Expression of desmin cDNA in PtK2 cells results in assembly of desmin filaments from multiple sites throughout the cytoplasm.
The assembly of intermediate filaments into a cytoplasmic network was studied by microinjecting into the nuclei and cytoplasms of PtK2 cells, plasmids that contained a full length desmin cDNA and an RSV promoter. Immunofluorescence was used to monitor the expression of desmin and its integration into the cells' vimentin intermediate filament network. We found that the expressed desmin co-localized with filaments of vimentin just as it does with fluorescently labelled desmin is microinjected into the cytoplasm of PtK2 cells. As early as two hours after microinjection of the plasmids, small discrete dots and short fragments of desmin could be detected throughout the cytoplasm of the cells. This initial distribution of desmin was superimposed on the filamentous pattern of vimentin in the cells. At 8 hours after microinjection of the plasmids, some of the desmin was present in long filaments that were coincident with vimentin filaments. By 18 hours, most of the desmin was in a filamentous network co-localizing with vimentin. There was no indication that desmin assembly began in the perinuclear region and proceeded toward the cell periphery. In some cells, excessively high levels of desmin were expressed. In these cases, overexpression led to clumping of desmin filaments as well as to an accumulation of diffusely distributed desmin protein in the center of the cells. This effect was apparent at approximately 18 hours after introduction of the plasmid. The native vimentin filaments in such cells were also aggregated around the nucleus, co-localizing with desmin. The microtubule networks in all injected cells appeared normal; microtubules were extended in typical arrays out to the periphery of the cells.
Mittal
B
B
Department of Genetics, Sanjay Gandhi Institute of Medical Science, Lucknow, India.
Danowski
B A
BA
Sanger
J M
JM
Sanger
J W
JW
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D003893:Desmin / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D007382:Intermediate Filaments / Q000737:chemistry*; D007614:Macropodidae; D008845:Microinjections; D014162:Transfection; D014746:Vimentin / Q000096:biosynthesis
D003893:Desmin; D014746:Vimentin; D004247:DNA