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. Author manuscript; available in PMC: 2010 Nov 7.
Published in final edited form as: Traffic. 2006 May;7(5):574–588. doi: 10.1111/j.1600-0854.2006.00410.x

Figure 3. Clathrin is recruited prior to Sla2p during endocytic patch formation in wild-type cells.

Figure 3

GFP-CLC1 SLA2-mRFP strain SL5121 was viewed by wide-field (A) and TIRF microscopy (B). Note many cortical patches of clathrin do not contain Sla2p, consistent with earlier recruitment of clathrin. (C) Time-lapse video of a representative cortical clathrin patch at 3-second intervals visualized using wide-field imaging. The bottom line is a reference for the starting point of the clathrin patch when it is still at the cortex. The clathrin patch moves upwards in the video approximately 200 nm, illustrating its movement away from the cell surface. (D) Time-lapse videos of patches with GFP-LC and Sla2p-mRFP shown at 6.9-second intervals using TIRFM. Note clathrin recruitment prior to Sla2p and simultaneous disappearance of Sla2p and clathrin. In videos with longer Sla2p/clathrin lifetimes, fluctuations in signal intensities were observed, possibly due to photobleaching. The video in the top panel in (D) was started after clathrin already appeared. Clathrin patch lifetimes cannot be calculated from such examples, but these events clearly demonstrate earlier recruitment of clathrin. (E) Bar graph showing the average patch lifetimes of Sla2p-mRFP and GFP-LC using TIRFM. Lifetimes of Sla2p and LC were significantly different (p< 0.001). Scale bar = 5 μm. Time scales (C & D) are in seconds.