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Biomedical subjects

N Hirokawa

Publications and source records attributed to N Hirokawa.

At least 145 records · Page 8Linked to original sources
PubMed1989

A novel heat-stable 205 kDa microtubule-associated protein is involved in the neural development of the rat brain.

A novel microtubule associated protein (MAP) was identified with a monoclonal antibody (mAb) among rat brain microtubule (MT) proteins. This MAP, which is found in a heat-stable MAP fraction, is co-polymerized with tubulin from rat brain homogenates both in cycled and in taxol-driven assembly purifications. When the heat-stable fraction of MT proteins and purified tubulin are assembled in vitro without taxol, 205 kDa MAP is identified in the MT pellet. Immunofluorescent studies using the mAb against this MAP on sections of a rat spinal cord, hippocampus, retina and cerebellar cortex, showed that 205 kDa MAP was localized in not only neural cell bodies, dendrites, and axons, but also in glial cells. This 205 kDa MAP is clearly distinct from previously reported MAPs as to molecular mass, heat stability, immunoreactivity, distribution among tissues or within a neuron, and the developmental aspect of the postnatal cerebellar cortex. As for development, the 205 kDa MAP has already been seen to appear at postnatal day 3. Throughout the postnatal development of the cerebellar cortex, 205 kDa MAP is expressed in developing Purkinje cells, with the staining reaction being particularly intense in the more superficially positioned parallel fiber profiles. This points out the possible important functions of 205 kDa MAP in the morphogenesis of parallel fiber axons.

Animals↗
PubMed1989

Purification and characterization of a Ca2+-dependent actin filament severing protein from bovine adrenal medulla.

We describe the purification of an actin regulatory protein from bovine adrenal medulla. This protein caused a dose-dependent decrease of the specific viscosity of actin solution within 30 s of its addition in a Ca2+-sensitive way. Sedimentation assays and the observation by electron microscopy showed that this effect was ascribable to the fragmentation of actin filaments. This protein apparently promoted nucleation of actin polymerization and increased the critical concentration of actin for polymerization nearly 5-fold, suggesting its binding to the barbed end of actin filaments. The inhibitory effect of this protein on the elongation of actin from the barbed end of the myosin subfragment S1-labeled actin seeds confirmed this suggestion. These properties are similar to those of gelsolin. However, the physicochemical properties of this protein having a single polypeptide chain with a molecular weight of 74,000, a Stokes radius of 3.9 nm, a sedimentation coefficient (s0(20),w) of 4.5 S, and an immunological characterization showed that this protein is different from gelsolin.

Actin Cytoskeleton↗
PubMed1989

Immunocytochemical localization of microtubule-associated proteins 1A and 2 in the rat retina.

We have studied the immunocytochemical localization of microtubule-associated proteins (MAPs) in rat retinal cells. Using biochemical and immunochemical methods we have identified microtubule-associated protein 1 (MAP1) and microtubule-associated protein 2 (MAP2) as major MAPs in the rat retina. With indirect immunofluorescence microscopy, the inner plexiform layer and the ganglion cell layer were stained with both anti-MAP1A antibody and anti-MAP2 antibody. Cells at the inner margin of the inner nuclear layer were prominently stained with anti-MAP2, but not with anti-MAP1A. Thin section-immunoelectron microscopy using colloidal gold-labeled secondary antibodies revealed MAP1A and MAP2 staining in the neuronal processes of the inner plexiform layer. A filamentous network between the microtubules in the neurites was stained with both antibodies. The developmental course of expression of MAP1A and MAP2 in rat retina was studied by indirect immunofluorescence microscopy. Although MAP2 was already present at 1 day postnatal, MAP1A was not detected until 7 days postnatal. These results indicate that: (1) retinal neurons are heterogeneous in their expression of MAPs; (2) retinal ganglion cells show the same intracellular distribution of MAP1A and MAP2 as typical nerve cells such as motor neurons; and (3) MAP1A and MAP2 are differentially expressed in developing rat retina.

