Actin Cytoskeleton May Modulate Diverse EGF Effects on Membrane Traffic in Lacrimal Acini

Abstract

Abstract: : Purpose: We have previously shown that acute stimulation of lacrimal gland acini with epidermal growth factor (EGF) can alter the subcellular distributions of various endomembrane proteins, apparently by transiently diverting traffic from the lysosomal pathway to a basal-lateral recycling endosome (Xie J, ARVO Abstract #3112, 2002). Actin microfilaments have been found to be linked to EGF-induced signaling cascades, and preliminary studies suggested that mechanical stresses can alter actin microfilament organization. The goal of this study was to determine whether the mechanical stress associated with cell centrifugation and resuspension disrupts microfilament organization and modulates the EGF effect on traffic. Methods: Rabbit lacrimal gland acinar cells were isolated and maintained in primary culture as reconstituted acini for 2 - 3 d. Reconstituted acini were incubated at 37° for 20 m or 120 m with and without 200 ng/ml [¹²⁵I]-EGF. Two EGF stimulation protocols were followed for 20 m incubations, i.e., [¹²⁵I]-EGF was added either during the initial 20 m of resuspension, or after the acini had been resuspended and incubated for 100 m without EGF. Cells were then lysed and analyzed by isopycnic centrifugation on sorbitol density gradients. In some experiments cells were fixed after EGF loading and actin filament organization evaluated by confocal fluorescence microscopy after staining with rhodamine-phalloidin. Results: The effects of EGF stimulation on endomembrane protein compartmental distributions were substantially smaller when EGF was added during the last 20 m after cell resuspension. However, the distribution of [¹²⁵I]-EGF did not differ between the two loading protocols, i.e., it was accumulated to the same extent in the basal-lateral recycling endosome. Microfilament organization was markedly disrupted during the first 20 m after resuspension but recovered during the subsequent 100 m. EGF treatment appeared to trigger diverse effects on actin microfilament organization. Conclusions: The magnitude of the effect of EGF on endomembrane protein compartmental distribution was determined by the assembly state of the actin cytoskeleton. The effect was prominent when the actin cytoskeleton was disrupted. Protein sorting changes that may influence exposure of cryptic autoantigens can be caused by EGF-generated signals that are mediated, at least in part, through the actin cytoskeleton.

Keywords: lacrimal gland • growth factors/growth factor receptors • cytoskeleton