APICONUCLEAR ORGANIZATION OF MICROTUBULES DOES NOT SPECIFY PROTEIN DELIVERY FROM THE TRANS-GOLGI NETWORK TO DIFFERENT MEMBRANE DOMAINS IN POLARIZED EPITHELIAL-CELLS

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi compl ex and extend outward to the cell periphery (perinuclear [PN] organiza tion). During development of epithelial cell polarity, microtubules re organize to form long cortical filaments parallel to the lateral membr ane, a meshwork of randomly oriented short filaments beneath the apica l membrane, and short filaments at the base of the cell; the Golgi bec omes localized above the nucleus in the subapical membrane cytoplasm ( apiconuclear [AN] organization). The AN-type organization of microtubu les is thought to be specialized in polarized epithelial cells to faci litate vesicle trafficking between the trans-Golgi Network (TGN) and t he plasma membrane. We describe two clones of MDCK cells, which have d ifferent microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi comple x to an AN-type during development of polarity, and clone II/J cells w hich maintain a PN-type organization. Both cell clones, however, exhib it identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly esta blish direct targeting pathways for newly synthesized gp80 and gp135/1 70, and E-cadherin between the TGN and apical and basolateral membrane , respectively; this occurs before development of the AN-type microtub ule/Golgi organization in clone II/G cells. Exposure of both clone II/ G and II/J cells to low temperature and nocodazole disrupts >99% of mi crotubules, resulting in: 1) 25-50% decrease in delivery of newly synt hesized gp135/170 and E-cadherin to the apical and basolateral membran e, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of po larity development; 2) similar to 40% decrease in delivery of newly sy nthesized gp80 to the apical membrane with significant missorting to t he basolateral membrane in newly established cultures of clone II/G an d II/J cells; and 3) variable and nonspecific delivery of newly synthe sized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their depen dence on intact microtubules for efficient and specific targeting betw een the Golgi and plasma membrane domains.