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N Hirokawa

Publications and source records attributed to N Hirokawa.

At least 127 records · Page 7Linked to original sources
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↗
PubMed1990

Molecular architecture of the neurofilament. I. Subunit arrangement of neurofilament L protein in the intermediate-sized filament.

Using the smallest subunit (NF-L) of a neurofilament and a glial fibrillary acidic protein, the subunit arrangement in intermediate filaments was studied by low-angle rotary shadowing. NF-L formed a pair of 70 to 80 nm rods in a low ionic strength solution at pH 6.8. Two 70 to 80 nm rods appeared to associate in an antiparallel manner with an overlap of about 55 nm, almost the same length as the alpha-helix-rich central rod domain of intermediate filament proteins. The overlap extended for three-beaded segments, present at 22 nm intervals along the pairs of rods. The observations that (1) 70 to 80 nm rods were a predominant structure in a low ionic strength solution at pH 8.5, (2) the molecular weights of the rod and the pair were measured by sedimentation equilibrium as 190,000 and 37,000 respectively, and (3) the rods formed from the trypsin-digested NF-L had a length of about 47 nm, indicated that the 70 to 80 nm rod is the four-chain complex and the pair of rods is the eight-chain complex. Similar structures were observed with glial fibrillary acidic protein, indicating that these oligomeric structures are common to other intermediate filament proteins. NF-L assembled into short intermediate-sized filaments upon dialysis against a low-salt solution containing 1 to 2 mM-MgCl2 at 4 degrees C. The majority of these short filaments possessed four or five-beaded segments, suggesting that the pair of rods were arranged in a half-staggered fashion in neurofilaments. On the basis of these observations, we propose the following model for the intermediate filament subunit arrangement. (1) The four-chain complex is the 70 to 80 nm rod, in which two coiled-coil molecules align in parallel and in register. (2) Two four-chain complexes form the eight-chain complex by associating in an antiparallel fashion with the overlap of the entire central rod domain. (3) The eight-chain complex is the building block of the intermediate filament. The eight-chain complexes are arranged in a half-staggered fashion within the intermediate filament.

Animals↗
PubMed1990

Molecular architecture of the neurofilament. II. Reassembly process of neurofilament L protein in vitro.

Reassembly of the neurofilament (NF) in vitro was studied by low-angle rotary shadowing electron microscopy. Various intermediate stages of the reassembly were reconstructed from the smallest molecular mass subunit (NF-L) under controlled reassembly conditions. NF-L in 6 M-urea took the form of spherical particles with a diameter of about 12 nm. NF-L aggregated into rodlets of 70 to 80 nm long in a low-salt solution at alkaline pH. By reducing the pH of the dialyzing solution to 6.6, a pair of rods was formed by association side-by-side. Increasing the temperature of low-salt solutions from 4 degrees C to 35 degrees C did not produce intermediate-sized filaments. The addition of Mg2+ to the dialyzing solution resulted in the formation of short intermediate-sized filaments even at 4 degrees C. Further dialysis of the short intermediate-sized filaments against reassembly solution containing both NaCl and MgCl2 at 37 degrees C failed to elongate them into longer filaments, suggesting that annealing does not contribute to the elongation of neurofilaments. Different roles for Mg+ and NaCl in neurofilament reassembly were indicated. While Mg2+ strengthened the lateral association between 70 to 80 nm rods, NaCl appeared to promote the end-to-end association of filaments preferentially. Longer filaments were formed by increasing the NaCl concentration. By dialyzing NF-L against a buffer containing 50 mM-NaCl in the absence of Mg2+, unraveled filaments were formed. The many unraveled filaments were composed of four 8 nm wide filaments, which have been called the subfilament or the protofibril. Time-course experiments of the reassembly were performed in the absence of Mg2+, in which condition the rate of neurofilament reassembly appeared to be reduced. Star-like clusters, about four protofibrils joined together at one end, were suggested to be the initial stage of the intermediate-sized filament formation. The following two-step elongation mechanism of neurofilaments was deduced from these results. The pairs of rods were added to the ends of the protofibrils of neurofilaments, and after all four protofibrils were elongated they were then packed into neurofilaments. Distribution of larger molecular mass subunits, NF-M and NF-H, was studied. Addition of NF-M or NF-H to NF-L did not change the assembly properties of neurofilaments. Unraveled filaments reconstituted from NF-L plus either NF-M or NF-H indicated that NF-M and NF-H are incorporated evenly into each protofibril.

Animals↗
PubMed1990

Turnover of fluorescently labelled tubulin and actin in the axon.

