Biomedical subjects

Jiro Usukura

Publications and source records attributed to Jiro Usukura.

At least 19 recordsLinked to original sources

PubMed2006

Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography.

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm-thick consecutive sections made approximately 100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models.

Actin Cytoskeleton↗

PubMed2006

LEDGF/DFS70, a major autoantigen of atopic dermatitis, is a component of keratohyalin granules.

Lens epithelium-derived growth factor/dense fine speckles 70 kDa protein (LEDGF/DFS70) is a transcriptional cofactor, a transcriptional activator, survival factor, and HIV-1 transporter. It is also a major autoantigen in patients with atopic dermatitis (AD), because autoantibodies to this protein are found in approximately 30% of AD patients. To better understand the role of autoantibodies and autoantigens in the pathogenesis of AD, we examined the distribution of LEDGF/DFS70 in the epidermis of normal human skin by light and electron microscopic immunocytochemistry. Increased amounts of LEDGF/DFS70 were located in the nuclei of cells in the basal layer, whereas the cytoplasm of cells in the granular layer stained for LEDGF/DFS70 by light microscopy. Using immunoelectron microscopy, we observed the accumulation of LEDGF/DFS70 in keratohyalin granules (KGs) in the cytoplasm of cells in the granular layer. In addition, Ig heavy chain-binding protein/glucose-regulated protein, 78-kDa (Bip/GRP78), a stress sensing protein in the endoplasmic reticulum, colocalized with LEDGF/DFS70 in the KGs. These results suggest that LEDGF/DFS70 is predominantly located in the nucleus of the basal epidermal cells and translocates into the cytoplasm during differentiation. Once in the cytoplasm, LEDGF/DFS70 accumulates in the KGs in the granular layer. Finally, LEDGF/DFS70, a "nuclear" autoantigen in AD, may play a functional role in the KGs.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Illuminated rhodopsin is required for strong activation of retinal guanylate cyclase by guanylate cyclase-activating proteins.

We have recently shown that activation of retinal guanylate cyclase (retGC) by GC-activating proteins (GCAPs) is much stronger than that previously reported and that preincubation of photoreceptor outer segment homogenates with ATP or its analogue, adenylyl imidodiphosphate (AMP-PNP), is required for the strong activation [Yamazaki, A., Yu, H., Yamazaki, M., Honkawa, H., Matsuura, I., Usukura, J., and Yamazaki, R. K. (2003) J. Biol. Chem. 278, 33150-33160]. Here we show that illuminated rhodopsin is essential for development of the AMP-PNP incubation effect. This was demonstrated by illumination of dark homogenates and treatments of illuminated homogenates with 11-cis-retinal and hydroxylamine prior to the AMP-PNP incubation and by measurement of the GCAP2 concentration required for 50% activation. We also found that the AMP-PNP incubation effect was not altered by addition of guanosine 5'-O-(3-thiotriphosphate), indicating that transducin activation is not required. It is concluded that illuminated rhodopsin is involved in retGC activation in two ways: to initiate the ATP incubation effect for preparation of retGC activation as shown here and to reduce the Ca2+ concentrations through cGMP phosphodiesterase activation as already known. These two signal pathways may be activated in a parallel and perhaps proportional manner and finally converge for strong activation of retGC by Ca2+-free GCAPs.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2006

Structural insight of human DEAD-box protein rck/p54 into its substrate recognition with conformational changes.

Human rck/p54, a product of the gene cloned at the breakpoint of t(11; 14) (q23;q32) chromosomal translocation on 11q23 in B-cell lymphoma, is a member of the DEAD-box RNA helicase family. Here, the crystal structure of Nc-rck/p54, the N-terminal core domain of rck/p54, revealed that the P-loop in motif I formed a closed conformation, which was induced by Asn131, a residue unique to the RCK subfamily. It appears that ATP does not bind to the P-loop. The results of dynamic light scattering revealed to ATP-induced conformational change of rck/p54. It was demonstrated that free rck/p54 is a distended molecule in solution, and that the approach between N-terminal core and C-terminal domains for ATP binding would be essential when unwinding RNA. The results from helicase assay using electron micrograph, ATP hydrolytic and luciferase assay showed that c-myc IRES RNA, whose secondary structure regulates IRES-dependant translation, was unwound by rck/p54 and indicated that it is a good substrate for rck/p54. Over-expression of rck/p54 in HeLa cells caused growth inhibition and cell cycle arrest at G2/M with down-regulation of c-myc expression. These findings altogether suggest that rck/p54 may affect the IRES-dependent translation of c-myc even in the cells.

