Figure 2.
Reconstitution of CLIP-170 dependent organelle recruitment in vitro. (A) Schematic of in vitro reconstitution assay with p50-GFP-vesicles, EB1, and CLIP-A or CLIP-E in COS-7 cell extract HSS. (B) Western blot of fractions from neuronal vesicle isolation from mouse brain shows p150Glued and DIC are enriched in the vesicle fraction. Most of the CLIP-170 is retained in the low speed pellet and only a faint band for endogenous CLIP-170 is found in the vesicle fraction. Cytosolic marker GAPDH is not enriched in the vesicle fraction. (C) Representative kymographs of p50-GFP-vesicles on GMPCPP microtubules show that they exhibit processive, bidirectional, and confined motility, as defined by mean squared displacement analysis of particle trajectories. (D) Representative kymographs of p50-GFP-vesicles on single GMPCPP microtubules from each of the three conditions (100nM EB1 + Mock-HSS or CLIP-A or CLIP-E in motility assay buffer). The estimated final concentration of TMR-labeled HT-CLIP in this assay was ~100nM. EB1 + Mock-HSS and EB1 + CLIP-E show a basal landing rate, whereas there is an increase in organelle recruitment with CLIP-A. At least three different preparations of cell lysates were used. (E) Quantification of the landing rates in each of the experimental conditions shows a significant increase in organelle recruitment only with EB + CLIP-A (n >= 22 movies per condition with each movie with > 500 events per movie; N >= 3 independent vesicle isolations; Kruskal-Wallis (KW) one-way ANOVA with Tukey post-tests) (F) Empirical cumulative distribution function (CDF) for microtubule dwell times of all non-fixed particles. The stair plot starts at 0.1s as this is the minimum observable dwell time. Double exponential fit obtained via Maximum Likelihood Estimation (MLE) with 100 bootstrapped fits to obtain 95% CI. The dwell time distributions for Mock-HSS, CLIP-A, CLIP-E were compared in pairs using a two sample Kolmogorov-Smirnov (KS) test with a Bonferroni correction (p > 0.20 for all comparisons; n > 1500 particles per condition; N >= 3).