Abstract. Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non-coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTPbinding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2-terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and a-, but not 3% adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over-expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis.

Five high-molecular-weight microtubule-associated proteins (MAPs) were identified in brain tissue in previous work from this laboratory (Bloom et al., 1984). These proteins were termed MAP 1A, 1B, 1C, 2A, and 2B. The MAP 1's differed from the MAP 2's, and showed little evidence of interrelationship on the basis of immunological and biochemical comparison. We report here that MAP 1A and MAP 1B are, in fact, related at the level of subunit composition. Immunoprecipitation of the individual MAPs showed that both contained low-molecular-weight subunits of Mr 30,000 and Mr 19,000 (light chains 1 and 3). An additional subunit, light chain 2 (Mr 28,000), was primarily found in preparations of MAP 1A. The light chains co-sedimented with microtubules after chymotryptic digestion of the MAPs. This suggested an association of the light chains with the microtubule binding domains of the MAPs, which are identified here as distinct fragments of Mr 60,000 for MAP 1A and 120,000 for MAP 1B. A panel of monoclonal anti- MAP 1A and anti-MAP 1B antibodies, including one that reacts with a common phosphorylated epitope, was used to examine the distribution of these proteins in the developing rat brain and spinal cord. MAP 1B was found to be abundant in the newborn brain and to decrease with development, in contrast to MAP 1A which increased with development. By immunohistochemistry MAP 1B was found to be highly concentrated in developing axonal processes in the cerebellar molecular layer, the corticospinal tract, the mossy fibers in the hippocampus, and the olfactory nerve. Of particular interest, the mossy fiber and olfactory nerve staining persisted in the adult, indicating continued outgrowth of the mossy fibers as well as olfactory nerve axons. MAP 1A staining was, in contrast, weak or absent in developing axonal fibers but moderate in mature axons and intense in developing and mature dendritic processes. Our results indicate that MAP 1A and MAP 1B are structurally related components of the neuronal cytoskeleton with complementary patterns of expression.

We prepared a monoclonal antibody to microtubule-associated protein 1 (MAP 1), one of the two major high molecular weight MAP found in microtubules isolated from brain tissue . We found that MAP 1 can be resolved by SDS PAGE into three electrophoretic bands, which we have designated MAP ]A, MAP 113, and MAP 1 C in order of increasing electrophoretic mobility. Our antibody recognized exclusively MAP 1A, the most abundant and largest MAP 1 polypeptide . To determine the distribution of MAP 1A in nervous system tissues and cells, we examined tissue sections from rat brain and spinal cord, as well as primary cultures of newborn rat brain by immunofluorescence microscopy. Anti-MAP 1 A stained white matter and gray matter regions, while a polyclonal anti-MAP 2 antibody previously prepared in this laboratory stained only gray matter . This confirmed our earlier biochemical results, which indicated that MAP 1 is more uniformly distributed in brain tissue than MAP 2 (Vallee, R . B., 1982, /. Cell Biol., 92 :435-442) . To determine the identity of cells and cellular processes immunoreactive with anti-MAP 1A, we examined a variety of brain and spinal cord regions . Fibrous staining of white matter by anti-MAP 1A was generally observed . This was due in part to immunoreactivity of axons, as judged by examination of axonal fiber tracts in the cerebral cortex and of large myelinated axons in the spinal cord and in spinal nerve roots . Cells with the morphology of oligodendrocytes were brightly labeled in white matter . Intense staining of Purkinje cell dendrites in the cerebellar cortex and of the apical dendrites of pyramidal cells in the cerebral cortex was observed . By double-labeling with antibodies to MAP 1A and MAP 2, the presence of both MAP in identical dendrites and neuronal perikarya was found. In primary brain cell cultures anti-MAP 2 stained predominantly cells of neuronal morphology . In contrast, anti-MAP 1A stained nearly all cells . Included among these were neurons, oligodendrocytes and astrocytes as determined by double-labeling with anti-MAP 1A in combination with antibody to MAP 2, myelin basic protein or glial fibrillary acidic protein, respectively . These results indicate that in contrast to MAP 2, which is specifically enriched in dendrites and perikarya of neurons, MAP lA is widely distributed in the nervous sytem .Microtubules purified from a variety of sources have been shown to consist of both tubulin, the principal structural protein, and a diverse group of microtubule-associated proteins, or MAP (5,6,27,34,45,46). The MAP have been implicated in the regulation ofmicrotubule assembly (26,46), and. in mediating the interaction of microtubules with other structural elements ofthe cytoplasm (1,3,12,13,15,22,32,33,35). In brain, the most prominent MAP are high molecular weight proteins . These proteins have been divided into two classes, MAP 1 and MAP 2, on the basis oftheir electrophoretic mobility (34). MAP 2 (M, 270,000) remains soluble when microtubules are exposed to elevated temperatur...

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Axons from rats treated with the neurotoxic agent ~,/3'-iminodipropionitrile (IDPN) were examined by quick-freeze, deep-etch electron microscopy. Microtubules formed bundles in the central region of the axons, whereas neurofilaments were segregated to the periphery. Most membrane-bounded organelles, presumably including those involved in rapid axonal transport, were associated with the microtubule domain. The high resolution provided by quick-freeze, deep-etch electron microscopy revealed that the microtubules were coated with an extensive network of fine strands that served both to cross-link the microtubules and to interconnect them with the membrane-bounded organelles. The strands were decorated with granular materials and were irregular in dimension. They appeared either singly or as an extensive anastomosing network in fresh axons. The microtubule-associated strands were observed in fresh, saponin-extracted, or aldehyde-fixed tissue.To explore further the identity of the microtubule-associated strands, microtubules purified from brain tissue and containing the high molecular weight microtubule-associated proteins MAP 1 and MAP 2 were examined by quick-freeze, deep-etch electron microscopy. The purified microtubules were connected by a network of strands quite similar in appearance to those observed in the IDPN axons. Control microtubule preparations consisting only of tubulin and lacking the MAPs were devoid of associated strands. To learn which of the MAPs were present in the microtubule bundles in the axon, sections of axons from IDPN-treated rats were examined by immunofluorescence microscopy using antibodies to MAP 1A, MAP 1B, MAP 2, and tubulin. Anti-MAP 2 staining was only marginally detectable in the IDPN-treated axons, consistent with earlier observations. Anti-MAP 1A and anti-MAP 1B brightly stained the IDPN-treated axons, with the staining exclusively limited to the microtubule domains. Furthermore, thin section-immunoelectron microscopy using colloidal gold-labeled second antibodies revealed that both anti-MAP 1A and anti-MAP 113 stained fuzzy filamentous structures between microtubules. In view of earlier work indicating that rapid transport is associated with the microtubule domain in the IDPN-treated axon, it now appears that MAP 1A and MAP 1B may play a role in this process. We believe that MAP 1A and MAP 1B are major components of the microtubule-associated fibrillar matrix in the axon.The axon contains a highly ordered cytoskeletal system composed primarily of microtubules and neurofflaments. Mem-