Adenosine Triphosphate↗

PubMed2005

ATP binding is required for physiological activation of retinal guanylate cyclase.

ATP bound to retinal guanylate cyclase (retGC)/membranes prior to the assay (pre-binding effect) and during the assay (direct effect) further enhances retGC activity stimulated by GC-activating proteins (GCAPs). Here we investigate differences between these two effects. We found that the pre-binding effect, but not the direct effect, was absent in membranes pre-washed with Mg(2+)-free hypotonic buffers, that the pre-binding effect, but not the direct effect, was strictly limited to GCAP-stimulated retGC activity, and that these two effects were independent and additive rather than being synergistic. Pre-incubation with amiloride enhanced GCAP2-activated retGC activity in a manner similar to that by ATP pre-binding; however, amiloride did not directly stimulate the retGC activity. These results indicate that these two effects are mechanistically different. Levels of retGC activation by these effects and conditions required for these effects indicate that only the mechanism involving ATP pre-binding is physiologically relevant to retGC activation.

Adenosine Triphosphate↗

PubMed2005

Type-3 ryanodine receptor involved in Ca2+-induced Ca2+ release and transmitter exocytosis at frog motor nerve terminals.

Ca(2+)-induced Ca2+ release (CICR) occurs in frog motor nerve terminals after ryanodine receptors (RyRs) are primed for activation by conditioning large Ca2+ entry. We studied which type of RyR exists, whether CICR occurs without conditioning Ca2+ entry and how RyRs are primed. Immunohistochemistry revealed the existence of RyR3 in motor nerve terminals and axons and both RyR1 and RyR3 in muscle fibers. A blocker of RyR, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) slightly decreased rises in intracellular Ca2+ ([Ca2+]i) induced by a short tetanus (50 Hz, 1-2s), but not after treatment with ryanodine. Repetitive tetani (50 Hz for 15s every 20s) produced repetitive rises in [Ca2+]i, whose amplitude overall waxed and waned. TMB-8 blocked the waxing and waning components. Ryanodine suppressed a slow increase in end-plate potentials (EPPs) induced by stimuli (33.3 Hz, 15s) in a low Ca2+, high Mg2+ solution. KN-62, a blocker of Ca(2+)/calmoduline-activated protein kinase II (CaMKII), slightly reduced short tetanus-induced rises in [Ca2+]i, but markedly the slow waxing and waning rises produced by repetitive tetani in both normal and low Ca2+, high Mg2+ solutions. Likewise, KN-62, but not KN-04, an inactive analog, suppressed slow increases in EPP amplitude and miniature EPP frequency during long tetanus. Thus, CICR normally occurs weakly via RyR3 activation by single impulse-induced Ca2+ entry in frog motor nerve terminals and greatly after the priming of RyR via CaMKII activation by conditioning Ca2+ entry, thus, facilitating transmitter exocytosis and its plasticity.

Animals↗

PubMed2005

Physiological function of S-cone system is not enhanced in rd7 mice.

