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N Hirokawa

Publications and source records attributed to N Hirokawa.

At least 109 records · Page 6Linked to original sources
PubMed1993

Mechanism of axonal transport. Identification of new molecular motors and regulations of transports.

New molecular motors associated with microtubules and actin have been uncovered very recently. Furthermore, studies of the mechanisms of bidirectional fast axonal transports have clarified new aspects of these processes, such as identification of a kinesin binding protein (kinectin) and regulation of kinesin dissociation from membranous organelles by phosphorylation. These will lead to a more precise understanding of the mechanisms of axonal transports. Concerning the mechanism of the slow transport of cytoskeletal proteins, new approaches have provided further evidence that the axonal cytoskeleton in mammalian systems is largely stationary while dynamic exchanges occur between polymer and a small pool of moving subunits.

Animals↗
PubMed1993

Reorganization of brain spectrin (fodrin) during differentiation of PC12 cells.

Fodrin has been shown to redistribute dynamically between cytoplasmic and plasma membrane-associated compartments upon the differentiation of T lymphocytes. We studied the changes of distribution of fodrin in PC12 cells upon neuronal differentiation induced by nerve growth factor. To visualize preferentially the elements that were tightly associated with cytoskeletal structures, we performed immunofluorescence and immunoelectron microscopy on saponin-extracted cells. In undifferentiated PC12 cells, fodrin was distributed mostly underneath the plasma membrane. However, after the administration of nerve growth factor, perinuclear spot-like aggregates of fodrin appeared. Double-labeling immunofluorescence revealed that the cytoplasmic fodrin spot was co-localized with the intermediate filament proteins, peripherin and neurofilament. Immunogold electron microscopy showed that fodrin and neurofilament were localized in close association in the perinuclear regions enriched with intermediate filaments. With prolonged exposure to nerve growth factor, fodrin and intermediate filaments spread to the cytoplasm and neurites. These results suggest that there is a dynamic reorganization of fodrin during differentiation of PC12 cells, and that fodrin is first recruited in the perinuclear region closely associated with intermediate filaments. This dynamic reorganization of fodrin may represent important, previously unrecognized aspects of the morphological differentiation of neurons.

Animals↗
PubMed1993

Localization of dynamin: widespread distribution in mature neurons and association with membranous organelles.

Tissue distribution and intracellular localization of dynamin by immunoblotting and immunocytochemistry is investigated in this study. Dynamin was widely expressed in all the neurons we examined, and was especially abundant in the central nervous system after maturation, although its expression presented regional heterogeneity. Dynamin was present most abundantly in cerebellar Purkinje cells and hippocampal pyramidal cells, and to a lesser extent in motor neurons and peripheral nerves. However, dynamin was nearly absent in cells such as anterior pituitary cells and adrenal medullary cells which secrete mainly dense cored vesicles. Dynamin was localized not only in cell bodies, axons, and synapses but also in dendrites. Subcellular fractionation indicated that dynamin existed in the membrane fraction as well as in the soluble fraction. In ligated peripheral nerves, dynamin colocalized with tubulovesicular membranous organelles transported mainly anterogradely. By transfection of dynamin cDNA into mouse fibroblast L-cells, we showed it colocalized with some membranous organelles but not with microtubules. Our results show that dynamin is associated with membranous organelles in vivo, although a certain amount of dynamin also exists in the soluble fraction and is distributed diffusely throughout mature neurons. The data suggest that dynamin's fundamental role involves membrane trafficking in neurons in the central nervous system rather than in sliding microtubules as a motor protein.

Amino Acid Sequence↗
PubMed1993

Axonal transport and the cytoskeleton.

Great advances in the field of axonal transport have been made in the past year, including the identification of new molecular motors associated with microtubules and actin. In addition, studies on the mechanisms of bidirectional fast axonal transport have clarified new aspects of this process, such as the isolation of a kinesin-binding protein, kinectin, and the finding that phosphorylation regulates kinesin's dissociation from membranous organelles. New approaches to studying slow transport of cytoskeletal proteins have provided further evidence that the axonal cytoskeleton in mammalian systems is largely stationary, although a dynamic exchange occurs between polymers and a small pool of moving subunits.