Aging↗
PubMed1989

Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration.

Kinesin is a microtubule-activated ATPase thought to transport membrane-bounded organelles along MTs. To illuminate the structural basis for this function, EM was used to locate submolecular domains on bovine brain kinesin. Rotary shadowed kinesin appeared rod-shaped and approximately 80 nm long. One end of each molecule contained a pair of approximately 10 x 9 nm globular domains, while the opposite end was fan-shaped. Monoclonal antibodies against the approximately 124 kd heavy chains of kinesin decorated the globular structures, while those specific for the approximately 64 kd light chains labeled the fan-shaped end. Quick-freeze, deep-etch EM was used to analyze MTs polymerized from tubulin and cross-linked to latex microspheres by kinesin. Microspheres frequently attached to MTs by arm-like structures, 25-30 nm long. The MT attachment sites often appeared as one or two approximately 10 nm globular bulges. Morphologically similar cross-links were observed by quick-freeze, deep-etch EM between organelles and MTs in the neuronal cytoskeleton in vivo. These collective observations suggest that bovine brain kinesin binds to MTs by globular domains that contain the heavy chains, and that the attachment sites for organelles are at the opposite, fan-shaped end of kinesin, where the light chains are located.

Adenosine Triphosphatases↗
PubMed1989

The molecular structure of adrenal medulla kinesin.

The molecular structure of bovine adrenal kinesin was studied by electron microscopy using the low-angle rotary shadowing technique. Adrenal kinesin exhibited either a folded or an extended configuration; the ratio of the two is dependent on the salt concentration. Almost all adrenal kinesin molecules were folded in a low-ionic solution, and the ratio of extended molecules increased to 40-50% in a solution containing 1 M ammonium acetate. Kinesin in the extended configuration displayed a rod-shaped structure with a mean length of about 80 nm. The morphologies of the ends were different; one end was composed of two globular particles, similar to the two-headed structure of myosin, while the other end had a more ill-defined structure, appearing either as a globular particle, an aggregate of two to four small granules, or a frayed, fan-like structure. The folded kinesin molecule possessed a hinge region in the middle of the rod, at about 32 nm from the neck of the two heads. In our preparations, the majority of adrenal kinesin molecules were folded at physiological salt concentrations. Adrenal kinesin bound to microtubules in the presence of adenylyl imidodiphosphate (AMP-PNP) also displayed a folded morphology.

Acetates↗
PubMed1989

Cytoskeletal architecture of neuromuscular junction: localization of vinculin.

Cytoskeletons underneath the postsynaptic membrane of neuromuscular junctions were studied by using a quick-freeze deep-etch method and immunoelectron microscopy of ultrathin frozen sections. In a quick-freeze deep-etched replica of fresh, unfixed muscles, 8.9 +/- 1.5-nm particles were present on the true postsynaptic membrane surface. Underneath this receptor-rich postsynaptic membrane, networks of fine filaments were observed. These cytoskeletal networks were more clearly observed in extracted samples. In these samples, diameters of the filaments which formed networks were measured. In the platinum replica, three kinds of filament were recognized--12 nm, 9 nm, and 7 nm in diameter. The 12-nm filament seemed to correspond to the intermediate filament. The other two filaments formed meshworks between intermediate filaments and plasma membrane. In ultrathin frozen sections vinculin label was localized just beneath the plasma membrane. Thirty-six percent of the label was within 18 nm from the cytoplasmic side of the plasma membrane and 50% was within 30 nm. Taking the size of the vinculin molecule into account, it was concluded that vinculin is localized just beneath the plasma membrane and might play some role in anchoring filaments which formed meshworks underneath the plasma membrane.

Animals↗
PubMed1989

The arrangement of actin filaments in the postsynaptic cytoplasm of the cerebellar cortex revealed by quick-freeze deep-etch electron microscopy.

