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. 2009 Apr 24;5(4):e1000394. doi: 10.1371/journal.ppat.1000394

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Cureton et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Split channel images of a single BSC-1 cell (left and also in Video S1) transiently expressing tom-LCa (grey) highlight 3 virus particles (blue, circled) during internalization by clathrin-coated pits (CCPs). Kymographs (right) of sections of the cell surface showing tom-LCa fluorescence over time for clathrin-coated vesicles (CCV) containing and lacking virus. (B) A graph of the % of virus particles captured by a CCP or internalized by a CCV was plotted for 146 particles that attached to 28 different cells during image acquisition. (C) A tile view of images of a BSC-1 cell co-expressing σ2-eGFP (green) and tom-LCa (red) cropped from a time-lapse movie (Video S2), showing VSV appearing at the cell surface (time, t = −18 s) relative to the point (t = 0) of clathrin detection above background. (D) A graph of the kinetics of AP-2 and clathrin recruitment to the CCPs of panel C. Fluorescence intensities were plotted relative to the time of clathrin detection, and are expressed as a % of the average maximum clathrin observed in all pits lacking virus. The points are a weighted average of fluorescence intensities calculated as described in Materials and Methods. (E) A graph of the average kinetics of LCa and σ2 recruitment to CCPs containing (right, from 4 cells) or lacking (left, from 2 of the 4 cells) virus. Average fluorescence intensity and time are expressed as a % relative to CCV lacking virus observed in the same cells. Fluorescence intensity was calculated at 8 equally-spaced intervals and is plotted+/−standard error. (F) A graph of the clathrin fluorescence relative to vesicle lifetime for CCPs lacking (open circles) or containing (blue) virus. Fluorescence was expressed as a % relative to the average maximum for tom-LCa in pits lacking VSV. The average lifetime for pits containing virus was 110+/−44 s (15 cells), which was statistically distinct (Student's t-test: p = 2e-12) to that for pits lacking virus (51+/−16 s from 8 of the same cells). The peak clathrin fluorescence intensities in pits containing and lacking virus was 155+/−69 and 100+/−34, respectively. The difference between these values is statistically significant (Student's t-test: p = 1e-6).