Intermediate Filament Protein

A novel intermediate filament cDNA, pG-IF, has been isolated from a Drosophila melanogaster embryonic expression library screened with a polyclonal antiserum produced against a 46 kDa cytoskeletal protein isolated from Kc cells. This 46 kDa protein is known to be immunologically related to vertebrate intermediate filament proteins. The screen resulted in the isolation of four different cDNA groups. Of these, one has been identified as the previously characterized Drosophila nuclear lamin cDNA, Dm0, and a second, pG-IF, demonstrates homology to Dm0 by cross hybridization on Southern blots. DNA sequence analysis reveals that pG-IF encodes a newly identified intermediate filament protein in Drosophila. Its nucleotide sequence is highly homologous to nuclear lamins with lower homology to cytoplasmic intermediate filament proteins. pG-IF predicts a protein of 621 amino acids with a predicted molecular mass of 69,855 daltons. In vitro transcription and translation of pG-IF yielded a protein with a SDS-PAGE estimated molecular weight of approximately 70 kDa. It contains sequence principles characteristic of class V intermediate filament proteins. Its near neutral pI (6.83) and the lack of a terminal CaaX motif suggests that it may represent a lamin C subtype in Drosophila. In situ hybridization to polytene chromosomes detects one band of hybridization on the right arm of chromosome 2 at or near 51A. This in conjunction with Southern blot analysis of various genomic digests suggests one or more closely placed genes while Northern blot analysis detects two messages in Kc cells.

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The presence or absence of a vimentin-type intermediate filament network affects the shape of the nucleus in human SW-13 cells

Journal of Cell Science ◽

10.1242/jcs.107.6.1593 ◽

1994 ◽

Vol 107 (6) ◽

pp. 1593-1607 ◽

Cited By ~ 7

Author(s):

A.J. Sarria ◽

J.G. Lieber ◽

S.K. Nordeen ◽

R.M. Evans

Keyword(s):

Electron Microscopy ◽

Intermediate Filament ◽

Nuclear Morphology ◽

Intermediate Filament Protein ◽

Mosaic Pattern ◽

Expression Plasmid ◽

Vimentin Expression ◽

Filament System ◽

Filament Protein ◽

Filament Network

Human SW-13 cells express the intermediate filament protein vimentin in a mosaic pattern (Hedberg, K. K. and Chen, L. B. (1986). Exp. Cell Res. 163, 509–517). We have isolated SW-13 clones that do (vim+) or do not (vim-) synthesize vimentin as analyzed using anti-intermediate filament immunofluorescence, electron microscopy and two-dimensional gel analysis of detergent-extracted preparations. Vimentin is the only cytoplasmic intermediate filament protein present in the vim+ cells, and the vim- cells do not contain any detectable cytoplasmic intermediate filament system. The presence or absence of intermediate filaments did not observably affect the distribution of mitochondria, endoplasmic reticulum, microtubules or actin stress fibers when these structures were visualized by fluorescence microscopy. However, electron microscopy and anti-lamin A/C immunofluorescence studies showed that nuclear morphology in vim- cells was frequently characterized by large folds or invaginations, while vim+ cells had a more regular or smooth nuclear shape. When vim- cells were transfected with a mouse vimentin expression plasmid, the synthesis of a mouse vimentin filament network restored the smooth nuclear morphology characteristic of vim+ cells. Conversely, when vim+ cells were transfected with a carboxy-terminally truncated mutant vimentin, expression of the mutant protein disrupted the organization of the endogenous vimentin filaments and resulted in nuclei with a prominently invaginated morphology. These results indicated that in SW-13 cells the vimentin filament system affects the shape of the nucleus.

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