Figure S2.
Endogenous vimentin organization is unaffected by vimentin-SunTag expression and exhibits co-mobility with dense vimentin-SunTag labeling. (A–C) Representative spinning disk confocal micrographs of fixed RPE cells showing immunostained vimentin and either (A) empty GFP channel in a nontransfected control, (B) vimentin-SunTag expressed under the standard full-length CMV promoter, or (C) vimentin-SunTag expressed under the truncated, inefficient CMV100 promoter. (D) RPE cells transfected with standard full-length CMV-vimentin-SunTag (green) and vimentin-mCherry (magenta), a snapshot from the time-lapse image (see Video 2). Inset indicates a region of highly saturated vimentin-SunTag labeling density, complicating efforts to localize single particles. (E) Pseudocolored maximum intensity time projection of dense-labeled vimentin-SunTag particles over 121 frames (60 s). The dashed line indicates the position of the line scan used to generate kymograph (right). Arrows indicate SunTag trajectories with both uniform velocity and constant relative spacing, a motility pattern consistent with multiple spots associated with a single motile filament. (F) Cartoon indicating predicted vimentin-SunTag local motility in either sparse (top) or dense (bottom) labeling regimes. Refer to the image caption for details.

Endogenous vimentin organization is unaffected by vimentin-SunTag expression and exhibits co-mobility with dense vimentin-SunTag labeling. (A–C) Representative spinning disk confocal micrographs of fixed RPE cells showing immunostained vimentin and either (A) empty GFP channel in a nontransfected control, (B) vimentin-SunTag expressed under the standard full-length CMV promoter, or (C) vimentin-SunTag expressed under the truncated, inefficient CMV100 promoter. (D) RPE cells transfected with standard full-length CMV-vimentin-SunTag (green) and vimentin-mCherry (magenta), a snapshot from the time-lapse image (see Video 2). Inset indicates a region of highly saturated vimentin-SunTag labeling density, complicating efforts to localize single particles. (E) Pseudocolored maximum intensity time projection of dense-labeled vimentin-SunTag particles over 121 frames (60 s). The dashed line indicates the position of the line scan used to generate kymograph (right). Arrows indicate SunTag trajectories with both uniform velocity and constant relative spacing, a motility pattern consistent with multiple spots associated with a single motile filament. (F) Cartoon indicating predicted vimentin-SunTag local motility in either sparse (top) or dense (bottom) labeling regimes.

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