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. Author manuscript; available in PMC: 2014 Aug 29.
Published in final edited form as: Cell Rep. 2013 Aug 15;4(4):10.1016/j.celrep.2013.07.017. doi: 10.1016/j.celrep.2013.07.017

Figure 2. Absence of Coat Dynamics in Chemically Arrested Mitotic Cells.

Figure 2

(A) Images obtained by spinning-disc confocal fluorescence microscopy from the top and bottom surfaces and middle equatorial planes of HeLa and BSC1 cells stably expressing σ2-EGFP undergoing natural mitosis (control), during the washout period after treatment with RO-3306, or after chemical arrest during mitosis by nocodazole or STLC treatment. Examples of AP2 spots (orange arrowheads) are shown. Note the complete absence of clathrin/AP2-coated pits and vesicles in mitotic cells arrested by nocodazole or STLC. RO-3306 treatment results in the formation of large intracellular vacuoles (white arrowheads). The scale bar represents 10 µm.

(B) Total number of clathrin/AP2 structures (CCS per cell) on the plasma membrane of the cell as determined by counting the number of fluorescent AP2 spots detected in the complete z stack of each cell obtained by 3D live-cell imaging. Averages ± SD; n, number of cells; ***p < 0.0001.

(C) Representative kymographs showing clathrin/AP2 dynamics from 4D time series obtained by live-cell spinning disc confocal acquired from the top of HeLa or bottom surfaces of BSC1 cells stably expressing σ2-EGFP during natural mitosis (control), mitotic cells imaged during the washout period after treatment with RO-3306, or mitotic cells arrested by incubation with nocodazole or STLC. Examples of AP2 tracks are highlighted (arrowhead).

(D) Upper panel: plot of the individual lifetimes of clathrin/AP2 pits from HeLa and BSC1 cells calculated from 4D time series obtained from the bottom and top surface of metaphase or from 2D and 4D time series from the top and bottom surfaces of interphase cells. At least five cells were imaged for each condition. Bars and numerical values are averages ± SD; n, number of AP2 spots. Lower panel: fraction of AP2 spots whose lifetime was longer than the time series (120 s) used to calculate the data in the upper panel (percentage of arrested pits). ***p < 0.0001; ns, not significant (p > 0.05).