Animals↗
PubMed1993

Thermal drift is enough to drive a single microtubule along its axis even in the absence of motor proteins.

One-dimensional diffusion of microtubules (MTs), a back-and-forth motion of MTs due to thermal diffusion, was reported in dynein motility assay. The interaction between MTs and dynein that allows such motion was implicated in its importance in the force generating cycle of dynein ATPase cycle. However, it was not known whether the phenomenon is special to motor proteins. Here we show two independent examples of one-dimensional diffusion of MTs in the absence of motor proteins. Dynamin, a MT-activated GTPase, causes a nucleotide dependent back-and-forth movement of single MT up to 1 micron along the longitudinal axes, although the MT never showed unidirectional consistent movement. Quantitative analysis of the motion and its nucleotide condition indicates that the motion is due to a thermal driven diffusion, restricted to one dimension, under the weak interaction between MT and dynamin. However, specific protein-protein interaction is not essential for the motion, because similar back-and-forth movement of MT was achieved on coverslips coated with only 0.8% methylcellulose. Both cases demonstrate that thermal diffusion could provide a considerable sliding of MTs only if MTs are restricted on the surface appropriately.

Animals↗
PubMed1993

Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C).

MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein-labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo.

Animals↗
PubMed1993

Do photobleached fluorescent microtubules move?: re-evaluation of fluorescence laser photobleaching both in vitro and in growing Xenopus axon.

We previously documented differences in the behavior of microtubules in growing axons of two types of neurons, adult mouse sensory neurons and Xenopus embryonal spinal cord neurons. Namely, the bulk of microtubules was stationary in mouse sensory neurons both by the method of photoactivation of caged-fluorescein-labeled tubulin and photobleaching of fluorescein-labeled tubulin, but the bulk of microtubules did translocate anterogradely by the method of photoactivation. Although these results indicated that the stationary nature of photobleached microtubules in mouse neurons is not an artifact derived from the high levels of energy required for the procedure, it has not yet been settled whether the photobleaching method can detect the movement of microtubules properly. Here we report photobleaching experiments on growing axons of Xenopus embryonal neurons. Anterograde movement of photobleached microtubules was observed at a frequency and translocation rate similar to the values determined by the method of photoactivation. Our results suggest that, under appropriate conditions, the photobleaching method is able to reveal the behavior of microtubules as accurately as the photoactivation method.

Animals↗
PubMed1993

Dynamics of the neuronal intermediate filaments.

We have analyzed the dynamics of neuronal intermediate filaments in living neurons by using the method of photobleaching of fluorescently-labeled neurofilament L protein and immunoelectron microscopy of incorporation sites of biotinylated neurofilament L protein. Low-light-level imaging and photobleaching of growing axons of mouse sensory neurons did not affect the rate of either axonal growth or the addition of intermediate filament structures at the axon terminal, suggesting that any perturbations caused by these optical methods would be minimal. After laser photobleaching, recovery of fluorescence did occur slowly with a recovery half-time of 40 min. Furthermore, we observed a more rapid fluorescence recovery in growing axons than in quiescent ones, indicating a growth-dependent regulation of the turnover rate. Incorporation sites of biotin-labeled neurofilament L protein were localized as numerous discrete sites along the axon, and they slowly elongated to become continuous arrays 24 h after injection. Collectively, these results indicate that neuronal intermediate filaments in growing axons turn over within the small area of the axoplasm possibly by the mechanism of lateral and segmental incorporation of new subunits.

Animals↗
PubMed1993

A novel member of the dynamin family of GTP-binding proteins is expressed specifically in the testis.

