Morphology of dynamin mutants.
Mutants that did not affect transferrin uptake showed a cytoplasmic distribution of dynamin by immunofluorescence with some enrichment at the plasma membrane. All mutants that inhibited transferrin uptake showed a more discrete distribution (K44A, S45N, T65A, R66A, T141Q, and K142A). Using electron microscopy we have only analysed the T65A mutant in depth as preliminary investigations did not reveal such striking phenotypes with the other mutants. T65A expressing cells showed membrane tubules of various lengths surrounded by the T-bar like structure characteristic of dynamin. Indeed immunogold directed against dynamin decorated these tubules (data not shown). Tubule lengths were frequently in excess of 0.9um and had a similar diameter to the in vitro lipid nanotubes on which we assembled dynamin (Fig. 4). By immunofluorescence we observe that the extent of tubulation is dependent on the expression level of the mutant dynamin (data not shown). EM analysis of other mutants has been much less extensive but our initial observations indicate that the mutants that give discrete staining have occasional short membrane tubules coated in T-bar-like structures. In the T141Q mutant we sometimes found self-assembled dynamin in the absence of lipid membrane. This effect was exaggerated at lower temperatures.
For the T65A mutant we confirmed by immunofluorescence the integrity of intracellular endosomal, lysosomal and cis-Golgi compartments.
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