Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.

Flow-dependent channel formation in clots by an erythrocyte-bound fibrinolytic agent

Studies in animal models have shown that plasminogen activators bound to erythrocytes (RBC-PA) have an extended lifetime in the circulation and are safer than free PAs. RBC-PAs incorporate into nascent thrombi, which are focally lysed from within, an attractive thromboprophylactic option. In static systems, RBC-PAs cleave surrounding fibrin fibers, forming pores larger than the cells themselves, and move around the pore edges, enlarging them until eventual clot dissolution. We hypothesized that under flow in blood vessels, RBC-PAs form functional patent channels before clot dissolution. Here we used perfusion chambers to study clot lysis by RBC-PAs under static versus arterial and venous flow conditions. We found that flow decelerates bulk clot lysis but quickly generates patent channels filled with passing RBCs, via pore enlargement and merging in the direction of flow. Formation of such channels by RBC-PAs may help rescue ischemic tissue before bulk dissolution of potentially occlusive clots.

The Six-Helix Bundle of Human Immunodeficiency Virus Env Controls Pore Formation and Enlargement and Is Initiated at Residues Proximal to the Hairpin Turn

ABSTRACT Residues that create the grooves of the human immunodeficiency virus type 1 (HIV-1) Env triple-stranded coiled coil (HR1) and the residues that pack into the grooves (HR2) to complete the formation of the six-helix bundle (6HB) were mutated. The extent and kinetics of fusion as well as pore enlargement were measured for each mutant. Mutations near the hairpin turns of each monomer of the 6HB were more important than those far from the turn, for both HR1 and HR2. This result is consistent with the idea that binding of HR2 to the HR1 grooves is initiated near the hairpin turn of each monomer. Mutations at the distal portions also reduced fusion, albeit to a smaller extent. An intermediate of fusion (temperature-arrested state [TAS]) was formed, and the consequences of mutation were compared; a mutant that exhibited less fusion also showed slower kinetics from TAS. This suggests that formation of the bundle is a rate-limiting step downstream of the intermediate state. The rate of enlargement of a fusion pore also correlated with the extent and kinetics of fusion. The rate of pore enlargement was severely reduced by mutation. This supports our prior conclusion that formation of the 6HB occurs after pore creation and strongly suggests that the free energy released by bundle formation is directly used to promote pore growth.