FIGURE 5:
starPEG-(KA7)4 bundles microtubules. (A) Representative images showing the microtubule organization of dynamic Alexa 647–labeled microtubules after 15 min incubation at 35°C in the absence or presence of the indicated peptides at given concentrations in BRB80. (B) Stills of Supplemental Movies S4.1–S4.3 showing different modes of starPEG-(KA7)4-TAMRA-mediated microtubule overlap formation; MT-zippering and (anti-)parallel guided polymerization. The respective top rows show the starPEG-(KA7)4-TAMRA (50 nM) signal only; the bottom rows show the starPEG-(KA7)4-TAMRA (cyan) together with Alexa 647–labeled tubulin (red). White arrowheads indicate the resulting microtubule overlaps. (C, D) Fluorescence images of starPEG-(KA7)4-TAMRA (top) on microtubule overlaps (bottom). Intensity profiles derived by line scans along the microtubule are shown in the graphs above. The starPEG-(KA7)4-TAMRA intensity profile is shown in light (5 nM) and dark blue (25 nM, average intensities in orange); the microtubule intensity profile is shown in gray (average intensities in magenta). Vertical lines depict the average intensity across the indicated range. Numbers are average ± SE. NR, normalized ratio (see also E) (E) Normalized intensity ratio of microtubule-bound starPEG-(KA7)4-TAMRA (at 5 and 25 nM), that is, the average starPEG-(KA7)4-TAMRA intensity on microtubule overlaps divided by the corresponding average intensity on single microtubules normalized by the same value ratio measured in the tubulin channel. Horizontal bars indicate median. Values above 1 mean accumulation within the overlap and values below 1 exclusion from the overlaps; n = 16 and 15, respectively. P value is derived from a Mann–Whitney test.