Recycling of Kinesin-1 Motors by Diffusion after Transport
Figure 3
Kinesin-1 returns to the cell body from neurite tips.
Differentiated CAD cells were transfected with a plasmid encoding PAGFP-tagged KHC. 48 hr later, the cells were imaged by confocal microscopy. Representative times series from three different imaging conditions are shown. (A) No photoactivation. GFP images of the entire field of view were collected over time. In the final image, the total level of expressed PAGFP-KHC was determined by photoactivation of the entire field of view (all-activ.). (B) One photoactivation event. GFP images were collected (pre-activ.) and then PAGFP-KHC in one neurite tip (white box) was photoactivated (post-activ.) followed by imaging of GFP fluorescence for the entire field of view over time. In the final image, the total level of expressed PAGFP-KHC was determined by photoactivation of the entire field of view (all-activ.). (C) Multiple photoactivation events. GFP images were collected (pre-activ.) and then PAGFP-KHC in one neurite tip (white box) was photoactivated (post-activ.) followed by imaging of GFP fluorescence for the entire field of view for 4 min. The neurite tip photoactivation and whole field GFP imaging was then repeated 9 times. In the final image, the total level of expressed PAGFP-KHC was determined by photoactivation of the entire field of view (all-activ.). Scale bar, 10 µm. (D) Quantification of the average PAGFP-KHC fluorescence in the cell body over time. At least 7 cells in two to three independent experiments were imaged for each condition. Error bars indicate SEM.