To assess the role of clathrin in the bulk endocytic flow of rat foetal fibroblasts, the rate of internalization of fluid-phase and membrane-lipid tracers were compared, under control conditions and after inhibition of endocytic clathrin-coated pit formation. After intracellular potassium depletion or upon cell transfer into 0.35 M NaCl, the rate of internalization of receptor-bound transferrin and the residual membrane area of plasmalemmal clathrin-coated pits and vesicles were similarly decreased by ~90%. In contrast, the initial rate (<5 min) of intracellular accumulation of the fluid-phase tracer HRP was not affected. Both in control and treated cells, the rate of HRP accumulation declined after ~5 min, and was twofold lower in treated cells, due to enhanced regurgitation. After correction for regurgitation, the endocytic rate constant was similar to measurements at shorter intervals and identical in control and treated cells. Similarly, the rate of internalization and the steady-state level of intracellular accumulation of two fluorescent lipid derivatives, 6-[N-(7-nitro-2,1,3- benzoxadiazol-4-yl)amino]hexanoylglucosylsphingosine (C6-NBD-GlcCer) and 1- [4-(trimethylamino)phenyl]-6-phenyl-hexa-1,3,5-triene (TMA-DPH), were not affected by potassium depletion, indicating that the endocytic membrane traffic was equally preserved. Finally, the size distribution of primary endocytic particles that were accessible to HRP within 15 s before glutaraldehyde fixation was also indistinguishable in control and potassium- depleted cells. The simplest explanation is that clathrin polymerization is necessary to concentrate receptor-bound ligands in primary endocytic vesicles, but superfluous to the basic endocytic machinery in rat foetal fibroblasts.
ASJC Scopus subject areas
- Cell Biology