The rd7mouse is a mutant mouse with a relatively late development of retinal degeneration. Earlier studies have shown that rd7 mice have a distinctive pattern of retinal dysplasia with an increased number of cone cells, particularly those with S (short wavelength)-opsin immunoreactivity. These alterations of the rd7 retina are caused by a mutation in the photoreceptor cell-specific nuclear receptor gene, Nr2e3, which is involved in the signaling pathway regulating photoreceptor cell differentiation, cell maintenance, and cell-cell interactions. The purpose of this study was to determine the physiological properties of the rd7 retina using electroretinographic (ERG) techniques. We found that the maximal a-wave amplitude of the ERG in rd7 mice was already reduced to half of the congenic controls at 6 weeks of age with normal phototransduction sensitivity. The photopic ERGs of rd7 mice were not supernormal, and the amplitudes of the S-cone ERGs were not significantly different from those recorded in controls. These results suggested that even though the number of cones expressing S-opsin is increased, the physiological function of the S-cone system is not enhanced in rd7 mice.

Animals↗

PubMed2005

Identification of trichoplein, a novel keratin filament-binding protein.

Keratins 8 and 18 (K8/18) are major components of the intermediate filaments (IFs) of simple epithelia. We report here the identification of a novel protein termed trichoplein. This protein shows a low degree of sequence similarity to trichohyalin, plectin and myosin heavy chain, and is a K8/18-binding protein. Among interactions between trichoplein and various IF proteins that we tested using two-hybrid methods, trichoplein interacted significantly with K16 and K18, and to some extent with K5, K6a, K8 and K14. In in vitro co-sedimentation assays, trichoplein directly binds to K8/18, but not with vimentin, desmin, actin filaments or microtubules. An antibody raised against trichoplein specifically recognized a polypeptide with a relative molecular mass of 61 kDa in cell lysates. Trichoplein was immunoprecipitated using this antibody in a complex with K8/18 and immunostaining revealed that trichoplein colocalized with K8/18 filaments in HeLa cells. In polarized Caco-2 cells, trichoplein colocalized not only with K8/18 filaments in the apical region but also with desmoplakin, a constituent of desmosomes. In the absorptive cells of the small intestine, trichoplein colocalized with K8/18 filaments at the apical cortical region, and was also concentrated at desmosomes. Taken together, these results suggest that trichoplein is a keratin-binding protein that may be involved in the organization of the apical network of keratin filaments and desmosomes in simple epithelial cells.

Amino Acid Sequence↗

PubMed2005

Akt/PKB regulates actin organization and cell motility via Girdin/APE.

The serine/threonine kinase Akt (also called protein kinase B) is well known as an important regulator of cell survival and growth and has also been shown to be required for cell migration in different organisms. However, the mechanism by which Akt functions to promote cell migration is not understood. Here, we identify an Akt substrate, designated Girdin/APE (Akt-phosphorylation enhancer), which is an actin binding protein. Girdin expresses ubiquitously and plays a crucial role in the formation of stress fibers and lamellipodia. Akt phosphorylates serine at position 1416 in Girdin, and phosphorylated Girdin accumulates at the leading edge of migrating cells. Cells expressing mutant Girdin, in which serine 1416 was replaced with alanine, formed abnormal elongated shapes and exhibited limited migration and lamellipodia formation. These findings suggest that Girdin is essential for the integrity of the actin cytoskeleton and cell migration and provide a direct link between Akt and cell motility.

Actins↗

PubMed2005

Both type-I hemidesmosomes and adherens-type junctions contribute to the cell-substratum adhesion system in myoepithelial cells.

Myoepithelial cells present in exocrine glands cause secretion from the glands by contraction. They have mixed characteristics with regard to cytoskeletal elements, containing both epithelial-type intermediate filaments and smooth muscle-type myofilaments. For further characterization, myoepithelial cells from bovine apocrine sweat glands and tracheal glands were here examined with special attention to the cell-substratum adhesion system. Immunofluorescence microscopy using a panel of antibodies against adherens-type junctional and hemidesmosomal proteins demonstrated two types of cell-substratum junctions in myoepithelial cells from both glands. Type-I hemidesmosomes (HDs) consisting of plectin, BP230, integrin alpha6beta4, and BP180 were thus observed as punctate arrays longitudinally arranged along myoepithelial cell surfaces, while adherens-type junctions were similarly evident as linear rib-like structures. Double-label immunofluoresence revealed the two junctions to be distributed in a mutually exclusive or independent manner. Electron microscopy further demonstrated that apocrine myoepithelial cells surround secretory epithelial cells completely, without any gaps, HDs being abundant along the basement membrane, but with no distinct structures in the inter-hemidesmosomal regions. Immunoelectron microscopy, however, revealed an interhemidesmosomal localization of vinculin, pointing to the existence of adherens-type junctions. Secretory epithelial cells in tracheal glands were found not to be completely covered with myoepithelial cells, so that more than half of them are directly attached to the basement membrane, where they form type II-HDs lacking BP230 and BP180, but no detectable adherens junctions, like epidermal basal cells and sebaceous gland cells. These observations demonstrate that, in addition to their cytoskeleton, myoepithelial cells have both epithelial- and smooth muscle-type cell-substratum adhesion structures, i.e. HDs and dense plaque-like adherens junctions.

