Fusion of exocytotic vesicles with the plasma membrane gives rise to an increase in membrane surface area, whereas the surface area is decreased when vesicles are internalized during endocytosis. Changes in membrane surface area, resulting from fusion and fission of membrane vesicles, can be followed by monitoring the corresponding proportional changes in membrane capacitance. Using the cell-attached configuration of the patch-clamp techniques we were able to resolve the elementary processes of endo-and exocytosis in yeast protoplasts at high temporal and spatial resolution. Spontaneous capacitance changes were predominantly in the range of 0.2-1 fF which translates to vesicle diameters of 90-200 nm. The size distribution revealed that endocytotic vesicles with a median at about 132 nm were smaller than exocytotic vesicles with a median at 155 nm. In energized and metabolizing protoplasts, endo-and exocytotic events occurred at frequencies of 1.6 and 2.7 events per minute, respectively. Even though these numbers appear very low, they are in good agreement with the observed growth rate of yeast cells and protoplasts.
cylindrical LPS from S. minnesota. Considering the role of TRPV4 in mechanosensation we hypothesized that TRPV4 senses the disturbances induced by insertion of LPS in the plasma membrane. By measuring membrane fluidity using fluorescence probes, we found that conically-shaped LPS from E. coli, but not cylindrical LPS from S. minnesota, causes a membrane phase shift towards gel-like state. This suggests that LPS structural differences are important in its ability to induce mechanical alterations in the membrane. We also found that LPS activates native TRPV4 in the human bronchial epithelial cell line 16HBE. Acute stimulation of TRPV4 in monolayer cultures of 16HBE cells induced a rapid increase of the transepithelial electrical resistance. Additionally, LPS-mediated TRPV4 activation increased colocalization of ZO-1 and Occludin in the plasma membrane. Altogether, these data suggest that LPS induces a TRPV4-dependent reorganization of tight junction proteins, leading to an increase in barrier function. We propose that TRPV4 participates in the orchestration of the innate immune response to the challenge with bacterial endotoxins.
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