The cytoskeletal architecture of the postsynaptic cytoplasm in the cerebellar cortex of mice and rats was observed by quick-freeze, deep-etch electron microscopy. The postsynaptic cytoplasm was mainly filled with a network of actin filaments (approximately 8 nm in width). The tips of the actin filaments were closely associated with the true inner side of the postsynaptic membranes. However, the organization of the actin filaments was distinct depending on the types of synapses. In axosomatic synapses the actin filaments tended to run randomly and form a network while in the postsynaptic spine, such as Purkinje cell dendritic spines, the actin filaments were mainly arranged parallel to the stalk of the spines. Only a few actin filaments were found in the postsynaptic cytoplasm of some axodendritic synapses such as mossy fiber-granule cell synapses. In most cases a mesh of fine strands (approximately 6 nm in width) and granular substances was observed just underneath the postsynaptic membranes which also associated with actin filaments. The arrangement of actin filaments in the spine does not support the possibility of constriction of spines as a basis for long-term depression (LTD).

Actins↗
PubMed1989

Microtubule-associated protein 1B: molecular structure, localization, and phosphorylation-dependent expression in developing neurons.

Two monoclonal antibodies, 5E6 and 1B6, were raised against microtubule-associated protein 1B (MAP1B), a major component of the neuronal cytoskeleton. 5E6 recognized the entire MAP1B population, while 1B6 detected only phosphorylated forms. Affinity-purified MAP1B appeared as a long, filamentous molecule (186 +/- 38 nm) with a small spherical portion at one end, forming long cross-bridges between microtubules in vitro. These results, together with in vivo data from immunogold methods, demonstrate that MAP1B is a component of cross-bridges between microtubules in neurons. By immunohistochemical analysis, phosphorylated forms were shown to exist mainly in axons, whereas unphosphorylated forms were limited to cell bodies and dendrites. Phosphorylated MAP1B was quite abundant in developing axons, suggesting its essential role in axonal elongation.

Animals↗
PubMed1989

Rapid turnover of microtubule-associated protein MAP2 in the axon revealed by microinjection of biotinylated MAP2 into cultured neurons.

We studied the mechanism of compartmentation of microtubule-associated protein 2 (MAP2) in the dendrites and cell bodies by using microinjection of biotin-labeled MAP2 into mature spinal cord neurons in culture. MAP2 molecules microinjected into the nerve cell body were distributed not only throughout the cytoplasm of the cell body and dendrites, but also in the axon as far as a few millimeters from the cell body within 24 hr after injection. However, when injected cells were incubated for more than 3 days, the amount of biotin-labeled MAP2 in the axon decreased remarkably compared with that in the dendrites. This indicates that there is no sorting mechanism in the cell body for the transport of MAP2 selectively into the dendrites but that the turnover rate of MAP2 in the axons differs from that in the dendrites. To further characterize the mechanism of MAP2 compartmentation, we performed immunoelectron microscopy of injected cells and detergent extraction of microinjected cells prior to immunocytochemistry with anti-biotin. The results strongly suggest that a large part of axonal MAP2 is not associated with cytoskeleton and that this weak association of MAP2 favors selective loss of MAP2 from the axon.

Animals↗
PubMed1989

The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1.