Adherens Junctions↗

PubMed2005

Phosphorylation by Rho kinase regulates CRMP-2 activity in growth cones.

Collapsin response mediator protein 2 (CRMP-2) enhances the advance of growth cones by regulating microtubule assembly and Numb-mediated endocytosis. We previously showed that Rho kinase phosphorylates CRMP-2 during growth cone collapse; however, the roles of phosphorylated CRMP-2 in growth cone collapse remain to be clarified. Here, we report that CRMP-2 phosphorylation by Rho kinase cancels the binding activity to the tubulin dimer, microtubules, or Numb. CRMP-2 binds to actin, but its binding is not affected by phosphorylation. Electron microscopy revealed that CRMP-2 localizes on microtubules, clathrin-coated pits, and actin filaments in dorsal root ganglion neuron growth cones, while phosphorylated CRMP-2 localizes only on actin filaments. The phosphomimic mutant of CRMP-2 has a weakened ability to enhance neurite elongation. Furthermore, ephrin-A5 induces phosphorylation of CRMP-2 via Rho kinase during growth cone collapse. Taken together, these results suggest that Rho kinase phosphorylates CRMP-2, and inactivates the ability of CRMP-2 to promote microtubule assembly and Numb-mediated endocytosis, during growth cone collapse.

Animals↗

PubMed2004

The switch mechanism of the cell death mode from apoptosis to necrosis in menadione-treated human osteosarcoma cell line 143B cells.

Time-dependent changes in the cell death mode from apoptosis to necrosis were studied in cultured 143B cells treated with menadione, an anti-cancerous drug, excluding a possible involvement of "secondary necrosis." The population of apoptotic cells judged by FITC-Annexin V and propidium iodide (PI) double staining reached its maximum at 6 hours after 100 microM menadione treatment followed by an abrupt decrease thereafter, while that of necrotic cells continuously increased reaching 90% at 24 hours. Electron microscopically, cells attached to the culture dish at 6 hours after the treatment consisted of two different types of cells: cells with typical apoptotic features occupying the major population and those with condensed nuclei and swollen cytoplasm. Cells attached to the culture dish at 8 hours after the treatment consisted exclusively of those with condensed nuclei and swollen cytoplasm. Mitochondria in these cells showed various structural changes: those swollen to various degrees with deposition of flocculent densities, or those with highly condensed matrix. Distinct decreases both in intracellular levels of ATP and caspase-3-like activities and remarkable elevations of intracellular levels of superoxide, which were partly suppressed by NAD(P)H oxidase inhibitors, occurred at 6 hours after the treatment. These results may suggest that distinct increases of the intracellular level of superoxide derived from plasma membrane NAD(P)H oxidase besides that from mitochondria have triggered the transition of cell death mode from apoptosis to necrosis. Transition of highly condensed mitochondria to extremely swollen ones may reflect necrotic processes in menadione-treated cells. The present study strongly suggests that time-dependent study is essential using the electron microscopic technique to analyze detailed processes in the changes of the cell death mode.

Acetophenones↗

PubMed2004

The reconstituted human Chl12-RFC complex functions as a second PCNA loader.

