Phosphatidylserine polarization is required for proper Cdc42 localization and for development of cell polarity.

We used genetically-encoded fluorescent probes to visualize the distribution of phosphatidylserine (PS) in live S. cerevisiae. The majority of the PS was found to reside in the cytosolic leaflet of the plasma membrane. Remarkably, PS was polarized, accumulating in bud necks, the bud cortex and the tips of mating projections. Polarization required vectorial delivery of PS-enriched secretory and recycling vesicles. Rapid dissipation of the PS gradient is prevented by the slow diffusion of lipids along the plasmalemmal inner leaflet, estimated by photobleaching recovery measurements to be over an order of magnitude slower than in mammalian cells. In mutants lacking PS-synthase the absence of PS was associated with, and likely responsible for impaired polarization of the Cdc42 complex, leading to inhibition of bud emergence, diminished growth rate and abolishment of mating. The results indicate that PS polarization is required for optimal Cdc42 targeting and activation during cell division and mating

Sensing Phosphatidylserine in Cellular Membranes

Phosphatidylserine, a phospholipid with a negatively charged head-group, is an important constituent of eukaryotic cellular membranes. On the plasma membrane, rather than being evenly distributed, phosphatidylserine is found preferentially in the inner leaflet. Disruption of this asymmetry, leading to the appearance of phosphatidylserine on the surface of the cell, is known to play a central role in both apoptosis and blood clotting. Despite its importance, comparatively little is known about phosphatidylserine in cells: its precise subcellular localization, transmembrane topology and intracellular dynamics are poorly characterized. The recent development of new, genetically-encoded probes able to detect phosphatidylserine within live cells, however, is leading to a more in-depth understanding of the biology of this phospholipid. This review aims to give an overview of the current methods for phosphatidylserine detection within cells, and some of the recent realizations derived from their use

Transmembrane disposition of the phlorizin binding protein of intestinal brush borders

Feu Estrada, Modest (arquitecte)Primer pla d'un edifici d'habitatges de planta baixa més cinc plantes pis amb coberta terrat. La planta baixa és un dels pocs exemples on no s'han produït intervencions i es conserva tal com ha estat des del seu origen

Role of CrkII in Fc gamma receptor-mediated phagocytosis

Phagocytosis of IgG-opsonized pathogens by Fc gamma receptors requires extensive remodeling of the actin cytoskeleton, a process regulated by the small GTPase Rac. Vav was thought to be the guanine nucleotide exchange factor responsible for the activation of Rac, but recent evidence indicates that Fc gamma receptor-mediated phagocytosis is unaffected in macrophages lacking all three isoforms of Vav. We therefore tested whether another GEF, DOCK180, participates in Fc gamma receptor-initiated phagocytosis. DOCK180 associates with the adaptor protein Crk, which mediates recruitment of the GEF to sites of tyrosine phosphorylation. CrkII and DOCK180 were found to accumulate at the phagocytic cup. Knockdown of Crk or DOCK180 in murine macrophages using small interfering RNA inhibited phagocytosis of IgG-opsonized particles. Moreover, transfection of dominant negative CrkII prevented both recruitment of DOCK180 and the activation of Rac at the phagocytic cup. This is the first report of a role for either Crk or DOCK180 in Fc gamma receptor-mediated phagocytosis. The Crk-DOCK180 complex is involved in the clearance of apoptotic cells, which unlike the ingestion of IgG-opsonized particles, is an anti-inflammatory process. The finding that CrkII-DOCK180 is also responsible, at least in part, for the effects of Fc gamma receptors implies that additional, parallel pathways must account for the associated pro-inflammatory effect

The chloride channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) uncouples mitochondria and increases the proton permeability of the plasma membrane in phagocytic cells

AbstractWe present evidence that the potent chloride channel blocker NPPB has protonophoric activity in the mitochondria and across the plasma membrane of phagocytic cells. The resting O2 consumption of murine peritoneal macrophages was stimulated up to 2.5-fold in the presence of NPPB, with a K0.5 of 15 μM. The stimulatory effect of NPPB also O2 consumption, like that of the classical protonophore CCCP, was prevented by the mitochondrial respiratory chain inhibitors antimycin A, rotenone or cyanide. NPPB also mediated rheogenic proton transport across the plasma membrane of human neutrophils and macrophages in the direction dictated by the electrochemical proton gradient. As a consequence of its protonophoric activity, NPPB uncoupled mitochondrial ATP synthesis, resulting in partial depletion of cellular ATP. These observations indicate that, at the concentrations frequently used for blockade of anion channels, NPPB acts as an effective protonophore, potentially disturbing cytosolic pH and mitochondrial ATP synthesis