Biomedical subjects

N Hirokawa

Publications and source records attributed to N Hirokawa.

At least 19 recordsLinked to original sources

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↗

PubMed1992

The 72-kDa microtubule-associated protein from porcine brain.

A microtubule-associated protein (MAP) with a molecular mass of 72-kDa that was purified from porcine brain by using its property of heat stability in a low pH buffer was characterized. Low-angle rotary shadowing revealed that the 72-kDa protein was a rodlike protein approximately 55-75 nm long. The 72-kDa protein bound to microtubules polymerized from phosphocellulose column-purified tubulin (PC-tubulin) with taxol and promoted the polymerization of PC-tubulin in the absence of taxol. Microtubules polymerized by the 72-kDa protein showed a tendency to form bundles of several microtubules. Quick-freeze, deep-etch electron microscopy revealed that the 72-kDa protein formed short crossbridges between microtubules. We performed peptide mapping to analyze the relationship of the 72-kDa protein to other heat-stable MAPs, and the results showed some resemblance of the 72-kDa protein to MAP2. Cross-reactivity with a monoclonal anti-MAP2 antibody further suggested that the 72-kDa protein and MAP2 are immunologically related. To study the relationship between the 72-kDa protein and MAP2C, a smaller molecular form of MAP2 identified in juvenile rat brain, we prepared the 72-kDa protein from rat brain by the same method as that used for porcine brain. The fact that the 72-kDa protein from juvenile rat brain was also stained with our monoclonal anti-MAP2 antibody also suggested that the 72-kDa protein is an MAP2C homologue of the porcine brain.

Animals↗

PubMed1992

Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau.

We previously transfected MAP2, tau and MAP1B cDNA into fibroblasts and have studied the effect of expression of these microtubule-associated proteins on microtubule organization. In this study, we examined some additional characteristics of microtubule bundles and arrays formed in fibroblasts transfected with these microtubule-associated proteins. It was found that microtubule bundles formed in MAP2c- or tau-transfected cells were stabilized against microtubule depolymerizing reagents and were enriched in acetylated alpha tubulin. When mouse MAP1B cDNA was expressed following transfection into COS cells, MAP1B was localized along microtubule arrays, but no extensive reorganization of microtubules such as bundle formation was observed, in agreement with our previous finding using HeLa and 3T3 cells. However, stabilization of microtubules was indicated: (a) microtubules in MAP1B-transfected cells were stabilized against a microtubule depolymerizing reagent, although stabilization was less efficient than that seen in MAP2c- or tau-transfected cells, and (b) microtubules in MAP1B-transfected cells were enriched in acetylated alpha tubulin. These results suggest that neuronal microtubule-associated proteins introduced into fibroblasts by cDNA transfection stabilize microtubules and affect the state of post-translational modification of tubulin.

Acetylation↗

PubMed1992

Ultrastructure of detergent-resistant cytoskeletons in the noncortical domain of sea urchin eggs as revealed by the quick-freeze deep-etch technique.

The ultrastructure of detergent-resistant cytoskeletons in the noncortical cytoplasm of sea urchin eggs was studied by quick-freeze, deep-etch electron microscopy. Two different cytoskeletal organizations were identified in the detergent-treated sea urchin eggs. They were distinguished by the presence or the absence of long actin filaments and probably correspond to the cortex and the noncortical cytoplasm, respectively. The non-cortical cytoplasm was composed of a complex network (designated here as the ground network) of filaments 6 to 13 nm in diameter, that interconnected aggregates of small globular materials, yolk granules and a meshwork of uniform filaments (8-9 nm in diameter). The 6 to 13 nm filaments comprising the ground network were branched and associated with filaments of the same or other sizes, resulting in the formation of an extremely complex network. The meshwork of 8-9 nm filaments was homogeneous in composition and constitutes a novel structure which has not been previously described. The 8-9 nm filaments were connected to one another at their ends, forming a meshwork of polygons. Meshworks, ranging up to 3 microns in diameter, were distributed throughout the non-cortical cytoplasm of the egg. Similar cytoplasmic structures were also observed in fertilized eggs.

Actins↗

PubMed1992

Organization of cortical cytoskeleton of cultured chromaffin cells and involvement in secretion as revealed by quick-freeze, deep-etching, and double-label immunoelectron microscopy.