We have examined the cytoskeletal architecture and its relationship with synaptic vesicles in synapses by quick-freeze deep-etch electron microscopy (QF.DE). The main cytoskeletal elements in the presynaptic terminals (neuromuscular junction, electric organ, and cerebellar cortex) were actin filaments and microtubules. The actin filaments formed a network and frequently were associated closely with the presynaptic plasma membranes and active zones. Short, linking strands approximately 30 nm long were found between actin and synaptic vesicles, between microtubules and synaptic vesicles. Fine strands (30-60 nm) were also found between synaptic vesicles. Frequently spherical structures existed in the middle of the strands between synaptic vesicles. Another kind of strand (approximately 100 nm long, thinner than the actin filaments) between synaptic vesicles and plasma membranes was also observed. We have examined the molecular structure of synapsin 1 and its relationship with actin filaments, microtubules, and synaptic vesicles in vitro using the low angle rotary shadowing technique and QF.DE. The synapsin 1, approximately 47 nm long, was composed of a head (approximately 14 nm diam) and a tail (approximately 33 nm long), having a tadpole-like appearance. The high resolution provided by QF.DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles, forming approximately 30-nm short strands. The head was on the actin and the tail was attached to the synaptic vesicle or actin filament. Microtubules were also cross-linked by a single synapsin 1, which also connected a microtubule to synaptic vesicles, forming approximately 30 nm strands. The spherical head was on the microtubules and the tail was attached to the synaptic vesicles or to microtubules. Synaptic vesicles incubated with synapsin 1 were linked with each other via fine short fibrils and frequently we identified spherical structures from which two or three fibril radiated and cross-linked synaptic vesicles. We have examined the localization of synapsin 1 using ultracryomicrotomy and colloidal gold-immunocytochemistry of anti-synapsin 1 IgG. Synapsin 1 was exclusively localized in the regions occupied by synaptic vesicles. Statistical analyses indicated that synapsin 1 is located mostly at least approximately 30 nm away from the presynaptic membrane. These data derived via three different approaches suggest that synapsin 1 could be a main element of short linkages between actin filaments and synaptic vesicles, and between microtubules and synaptic vesicles, and between synaptic vesicles in the nerve terminals.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1989

Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA.

Tau proteins are a class of low molecular mass microtubule-associated proteins that are specifically expressed in the nervous system. A cDNA clone of adult rat tau was isolated and sequenced. To analyze functions of tau proteins in vivo, we carried out transfection experiments. A fibroblast cell line, which was transfected with the cDNA, expressed three bands of tau, while six bands were expressed in rat brain. After dephosphorylation, one of the three bands disappeared, demonstrating directly that phosphorylation was involved in the multiplicity of tau. Morphologically, we observed a thick bundle formation of microtubules in the transiently and stably tau-gene-transfected cells. In addition, we found that the production of tubulin was prominently enhanced in the stably transfected cells. Thus, we suppose that tau proteins promote polymerization of tubulin, form bundles of microtubules in vivo, and play important roles in growing and maintaining nerve cell processes.

Amino Acid Sequence↗
PubMed1989

Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study.

We investigated the mechanism of turnover of an actin microfilament system in fibroblastic cells on an electron microscopic level. A new derivative of actin was prepared by labeling muscle actin with biotin. Cultured fibroblastic cells were microinjected with biotinylated actin, and incorporated biotin-actin molecules were detected by immunoelectron microscopy using an anti-biotin antibody and a colloidal gold-labeled secondary antibody. We also analyzed the localization of injected biotin-actin molecules on a molecular level by freeze-drying techniques. Incorporation of biotin-actin was rapid in motile peripheral regions, such as lamellipodia and microspikes. At approximately 1 min after injection, biotin-actin molecules were mainly incorporated into the distal part of actin bundles in the microspikes. Heavily labeled actin filaments were also observed at the distal fringe of the densely packed actin networks in the lamellipodium. By 5 min after injection, most actin polymers in microspikes and lamellipodia were labeled uniformly. These findings suggest that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. At earlier times after injection, we often observed that the labeled segments were continuous with unlabeled segments, suggesting the incorporation of new subunits at the ends of preexisting filaments. Actin incorporation into stress fibers was a slower process. At 2-3 min after injection, microfilaments at the surface of stress fibers incorporated biotin-actin, but filaments in the core region of stress fibers did not. At 5-10 min after injection, increasing density of labeling along stress fibers toward their distal ends was observed. Stress fiber termini are generally associated with focal contacts. There was no rapid nucleation of actin filaments off the membrane of focal contacts and the pattern of actin incorporation at focal contacts was essentially identical to that into distal parts of stress fibers. By 60 min after injection, stress fibers were labeled uniformly. We also analyzed the actin incorporation into polygonal nets of actin bundles. Circular dense foci, where actin bundles radiate, were stable structures, and actin filaments around the foci incorporated biotin-actin the slowest among the actin-containing structures within the injected cells. These results indicate that the rate and pattern of actin subunit incorporation differ in different regions of the cytoplasm and suggest the possible role of rapid actin polymerization at the leading margin on the protrusive movement of fibroblastic cells.

