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Hideaki Fujita

Publications and source records attributed to Hideaki Fujita.

At least 19 recordsLinked to original sources
PubMed2005

Auto-oscillations of skinned myocardium correlating with heartbeat.

Skinned myocardium (or myofibrils) exhibits auto-oscillations of sarcomere length and developed force called SPOC (SPontaneousOscillatoryContraction) under partial activation conditions. In SPOC, each sarcomere repeats the cycle of slow shortening and rapid lengthening, and the lengthening phase propagates sequentially to the adjacent sarcomeres in waves (SPOC wave). In this study, we analyzed the sarcomeric oscillation in SPOC in skinned myocardium of various animal species (rat, rabbit, dog, pig, and cow) with different heart rates. The period of oscillation, the sarcomere shortening velocity, and the velocity of SPOC wave, strongly correlated with the resting heart rate of the animal species. The shortening velocity in particular was proportional to the resting heart rate. We then examined the motile activity of each cardiac myosin by an in vitro motility assay. The sliding velocity of actin filaments, which is an index of the motile activity of myosin, also correlated with the resting heart rate but the relationship was not proportional. As a result, the ratio of sarcomere shortening velocity in SPOC to the sliding velocity of actin filaments was not constant but became higher with a higher heart rate. This suggests that the sarcomere shortening velocity in SPOC is modulated by some additional factors besides the motile activity of myosin, resulting in the proportional relationship between the shortening velocity of the sarcomere and the resting heart rate.

Actin Cytoskeleton↗
PubMed2005

A new muscle contractile system composed of a thick filament lattice and a single actin filament.

To bridge the gap between the contractile system in muscle and in vitro motility assay, we have devised an A-band motility assay system. A glycerinated skeletal myofibril was treated with gelsolin to selectively remove the thin filaments and expose a single A-band. A single bead-tailed actin filament trapped by optical tweezers was made to interact with the inside or the outer surface of the A-band, and the displacement of the bead-tailed filament was measured in a physiological ionic condition by phase-contrast and fluorescence microscopy. We observed large back-and-forth displacement of the filament accompanied by a large change in developed force. Despite this large tension fluctuation, we found that the average force was proportional to the overlap inside and outside the A-band up to approximately 150 nm and 300 nm from the end of the A-band, respectively. Consistent with the difference in the density of myosin molecules, the average force per unit length of the overlap inside the A-band (the time-averaged force/myosin head was approximately 1 pN) was approximately twice as large as that outside. Thus, we conclude that the A-band motility assay system described here is suitable for studying force generation on a single actin filament, and its sliding movement within a regular three-dimensional thick filament lattice.

Actin Cytoskeleton↗
PubMed2005

The NH(2)-terminal transmembrane and lumenal domains of LGP85 are needed for the formation of enlarged endosomes/lysosomes.

LGP85 is a lysosomal membrane protein possessing a type III topology and is also known as a member of the CD36 superfamily of proteins, such as CD36 and the scavenger-receptor BI (SR-BI). We have recently demonstrated that overexpression of LGP85 in various mammalian cell lines causes the enlargement of endosomal/lysosomal compartments (ELCs). Using chimeras and deletion mutants, we show here that the lumenal region of LGP85 is necessary, but not sufficient, for the development of ELCs. Effective formation of enlarged ELC was largely dependent on the presence of a preceding NH(2)-terminal transmembrane segment. Analyses of deletion mutants within the lumenal domain further revealed a requirement of the NH(2)-terminal transmembrane proximal lumenal region, with high sequence similarity with SR-BI for the enlargement of ELC. These results suggest that an interaction of the NH(2)-terminal transmembrane proximal lumenal domain of LGP85 with the inner leaflet of endosomal/lysosomal membranes through the connection with the transmembrane domain is an essential determinant for the regulation of endosomal/lysosomal membrane traffic. Interestingly, although the NH(2)-terminal transmembrane domain itself was not sufficient for the enlargement of ELCs, it appeared to be required for direct targeting of LGP85 from the trans-Golgi network to late endosomes/lysosomes. Taken together, these results indicate the involvement of distinct domain of LGP85 in the targeting to, and biogenesis and maintenance of, ELC.

