ULTRASTRUCTURAL STUDIES OF CARBOXYL-TERMINAL TRUNCATION MUTANTS OF THE NEURONAL INTERMEDIATE FILAMENT PROTEIN PERIPHERIN

We have prepared carboxyl-terminal truncation mutants of the neuronal intermediate filament (IF) protein peripherin and examined the assembl y characteristics of these mutant proteins in SW13 cells in the presen ce and absence of vimentin. In the absence of vimentin, tailless perip herin protein (Per-C424) self-assembles into bundles and clumps as obs erved by immunofluorescence, whereas a peripherin mutant that is missi ng the tail as well as a small portion of the rod (Per-C356) appears a s spherical aggregates. Similar phenotypes are observed when vimentin- positive cells are transfected with Per-C424 or Per-C356. In these cel ls, the entire IF network is disrupted, and vimentin colocalizes with the mutant peripherin proteins. To examine the morphology of the bundl es and clumps formed by Per-C424 at the electron microscopic level, we prepared stable cell lines expressing different levels of this mutant protein. By immunofluorescence, Per-C424 appears as either clumps or bundles of filaments depending on the expression level of the mutant p rotein. However, under electron microscopy, it is apparent that both c lumps and bundles are composed of tightly packed Ifs. We were unable t o obtain stable cell lines expressing Per-C356, indicating that this m utant may prevent cell proliferation. Using a vector containing an int ernal ribosomal entry site, we prepared a construct that expresses Per -C356 and green fluorescent protein as a single mRNA, and we were able to isolate cells that expressed Per-C356 by fluorescence-activated ce ll sorting. Electron microscopic analysis of these cells showed that t hese aggregates are solid and contain no obvious filamentous structure s.