The sister chromatid cohesion factor Chl12 shares amino acid sequence similarity with RFC1, the largest subunit of replication factor C (RFC), and forms a clamp loader complex in association with the RFC small subunits RFCs2-5. It has been shown that the human Chl12-RFC complex, reconstituted with a baculovirus expression system, specifically interacts with human proliferating cell nuclear antigen (PCNA). The purified Chl12-RFC complex is structurally indistinguishable from RFC, as shown by electron microscopy, and it exhibits DNA-stimulated ATPase activity that is further enhanced by PCNA, and by DNA binding activity on specific primer/template DNA structures. Furthermore, the complex loads PCNA onto a circular DNA substrate, and stimulates DNA polymerase delta DNA synthesis on a primed M13 single-stranded template in the presence of purified replication proteins. However, it cannot substitute for RFC in promoting simian virus 40 DNA replication in vitro with crude fractions. These results demonstrate that the human Chl12-RFC complex is a second PCNA loader and that its roles in replication are clearly distinguishable from those of RFC.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2004

Interaction of Rho-kinase with myosin II at stress fibres.

Rho-kinase and myosin phosphatase cooperatively regulate the phosphorylation level of myosin light chain and are involved in the formation of stress fibres and smooth muscle contraction. Rho-kinase has been known to be localized at stress fibres, but little is known about the mechanism of its localization. Here we identified non-muscle myosin heavy chain IIA and IIB as the pleckstrin homology domain-interacting molecules by affinity column chromatography. The pleckstrin homology domain of Rho-kinase binds to myosin II directly in in vitro cosedimentation assay. The C-terminal region of the pleckstrin homology domain was important for this interaction, and the point mutations in the pleckstrin homology domain mutant (W1170A, W1340L) resulted in a decrease in the binding. We also found that the pleckstrin homology domain, but not the pleckstrin homology domain mutant (W1170A, W1340L), was localized at stress fibres in fibroblasts. These results indicate that Rho-kinase is localized at stress fibres through binding of the pleckstrin homology domain to myosin II.

Animals↗

PubMed2003

Characterization of mammalian synemin, an intermediate filament protein present in all four classes of muscle cells and some neuroglial cells: co-localization and interaction with type III intermediate filament proteins and keratins.

Using a monoclonal antibody, we have detected a high molecular weight muscle protein, co-localized and co-isolating with desmin. Searching a human cDNA database with partial amino acid sequences of the protein, we found a cDNA clone encoding a 1565-amino-acid polypeptide, identified as a mammalian (human) synemin, a member of the intermediate filament (IF) protein family. Immunoblotting showed the presence of a 180-kDa polypeptide in skeletal muscle and 180- and 200-kDa polypeptides in cardiac and smooth muscles. Interestingly, synemin was also found in myoepithelial cells, which have keratin filaments instead of desmin. Moreover, synemin was also found in astrocytes of optic nerves and non-myelin-forming Schwann cells, together with glial fibrillary acidic protein (GFAP) and vimentin. Blot overlays pointed to molecular interactions of synemin with desmin, vimentin, GFAP and keratin 5 and 6, but not with keratin 14. The experimental data also suggested a possible link with nebulin, a skeletal muscle protein. Purified synemin was coassembled with desmin in different molar ratios, and at 1:25, as typically found in vivo, IFs were formed which were comparable in length to desmin filaments. However, at molar ratios of 3:25 and 6:25, much shorter and irregular shaped filamentous polymers were generated. The fact that synemin is present in all four classes of muscle cells and a specific type of glial cells is indicative of important functions. Its incorporation may give structural and functional versatility to the IF cytoskeleton.

Animals↗

PubMed2003

A critical role for ATP in the stimulation of retinal guanylyl cyclase by guanylyl cyclase-activating proteins.