Amino Acid Sequence↗
PubMed2004

Mammalian class E Vps proteins, SBP1 and mVps2/CHMP2A, interact with and regulate the function of an AAA-ATPase SKD1/Vps4B.

SKD1 belongs to the AAA-ATPase family and is one of the mammalian class E Vps (vacuolar protein sorting) proteins. Previously we have reported that the overexpression of an ATPase activity-deficient form of SKD1 (suppressor of potassium transport growth defect), SKD1(E235Q), leads the perturbation of membrane transport through endosomes and lysosomes, however, the molecular mechanism behind the action of SKD1 is poorly understood. We have identified two SKD1-binding proteins, SBP1 and mVps2, by yeast two-hybrid screening and we assign them as mammalian class E Vps proteins. The primary sequence of SBP1 indicates 22.5% identity with that of Vta1p from Saccharomyces cerevisiae, which was recently identified as a novel class E Vps protein binding to Vps4p. In fact, SBP1 binds directly to SKD1 through its C-terminal region (198-309). Endogenous SBP1 is exclusively localized to cytosol, however it is redirected to an aberrant endosomal structure, the E235Q compartment, in the cells expressing SKD1(E235Q). The ATPase activity of SKD1 regulates both the membrane association of, and assembly of, a large hetero-oligomer protein complex, containing SBP1, which is potentially involved in membrane transport through endosomes and lysosomes. The N-terminal half (1-157) of human SBP1 is identical to lyst-interacting protein 5 and intriguingly, SKD1 ATPase activity significantly influences the membrane association of lyst protein. The SKD1-SBP1 complex, together with lyst protein, may function in endosomal membrane transport. A primary sequence of mVps2, a mouse homologue of human CHMP2A/BC-2, indicates 44.4% identity with Vps2p/Did4p/Chm2p from Saccharomyces cerevisiae. mVps2 also interacts with SKD1 and is localized to the E235Q compartment. Intriguingly, the N-terminal coiled-coil region of mVps2 is required for the formation of the E235Q compartment but not for binding to SKD1. We propose that both SBP1 and mVps2 regulate SKD1 function in mammalian cells.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

3-Methyladenine specifically inhibits retrograde transport of cation-independent mannose 6-phosphate/insulin-like growth factor II receptor from the early endosome to the TGN.

3-Methyladenine (3-MA), a well-known inhibitor of autophagic sequestration, can also prevent class III phosphatidylinositide (PI) 3-kinase activity, which is required for many processes in endosomal membrane trafficking. Although much is known about the effects of other PI 3-kinase inhibitors, such as wortmannin and LY294002, on endosomal membrane trafficking, little is known about those of 3-MA. Here we show that the treatment of cells with 3-MA results in a specific redistribution of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR300) from the trans-Golgi network (TGN) to early/recycling endosomal compartments containing internalized transferrin. Importantly, in contrast to wortmannin and LY294002, 3-MA did not cause the enlargement of late endosomal/lysosomal compartments. The results suggest that the effect of 3-MA is restricted to the retrieval of MPR300 from early/recycling endosomes.

Adenine↗
PubMed2004

Analysis of post-lysosomal compartments.

Lysosomes are acidic intracellular compartments and are regarded as degradative and the end point, of the endocytic pathway. Here we provide evidence for the generation of acid hydrolase poor and non-acidic post-lysosomal compartments in NRK cells that have accumulated non-digestible macromolecules, Texas red-dextran (TR-Dex), within lysosomes. When TR-Dex was fed to the cells for 6h, most of the internalized TR-Dex colocalized with a lysosomal enzyme, cathepsin D. With an increase in the chase period, however, the internalized TR-Dex gradually accumulated in cathepsin D-negative vesicles. These vesicles were positive for a lysosomal membrane protein, LGP85, and their formation was inhibited by treatment of the cells with U18666A, which impairs membrane transport out of late endosomal/lysosomal compartments, thereby suggesting that the vesicles are derived from lysosomes. Interestingly, these compartments are non-acidic as judged for the DAMP staining. The results, therefore, suggest that the excess accumulation of non-digestible macromolecules within lysosomes induces the formation of acid hydrolase poor and non-acidic post-lysosomal compartments. The fact that treatment of the cells with lysosomotropic amines or a microtubule-depolymerization agent resulted in extensive colocalization of TR-Dex with cathepsin D further indicates that the formation of the post-lysosomal compartments depends on the lysosomal acidification and microtubule organization. Furthermore, these results suggest bi-directional membrane transport between lysosomes and the post-lysosomal compartments, which implies that the latter are not resting compartments.

