BRAF increases endothelial cell stiffness through reorganization of the actin cytoskeleton.

The dynamics of the actin cytoskeleton and its connection to endothelial cell-cell junctions determine the barrier function of endothelial cells. The proper regulation of barrier opening/closing is necessary for the normal function of vessels, and its dysregulation can result in chronic and acute inflammation leading to edema formation. By using atomic force microscopy, we show here that thrombin-induced permeability of human umbilical vein endothelial cells, associated with actin stress fiber formation, stiffens the cell center. The depletion of the MEK/ERK kinase BRAF reduces thrombin-induced permeability prevents stress fiber formation and cell stiffening. The peripheral actin ring becomes stabilized by phosphorylated myosin light chain, while cofilin is excluded from the cell periphery. All these changes can be reverted by the inhibition of ROCK, but not of the MEK/ERK module. We propose that the balance between the binding of cofilin and myosin to F-actin in the cell periphery, which is regulated by the activity of ROCK, determines the local dynamics of actin reorganization, ultimately driving or preventing stress fiber formation.

A myosin II nanomachine mimicking the striated muscle.

The contraction of striated muscle (skeletal and cardiac muscle) is generated by ATP-dependent interactions between the molecular motor myosin II and the actin filament. The myosin motors are mechanically coupled along the thick filament in a geometry not achievable by single-molecule experiments. Here we show that a synthetic one-dimensional nanomachine, comprising fewer than ten myosin II dimers purified from rabbit psoas, performs isometric and isotonic contractions at 2 mM ATP, delivering a maximum power of 5 aW. The results are explained with a kinetic model fitted to the performance of mammalian skeletal muscle, showing that the condition for the motor coordination that maximises the efficiency in striated muscle is a minimum of 32 myosin heads sharing a common mechanical ground. The nanomachine offers a powerful tool for investigating muscle contractile-protein physiology, pathology and pharmacology without the potentially disturbing effects of the cytoskeletal-and regulatory-protein environment.

Alterations in the properties of the cell membrane due to glycosphingolipid accumulation in a model of Gaucher disease.

Gaucher disease is a lysosomal storage disease characterized by the malfunction of glucocerebrosidase resulting in the accumulation of glucosylceramide and other sphingolipids in certain cells. Although the disease symptoms are usually attributed to the storage of undigested substrate in lysosomes, here we show that glycosphingolipids accumulating in the plasma membrane cause profound changes in the properties of the membrane. The fluidity of the sphingolipid-enriched membrane decreased accompanied by the enlargement of raft-like ordered membrane domains. The mobility of non-raft proteins and lipids was severely restricted, while raft-resident components were only mildly affected. The rate of endocytosis of transferrin receptor, a non-raft protein, was significantly retarded in Gaucher cells, while the endocytosis of the raft-associated GM1 ganglioside was unaffected. Interferon-γ-induced STAT1 phosphorylation was also significantly inhibited in Gaucher cells. Atomic force microscopy revealed that sphingolipid accumulation was associated with a more compliant membrane capable of producing an increased number of nanotubes. The results imply that glycosphingolipid accumulation in the plasma membrane has significant effects on membrane properties, which may be important in the pathogenesis of Gaucher disease.

The growth determinants and transport properties of tunneling nanotube networks between B lymphocytes.

Tunneling nanotubes (TNTs) are long intercellular connecting structures providing a special transport route between two neighboring cells. To date TNTs have been reported in different cell types including immune cells such as T-, NK, dendritic cells, or macrophages. Here we report that mature, but not immature, B cells spontaneously form extensive TNT networks under conditions resembling the physiological environment. Live-cell fluorescence, structured illumination, and atomic force microscopic imaging provide new insights into the structure and dynamics of B cell TNTs. Importantly, the selective interaction of cell surface integrins with fibronectin or laminin extracellular matrix proteins proved to be essential for initiating TNT growth in B cells. These TNTs display diversity in length and thickness and contain not only F-actin, but their majority also contain microtubules, which were found, however, not essential for TNT formation. Furthermore, we demonstrate that Ca2+-dependent cortical actin dynamics exert a fundamental control over TNT growth-retraction equilibrium, suggesting that actin filaments form the TNT skeleton. Non-muscle myosin 2 motor activity was shown to provide a negative control limiting the uncontrolled outgrowth of membranous protrusions. Moreover, we also show that spontaneous growth of TNTs is either reduced or increased by B cell receptor- or LPS-mediated activation signals, respectively, thus supporting the critical role of cytoplasmic Ca2+ in regulation of TNT formation. Finally, we observed transport of various GM1/GM3+ vesicles, lysosomes, and mitochondria inside TNTs, as well as intercellular exchange of MHC-II and B7-2 (CD86) molecules which may represent novel pathways of intercellular communication and immunoregulation.