Self-Organization of Muscle Cell Structure and Function

The organization of muscle is the product of functional adaptation over several length scales spanning from the sarcomere to the muscle bundle. One possible strategy for solving this multiscale coupling problem is to physically constrain the muscle cells in microenvironments that potentiate the organization of their intracellular space. We hypothesized that boundary conditions in the extracellular space potentiate the organization of cytoskeletal scaffolds for directed sarcomeregenesis. We developed a quantitative model of how the cytoskeleton of neonatal rat ventricular myocytes organizes with respect to geometric cues in the extracellular matrix. Numerical results and in vitro assays to control myocyte shape indicated that distinct cytoskeletal architectures arise from two temporally-ordered, organizational processes: the interaction between actin fibers, premyofibrils and focal adhesions, as well as cooperative alignment and parallel bundling of nascent myofibrils. Our results suggest that a hierarchy of mechanisms regulate the self-organization of the contractile cytoskeleton and that a positive feedback loop is responsible for initiating the break in symmetry, potentiated by extracellular boundary conditions, is required to polarize the contractile cytoskeleton

Sphingomyelin Functions as a Novel Receptor for Helicobacter pylori VacA

The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells

Chimeric Vitronectin : Insulin-like Growth Factor Proteins Enhance Cell Growth and Migration through Co-Activation of Receptors

Complexes comprised of IGF-I, IGF-binding proteins and the ECM protein vitronectin (VN) stimulate cell migration and growth and can replace the requirement for serum for the ex vivo expansion of cells, as well as promote wound healing in vivo. Moreover, the activity of the complexes is dependent on co-activation of the IGF-I receptor and VN-binding integrins. In view of this we sought to develop chimeric proteins able to recapitulate the action of the multiprotein complex within a single molecular species. We report here the production of two recombinant chimeric proteins, incorporating domains of VN linked to IGF-I, which mimic the functions of the complex. Further, the activity of the chimeric proteins is dependent on co-activation of the IGF-I- and VN-binding cell surface receptors. Clearly the use of chimeras that mimic the activity of growth factor:ECM complexes, such as these, offer manufacturing advantages that ultimately will facilitate translation to cost-effective therapies

Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction

Cardiac muscle cells are known to adapt to their physical surroundings, optimizing intracellular organization and contractile function for a given culture environment. A previously developed in vitro model system has shown that the inclusion of discrete microscale domains (or microrods) in three dimensions (3D) can alter long-term growth responses of neonatal ventricular myocytes. The aim of this work was to understand how cellular contact with such a domain affects various mechanical changes involved in cardiac muscle cell remodeling. Myocytes were maintained in 3D gels over 5 days in the presence or absence of 100 – μm-long microrods, and the effect of this local heterogeneity on cell behavior was analyzed via several imaging techniques. Microrod abutment resulted in approximately twofold increases in the maximum displacement of spontaneously beating myocytes, as based on confocal microscopy scans of the gel xy-plane or the myocyte long axis. In addition, microrods caused significant increases in the proportion of aligned myofibrils (≤20° deviation from long axis) in fixed myocytes. Microrod-related differences in axial contraction could be abrogated by long-term interruption of certain signals of the RhoA-/Rho-associated kinase (ROCK) or protein kinase C (PKC) pathway. Furthermore, microrod-induced increases in myocyte size and protein content were prevented by ROCK inhibition. In all, the data suggest that microdomain heterogeneity in 3D appears to promote the development of axially aligned contractile machinery in muscle cells, an observation that may have relevance to a number of cardiac tissue engineering interventions