Androstenes↗
PubMed2004

The effect of tropomyosin on force and elementary steps of the cross-bridge cycle in reconstituted bovine myocardium.

The role of tropomyosin (Tm) in the elementary steps of the cross-bridge cycle in bovine myocardium was investigated. The thin filament was selectively removed using gelsolin (thin filament severing protein), and the actin filament was reconstituted from G-actin. Tm was further reconstituted without troponin (Tn), and the kinetic constants of the elementary steps of the cross-bridge cycle were deduced using sinusoidal analysis at pCa </= 4.66, pH 7.00, and 25 degrees C. The association constant of MgATP to cross-bridges (K(1)) after reconstitution of Tm was 20.7 +/- 2.3 mm(-1), which was about 2 x the control (untreated) myocardium (9.1 +/- 1.3 mm(-1)). Following reconstitution of Tm, the equilibrium constant of the cross-bridge detachment step (K(2)), the phosphate (P(i)) association constant (K(5)) and the equilibrium constant of the force-generation step (K(4)), which significantly changed in the actin filament-reconstituted myocardium, recovered to those of the control myocardium. Active tension after reconstitution of Tm was 0.69 x the control myocardium, a value between the control (1.00 x) and the actin filament-reconstituted myocardium (0.59 x). Tm-reconstituted myocardium was further reconstituted with Tn, and the effect of MgATP on the rate constants (K(1), K(2)) was studied. Following reconstitution with Tn, the myocardium regained the Ca(2+)-sensitivity and the active tension became 0.83 x the control myocardium. In addition, K(1) recovered to the value of the control myocardium with Tn reconstitution. These results indicate that both Tm and Tn enhance the force generated by each cross-bridge, and that Tm is primarily responsible for the change in the kinetic constants of the elementary steps of the cross-bridge cycle.

Actin Cytoskeleton↗
PubMed2004

Titin isoform-dependent effect of calcium on passive myocardial tension.

We studied the effects of Ca2+ on titin (connectin)-based passive tension in skinned myocardium expressing either predominantly N2B titin (rat right ventricle, RRV) or predominantly N2BA titin (bovine left atrium, BLA). Actomyosin-based tension was abolished to undetectably low levels by selectively removing the thin filaments with a Ca2+-insensitive gelsolin fragment (FX-45). Myocardium was stretched in the presence and absence of Ca2+, and passive tension was measured. Ca2+ significantly increased passive tension during and after stretch in the BLA. The increase was insensitive to the actomyosin inhibitor 2,3-butanedione 2-monoxime, supporting the conclusion that the effect is titin based. Passive tension did not respond to calcium in the RRV, indicating that passive tension developed by N2B titin is calcium insensitive. Western blot analysis and immunofluorescence studies indicated that N2BA titin expresses E-rich PEVK motifs, whereas they are absent from N2B titin, supporting earlier single molecule studies that reported that E-rich motifs are required for calcium sensitivity. We conclude that calcium affects passive myocardial tension in a titin isoform-dependent manner.

Actin Cytoskeleton↗
PubMed2003

Calcium-dependent molecular spring elements in the giant protein titin.

Titin (also known as connectin) is a giant protein with a wide range of cellular functions, including providing muscle cells with elasticity. Its physiological extension is largely derived from the PEVK segment, rich in proline (P), glutamate (E), valine (V), and lysine (K) residues. We studied recombinant PEVK molecules containing the two conserved elements: approximately 28-residue PEVK repeats and E-rich motifs. Single molecule experiments revealed that calcium-induced conformational changes reduce the bending rigidity of the PEVK fragments, and site-directed mutagenesis identified four glutamate residues in the E-rich motif that was studied (exon 129), as critical for this process. Experiments with muscle fibers showed that titin-based tension is calcium responsive. We propose that the PEVK segment contains E-rich motifs that render titin a calcium-dependent molecular spring that adapts to the physiological state of the cell.

