Figure 1.
Nav1.6 is distributed heterogeneously in the somatic membrane. (A) Nav1.6-BAD surface expression in DIV10 rat hippocampal neurons is highly enriched at the AIS, as indicated by live-cell labeling with SA-CF640R (red). (B) Same image as (A) but without the DIC overlay. Channels on the somatic surface are barely visible at this contrast. (C) An enlargement of the boxed region shown in (B). The white arrows point to Nav1.6-BAD surface nanoclusters. The image contrast is enhanced relative to that in (B) and a different time point is presented. (D) DIC and surface labeling of a transfected glial cell within the neuronal culture. (E) The surface expression pattern for Nav1.6-BAD in the glial cell shown in (D). (F) An enlargement of the boxed region in (E). Note the absence of the neuronal nanoclusters. The heterogeneity in single-channel intensity is due to variability in CF640R labeling of the streptavidin in addition to single-fluorophore photobleaching during imaging. (G) Left: An enlargement of the white box in (C) with the CF640R fluorescence pseudocolored magenta. Contrast has been enhanced to visualize individual (orange arrows) and clustered (white arrow) somatic channels. Middle: The same field imaged 10 s later and with the SA-CF640R fluorescence now pseudocolored green. Right: Overlay of the two time frames, where colocalization appears white. The brightest punctum, i.e., nanocluster, appears in the same location in both image sequences (white arrow), whereas smaller puncta demonstrate mobility (orange arrows). (H) Same temporal analysis as in (G) but performed with the glial cell shown in (D)–(F). Note that most particles moved during the 10 s time period. (I) Stability of Nav1.6 puncta. Puncta were detected in frame 1, and in subsequent frames the fraction of puncta remaining in the same location was determined. Summed data from three neurons and three glial cells are presented. The total puncta number in both cell types remained constant during this 15 s imaging period.