Fibrotic rigidification following a myocardial infarct is known to impair cardiac output, and it is also known that cardiomyocytes on rigid culture substrates show a progressive loss of rhythmic beating. Here, isolated embryonic cardiomyocytes cultured on a series of flexible substrates show that matrices that mimic the elasticity of the developing myocardial microenvironment are optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1-Hz beating. On hard matrices that mechanically mimic a post-infarct fibrotic scar, cells overstrain themselves, lack striated myofibrils and stop beating; on very soft matrices, cells preserve contractile beating for days in culture but do very little work. Optimal matrix leads to a strain match between cell and matrix, and suggests dynamic differences in intracellular protein structures. A `cysteine shotgun' method of labeling the in situ proteome reveals differences in assembly or conformation of several abundant cytoskeletal proteins, including vimentin, filamin and myosin. Combined with recent results, which show that stem cell differentiation is also highly sensitive to matrix elasticity, the methods and analyses might be useful in the culture and assessment of cardiogenesis of both embryonic stem cells and induced pluripotent stem cells. The results described here also highlight the need for greater attention to fibrosis and mechanical microenvironments in cell therapy and development.

Summary Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca2+, but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined; previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes, but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion.

To identify cytoskeletal proteins that change conformation or assembly within stressed cells, in situ labeling of sterically shielded cysteines with fluorophores was analyzed by fluorescence imaging, quantitative mass spectrometry, and sequential two-dye labeling. Within red blood cells, shotgun labeling showed that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin were increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. Within mesenchymal stem cells-as a prototypical adherent cell-nonmuscle myosin IIA and vimentin are just two of the cytoskeletal proteins identified that show differential labeling in tensed versus drug-relaxed cells. Cysteine labeling of proteins within live cells can thus be used to fluorescently map out sites of molecular-scale deformation, and the results also suggest means to colocalize signaling events such as phosphorylation with forced unfolding.Force-induced changes in protein conformation have long been postulated to contribute to the deformability of cells (1,2). Likewise, in cell adhesion, forces of pico-Newton magnitude that result from cells pulling on matrix (3) are believed to induce conformational changes that initiate essential anchorage signals (4-8). Single-molecule measurements indeed show that domain unfolding occurs in reversible extension of purified cytoskeletal, motor, and matrix adhesion proteins (9-12), and simulations of the molecular dynamics of protein extension have helped to clarify mechanisms (13-15). Direct cell-level evidence is lacking or even contrary to forced unfolding (16), although cytoskeletal association of a large and rare conformationsensitive antibody has suggested extension of a proline-rich region in one protein within spread, fixed cells (17). The more broadly directed "shotgun" approach here is applied to live cells under physiological stresses and exploits small thiol-reactive probes that permanently label force-sensitive domains.Cysteine (Cys) is a moderately hydrophobic amino acid that is frequently shielded by tertiary or quaternary protein structure. Labeling of cysteine's SH moiety has been exploited in solution denaturation studies on a few small purified proteins (18,19), as well as in an anemia-causing proline mutation in the red blood cell (RBC) protein spectrin (20). In addition, forced unfolding of single proteins with core-sequestered disulfides demonstrates reduction of the S-S within seconds by reactive thiols in the medium (21,22). We show here, in intact cells, that forceinduced changes in protein structure can also expose-for relatively rapid reaction-specific buried Cys (Fig. 1) in a number of key cytoskeletal proteins. Sequential n-dye-labeling with different color fluorophores (n = 2 here) proves to be a facile approach to amplifying signals ¶To whom correspondence should be addressed. E-mail: discher@seas.upenn.edu. * These authors conducted experiments. † These authors designed, refined, and analyzed experiments. ‡ These authors ...

Evidence suggests that protein kinase C (PKC) and intracellular calcium are important for amphetaminestimulated outward transport of dopamine in rat striatum. In this study, we examined the effect of select PKC isoforms on amphetamine-stimulated dopamine efflux, focusing on Ca 2؉ -dependent forms of PKC. Efflux of endogenous dopamine was measured in superfused rat striatal slices; dopamine was measured by high performance liquid chromatography. The non-selective classical PKC inhibitor Gö 6976 inhibited amphetamine-stimulated dopamine efflux, whereas rottlerin, a specific inhibitor of PKC␦, had no effect. A highly specific PKC␤ inhibitor, LY379196, blocked dopamine efflux that was stimulated by either amphetamine or the PKC activator, 12-O-tetradecanoylphorbol-13-acetate. None of the PKC inhibitors significantly altered [3 H]dopamine uptake. PKC␤ I and PKC␤ II , but not PKC␣ or PKC␥, were coimmunoprecipitated from rat striatal membranes with the dopamine transporter (DAT). Conversely, antisera to PKC␤ I and PKC␤ II but not PKC␣ or PKC␥ were able to co-immunoprecipitate DAT. Amphetamine-stimulated dopamine efflux was significantly enhanced in hDAT-HEK 293 cells transfected with PKC␤ II as compared with hDAT-HEK 293 cells alone, or hDAT-HEK 293 cells transfected with PKC␣ or PKC␤ I . These results suggest that classical PKC␤ II is physically associated with DAT and is important in maintaining the amphetamine-stimulated outward transport of dopamine in rat striatum.