The cytoskeleton has an important role in the generation and maintenance of the structure of the axon. Microtubules, neurofilaments and actin, together with various kinds of associated proteins, form highly organized dynamic cytoskeletal structures. Because tubulin and actin molecules are essential cytoskeletal components and are transported down the axon, it is important to understand their dynamic behaviour within the axon. Although previous pulse-labelling studies have indicated that the axonal cytoskeleton is a static complex travelling down the axon, this view has been challenged by the results of several recent experiments. We have now addressed this question by analysing the recovery of fluorescence after photobleaching fluorescent analogues of tubulin and actin in the axons of cultured neurons. We did not observe movement or spreading of bleached zones along the axon, both in neurons injected with fluorescein-labelled tubulin and actin. All bleached zones recovered their fluorescence gradually, however, indicating that microtubules and actin filaments are not static polymers moving forward within the axon, but are dynamic structures that continue to assemble along the length of the axon.

Actins↗
PubMed1990

Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry.

Calpactin I complex, a calcium-dependent phospholipid-binding protein, promotes aggregation of chromaffin vesicles at physiological micromolar calcium ion levels. Calpactin I complex was found to be a globular molecule with a diameter of 10.7 +/- 1.7 (SD) nm on mica. When liposomes were aggregated by calpactin, quick-freeze, deep-etching revealed fine thin strands (6.5 +/- 1.9 [SD] nm long) cross-linking opposing membranes in addition to the globules on the surface of liposomes. Similar fine strands were also observed between aggregated chromaffin vesicles when they were mixed with calpactin in the presence of Ca2+ ion. In cultured chromaffin cells, similar cross-linking short strands (6-10 nm) were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structures approximately 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy on frozen ultrathin sections showed that calpactin I was closely associated with the inner face of the plasma membranes and was especially conspicuous between plasma membranes and adjacent vesicles in chromaffin cells. These in vivo and in vitro data strongly suggest that calpactin I complex changes its conformation to cross-link vesicles and the plasma membrane after stimulation of cultured chromaffin cells.

Adrenal Medulla↗
PubMed1990

Brain dynein (MAP1C) localizes on both anterogradely and retrogradely transported membranous organelles in vivo.

Brain dynein is a microtubule-activated ATPase considered to be a candidate to function as a molecular motor to transport membranous organelles retrogradely in the axon. To determine whether brain dynein really binds to retrogradely transported organelles in vivo and how it is transported to the nerve terminals, we studied the localization of brain dynein in axons after the ligation of peripheral nerves by light and electron microscopic immunocytochemistry using affinity-purified anti-brain dynein antibodies. Different classes of organelles preferentially accumulated at the regions proximal and distal to the ligated part. Interestingly, brain dynein accumulated both at the regions proximal and distal to the ligation sites and localized not only on retrogradely transported membranous organelles but also on anterogradely transported ones. This is the first evidence to show that brain dynein associates with retrogradely transported organelles in vivo and that brain dynein is transported to the nerve terminal by fast flow. This also suggests that there may be some mechanism that activates brain dynein only for retrograde transport.

Adenosine Triphosphatases↗
PubMed1990

Effects of phosphorylation of the neurofilament L protein on filamentous structures.

Effects of phosphorylation of the neurofilament L protein (NF-L) on the reassembly system were studied by both sedimentation experiments and low-angle rotary shadowing. Bovine spinal cord NF-L was phosphorylated with 3-4 mol/mol protein by either the catalytic subunit of cAMP-dependent protein kinase or protein kinase C. Phosphorylated NF-L could not assemble into filaments. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C inhibited the same step of the reassembly process. Phosphorylated NF-L remained as an 8-chain complex even in favorable conditions for reassembly. The extent of the effect of phosphorylation on the filamentous structure of NF-L was also investigated by using the catalytic subunit of cAMP-dependent protein kinase. The amount of unassembled NF-L increased linearly with increased phosphorylation in the sedimentation experiments. Structural observations indicated that 1 or 2 mol of phosphorylation is enough to inhibit reassembly and to induce disassembly, and the disassembly process was also observed. The filaments were shown to unravel with disassembly. Star-like clusters, which we reported as being the initial stage of reassembly, were also identified.

Animals↗
PubMed1990

Developmental changes of synapsin I subcellular localization in rat cerebellar neurons.

Synapsin I, one of the major synaptic proteins, is thought to associate with synaptic vesicles and to play a regulatory role in neurotransmitter release. In mature neurons, it is concentrated almost exclusively in presynaptic nerve endings. Here, we studied the subcellular localization of synapsin I during the development of rat cerebellar cortices by immunocytochemistry, using anti-synapsin I antibodies and found that during the development of rat cerebellar cortices it tentatively exists in the dendritic growth cones of immature internal granule cells and in the axonal growth cones of mossy fibers as well as mature presynaptic endings. Also, we found that synapsin I, in the axonal and dendritic growth cones does not necessarily associate with vesicles, but rather with fuzzy filamentous structures in the cytoplasm. In search of the structure of synapsin I in vivo, we employed the quick-freeze, deep-etch technique after immunogold labeling. Synapsin I seems to thereby connect synaptic vesicles or anchor them to cytoskeletons in presynaptic endings.

Animals↗