21] Morphological studies of formation of coated pits and coated vesicles in broken cells

Abstract

This chapter describes the morphological methods used to measure the formation of coated pits and vesicles from the plasma membrane of broken A431 cells. Coated pits are not accessible to direct experimental manipulation in intact cells. Attempts to overcome this problem includes the introduction of anti-clathrin antibodies into living cells or the use of inhibitors that affect the normal functioning of coated pits. The numbers of coated pits or vesicles per cell have never been estimated before in cultured mammalian cells. In addition, sampling with the disector is not affected by the size or shape of the particles to be counted. To test this, the number of coated pits sampled using a two-dimensional sampling frame placed on the first section in each stack of three sections has been divided by the number of coated pits sampled in the same stack using the disector. Sampling using a two-dimensional sampling frame, therefore led to a threefold reduction in the number of coated pits found on broken cells following incubation at 37° ,instead of observing a relatively constant number of coated structures (coated pits plus coated vesicles) during incubation as was the case when using the disector. It illustrates the importance of choosing the right stereological method when using electron microscopy as a tool to solve problems in cell biology.