Dynamin is a member of a new GTPase family, which includes the mouse Mx protein, the yeast VPS1 and the Drosophila shibire gene product. A high homology with the shibire product suggests a role for dynamin in the endocytotic process, but it is expressed only in mature neurons. We identified two additional dynamin-like proteins in rats, by using the polymerase chain reaction with degenerate primers corresponding to the GTP-binding areas conserved between dynamin and VPS1. The full coding sequence of one of them, dynamin-2, revealed that it has 848 amino acids and has great similarity with brain dynamin and the shibire product. Northern blot analysis and in situ hybridization revealed its expression to be specific to the seminiferous tubules in the testis. Dynamin-2 (testis type dynamin) was expressed in germ-cell-depleted testis as well, indicating its expression in Sertoli cells. Our data imply that a number of dynamin family proteins, which are products of distinct genes, may play different roles specific to each cell type in the same rat.

Amino Acid Sequence↗
PubMed1992

Projection domains of MAP2 and tau determine spacings between microtubules in dendrites and axons.

Neurons develop a highly polarized morphology consisting of dendrites and a long axon. Both axons and dendrites contain microtubules and microtubule-associated proteins (MAPs) with characteristic structures. Among MAPs, MAP2 is specifically expressed in dendrites whereas MAP2C and tau are abundant in the axon. But the influence of MAP2, MAP2C and tau on the organization of microtubule domains in dendrites versus axons is unknown. Both MAP2 and tau induce microtubule bundle formation in fibroblasts after transfection of complementary DNAs, and a long process resembling an axon is extended in Sf9 cells infected with recombinant baculovirus expressing tau. We have now expressed MAP2 and MAP2C in Sf9 cells in order to compare their morphology and the arrangement of their microtubules to that found in Sf9 cells expressing tau. We report here that the spacing between microtubules depends on the MAP expressed: in cells expressing MAP2, the distance is similar to that found in dendrites, whereas the spacing between microtubules in cells expressing MAP2C or tau is similar to that found in axons.

Animals↗
PubMed1992

The phosphorylation of kinesin regulates its binding to synaptic vesicles.

Membrane organella are transported bidirectionally in cells, and the axonal transport system has provided an ideal model system for studying this bidirectional transport. Kinesin and cytoplasmic dynein were identified as candidates for the motor molecules of fast axonal transport, which transport organella along microtubules anterogradely and retrogradely. However, the mechanism that controls this bidirectional transport is unknown. Our previous work revealed that kinesin in axons was associated abundantly with anterogradely transported membranous organella, most of which are believed to be precursors of synaptic vesicles and axonal plasma membranes, while the fractions bound to retrogradely transported ones were very small (Hirokawa, N., Sato-Yoshitake, R., Kobayashi, N., Pfister, K. K., Bloom, G. S., and Brady, S. T. (1991) J. Cell Biol. 114, 295-302). Here we demonstrated in vitro that the binding of kinesin to synaptic vesicles was concentration-dependent and saturable and could be released by high salt concentration. When kinesin was phosphorylated by cAMP-dependent protein kinase, its binding to symaptic vesicles was significantly reduced. By motility assay and by statistical analysis using electron microscopy, we further revealed that synaptic vesicles preincubated with phosphorylated kinesin associated less frequently with microtubules than synaptic vesicles preincubated with unphosphorylated kinesin. The phosphorylation of kinesin should therefore play an essential role in regulating the direction of fast axonal transport by inhibiting its binding to membrane organella, thus releasing it from membrane organella at nerve terminals.

Animals↗
PubMed1992

Chronological expression of microtubule-associated proteins (MAPs) in EC cell P19 after neuronal induction by retinoic acid.

