Journal: Current biology : CB

Article Title: Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse

doi: 10.1016/j.cub.2018.11.065

Figure Lengend Snippet: A) KIF5C(1-560)-HaloTag motility on dynamic microtubules. Left panel shows full kymograph of dynamic microtubule channel. Insets: upper panel shows early time point when the microtubule is short; Left, overlay of dynamic microtubule (cyan) and GMPCPP-stabilized microtubule seed (magenta) channels, (−) and (+) represent the microtubule minus- and plus-end; Center, KIF5C channel; Right, overlay of a line representing the growing microtubule plus-end on the KIF5C channel. Horizontal scale bars: 10 μm; Vertical scale bars: 30 sec. The plot in the middle represents the location of KIF5C run terminations (blue circles) along a growing dynamic microtubule (dark blue line; microtubule plus-end), respective to the distance to the minus-end of that microtubule. The plot on the right shows the length of KIF5C runs (blue lines) that terminated at the plus-end (dark blue line). The numbers refer to the maximum KIF5C run-length observed in 120 seconds periods over 10 minutes. B) same as (A), but for KIF1A(1-393)-HaloTag. C) Percentage of runs initiating and D) terminating within 2μm of a microtubule plus-end (n=1035-1755 runs from 4-6 microtubules; average±95%CI). E) Circles represent KIF1A (red) and KIF5C (blue) run lengths observed on three representative microtubules and normalized to the maximum length of the microtubule they were observed on. The line traces show the dynamic profile of the representative microtubules along time. See also Figure S5 and S6.

Article Snippet: GMPCPP-microtubule seeds for assays with dynamic microtubules were prepared as above but with 5% biotinylated (Cytoskeleton) and 5% HyLite-647-labeled tubulin (Cytoskeleton).

Techniques:

Journal: Current biology : CB

Article Title: Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse

doi: 10.1016/j.cub.2018.11.065

Figure Lengend Snippet: A) Binding and B) quantification of KIF1A, KIF5B, and KIF5C to GMPCPP- and GDP-taxol-stabilized microtubules in the presence of AMP-PNP (n=1004-5855 microtubules per condition; n.s., non-significant, ***p<0.0001; one-way ANOVA with Sidak’s post-hoc test). C) Binding and D) quantification of KIF1A binding to GMPCPP- and GDP-taxol-stabilized microtubules under increasing ionic strength. Circles show mean intensity values; the 95% confidence intervals are too narrow and not visible in the graph. (n=398-2491 microtubules). E) Binding and F) quantification of KIF1A binding to fully tyrosinated and detyrosinated GMPCPP-stabilized microtubules G) under increasing ionic strength. Circles show mean intensity values; the 95% confidence intervals are too narrow and not visible in the graph. (n=2709-6737 microtubules). H) Binding and I) quantification of KIF1A binding to GMPCPP-stabilized microtubules in the presence or absence of EB3 (n=13569-14309 microtubules). J) Representative kymographs and stills depictingKIF1A motors rapidly detaching from a microtubule at the EB1 comet region (asterisks). The stills on the right show one of these events (each time point integrates a 150 msec interval).

Article Snippet: GMPCPP-microtubule seeds for assays with dynamic microtubules were prepared as above but with 5% biotinylated (Cytoskeleton) and 5% HyLite-647-labeled tubulin (Cytoskeleton).

Techniques: Binding Assay

Journal: Current biology : CB

Article Title: Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse

doi: 10.1016/j.cub.2018.11.065

Figure Lengend Snippet: A) KIF1A motor domain. The T258M mutation affects a residue located in KIF1A motor domain loop 11. B) Panels and quantification showing KIF1A-WT and -T258M(1-393)-HaloTag binding to GMPCPP- and GDP-taxol-stabilized microtubules. (n=2303-2351 microtubles per group; average±95%CI; **p<0.01,****p<0.0001; Kruskal-Wallis with Dunn’s post-hoc test). C) same as Figure 3A-B and D) same as Figure 3E but for KIF1A-T258M(1-393)-HaloTag, which is represented in orange. E) eCDF showing the location of KIF1A-WT (red) and KIF1A-T258M (orange) run initiation, respective to the plus-end tip. X-intercepts represent the location on the microtubule where run initiations starts to follow a random pattern. (KIF1A-WT, n=1035 runs from 6 microtubules; KIF1A-T258M, n=1628 runs from 5 microtubules). F) Correlation between GDP-taxol/GMPCPP microtubules binding ratio and distance of run initiation to the plus-end tip. (KIF5C: n=1755 runs from 4 microtubules; KIF1A-WT: n=1035 runs from 6 microtubules; KIF1A-A255V: n=702 runs from 4 microtubules; KIF1A-T258M: n=1628 runs from 5 microtubules; KIF1A-R350G: n=596 runs from 4 microtubules; GDP/GMPCPP MT binding ratio – KIF5C-WT: n=5855/4000; KIF1A-WT: n=2491/2303; KIF1A-A255V: n=2130/1046; KIF1A-T258M: n=2351/2351; KIF1A-R350G: n=2106/1162). G) KIF1A-WT rapidly detaches from microtubules once it reaches the microtubule plus-end, whereas KIF1A-T258M frequently lingers at the microtubule plus-end before detaching. See also Figure S5, S6, and Table S1.

Article Snippet: GMPCPP-microtubule seeds for assays with dynamic microtubules were prepared as above but with 5% biotinylated (Cytoskeleton) and 5% HyLite-647-labeled tubulin (Cytoskeleton).

Techniques: Mutagenesis, Binding Assay