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H Hotani

Publications and source records attributed to H Hotani.

At least 37 records · Page 2Linked to original sources
PubMed1993

Flagellar growth in a filament-less Salmonella fliD mutant supplemented with purified hook-associated protein 2.

Bacterial flagellum consists of a basal body, a hook, HAP1 (hook-associated protein 1), HAP3, a long helical filament, and a cap (composed of HAP2), all connected in series. The mutant deficient in the HAP2 structural gene (fliD) of Salmonella typhimurium has flagella composed of only hook-HAP1-HAP3 and excretes flagellin monomers into the culture medium. However, when purified HAP2 was added to this mutant, the flagellin stopped leaking out and flagellar filaments grew. Turnover of HAP2 was not necessary for the growth of a filament. Therefore HAP2 facilitates the polymerization of endogenous flagellin, apparently without falling off the filament tip. This experimental system with exogenous HAP2 allowed us to synchronize filament growth; the average rate of filament growth can be estimated by measuring the length of grown filaments at various time periods in electron micrographs. The initial growth rate was about 30 nm/min, which corresponds to one flagellin per second.

Bacterial Proteins↗
PubMed1992

Morphological changes in liposomes caused by polymerization of encapsulated actin and spontaneous formation of actin bundles.

Spherical giant liposomes that had encapsulated skeletal-muscle G-actin were made by swelling a dried lipid mixture of dimyristoyl phosphatidylcholine/cardiolipin, 1:1 (wt/wt), in a solution of G-actin/CaCl2 at 0 degree C. Polymerization of the encapsulated G-actin into actin filaments was achieved by raising the temperature to 30 degrees C. We observed the subsequent shape changes of the liposomes by dark-field and differential interference-contrast light microscopy. After approximately 40 min, which was required for completion of actin polymerization, two shapes of liposome were evident: dumbbell and disk. Elongation of the dumbbell-shaped liposomes was concomitant with actin polymerization. Polarization microscopy showed that actin filaments formed thick bundles in the liposomes and that these filaments lay contiguous to the periphery of the liposome. Localization of actin filaments in the liposomes was confirmed by observation of rhodamine phalloidin-conjugated actin filaments by fluorescence microscopy. Both dumbbell- and disk-shaped liposomes were rigid and kept their shapes as far as actin filaments were stabilized. In contrast, liposomes containing bovine serum albumin were fragile, and their shapes continually fluctuated from Brownian motion, indicating that the actin bundles served as mechanical support for the liposome shapes.

Actin Cytoskeleton↗
PubMed1992

Interaction of the Golgi membranes isolated from rabbit liver with microtubules in vitro.

We have developed a reconstituted model system to study the interaction of the Golgi membranes isolated from rabbit liver with taxol-stabilized bovine-brain microtubules without microtubule-associated proteins (MAPs). The Golgi membranes are associated with microtubules. The sheets of vesicles and the membranous tubules are observed along microtubules by direct visualization using differential-interference-contrast, dark field, or fluorescence microscopy. The monoclonal antibody against Golgi membranes suggests that the Golgi membranes, but not the contaminating vesicles, are interacting with microtubules. The degree of association is assayed quantitatively using rhodamine-labeled microtubules after separation of the complex from unbound microtubules by centrifugation upon sucrose gradient. The association is inhibited by crude MAPs, purified MAP2, or 1.0 mM ATP. However, the association neither requires the cytosol from rat liver or bovine brain nor N-ethylmaleimide, brefeldin A, or GTP-gamma-S. The association is mediated by trypsin-sensitive peripheral protein(s) on the Golgi membranes.

Adenosine Triphosphate↗
PubMed1991

The morphogenesis of liposomes viewed from the aspect of bending energy.