Pluripotent murine embryonal carcinoma (EC) P19 cells are induced at a high rate into neural cells using retinoic acid and serum-free medium. EM observation revealed great increase of microtubules (MTs) after neuronal induction. To study the expression of microtubule-associated proteins (MAPs), immunoblotting and immunocytochemistry were performed with phosphorylated MAP1B (pMAP1B)-, MAP2-, and MAP1A-specific monoclonal antibodies. They did not stain undifferentiated cells. Early MAPs (pMAP1B and MAP2C) appeared 12 h after the neuronal induction, changing to late MAPs (MAP1A and MAP2A/B) at 3-5 days. These expression patterns are quite similar to those of neural cells in vivo. Anti-pMAP1B stained not only neurites but also the cell body and varicosities. But after extraction of the soluble component by permeabilization, pMAP1B was found in only MT-domains of the neurites at LM and EM levels, indicating that some part of pMAP1B is a structural component of neurite MTs and others exist in a soluble form. After culturing for more than 5 days, pMAP1B disappeared from the soma, but still remained in the distal ends of neurites. Here we showed that P19 is a good model system for studying the expression of MAPs on the continuous course of neuronal differentiation.

Animals↗
PubMed1992

Immunocytochemical localization of 205 kDa microtubule-associated protein (205 kDa MAP) in the guinea pig organ of Corti.

We have studied the immunocytochemical localization of microtubule-associated proteins (MAPs) in the guinea pig organ of Corti. Using immunological methods with antibodies against MAP1A, MAP1B, MAP2, tau and 205 kDa MAP, we have identified 205 kDa MAP as a major MAP of the sensory epithelium in the organ of Corti. Immunoperoxidase microscopic study has shown that both cochlear hair cells and supporting cells reacted with anti-205 kDa MAP antibody. Immunoelectron microscopy revealed that 205 kDa MAP was associated with most microtubules in the sensory epithelial cells. It was also associated with the microtubules of bundle structures within supporting cells, suggesting that this MAP might form a part of cross-bridges between microtubules and between microtubules and actin filaments in the bundle structure. In contrast, MAP1A, MAP1B and tau, which are known to be expressed in neuronal tissue, were localized only in nerve fibers in the organ of Corti, not in the sensory epithelium. MAP2, which is known to be localized in dendrites and soma of nerve cells, was not distributed in nerve fibers in the organ of Corti. These results suggest possible roles of the 205 kDa MAP in the formation and maintenance of the highly polarized morphology of the epithelial cells of the organ of Corti, through stabilization and modulation of microtubule networks of these cells.

Animals↗
PubMed1992

Microtubule bundling by tau proteins in vivo: analysis of functional domains.

Tau varies both in the N-terminal region (three types) and in the C-terminal repeated microtubule binding domain (two types), generating six isoforms through alternative splicing. To understand the differences between the isoforms and to determine which domains are important for microtubule bundling, we performed transfection studies on fibroblasts using tau isoforms and deletion mutants to quantify their ability to bundle microtubules. By comparing the isoforms, we found that a longer N-terminal region induced microtubule bundling more efficiently, but changes in the microtubule binding domain did not. Mutants lacking the proline rich region or the repeated domain did not bind to microtubules. Although all the other mutants could bind to and bundle microtubules, deletion in the N-terminal neutral region or the first half of the C-terminal tail caused a significant decrease in microtubule bundling, indicating the importance of these regions in microtubule bundling.

Animals↗
PubMed1992

Differential behavior of photoactivated microtubules in growing axons of mouse and frog neurons.

To characterize the behavior of axonal microtubules in vivo, we analyzed the movement of tubulin labeled with caged fluorescein after activation to be fluorescent by irradiation of 365-nm light. When mouse sensory neurons were microinjected with caged fluorescein-labeled tubulin and then a narrow region of the axon was illuminated with a 365-nm microbeam, photoactivated tubulin was stationary regardless of the position of photoactivation. We next introduced caged fluorescein-labeled tubulin into Xenopus embryos and nerve cells isolated from injected embryos were analyzed by photoactivation. In this case, movement of the photoactivated zone toward the axon tip was frequently observed. The photoactivated microtubule segments in the Xenopus axon moved out from their initial position without significant spreading, suggesting that fluorescent microtubules are not sliding as individual filaments, but rather translocating en bloc. Since these observations raised the possibility that the mechanism of nerve growth might differ between two types of neurons, we further characterized the movement of another component of the axon structure, the plasma membrane. Analysis of the position of polystyrene beads adhering to the neurites of Xenopus neurons revealed anterograde movement of the beads at the rate similar to the rate of microtubule movement. In contrast, no movement of the beads relative to the cell body was observed in mouse sensory neurons. These results suggest that the mode of translocation of cytoskeletal polymers and some components of the axon surface differ between two neuron types and that most microtubules are stationary within the axon of mammalian neurons where the surface-related motility of the axon is not observed.