We have studied the organization of the cytoskeleton in both unstimulated and stimulated cultured chromaffin cells, as well as its relationship with their secretory process by exocytosis. We found the spatial heterogeneity in the intensity of cortical rhodamine-phalloidin staining within a cell. The overall staining pattern or intensity was minimally altered after stimulation, although dopamine-beta-hydroxylase (DBH) antigen, a marker for the chromaffin granule membrane, was exposed preferentially on the plasma membrane areas with lower intensity of rhodamine-phalloidin staining. Using the quick-freeze, deep-etch technique, we found the heterogeneity in the organization of cortical cytoskeletal networks--some regions have actin filament bundles running parallel to the plasma membrane interspersed between granules and the plasma membrane, while others have few actin filaments beneath the plasma membrane before stimulation. Actin filaments were rarely observed in the inner cytoplasm. We did not observe the overall change in its organization after stimulation. Double-label immunogold EM using anti-DBH antibody and anti-actin antibody combined with statistical analysis showed that (1) DBH was exposed on the plasma membrane preferentially where actin was sparse after stimulation (significant at less than 0.1%), although (2) regions having sparse actin were not always the sites for DBH exposure, and (3) the cortical actin zone was sometimes disrupted at the DBH-exposed sites after stimulation. The present data suggested that (1) secretion is related to heterogeneous organization of cortical cytoskeleton after stimulation and (2) massive synchronized reorganization of the cytoskeleton in the whole cell is not necessary for secretion, although small changes of the cytoskeleton might occur under local regulation at each exocytotic site at the moment of the release.

Actins↗

PubMed1991

In situ localization of tau mRNA in developing rat brain.

A microtubule-associated protein, tau, promotes microtubule assembly, forms characteristic short cross-bridges (less than 20 nm) between microtubules, and switches isoforms from juvenile to adult at the end of the first postnatal week in the rat brain. The developmental expression of tau was studied in rat central nervous system, mainly the cerebrum and cerebellum, by in situ hybridization. Tau mRNAs were localized in a wide variety of neural cells. The expression of tau mRNAs in the spinal cord appeared to precede that in the brain, and the expression in the brainstem appeared to precede that in the cerebral cortex and cerebellum. On neural cells throughout the cortical plate of the cerebral cortex, tau mRNAs were expressed in large amounts during the first postnatal week, but by the third postnatal week the expression had become reduced. In the cerebellum, tau mRNAs were enriched in granule cells. The expression in the internal granular layer peaked during the second and third postnatal weeks, and the relatively high level of expression persisted to young adulthood. Thin section transmission electron microscopic study revealed that the proportion of neighboring microtubules in parallel fiber axons of cerebellar granule cells with the distance less than 20 nm was as low as 10% at the end of the first postnatal week, but this proportion increased to as high as 35% at the end of the second postnatal week. Northern blot analysis showed that tau mRNAs were congruent to 6 kb as was reported previously, and those detected in the first postnatal week were three- to five-fold more abundant and approximately 0.2 kb smaller than those detected in the second or third postnatal weeks. The data suggest that (a) tau mRNAs are abundantly expressed in a wide variety of neurons in the central nervous system at the stage of neurite formation, and (b) tau mRNAs are expressed in more basal levels at later stages, but may be important in the formation and maintenance of characteristic microtubule bundles typically found in parallel fiber axons and in other axons.

Animals↗

PubMed1991

Predominant and developmentally regulated expression of dynamin in neurons.

We have cloned a cDNA for dynamin, a 100 kd microtubule-associated motor protein whose 5' region contains a GTP-binding motif homologous to that of the Mx proteins, from a rat brain library and analyzed its expression. Dynamin mRNA is 3.6 kb and is preferentially expressed in the brain after postnatal day 7, parallel to the developmental increase of the protein. In situ hybridization revealed high levels of dynamin transcripts in neural cells in the cerebellar cortex, hippocampus (particularly in the CA3 area), and cerebral cortex. The transcripts appeared in cerebellar granular cells only after they had ceased dividing and had migrated to the inner granular layer. We show that dynamin is expressed predominantly in neural cells after elongation of their processes, suggesting a role especially in mature neurons.

Adrenal Gland Neoplasms↗

PubMed1991

Kinesin associates with anterogradely transported membranous organelles in vivo.

Biochemical, pharmacological and immunocytochemical studies have implicated the microtubule-activated ATPase, kinesin, in the movement of membrane bounded organelles in fast axonal transport. In vitro studies suggested that kinesin moves organelles preferentially in the anterograde direction, but data about the function and precise localization of kinesin in the living axon were lacking. The current study was undertaken to establish whether kinesin associates with anterograde or retrograde moving organelles in vivo. Peripheral nerves were ligated to produce accumulations of organelles moving in defined directions. Regions proximal (anterograde) and distal (retrograde) to the ligation were analyzed for kinesin localization by immunofluorescence, and by immunogold electron microscopy using ultracryomicrotomy. Substantial amounts of kinesin were associated with anterograde moving organelles on the proximal side, while significantly less kinesin was detected distally. Statistical analyses indicated that kinesin was mostly associated with membrane-bounded organelles. These observations indicate that axonal kinesin is primarily associated with anterograde moving organelles in vivo.

