Biochemical and immunochemical analysis of rat brain dynamin interaction with microtubules and organelles in vivo and in vitro.
We have purified a 100-kD rat brain protein that has microtubule cross-linking activity in vitro, and have determined that it is dynamin, a putative microtubule-associated motility protein. We find that dynamin appears to be specific to neuronal tissue where it is present in both soluble and particulate tissue fractions. In the cytosol it is abundant, representing as much as 1.5% of the total extractable protein. Dynamin appears to be in particulate material due to association with a distinct subcellular membrane fraction. Surprisingly, by immunofluorescence analysis of PC12 cells we find that dynamin is distributed uniformly throughout the cytoplasm with no apparent microtubule association in either interphase, mitotic, or taxol-treated cells. Upon nerve growth factor (NGF) induction of PC12 cell differentiation into neurons, dynamin levels increase approximately twofold. In the cell body, the distribution of dynamin again remains clearly distinct from that of tubulin, and in axons, where microtubules are numerous and ordered into bundles, dynamin staining is sparse and punctate. On the other hand, in the most distal domain of growth cones, where there are relatively few microtubules, dynamin is particularly abundant. The dynamin staining of neurites is abolished by extraction of the cells with detergent under conditions that preserve microtubules, suggesting that dynamin in neurites is associated with membranes. We conclude that dynamin is a neuronal protein that is specifically associated with as yet unidentified vesicles. It is possible, but unproven, that it may link vesicles to microtubules for transport in differentiated axons.
Scaife
R
R
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Margolis
R L
RL
D000255:Adenosine Triphosphate; D000818:Animals; D001369:Axons / Q000201:enzymology; D001921:Brain / Q000201:enzymology*; D017301:Ca(2+) Mg(2+)-ATPase / Q000302:isolation & purification / Q000502:physiology*; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D034281:Dynamins; D005455:Fluorescent Antibody Technique; D007106:Immune Sera; D007425:Intracellular Membranes / Q000201:enzymology; D008870:Microtubules / Q000201:enzymology*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification; D009928:Organ Specificity; D015388:Organelles / Q000201:enzymology; D051381:Rats
D007106:Immune Sera; D009419:Nerve Tissue Proteins; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D034281:Dynamins