(
A) Workflow for GFP (monomer reference) and KIF21B-GFP single-molecule counting. Imaging was performed in a dual-chamber microscope slide in a single imaging session with identical microscope settings. Monomeric GFP was expressed in HEK293T cells and lysates were diluted in PBS to single-molecule densities in the first chamber. KIF21B-KO #2 Jurkat T cell line with stable overexpression of KIF21B-GFP were added to the second chamber, which was coated with anti-CD3 antibodies. Single molecules were detected using DoM ImageJ plugin and for KIF21B-GFP motors in cells, single molecules were only analyzed from kinesin tracks with a duration of >1 s. (
B) Lognormal fits of peak-fitted fluorescence intensities of monomeric GFP immobilized on glass (green dots) and KIF21B-GFP motors imaged in Jurkat T cells corrected for a hypothetical range of distances between molecule and coverslip. The evanescent field decays exponentially and KIF21B-GFP fluorescence is corrected for that given the TIRF laser angle was fixed at a penetration depth d of 180 nm. (
C) Kymograph illustrating KIF21B-GFP motors switching from walking to a pausing state. Growing microtubule (MT) end is visualized with EB3-mCherry. A static plus end undergoes a catastrophe after a KIF21B-GFP pausing event (right). (
D) Quantification of MT plus-end state when a KIF21B-GFP motor reaches the EB3-mCherry-labeled MT plus end and transitions to a paused state, as illustrated in
Figure 3D and C. Numbers relate to the ‘Pausing’ and ‘Tip-tracking + pausing’ category of
Figure 3G. n = 149 events from two independent experiments. (
E) Kymographs illustrating a KIF21B-GFP motor reaching a growing EB3-mCherry MT plus end where motor dissociation from the MT coincides with MT pausing (left) or a catastrophe event (right). (
F) Quantification of MT plus-end fate when a KIF21B-GFP motor reaches a growing EB3-mCherry-labeled plus end and dissociates, as illustrated in
Figure 3F, E. n = 75 events from two independent experiments. (
G) Kymograph illustrating a KIF21B-GFP motor directly associating to an EB3-mCherry-labeled growing MT plus end and affecting MT growth until KIF21B-GFP dissociates.