Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells.

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Abstract
Microtubule-associated proteins (MAP) from calf brain were fluorescently labeled with 6-iodoacetamido fluorescein (1-AF). The modified MAP (especially enriched for MAP2) were fully active in promoting tubulin polymerization in vitro and readily associated with cytoplasmic filaments when microinjected into living cultured cells. Double-labeling experiments indicated that the microinjected AF-MAP were incorporated predominantly into cytoplasmic microtubules in untreated cells or paracrystals induced within vinblastine-treated cells. Similar results were obtained with different cell types (neuronal, epithelial and fibroblastic) of diverse origin (man, mouse, chicken and rat kangaroo). Mobility measurements of the microinjected AF-MAP using the method of fluorescence-photobleaching recovery (FPR) revealed 2 populations of AF-MAP with distinct dynamic properties. One fraction represents the soluble pool of MAP and is mobile with a diffusion coefficient of D = 3 .times. 10-9 cm2/s. The other fraction of MAP is associated with the microtubules and is essentially immobile on the time scale of FPR experiments. However, it showed slow fluorescence recovery with an apparent half time of .apprx. 5 min. The slow recovery of fluorescence on defined photobleached microtubules occurred most probably by the incorporation of AF-MAP from the soluble cytoplasmic pool into the bleached area. The bleached spot on defined microtubules remained essentially immobile during the slow recovery phase. These results suggest that MAP can associate in vivo with microtubules of diverse cell types and that treadmilling MAP2-containing microtubules in vivo, if it exists, is slower than 4 .mu.m/h.