It is known that liposomes transform their shapes sequentially through one of several transformation pathways. Using the mechanical principle of the least bending energy of membranes, we investigate the stability and shape transformation of liposomes with geometrical symmetry. We have done this by computer simulations and theoretical analyses, in which three-dimensional liposome shapes have been generated by the modified Cassini equation. We show first that there are energetically stable liposome shapes having intrinsic geometrical symmetry. We find that by reducing the volume, the stable shape can change from a circular biconcave shape as in red blood cells, to elliptical, triangular, square, and other polygonal shapes. It is also found that the preceding two results hold true irrespective of the overall surface area of liposome.

Animals↗
PubMed1991

Rapid changes in flagellar rotation induced by external electric pulses.

The bacterial flagellar motor is the only molecular rotary machine found in living organisms, converting the protonmotive force, i.e., the membrane voltage and proton gradients across the cell membrane, into the mechanical force of rotation (torque). We have developed a method for holding a bacterial cell at the tip of a glass micropipette and applying electric pulses through the micropipette. This method has enabled us to observe the dynamical responses of flagellar rotation to electric pulses that change the membrane voltage transiently and repeatedly. We have observed that acceleration and deceleration of motor rotation are induced by application of these electric pulses. The change in the rotation rate occurred within 5 ms after pulse application.

Cell Membrane↗
PubMed1990

Dynamic features of microtubules as visualized by dark-field microscopy.

We have reviewed recent progress in the dynamic features of microtubules in vitro as visualized by dark-field light microscopy using high intensity illumination. Observations of individual microtubules in real-time have made it possible to analyze the unique characteristics of microtubules exactly. The following three topics have been discussed: (1) treadmilling, i.e., the simultaneous assembly of tubulin at one end and disassembly at the other end on a single microtubule at a steady state. (2) Dynamic instability, i.e., the very unusual phenomenon in which two populations of microtubules coexist: those in one population elongating while those in the other shortening in the absence of MAPs. Both ends of the microtubules exist either in the growing or the shortening phase, and alternate between the two phases in a stochastic manner. (3) Morphogenesis of liposomes by microtubule growth. Tubulin is encapsulated into model membrane vesicles, liposomes. Polymerization of the encapsulated tubulin causes a change in shape of the spherical liposomes to form bipolar or multipolar vesicles, suggesting that microtubules have an active function in the morphogenesis of membranous organelles and cells.

Animals↗
PubMed1989

Spatial patterns from oscillating microtubules.

Microtubules are fibers of the cytoskeleton involved in the generation of cell shape and motility. They can be highly dynamic and are capable of temporal oscillations in their state of assembly. Solutions of tubulin (the subunit protein of microtubules) and guanosine triphosphate (GTP, the cofactor required for microtubule assembly and oscillations) can generate various dissipative structures. They include traveling waves of microtubule assembly and disassembly as well as polygonal networks. The results imply that cytoskeletal proteins can form dynamic spatial structures by themselves, even in the absence of cellular organizing centers. Thus the microtubule system could serve as a simple model for studying pattern formation by biomolecules in vitro.

Biopolymers↗
PubMed1988

Dynamics of microtubules visualized by darkfield microscopy: treadmilling and dynamic instability.

Individual microtubules undergoing treadmilling in vitro were visualized by darkfield light microscopy, and the relationship between treadmilling and dynamic instability was studied as a function of microtubule-associated proteins (MAPs). In order to demonstrate treadmilling directly by real-time observation, we constructed three-block microtubules, the center-block of which was decorated with Tetrahymena dynein. The decorated block can easily be distinguished from undecorated blocks in the darkfield microscope because the decorated one appears much thicker. At steady-state conditions, the length of an undecorated block at one end increased and that at another end decreased, while the decorated center-block did not change in its length. The results from these direct observations show that calf brain 3X-microtubules exhibit a treadmilling flux of 0.9 micron/h. Using a similar microscopy technique, we previously demonstrated that phosphocellulose PC-microtubules existed in either the growing or the shortening phase and alternated quite frequently at steady-state conditions (dynamic instability). How does treadmilling relate to dynamic instability? An image recording of individual 3X-microtubules containing MAPs revealed that the microtubules undergo treadmilling and do not exhibit any dynamic instability. This evidence shows that MAPs suppress the dynamic instability of microtubules. That is, treadmilling can take place in the steady state only after microtubules have been stabilized by MAPs.