Adenosine Triphosphatases↗

PubMed1991

Actin dynamics in growth cones.

The mechanism of actin incorporation and turnover in the nerve growth cone was examined by immunoelectron microscopy and low-light-level video microscopy of cultured neurons injected with biotin-labeled actin or fluorescently labeled actin. We first determined the sites of actin incorporation into the cytoskeleton of growth cones by immunoelectron microscopy of cultured neurons injected with biotin-labeled actin and reacted with an anti-biotin antibody and a gold-labeled secondary antibody. Shortly after the injection, biotin-actin molecules incorporated into the cytoskeleton were localized in the distal part of actin bundles in the filopodia and at the membrane-associated fringe of the actin filament network. With longer incubation, most actin polymers in the growth cones were labeled uniformly, suggesting that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. We then determined whether actin filaments translocate within the growth cones by low-light-level video microscopy of living neurons injected with fluorescently labeled actin and photobleached with a laser beam. When actin fluorescence at the leading edge of a growth cone was bleached, a rearward translocation of the bleached spot toward the base of the growth cone was observed. This observation suggests the presence of a rearward flow of actin polymers within growth cones. Taken together, these results indicate that there is a continuous addition of actin monomers at the leading edge of the growth cone and a successive rearward translocation of the assembled filaments.

Actins↗

PubMed1990

Dephosphorylation-induced interactions of neurofilaments with microtubules.

Effects of dephosphorylation on interactions of neurofilaments (NFs) with microtubules (MTs) were studied by the cosedimentation method. Centrifugation conditions were chosen so that MTs pelleted but NFs did not. While NFs isolated from bovine spinal cords did not cosediment with MTs polymerized in the presence of taxol, NFs dephosphorylated with Escherichia coli alkaline phosphatase began to coprecipitate with MTs. The dephosphorylated NFs bound to MTs but not to the unpolymerized tubulin dimer. The binding was not observed in the presence of high salt or with MTs containing microtubule-associated proteins. The cosedimentation experiments using purified NF subunit proteins showed that the dephosphorylation-induced binding of NFs to MTs was mediated by the largest subunit of NF (NF-H). Negative staining electron microscopy confirmed bindings of the dephosphorylated NFs and NF-H to MTs. Densitometric measurement of the bound and unbound NF-H after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the binding of the dephosphorylated NF-H to MT was saturable and gave the following binding parameters. Approximately 1 mol of NF-H bound per 10 mol of tubulin dimer with a high affinity site (Kd = 3.8 x 10(-8) M) and per 16 mol of tubulin dimer with a low affinity site (Kd = 1.1 x 10(-7) M).

Animals↗

PubMed1990

Localization and subcellular distribution of smg p25A, a ras p21-like GTP-binding protein, in rat brain.

We have made a monoclonal antibody which specifically recognizes smg p25A among many ras p21/ras p21-like GTP-binding proteins thus far purified from bovine brain membranes. By use of this antibody, we have investigated the localization and subcellular distribution of smg p25A in rat brain by light and electron microscopic immunocytochemistry and by immunoblotting. By light microscopic immunocytochemistry, specific immunoreactivity is widely distributed, most abundant in neuropil, weak in neuronal somata, and absent from white matter. By electron microscopic immunocytochemistry, intense labeling is demonstrated on most of the synapses and concentrated in the presynaptic area where synaptic vesicles are observed. Presynaptic plasma membranes are weakly labeled but mitochondria, postsynaptic plasma membranes, and postsynaptic densities are unlabeled. In subcellular fractionation analysis of cerebrum, about one-fifth of smg p25A is found in the soluble cytosol fraction and the rest is found in the particulate fraction. About half of the particulate-bound smg p25A is recovered in the P2 fraction containing synaptosomes, mitochondria, and myelin, among which a major portion of smg p25A is recovered in the synaptosomal fraction. In the synaptosomal fraction, smg p25A is concentrated about 8-fold in the fraction containing synaptic vesicles and about 3-fold in the fraction containing synaptic plasma membranes compared with the original homogenate. smg p25A is present at a low level in the fraction containing synaptosomal soluble substances but almost absent from the fractions containing intrasynaptosomal mitochondria or post-synaptic densities. These results suggest that smg p25A plays important roles in the regulation of synaptic functions such as exo-endocytotic recycling of synaptic vesicles during neurotransmitter release.

Animals↗