Characterization of fluorescently derivatized bovine tau protein and its localization and functions in cultured Chinese hamster ovary cells.
Bovine brain tau protein was tagged with the fluorescent dye 5 (and 6)-carboxyx-rhodamine-succinimidyl ester and the functional properties of the fluorescent analog were tested in vitro by kinetic measurement and SDS gel electrophoresis. X-rhodamine tau was competent to bind to microtubules and promote microtubule assembly in vitro. Labeled tau was further characterized by microinjection of cultured Chinese hamster ovary (CHO) cells to study its intracellular distribution and potential new functions. X-rhodamine tau incorporated rapidly into centrosomes within seconds after microinjection. It distinctly labeled the microtubule network as early as 5 to 10 minutes following miroinjection. In addition, X-rhodamine tau was transported into the nucleus and labeled the nucleolus specifically. Double labeling of the injected cells with DiC6(3) indicated that in some cases, fluorescent tau may associate with the endoplasmic reticulum. The concentrations of injected X-rhodamine tau ranged from 1.7 to 5.0 mg/ml, yet distinct bundling of microtubules was not observed. Studies of nocodazole effects on the microtubules established that X-rhodamine tau stabilized microtubules against depolymerization conditions. We conclude that this fluorescent analog of tau is associated with microtubules, the nucleolus, and other microtubule-related structures in living cells, and is competent to stabilize microtubules against microtubule depolymerizing drug treatment. This approach provides a useful model system for the study of modified tau in neurodegenerative disease.
Lu
Q
Q
Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322.
Wood
J G
JG
D000818:Animals; D016466:CHO Cells / Q000378:metabolism / Q000648:ultrastructure; D002417:Cattle; D002466:Cell Nucleolus / Q000378:metabolism*; D006224:Cricetinae; D005456:Fluorescent Dyes; D007700:Kinetics; D008845:Microinjections; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D016875:tau Proteins / Q000032:analysis* / Q000502:physiology
D005456:Fluorescent Dyes; D016875:tau Proteins; D015739:Nocodazole