Figure 6.

Basolateral secretion and WDR1 distributions are actin dependent. (A) WIF-B cells were treated in the absence or presence of 5 μM latrunculin B (lat B) for 60 min and immunolabeled for WDR1. In a and b, WIF-B cells in the middle of the monolayer are shown whereas in c and d, cells at the edges of the monolayer were imaged. Arrowheads are pointing to the actin-associated WDR1 present at the basolateral cell surface (a) or on plasma membrane patches (c). In panels b and d, arrowheads are marking the cell membrane lacking WDR1 labeling. Asterisks are marking the bile canalicular spaces. Bar = 10 μm (B), Clone 9 cells were treated in the absence or presence of 5 μM latrunculin B (lat B) for 30 min and immunolabeled for WDR1. Arrowheads are pointing to the actin-associated WDR1 present at the cell surface in panel a. Arrowheads in panel b are marking the cell periphery with no detectable WDR1. Bar = 10 μm (C), Cells were scored for the presence or absence of cell surface WDR labeling and the percent of total cells with surface labeling was plotted. Values represent the mean ± SEM from three independent experiments. *p ≤ 0.001 (D), WIF-B cells were pretreated with 10 μM latrunculin B (lat b) for 30 (short) or 60 min (long) at 37 °C. Cells were rinsed five times with prewarmed serum-free medium and then reincubated in serum-free medium. At 0 and 15 min after reincubation, aliquots of media were collected and analyzed for albumin secretion by immunoblotting (bottom panels). Whole cell extracts harvested after the secretion assay was performed were harvested and immunoblotted for tubulin (tub) to serve as a loading control (upper panels). (D) The relative levels of albumin secreted into the medium was calculated from densitometric analysis of immunoreactive bands on blots as shown in (A). Control values were set to 100%. Values are expressed as the mean ± SEM from three independent experiments. *p ≤ 0.029, **p ≤ 0.015.