Membrane Redistribution in Isolated Amphibian Urocytes

Abstract

The epithelium of amphibian urinary bladder is readily dissociated into single cells by exposure to solutions of low Ca²⁺ activity. Using a novel device for mounting bladders on the microscope stage, we studied the net movement of membrane material in freshly dissociated viable cells. Fluorescent labelling of apical and basolateral membrane glycoconjugates and proteins with Lucifer yellow CH, tetramethyl rhodamine B isothiocyanate (TRITC), and lectin-TRITC showed pronounced redistribution. In response to opening of the tight junctions and subsequent cell dissociation, apical label expanded over most of the cell surface. In contrast, the larger part of the basolateral membrane was invaginated and became the limiting membrane of internalized vesicles. The vesicles fused and expanded into large vacuoles within 2 h. Cell rounding and vacuolation were inhibited by cytochalasin B, i.e. dependent on functions of microfilaments. We suggest that the membrane redistribution occurs by disruption of recycling pathways, initiated by withdrawal of the tight junction: the trafficking of apical material appears interrupted at the internalization step, that of basolateral material at the reinsertion step.

Vacuolation of Isolated Amphibian Urocytes

“…To dissociate urocytes from the epithelium, we fol lowed the procedure described before [2]. In brief, both the mucosal and the serosal sides of the bladder were exposed to Ca2M'rce Ringer's solution containing I m.V/ EGTA.…”

“…Cells incubated in dextrose-containing Ringer's solution were used for electron microprobc analysis only. Vacuolation was observed in the light microscope at different incubation periods, as described before [2].…”

“…For fluorescence micros copy (excitation at 365 nm). urinary bladders were mounted using the tissue stand described previously [2], For the localization of Na+/K+ ATPase in isolated urocytes, the epithelium was first labelled with an throyl-ouabain, then dissociated into single cells as described above.…”

“…Most of the isolated cells devel oped intracellular vacuoles within 2 h [2]. En ergy dependence of vacuolar development was demonstrated by incubation of isolated urocytes in solutions with differing contents of metabolic substrates: i.e.…”

“…In isolated amphibian urocytes, intracellu lar vacuoles develop from internalized basola teral membranes [1,2]. The vacuoles enlarge continuously.…”