Regulation of vesicle transport in CV-1 cells and extracts

Abstract

To investigate the regulation of microtubule-dependent vesicle motility, we have studied the effects of pharmacological agents on the frequency and velocity of small vesicle movements in intact CV-1 cells. Nocodazole, but not cytochalasin B or D, abolished vesicle movements, indicating that these movements were microtubule and not actin-dependent. The frequency of vesicle movements was stimulated maximally sixfold by okadaic acid from a resting value of 1.6 movements/min per μm2 in serum-starved CV-1 cells. Other activators in decreasing order of effectiveness are fetal calf serum, dibutyryl cAMP, cholera toxin, genistein, A23187, and trental. On the other side, taxol inhibited vesicle movements by a factor of four. The activators, okadaic acid, fetal calf serum, and dibutyryl cAMP, also increased vesicle velocity and run length, while taxol decreased vesicle velocity. Although modulation of the frequency of vesicle movements over a >20-fold range was observed, under all conditions the fraction of vesicles moving inward versus outward did not significantly change. Only in the case of taxol was the distribution of microtubules altered within this same time period. Both inward and outward microtubule-dependent vesicle movements therefore appear to be coordinately regulated. The enhanced vesicle motility elicited by fetal calf serum in intact cells correlated with in vitro measurements of vesicle motility and velocity on purified microtubules using microtubule affinity-purified motors and carbonate-washed vesicles from cells treated with fetal calf serum. This suggests that the amount of vesicular intracellular membrane traffic is coordinately regulated with microtubule-dependent motor activity.