Animals↗
PubMed1992

Kinesin family in murine central nervous system.

In neuronal axons, various kinds of membranous components are transported along microtubules bidirectionally. However, only two kinds of mechanochemical motor proteins, kinesin and brain dynein, had been identified as transporters of membranous organelles in mammalian neurons. Recently, a series of genes that encode proteins closely related to kinesin heavy chain were identified in several organisms including Schizosaccharomyces pombe, Aspergillus niddulans, Saccharomyces cerevisiae, Caenorhabditus elegans, and Drosophila. Most of these members of the kinesin family are implicated in mechanisms of mitosis or meiosis. To address the mechanism of intracellular organelle transport at a molecular level, we have cloned and characterized five different members (KIF1-5), that encode the microtubule-associated motor domain homologous to kinesin heavy chain, in murine brain tissue. Homology analysis of amino acid sequence indicated that KIF1 and KIF5 are murine counterparts of unc104 and kinesin heavy chain, respectively, while KIF2, KIF3, and KIF4 are as yet unidentified new species. Complete amino acid sequence of KIF3 revealed that KIF3 consists of NH2-terminal motor domain, central alpha-helical rod domain, and COOH-terminal globular domain. Complete amino acid sequence of KIF2 revealed that KIF2 consists of NH2-terminal globular domain, central motor domain, and COOH-terminal alpha-helical rod domain. This is the first identification of the kinesin-related protein which has its motor domain at the central part in its primary structure. Northern blot analysis revealed that KIF1, KIF3, and KIF5 are expressed almost exclusively in murine brain, whereas KIF2 and KIF4 are expressed in brain as well as in other tissues. All these members of the kinesin family are expressed in the same type of neurons, and thus each one of them may transport its specific organelle in the murine central nervous system.

Amino Acid Sequence↗
PubMed1992

Interaction of dynamin with microtubules: its structure and GTPase activity investigated by using highly purified dynamin.

We purified a large amount of dynamin with high enzymatical activity from rat brain tissue by a new procedure. Dynamin 0.48 mg was obtained from 20 g of rat brain. The purity of dynamin was almost 98%. Dynamin plays a role of GTPase rather than ATPase. In the absence of microtubules, Michaelis constant (Km) and maximum velocity (Vmax) for dynamin GTPase were 370 microM and 0.25 min-1, respectively, and in their presence, both were significantly accelerated up to 25 microM and 5.5 min-1. On the other hand, the ATPase activity was very low in the absence of microtubules, and even in their presence, Km and Vmax for dynamin ATPase were 0.2 mM and 0.91 min-1. Despite slow GTPase turnover rate in the absence of microtubules, binding of GTP and its nonhydrolizing analogues was very fast, indicating that GTP binding step is not rate limiting. Dynamin did not cause a one-directional consistent microtubule sliding movement just like kinesin or dynein in the presence of 2 mM ATP or 2 mM GTP. We observed the molecular structure of dynamin with low-angle rotary shadowing technique and revealed that the dynamin molecule is globular in shape. Gel filtration assay revealed that these globules were the oligomers of 100-kDa dynamin polypeptide. Dynamin bound to microtubules with a 1:1 approximately 1.2 molar ratio in the absence of GTP. Quick-freeze deep-etch electron microscopy of the dynamin-microtubule complex showed that dynamin decorates the surface of microtubules helically, like a screw bolt, very orderly and tightly with 11.4 +/- 0.9 (SD)nm period. Contrary to the previous report, microtubules make bundles by the attachment of the dynamin helixes around each adjacent microtubule, and no cross-bridge formation was observed.

Amino Acid Sequence↗