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 assembly characteristics of these mutant proteins in SW13 cells in the presence and absence of vimentin. In the absence of vimentin, tailless peripherin protein (Per-C424) self-assembles into bundles and clumps as observed by immunofluorescence, whereas a peripherin mutant that is missing the tail as well as a small portion of the rod (Per-C356) appears as spherical aggregates. Similar phenotypes are observed when vimentin-positive cells are transfected with Per-C424 or Per-C356. In these cells, the entire IF network is disrupted, and vimentin colocalizes with the mutant peripherin proteins. To examine the morphology of the bundles 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 protein. However, under electron microscopy, it is apparent that both clumps and bundles are composed of tightly packed IFs. We were unable to obtain stable cell lines expressing Per-C356, indicating that this mutant may prevent cell proliferation. Using a vector containing an internal 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 cell sorting. Electron microscopic analysis of these cells showed that these aggregates are solid and contain no obvious filamentous structures.
Ho
C L
CL
Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Chien
C L
CL
Brown
K
K
Liem
R K
RK
D002460:Cell Line; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure*; D008562:Membrane Glycoproteins; D008854:Microscopy, Electron; D009154:Mutation / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure*; D064531:Peripherins; D014162:Transfection
D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C578994:PRPH protein, human; D064531:Peripherins