Figure 6.

Role of intact microtubules in the targeting of newly synthesized E-cadherin to the plasma membrane during the development of polarity in MDCK clone II/G and II/J cells. Confluent MDCK clone II/G and II/J cultures were established as described in MATERIALS AND METHODS. As diagrammed in Figure 3A, cultures were incubated in either the absence (−noc) or presence (+noc) of nocodazole. During the last hour at 37°C, cultures were metabolically labeled with [³⁵S]methionine/cysteine. At the indicated times after the induction of cell–cell contacts (10–120 h), duplicate filters were labeled on either the apical (Ap) or basolateral (BL) membrane with NHS-SS-biotin. Cells were than extracted in RIPA buffer as described in MATERIALS AND METHODS. Samples were sequentially precipitated with an antiserum to E-cadherin followed by avidin agarose. Immunoprecipitates were then processed for SDS-PAGE and fluorography. (A) Fluorography results from representative samples for each time point are presented. The amount of labeled E-cadherin in gels was determined directly using a Molecular Dynamics phosphor imager (model 820). Scanning densitometry results of protein from MDCK clone II/G (B) and clone II/J (C) cells are presented as a percentage of the total newly synthesized E-cadherin delivered to the plasma membrane in the absence of nocodazole (Percent Delivery). The bar graphs depict the mean of one to three independent experiments, which are represented by one of the symbols in the scatter plots.