Actins↗
PubMed1989

Localization of 4.1 related proteins in cerebellar neurons.

Localization of 4.1 related proteins in neurons was studied with immunofluorescence microscopy and with immunoelectron microscopy on ultrathin cryosections. In rat cerebellum, 4.1 immunoreactive proteins were demonstrated in Purkinje cell bodies, dendrites and other neurons in the cerebellar cortex. Some glial cells showed staining, but no labeling was found in myelinated axons of the white matter and of the glomeruli in the granule cell layer. At the ultrastructural level, the 4.1 related proteins were localized mainly in the cytoplasmic matrix, while some labeling was found underneath the plasma membrane. To determine whether 4.1 related proteins in neuronal cytoplasm exist as part of the cytoskeleton or not, PC12 cells cultured in the presence of nerve growth factor were stained with the anti-4.1 antibody. Since cytoplasmic staining was retained after detergent treatment, the 4.1 related proteins seem to exist as a component of the neural cell cytoskeleton. Localization of 4.1 related proteins during the postnatal development of the cerebellum was also studied. In Purkinje cells, localization of 4.1 related proteins changed according to the stages of the postnatal development. The present data suggest that 4.1 related proteins in neurons localized mainly in the cytoplasm and may play some role in organizing cytoskeletal networks in the cytomatrix. Their distribution is developmentally regulated in some neurons, possibly in relationship to their maturation in the cytoskeleton.

Animals↗
PubMed1989

The effects of dephosphorylation on the structure of the projections of neurofilament.

Carboxy-terminal tail domains of larger molecular mass subunits (NF-M and NF-H) of neurofilaments (NFs), which are the highly phosphorylated moieties, were observed as thin flexible filaments projecting from NF core filaments by rotary shadowing (Hisanaga and Hirokawa, 1988). Dephosphorylation of NFs has been suspected to affect the structures and the functions of the carboxy-terminal tail projections. We report here the effects of the dephosphorylation on the structure of NFs studied by electron microscopy. (1) The structures of carboxy-terminal tail projections after dephosphorylation were compared with those of the control NFs by low-angle rotary shadowing. This was examined with 2 samples; the isolated neurofilaments and the short filaments assembled from NF-H. Both the dephosphorylated NFs and the short filaments showed many projections laterally extending from core filaments similar to those observed in the control samples. (2) With respect to the structure of NF in physiological solution, the density of NFs in the precipitates was examined by thin-section electron microscopy. No difference in the density was noted between control and dephosphorylated NFs. (3) The ability to form cross-bridges in vitro was examined by quick-freeze, deep-etch electron microscopy. The structure and frequency of cross-bridges appeared to be similar in both control and dephosphorylated NFs. (4) Phosphate determination revealed that about 90% of the phosphate groups of NF-H subunit were removed by treatment with E. coli alkaline phosphatase. These results indicated that the dephosphorylation of NF did not affect the structure and the ability to form cross-bridges of the carboxy-terminal tail projections in vitro.

Animals↗
PubMed1989

Quick-freeze, deep-etch electron microscopy.

This chapter briefly introduces the quick-freeze replica studies carried out mainly by Japanese scientists. Focus is on this technique's contribution to revealing three-dimensional organization of the cytoskeleton, and membrane with associated structures.

Animals↗