SUMMARY Synaptotagmin-I (syt) is a Ca2+ sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of syt was its ability to aggregate membranes in response to Ca2+. However, the mechanism and function of syt-mediated membrane aggregation are poorly understood. Here, we demonstrate that syt-mediated vesicle aggregation is driven by trans interactions between syt molecules bound to different membranes. We observed a strong correlation between the ability of Ca2+-syt to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes - using non-syt proteins - also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that syt drives the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion competent heterodimers, in an aggregation-independent manner. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of Ca2+-syt-regulated, SNARE-catalyzed fusion reactions.

Mutations in otoferlin are linked to human hearing loss. New research defines a function for this C2 domain–containing protein in synaptic vesicle exocytosis in cochlear hair cells.

Molecular force measurements quantified the impact of polysialylation on the adhesive properties both of membrane-bound neural cell adhesion molecule (NCAM) and of other proteins on the same membrane. These results show quantitatively that NCAM polysialylation increases the range and magnitude of intermembrane repulsion. The repulsion is sufficient to overwhelm both homophilic NCAM and cadherin attraction at physiological ionic strength, and it abrogates the protein-mediated intermembrane adhesion. The steric repulsion is ionic strength dependent and decreases substantially at high monovalent salt concentrations with a concomitant increase in the intermembrane attraction. The magnitude of the repulsion also depends on the amount of polysialic acid (PSA) on the membranes, and the PSA-dependent attenuation of cadherin adhesion increases with increasing PSA-NCAM:cadherin ratios. These findings agree qualitatively with independent reports based on cell adhesion studies and reveal the likely molecular mechanism by which NCAM polysialylation regulates cell adhesion and intermembrane space.Polysialic acid (PSA) 1 is a long, linear ␣2,8-linked carbohydrate composed of N-acetylneuraminic acid (Neu5Ac) residues (1). This carbohydrate is added post-translationally to the neural cell adhesion molecule (NCAM), which is responsible for a variety of functions, including axon pathfinding, synaptogenesis, and tissue formation in the central nervous system (2). The expression of the polysialylated form of NCAM (PSA-NCAM) peaks early in development and decreases with age. In some exceptions, such as the hippocampus, cells continue to express PSA-NCAM throughout the life of the organism. These regions of PSA expression are also associated with neural plasticity and the remodeling of neural connections (1, 2). Aberrant expression of PSA-NCAM is associated with tumor malignancy and metastasis, and the expression of PSA-NCAM has been detected in small cell carcinoma, neuroblastomas, and Wilm's tumor (3).Polysialic acid is thought to facilitate cell migration and plasticity by inhibiting cell adhesion to other cells and to the extracellular matrix, as a result of the large excluded volume of the polymer (4, 5). Electron microscopy images showed that PSA expression increased intercellular spacing by 10 -15 nm (4). The latter could be because of the inactivation of adhesion proteins or to the increased inter-membrane repulsion resulting from the confinement of the carbohydrate chains. Light scattering studies demonstrated that NCAM polysialylation doubles the hydrodynamic radius of NCAM. However, the latter results were based on calculations, using light scattering data and the assumption that the rod-like proteins were spherical. While this indicates the approximate size of the protein, the hydrodynamic radius does not quantify the effect of the carbohydrates on NCAM-mediated adhesion. In one proposed mechanism, for example, the increased repulsive pressure between the membranes is hypothesized to push the cells apart (6). Such a shif...

The extracellular regions of adhesion proteins of the Ig superfamily comprise multiple, tandemly arranged domains. We used directforce measurements to investigate how this modular architecture contributes to the adhesive interactions of the neural cell adhesion molecule (NCAM), a representative of this protein class. The extracellular region of NCAM comprises five immunoglobulin and two fibronectin domains. Previous investigations generated different models for the mechanism of homophilic adhesion that each use different domains. We use force measurements to demonstrate that NCAM binds in two spatially distinct configurations. Igdomain deletion mutants identified the domains responsible for each of the adhesive bonds. The measurements also confirmed the existence of a flexible hinge that alters the orientation of the adhesive complexes and the intermembrane distance. These results suggest that a combination of multiple bound states and internal molecular flexibility allows for sequentially synergistic bond formation and the ability to accommodate differences in intercellular space.