Amino Acid Motifs↗
PubMed2003

Distribution and trafficking of MPR300 is normal in cells with cholesterol accumulated in late endocytic compartments: evidence for early endosome-to-TGN trafficking of MPR300.

It has been reported that an accumulation of cholesterol within late endosomes/lysosomes in Niemann-Pick type C (NPC) fibroblasts and U18666A-treated cells causes impairment of retrograde trafficking of the cation-independent mannose 6-phosphate/IGF-II receptor (MPR300) from late endosomes to the trans-Golgi network (TGN). In apparent conflict with these results, here we show that as in normal fibroblasts, MPR300 localizes exclusively to the TGN in NPC fibroblasts as well as in normal fibroblasts treated with U18666A. This localization can explain why several lysosomal properties and functions, such as intracellular lysosomal enzyme activity and localization, the biosynthesis of cathepsin D, and protein degradation, are all normal in NPC fibroblasts. These results, therefore, suggest that the accumulation of cholesterol in late endosomes/lysosomes does not affect the retrieval of MPR300 from endosomes to the TGN. Furthermore, treatment of normal and NPC fibroblasts with chloroquine, which inhibits membrane traffic from early endosomes to the TGN, resulted in a redistribution of MPR300 to EEA1 and internalized transferrin-positive, but LAMP-2-negative, early-recycling endosomes. We propose that in normal and NPC fibroblasts, MPR300 is exclusively targeted from the TGN to early endosomes, from where it rapidly recycles back to the TGN without being delivered to late endosomes. This notion provides important insights into the definition of late endosomes, as well as the biogenesis of lysosomes.

Androstenes↗
PubMed2003

Cerebral microbleeds in Binswanger's disease: a gradient-echo T2*-weighted magnetic resonance imaging study.

We investigated the incidence and the number of microbleeds (MBs) on T2*-weighted gradient-echo magnetic resonance imaging in 30 Binswanger's disease (BD) patients with diffuse white matter lesions and a varying degree of lacunar infarction, 51 multiple lacunar stroke (MLS) patients with multiple lacunar infarction and no or mild white matter lesions, and 59 elderly controls. MBs were found in 23 (77%) patients with BD, 26 (51%) patients with MLS, and 5 (8%) controls, and the incidence and the number of MBs were significantly greater in the BD group compared with MLS and control groups. Patients with BD had a more widespread location of MBs. More specifically, MBs were commonly found in areas within or surrounded by white matter lesions of the patients with BD. When 81 patients from both the BD and MLS groups were analyzed, logistic regression analysis showed that number of lacunar infarct, severity of white matter hyperintensity, and use of antiplatelet agents were significantly associated with MBs. Patients with BD exhibited a high frequency and number of MBs, indicating advanced bleeding-prone microangiopathy within the brain, which should be taken into account in treatment and management.

Aged↗
PubMed2003

Two lysosomal membrane proteins, LGP85 and LGP107, are delivered to late endosomes/lysosomes through different intracellular routes after exiting from the trans-Golgi network.

Lysosomal membrane proteins are delivered from their synthesis site, the endoplasmic reticulum (ER) to late endosomes/lysosomes through the Golgi complex. It has been proposed that after leaving the Golgi they are transported either directly or indirectly (via the cell surface) to late endosomes/lysosomes. In the present study, we examined the transport routes taken by two structurally different lysosomal membrane proteins, LGP85 and LGP107, in rat 3Y1-B cells. Here we show that newly synthesized LGP85 and LGP107 are delivered to late endosomes/lysosomes via a direct route without passing through the cell surface. Interestingly, although LGP107 is delivered from the Golgi to early endosomes containing internalized horseradish peroxidase-conjugated transferrin (HRP-Tfn) en route to lysosomes, LGP85 does not pass through the HRP-Tfn-positive early endosomes. These results suggest, therefore, that LGP85 and LGP107 are sorted into distinct transport vesicles at the post-Golgi, presumably the trans-Golgi network (TGN), after which LGP85 is delivered directly to late endosomes/lysosomes, but significant fractions of LGP107 are targeted to early endosomes before transport to late endosomes/lysosomes. This study provides the first evidence that after exiting from the Golgi, LGP85 and LGP107 are targeted to late endosomes/lysosomes via a different pathway.

Animals↗
PubMed2003

A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments: class E vps phenotype in mammalian cells.

