techniques: by collapsing gas vesicles in rare species of bacteria [4], by AFM indentation [5, 6], and by calculating the total chemical content of the cytoplasm [7]. The estimated pressure values vary by more than an order of magnitude, from 10 4 to 3 × 10 5 Pa. While mechanical experiments, such as AFM indentation, are the most direct probes, separating the mechanical contributions of the wall and pressure has not been previously possible and thus these experiments may only provide an upper bound on the true turgor pressure.Similarly, the elasticity of the cell wall has been difficult to probe in individual, live cells. Most previous mechanical measurements on the cell wall have been performed using chemically isolated walls, termed sacculi, that may be altered from the native state, or on large bundles of cells [8]. Yao et al. reported an anisotropic elasticity of 25 MPa and 45 MPa in the axial and circumferential directions relative to a cell's rod-shape using single flattened E. coli sacculi adhered to a substrate [9]. Thwaites and coauthors probed the elastic modulus of macroscopic threads of many Bacillus subtilis sacculi in humid air and found that the modulus varied from 10 to 30 MPa depending on the humidity and salt concentration [10][11][12]. Attempts to probe whole-cell elasticity have also been made using AFM indentation of Myxococcus xanthus cells [13] and optical-tweezer bending of Borrelia burgdorf...

Whereas recent studies suggest that cholesterol plays important role in the regulation of membrane proteins, its effect on the interaction of the cell membrane with the underlying cytoskeleton is not well understood. Here, we investigated this by measuring the forces needed to extract nanotubes (tethers) from the plasma membrane, using atomic force microscopy. The magnitude of these forces provided a direct measure of cell stiffness, cell membrane effective surface viscosity and association with the underlying cytoskeleton. Furthermore, we measured the lateral diffusion constant of a lipid analog DiIC12, using fluorescence recovery after photobleaching, which offers additional information on the organization of the membrane. We found that cholesterol depletion significantly increased the adhesion energy between the membrane and the cytoskeleton and decreased the membrane diffusion constant. An increase in cellular cholesterol to a level higher than that in control cells led to a decrease in the adhesion energy and the membrane surface viscosity. Disassembly of the actin network abrogated all the observed effects, suggesting that cholesterol affects the mechanical properties of a cell through the underlying cytoskeleton. The results of these quantitative studies may help to better understand the biomechanical processes accompanying the development of atherosclerosis.

Background: MG53 is a membrane repair gene whose role in wound healing has not been studied. Results: Topical administration of MG53 protein facilitates wound healing and reduces scar formation. Conclusion: This study establishes MG53 as facilitator of injury repair and inhibitor of myofibroblast differentiation during wound healing. Significance: MG53 has therapeutic benefits in treating wounds and fibrotic diseases.

Background The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked with cardiac pathologies including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction in cardiac electrical activity is not well understood, and often overlooked in the cardiac arrhythmia field. Methods and Results Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that βII spectrin, an actin-associated molecule, is essential for the post-translational targeting and localization of critical membrane proteins in heart. βII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/βII spectrin interaction leading to severe human arrhythmia phenotypes. Mice lacking cardiac βII spectrin display lethal arrhythmias, aberrant electrical and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, βII spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes that include the Na/Ca exchanger, RyR2, ankyrin-B, actin, and αII spectrin. Finally, we observe accelerated heart failure phenotypes in βII spectrin-deficient mice. Conclusions Our findings identify βII spectrin as critical for normal myocyte electrical activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.

The formation of a collectively moving group benefits individuals within a population in a variety of ways. The surface-dwelling bacterium Myxococcus xanthus forms dynamic collective groups both to feed on prey and to aggregate during times of starvation. The latter behaviour, termed fruiting-body formation, involves a complex, coordinated series of density changes that ultimately lead to three-dimensional aggregates comprising hundreds of thousands of cells and spores. How a loose, two-dimensional sheet of motile cells produces a fixed aggregate has remained a mystery as current models of aggregation are either inconsistent with experimental data or ultimately predict unstable structures that do not remain fixed in space. Here, we use high-resolution microscopy and computer vision software to spatiotemporally track the motion of thousands of individuals during the initial stages of fruiting-body formation. We find that cells undergo a phase transition from exploratory flocking, in which unstable cell groups move rapidly and coherently over long distances, to a reversal-mediated localization into onedimensional growing streams that are inherently stable in space. These observations identify a new phase of active collective behaviour and answer a long-standing open question in Myxococcus development by describing how motile cell groups can remain statistically fixed in a spatial location.

Background: The endolysosomal TPC2 ion channel interacts with mTOR to regulate cellular energy utilization.Results: Mice lacking TPC2 display muscle atrophy phenotype with reduced muscle endurance, altered autophagy, and lysosomal enzymatic activities.Conclusion: TPC2 regulates autophagic signaling in skeletal muscle.Significance: TPC2 impacts protein turnover via regulating autophagy signaling in the process of tissue homeostasis and aging.