Figure 2.

ESCRT-III resides in two spatially distinct pools in intercellular bridges undergoing abscission. (a) Synchronized MDCK cells expressing Flag-tagged versions of CHMP4B, CHMP2A, or CHMP5 (from left to right) were fixed, stained with anti-α-tubulin (red) and anti-Flag (green) antibodies and imaged using a spinning disk confocal microscope. CHMP4B, CHMP2A, and CHMP5 localized in two spatially distinct pools; an initial pool located to the edge of the midbody dark zone (solid arrows) and a peripheral pool located at the site of microtubule constriction (dashed arrows). (b–d) Synchronized MDCK cells expressing CHMP4B-mCherry (red) together with either CHMP4B-Flag (b), CHMP2A-Flag (c), or CHMP5-Flag (d) were stained with anti-Flag antibodies (green) and imaged. Anti-α-tubulin staining was used to identify the intercellular bridges (not shown). Data show a high degree of overlap between the localization of CHMP4B-mCherry and either CHMP2A or CHMP5 on both the initial pool (solid arrows) and the peripheral pool (dashed arrows). Right panels show intensity profiles of a line drawn along the intercellular bridge between the ESCRT-III peripheral and initial pools. Similar data were obtained for all the ESCRT-III components examined. Scale bar = 2 μm. n (for each panel) = 10. Similar localization pattern and overlap between the pools were observed in all cells examined. Data were obtained from two independent experiments.