It has been believed that retinal guanylyl cyclase (retGC), a key enzyme in the cGMP recovery to the dark state, is solely activated by guanylyl cyclase-activating proteins (GCAPs) in a Ca2+-sensitive manner. However, a question has arisen as to whether the observed GCAP stimulation of retGC is sufficient to account for the cGMP recovery because the stimulated activity measured in vitro is less than the light/GTP-activated cGMP phosphodiesterase activity. Here we report that the retGC activation by GCAPs is larger than previously reported and that a preincubation with adenine nucleotide is essential for the large activation. Under certain conditions, ATP is two times more effective than adenylyl imidodiphosphate (AMP-PNP), a hydrolysis-resistant ATP analog; however, this study mainly used AMP-PNP to focus on the role of adenine nucleotide binding to retGC. When photoreceptor outer segment homogenates are preincubated with AMP-PNP (EC50 = 0.65 +/- 0.20 mM), GCAP2 enhanced the retGC activity 10-13 times over the control rate. Without AMP-PNP, GCAP2 stimulated the control activity only 3-4-fold as in previous reports. The large activation is due to a GCAP2-dependent increase in Vmax without an alteration of retGC affinity for GCAP2 (EC50 = 47.9 +/- 2.7 nM). GCAP1 stimulated retGC activity in a similar fashion but with lower affinity (EC50 = 308 nM). In the AMP-PNP preincubation, low Ca2+ concentrations are not required, and retGC exists as a monomeric form. This large activation is accomplished through enhanced action of GCAPs as shown by Ca2+ inhibition of the activity (IC50 = 178 nM). We propose that retGC is activated by a two-step mechanism: a conformational change by ATP binding to its kinase homology domain under high Ca2+ concentrations that allows large enhancement of GCAP activation under low Ca2+ concentrations.

Adenine↗

PubMed2003

A tumour-associated DEAD-box protein, rck/p54 exhibits RNA unwinding activity toward c-myc RNAs in vitro.

BACKGROUND: The rck/p54 protein of 473 amino acids belongs to the family of DEAD-box/putative RNA helicase proteins. DEAD-box proteins have been implicated in a wide variety of cellular processes ranging from the initiation of protein synthesis and ribosome biosynthesis to premRNA splicing by means of modifying the RNA structure. Our previous data suggested that rck/p54 positively affected the translation initiation of c-myc mRNA. RESULTS: The data obtained from morphological studies and surface plasmon resonance assays clearly indicated that the protein specifically bound to c-myc RNA transcripts (RNAs) and exhibited RNA unwinding activity toward c-myc RNAs in the presence of ATP in vitro. Experiments using a deletion mutant of rck/p54 retaining only its N-terminal 289 amino acids demonstrated that the deleted C-terminal 184 amino acid domain is involved in the RNA unwinding activity. CONCLUSION: These findings strongly suggest that rck/p54 may play an important role in translation initiation by restructuring mRNAs even in the cell and contribute to carcinogenesis.

DEAD-box RNA Helicases↗

PubMed2003

Ultrastructural basis for the transition of cell death mode from apoptosis to necrosis in menadione-treated osteosarcoma 143B cells.

Time-dependent ultrastructural changes of menadione-treated human osteosarcoma 143B cells were correlated with those in their stainability to Annexin V and propidium iodide (PI). Populations of both apoptotic (Annexin V(+)/PI(-)) and necrotic (Annexin V(+)/PI(+)) cells, judged by flow cytometry, began to increase at 2 h after menadione treatment. The former reached a maximum at 6 h followed by abrupt decreases thereafter, while the latter continued to increase. Electron microscopically, cells obtained at 6 h after the menadione treatment consisted of mixed populations of cells with typical apoptotic features and those with a mixture of apoptotic and necrotic features, while cells obtained at 8-24 h consisted exclusively of cells with a mixture of apoptotic and necrotic features. Thus, necrotic cells, as judged by flow cytometry, were in a transitional state of cell death mode from apoptosis to necrosis and are thus designated as 'intermediate cells'. Lack of apoptotic bodies, judged by flow cytometric analysis on sub-G1 nuclei and by electron microscopy in menadione-treated cells, suggested that the transition of cell death mode from apoptosis to necrosis occurred before the apoptotic processes were completed. Effects of N-acetylcysteine and Z-VAD-fmk on menadione-induced ultrastructural changes were also studied.

Antifibrinolytic Agents↗