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. 2011 Mar 1;22(5):634–650. doi: 10.1091/mbc.E10-06-0514

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© 2011 Barfod et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — GFP-MLCK rings appear to be associated with discrete membrane structures at the base of the cell. (A) Stills taken of cells expressing GFP-MLCK (green) and RFP-farnesyl (red) for different times (in min) after hypotonic treatment. Different angled arrows mark different MLCK rings displayed in the following two panels. Scale bars are 15 μm. (B) Sequential stills of the ring marked by an arrow in the upper right corner of panel A from just before to 2.5 min after hypotonic treatment. The top row shows GFP-MLCK (green) and RFP-farnesyl (red). The middle row shows signal from MLCK, and the bottom row shows signal from farnesyl. The arrow indicates a time (1.2 min after hypotonic exposure) when both MLCK and some of the membrane marker appear as rings. Scale bars are 5 μm. (C) Sequential stills of the ring marked by an arrow in the upper left corner of panel A from 0.7 min to 3.5 min after hypotonic treatment. The top row shows GFP-MLCK (green) and RFP-farnesyl (red). The middle row shows MLCK signal, and the bottom row shows farnesyl signal. The arrow indicates a time (1.8 min) when the lumen of the MLCK ring is filled with membrane marker. Scale bars are 5 μm. (D) Stills from live imaging of cells transfected with GFP-MLCK (green) exposed to exogenous FM4-64X (red) for 2 min during hypotonic treatment and then washed out for the remaining recovery. An arrow marks an area where membrane dye has been internalized, such that GFP-MLCK signal colocalizes with an apparent FM4-64X ring. The adjacent panels depict magnified images of the MLCK signal for subsequent times. Over time, the MLCK ring appears to become a patch. The scale bar is 15 μm.