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Real‐time confocal imaging of caveolae trafficking in endothelial cells
Author(s) -
Husain Yawer S.,
Vogel Stephen M.,
Malik Asrar B.,
Minshall Richard D.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a843-d
Subject(s) - caveolae , vesicle , transcytosis , microbiology and biotechnology , caveolin 1 , green fluorescent protein , confocal microscopy , confocal , biology , caveolin , chemistry , endocytosis , membrane , biochemistry , signal transduction , cell , geometry , mathematics , gene
Caveolae are cholesterol‐rich membrane domains that function as signaling hubs and vesicular transporters. Caveolin‐1‐GFP was expressed in rat or CAV1 − / − mouse lung endothelial cells to label caveolae, which were then tracked by spinning disc confocal microscopy to investigate caveolae trafficking. Images were acquired in 0.2 μm step increments and 4D data sets ( x, y, z, time ) were analyzed with particle‐tracking software to quantify the number, size, velocity, and directional movement of vesicles. At 25°C, 15 ± 5 % of GFP‐caveolin‐1 labeled vesicles (250 ± 80 vesicles) were mobile. Caveolae that were <0.1 μm 3 (85%) and 0.1‐1.0 μm 3 (15%) in volume moved with an average velocity of 0.30 ± 0.005 and 0.21 ± 0.01 μm/sec, respectively. 10 ± 2 % of vesicles moved directly to the opposite (basal or apical) membrane (0.55 ± 0.098 μm/sec), 65 ± 15 % made intermediate stops (0.35 ± 0.07 μm/sec), and 10 ± 3% did not move in the z direction (0.32 ± 0.05 μm/sec). Direct routes of caveolae trafficking between apical and basal membranes may represent the transcytosis pathway involved in the regulation of transendothelial albumin permeability, while other trafficking pathways may reflect the processing of constituents carried in caveolae. At 37°C, phospho‐mimicking (Y14D)‐caveolin‐1‐GFP labeled vesicles were twice as mobile (59±5%) as wild‐type caveolin‐1‐GFP vesicles (31±8%). The phosphorylation dependence of plasmalemma vesicle trafficking is consistent with the hypothesis that tyrosine phosphorylation of caveolin‐1 regulates caveolae‐mediated endocytosis and transcellular transport in endothelial cells. Supported by NIH R01 HL71626, P01 HL60678, and The University of Illinois Intercampus Research Initiative in Biotechnology.