Assembly of amino-terminally deleted desmin in vimentin-free cells.
To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin-free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.
Raats
J M
JM
Department of Biochemistry, University of Nijmegen, The Netherlands.
Pieper
F R
FR
Vree Egberts
W T
WT
Verrijp
K N
KN
Ramaekers
F C
FC
Bloemendal
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers; D002460:Cell Line; D002872:Chromosome Deletion; D006224:Cricetinae; D003893:Desmin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism* / Q000648:ultrastructure; D007633:Keratins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014746:Vimentin / Q000502:physiology
D001704:Biopolymers; D003893:Desmin; D014746:Vimentin; D007633:Keratins