The reversible action of alpha-amanitin on nuclear structure and molecular composition

The effects of brief trypsin treatment on cell shape, surface topography, cytoskeletal components and the freeze-fracture distribution of plasma membrane intramembranous particles (PMP) are examined in contact-inhibited 3T3 cells. Untreated 3T3 cells are flat without many surface modifications, have highly organized microfilaments and microtubules and have clusters of PMP. Within seconds of trypsin treatment using a concentration of trypsin used for routine passage numerous zeiotic blebs appear on the cell surface which appear to “cap” or coalesce on the central superior aspect of the cell. In later stages retraction fibers are observed and the cells then round up and detach from the substrate. Using thin section transmission electron microscopy (TEM) the zeiotic blebs are observed and various constituents of the cytosol, including lysosomes, ribosomes, etc. pinch off and are surrounded by plasma membrane. In general there is a profound disruption of microfilaments and cortical microtubules. There is, however, a focal coalescence of microfilaments subjacent to the zones of the “capped” zeiotic blebs and some relative resistance of centriolar associated microtubules to tryptic degradation. Trypsin also causes profound changes in the nucleus which transforms from the normal round shape to a very convoluted and irregular shape. In addition to the zeiotic bleb formation which can be visualized by freeze-fracture the distribution of PMP goes from the normally clustered state to a more uniform distribution following treatment with trypsin. These studies suggest that there may be some coordinated control of the changes in cell surface topography, cytoskeletal components and intramembranous particle distribution.