Selective and polarization-dependent secretory trafficking by kinesins in epithelial cells.

摘要

Microtubule-based, vesicular transport is well documented in epithelial cells but the specific motors involved and their regulation during polarization are largely unknown. Here we demonstrate that KIF5B mediates post-Golgi transport of an apical protein in epithelial cells, but only after polarity has developed. Time-lapse microscopy revealed that expression of a KIF5B dominant-negative or microinjection of function-blocking KIF5 antibodies inhibited selectively post-Golgi transport of the apical marker, p75-GFP, after polarization of MDCK cells. Expression of other KIF dominant-negatives did not alter p75-GFP trafficking. Immunoprecipitation experiments demonstrated an interaction between KIF5B and p75-GFP in polarized, but not in sub-confluent, MDCK cells. Our results demonstrate that apical protein transport depends on selective microtubule motors and that epithelial cells switch kinesins for post-Golgi transport during acquisition of polarity.;To identify the kinesin(s) responsible for p75 trafficking in non-polarized MDCK cells, we expressed KIF-specific dominant-negative constructs and assayed for changes in post-Golgi transport of p75 by time-lapse fluorescence microscopy. Over-expression of the tail domains of kinesin-3 family members that contain a C-terminal pleckstrin homology (PH) domain, KIF1A or KIF1Bbeta, attenuated the rate of p75 exit from the Golgi in non-polarized MDCK cells, but not in polarized cells. Analysis of p75 post-Golgi transport in cells expressing KIF1A or KIF1Bbeta with their PH domains deleted revealed that vesicle transport by these motors is mediated by the kinesin PH domains. Knock-down of canine KIF1A also inhibited exit of p75 from the Golgi and was rescued by expression of human KIF1A. Furthermore, purified KIRA and KIF1Bbeta tails interact with p75 vesicles and these interactions require the PH domain. These data demonstrate that post-Golgi transport of p75 is mediated, in a PH domain-dependent process, by kinesin-3 family motors before epithelial polarization.