SKD1 is a member of the family of ATPases associated with cellular activities whose yeast homologue Vps4p has been implicated in endosomal/vacuolar membrane transports. When a mutant of SKD1 that lacks ATPase activity [SKD1(E235Q)] was overexpressed in mammalian cells, it induced a dominant negative phenotype characterized by aberrant endosomal structures (denoted as E235Q compartments). Expression of SKD1(E235Q) caused an accumulation of basolateral recycling receptors, such as asialoglycoprotein receptor and low-density lipoprotein in polarized hepatocytes and Madin-Darby canine kidney cells, respectively, in E235Q compartments. In addition, SKD1(E235Q) also abrogated, via endosomes, transport to the trans-Golgi network, as indicated by an accumulation of TGN38 in E235Q compartments. Three lines of evidence further demonstrated that SKD1 participates in the membrane transport from early endosomes to late endosomes/lysosomes: (1) a redistribution of a late endosomal and lysosomal membrane protein endolyn in E235Q compartments; (2) an inhibition of epidermal growth factor receptor degradation, due to an accumulation of the receptors in E235Q compartments; and (3) a mis-sorting of and defect in the proteolytic processing of newly synthesized cathepsin D. An intriguing finding was that the expression of SKD1(E235Q) caused the number of lysosomes to decrease (to one-sixth of control numbers) but their size to increase (2.4-fold larger in diameter than control lysosomes). Indeed, an ultrastructural analysis revealed that the expression of SKD1(E235Q) causes an accumulation of hybrid organelles formed by direct fusion between late endosomes and lysosomes. We conclude that SKD1 regulates multiple steps of membrane transport out of early endosomes and the reformation of lysosomes from a hybrid organelle.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2003

Bio-nanomuscle project: contractile properties of single actin filaments in an A-band motility assay system.

We have developed a new microscopic technique to measure the force generated on a single actin filament (FA) in the A-band in which the intact lattice structure composed of myosin thick filaments is maintained; we call this newly developed system "Bio-nanomuscle (or an A-band motility assay system)". The A-bands were prepared by selective removal of thin filaments from rabbit skeletal glycerinated myofibrils under optical microscope with the use of gelsolin (a severing and barbed (B)-end capping protein of FA) that was prepared from bovine serum. A polystyrene bead of 1 microm in diameter attached to the B-end of FA (through a gelsolin molecule attached to the surface of the bead) was trapped and manipulated with optical tweezers. The displacement of the bead up to 200 nm (corresponding to the force of approximately 40 pN) was determined by phase-contrast image analysis. At the initial stage of this study, the overlapping length of an FA with the A-band was determined from the fluorescence image of FA labeled with rhodamine-phalloidin (Rh-Ph) and the phase-contrast image, but we later improved the method of determination by moving the sample stage stepwise using the piezo actuator. The average force per overlap was subsequently estimated and the histogram was fitted with two Gaussian distributions. Each peak is supposed to correspond to the force developed by FA interacting outside or inside the A-band, and the peak value of the latter was estimated to be 140 pN/microm. From this value, the average force developed per each cross-bridge (CB; a two-headed myosin molecule) was determined to be 1.3 pN.

Actin Cytoskeleton↗
PubMed2003

15O Radioactivity clearance is faster after intracarotid bolus injection of 15O-labeled oxyhemoglobin than after 15O-water injection.

The authors tested the hypothesis that the oxygen content of brain tissue is negligible by injecting an intracarotid bolus of 15O-labeled tracer into rats. Under the hypothesis, the clearance rates of 15O radioactivity from the brain after injections of both 15O-labeled water (H(2)15O) and 15O-labeled oxyhemoglobin (HbO15O) should be identical. However, the logarithmic slope of the 15O radioactivity curve after HbO15O injection (0.494 +/- 0.071 min-1) was steeper than that after H(2)15O injection (0.406 +/- 0.038 min-1) (P<0.001, n = 13), where the time range used in the comparison was between 60 and 120 seconds after the injection. A possible interpretation of this result is that nonmetabolized O15O may dwell in the brain tissue for a finite period of time before it is eventually metabolized or returned to the blood stream unaltered. These findings contradict assumptions made by models currently used to measure cerebral oxygen metabolism.

Animals↗