Visualization of intermediate filaments in living cells using fluorescently labeled desmin.
Fluorescently labeled desmin was incorporated into intermediate filaments when microinjected into living tissue culture cells. The desmin, purified from chicken gizzard smooth muscle and labeled with the fluorescent dye iodoacetamido rhodamine, was capable of forming a network of 10-nm filaments in solution. The labeled protein associated specifically with the native vimentin filaments in permeabilized, unfixed interphase and mitotic PtK2 cells. The labeled desmin was microinjected into living, cultured embryonic skeletal myotubes, where it became incorporated in straight fibers aligned along the long axis of the myotubes. Upon exposure to nocodazole, microinjected myotubes exhibited wavy, fluorescent filament bundles around the muscle nuclei. In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays. Exposure of the injected cells to nocadazole or acrylamide caused the desmin network to collapse and form a perinuclear cap that was indistinguishable from vimentin caps in the same cells. During mitosis, labeled desmin filaments were excluded from the spindle area, forming a cage around it. The filaments were partitioned into two groups either during anaphase or at the completion of cytokinesis. In the former case, the perispindle desmin filaments appeared to be stretched into two parts by the elongating spindle. In the latter case, a continuous bundle of filaments extended along the length of the spindle and appeared to be pinched in two by the contracting cleavage furrow. In these cells, desmin filaments were present in the midbody where they gradually were removed as the desmin filament network became redistributed throughout the cytoplasm of the spreading daughter cells.
Mittal
B
B
Department of Anatomy, University of Pennsylvania School of Medicine, Philadelphia 19104-6058.
Sanger
J M
JM
Sanger
J W
JW
D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D002645:Chickens; D003599:Cytoskeleton / Q000032:analysis*; D003893:Desmin / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D007382:Intermediate Filaments / Q000032:analysis* / Q000187:drug effects / Q000648:ultrastructure; D007399:Interphase; D008845:Microinjections; D008938:Mitosis; D015739:Nocodazole; D014741:Video Recording
D001562:Benzimidazoles; D003893:Desmin; D015739:Nocodazole