Animals↗
PubMed1988

Formation of membrane networks in vitro by kinesin-driven microtubule movement.

Certain intracellular organelles such as the endoplasmic reticulum (Terasaki, M., L. B. Chen, and K. Fujiwara. 1986. J. Cell Biol. 103:1557-1568) and lysosomes (Swanson, J., A. Bushnell, and S. C. Silverstein. Proc. Natl. Acad. Sci. USA. 84:1921-1925) form tubular networks that are closely aligned with microtubules. Here we describe the formation of polygonal networks composed of interconnected membrane tubules that occurs when a preparation of microtubule affinity-purified squid kinesin is combined with microtubules and ATP on a glass surface. The membrane, which is a minor contaminant in the microtubule affinity-purified kinesin preparation, binds to microtubules translocating along kinesin-coated glass surfaces. Force exerted by kinesin upon the microtubule is transmitted to the membrane and a tubular extension of the membrane is produced. As the membrane tubule elongates, membrane tension exerts an opposing force upon the translocating microtubule that can alter its direction of movement by dissociating or partially dissociating the microtubule from the kinesin-coated surface. Membrane tubules that come in contact appear to fuse with one another, and thus give rise to two-dimensional polygonal networks of tubules that have similar features to endoplasmic reticulum networks in cells. Artificial liposomes composed of dimyristoylphosphatidylcholine and yolk phosphatidylglycerol also form stable tubular structures when subjected to shear forces, but do not interact with microtubules or form polygonal networks, suggesting that such phenomena may require membrane-associated proteins. These findings indicate that kinesin generates sufficient force to form tubular membrane extensions in vitro and suggest that this microtubule-based motility protein may also be responsible for creating tubular membrane networks within cells.

Animals↗
PubMed1984

Transformation pathways of liposomes.

Liposomes undergoing transformation were observed by dark-field light microscopy in order to study the role of lipid in morphogenesis of biological vesicular structures. Liposomes were found to transform sequentially in a well-defined manner through one of several transformation pathways. A circular biconcave form was an initial shape in all the pathways and it transformed into a stable thin flexible filament or small spheres via a variety of regularly shaped vesicles which possessed geometrical symmetry. The transformation was reversible up to a certain point in each pathway. Osmotic pressure was found to be the driving force for the transformations. Biological membrane vesicles such as trypsinized red cell ghosts also transformed by similar pathways.

Cell Membrane↗
PubMed1982

Polymorphic transition in bacterial flagella.

The basic structure of the shaft of the bacterial flagellum is described and an account is given of work on polymorphism to date. The flagellum of wild-type Salmonella SJ670 has a characteristic left-hand helical form from pH 8 to pH 6, but undergoes two sharp transitions to other definite forms as the pH is lowered. The first transition, to a tight coil, can be followed by flow-birefringence. The flow-birefringence curve shows no evidence of hysteresis. Other details of the polymorphism, including the transitions which occur at high pH and at various KCl concentrations, are shown in a 'phase diagram'. Details of the transformation were studied by observing individual reconstituted flagella under dark-field light microscopy, while varying the bathing medium. Under some conditions, stress due to flow of the medium influences the transition. Under constant flow, alteration between two helical forms of opposite hand was sometimes observed. The two-state model is introduced to account for the helical structure and the polymorphism. Its prediction, that the two possible straight forms should have definite and opposite values of twist, is shown to agree with optical diffraction studies. Certain straight mutants are mentioned which show a novel feature in the diffraction pattern, probably due to a periodic perturbation of the helical lattice. The co-polymerisation of flagellins from straight mutants of the two different types yields a range of forms similar to the natural polymorphs. The implications of this finding are discussed.

Bacteria↗