Hirokazu Hotani

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.

Micro-video study of moving bacterial flagellar filaments: III. Cyclic transformation induced by mechanical force

Abstract Dynamic images of isolated bacterial flagellar filaments undergoing cyclic transformations were recorded by dark-field light microscopy and an ultrasensitive video camera. Flagellar filaments derived from Salmonella SJ25 sometimes stick to a glass surface by short segments near one end. When such a filament, which is a left-handed helix, was subjected to a steady flow of a viscous solution of methylcellulose, its free portion was found to transform cyclically between left-handed (normal) and right-handed (curly or semi-coiled) helical forms. The transformations did not occur simultaneously throughout the whole length of a filament, but occurred at a transition point, which proceeded along the filament. Each transformation process consisted of three phases: initiation, growth and travel. The magnitudes of the mechanical forces, torque and tension, which were generated on a filament by the viscous flow, were obtained by quantitative hydrodynamic analyses. The torque was found responsible for initiating the transformation. The critical magnitude of torque required to induce the normal to semi-coiled transformation was −11 × 10−19 N m and that for the reverse transformation from the semi-coiled to the normal form was 4 × 10−19 N m. Therefore, the filaments showed the characteristics of hysteresis during the cyclic transformation. New types of unstable right-handed helical forms (medium and large) were also induced by mechanical force.

Light microscope study of mixed helices in reconstituted Salmonella flagella

Abstract In this morphological study of bacterial flagella, single flagellar filaments in solutions were photographed by dark-field light microscopy to determine parameters to describe their intact shapes. First, I measured pitches of helices I, II and III assumed by copolymers of flagellins from Salmonella strains SJ25 and SJ814 (Asakura & Iino, 1972), and combined the data with electron microscopic data of the contour length per period of each filament to calculate the pitch angles of the three helices. (The pitch angle is the angle between a tangent to the filament and the helical axis.) Secondly, filaments which consisted of two blocks assuming different helices were prepared by two-step copolymerization of SJ25 and SJ814 flagellins and the configurations of these mixed-type filaments were examined. In filaments of any mixed type, the axes of the constituent blocks were oriented at the same angle, called the “block angle” Ψ. This angle was found to be approximated by Ψ = 180 ° − ¦θ1 − θ2¦, where θ1 and θ2 are the pitch angles of the mixed helices. On the basis of this finding, the morphology of mixed-type filaments is discussed.

Flagellin from Escherichia coli K12: Polymerization and molecular weight in comparison with Salmonella flagellins

Abstract 1. 1. The isolation and purification procedures of flagellar filaments from Escherichia coli K12 are described. E. coli flagellar filaments were dissociated either by heat or acid treatment, and were reconstituted by adding seed or by salting out. The polymerization was much slower than that of Salmonella. 2. 2. Molecular weight of E. coli flagellin was determined by sodium dodecyl-sulphate-polyacrylamide gel electrophoresis in comparison with Salmonella flagellins of various strains: E. coli flagellin has an apparent molecular weight of about 60 000 and Salmonella flagellins have lower molecular weights ranging from 51 000 (i) to 57 000 (g, m and g, p). Sedimentation of flagellins in a sucrose gradient was consistent with the above results. 3. 3. In spite of the relatively large difference in the molecular weights, E. coli and Salmonella flagellins copolymerized without any deformation in the overall shape. 4. 4. The kinetic parameters (V and Km) of the polymerization of E. coli and Salmonella (1, 2) flagellins were determined: they depend on both the kind of monomer and seed.

Micro-video study of moving bacterial flagellar filaments: I. Passive rotation by hydro dynamic force in vitro

Abstract Moving images of reconstituted single bacterial flagellar filaments in a dark-field microscope were recorded by an ultrasensitive video camera, and then transferred to 16 mm cinefilm for quantitative analysis of the dynamic properties of the filaments. Flagellar filaments are found to attach to a glass surface at only one end (the H -end). When attached helical filaments were subjected to viscous flow of methylcellulose solution, they rotated as a result of the hydrodynamic torque generated. Occasionally, two filaments associated into a bundle and rotated coordinately in the viscous flow, even though each filament was separately attached to the glass surface. In addition, we have observed partly rotating filaments which consisted of two portions, the rotating portion being connected end-to-end to the non-rotating portion. The magnitude of the hydrodynamic torque depended on the rotational friction which was determined by the manner of attachment. Based on hydrodynamic calculations, values of −5 × 10−12 and −1 × 10−13 dyne cm were obtained for the average torque for rotating filaments on glass and partly rotating filaments, respectively, in viscous fluid at a flow rate of 15 μm/s.

Micro-video study of moving bacterial flagellar filaments. II. Polymorphic transition in alcohol.

Abstract Isolated bacterial flagellar filaments were found to display several distinct types of helix depending on the concentrations of alcohol. Transformations of helical type were achieved by manipulating the concentration of ethylene glycol or methanol in the specimen of a dark-field light microscope. The dynamic images of a variety of filaments undergoing transformation were recorded by use of an ultrasensitive video camera. Each transition began at the end of, and then proceeded along the filament.

Growth-saturation in vitro of Salmonella flagella.

Abstract At physiological ionic strength and pH, short fragments of Salmonella flagella (seeds) grow longer in the presence of monomeric flagellin and there exists a one-to-one correspondence between the seeds and fully grown filaments (Asakura et al. , 1964). In this study it was shown that when monomer and seed derived from a preparation of flagella (strain SJ25) were mixed in a protein ratio r larger than 20, the filaments stopped growing or became inactive for a long period of time, and the average length of inactive filaments was independent of the value of r . The phenomenon was called growth-saturation . The antibody-labelling technique (Asakura et al. , 1968) made it possible to show that, though active filaments having equal lengths grew at various rates ranging between 0 and 0.16 μm/min, the average value of growth rate depended little on length. On the other hand, it was found that the proportion of inactive filament in the total filament increased rapidly as the value of r was increased continuously from 0 to 10. The dependence of the proportion of inactive filament on r suggested that filaments became inactive with a probability independent of their length. The rate of inactivation (or the probability with which a filament becomes inactive during growth by a unit length) had various values when different preparations of flagella were used as starting materials. The distribution of length for an assembly of inactive filaments was determined by low-magnification electron microscopy. The result could be approximated by an exponential distribution: the number-average length was 4.54 μm and the rate of inactivation was 0.224 μm −1 .

Biochemical evidence for identical primary structure of P-filament and flagellin

Abstract 1. 1. A membrane fraction derived from deflagellated cells of Salmonella promotes polymerization of flagellin into “P-filaments” (ref. 1). The present study was undertaken to investigate whether or not changes in primary structure of flagellin are prerequisite to this polymerization. 2. 2. In this study we used the “soluble fraction” instead of “membrane fraction” for the initiation of polymerization2. Isotope experiments showed that with addition of the soluble fraction more than 90% of the total flagellin polymerizes into P-filaments. 3. 3. Quantitative analysis of amino terminal residues showed that polymerization is not associated with cleavage of peptide bonds in flagellin. 4. 4. P-filament and intact flagellin were compared as to amino acid composition, amino and carboxyl terminal residues and tryptic peptide map; in these respects they were indistinguishable. 5. 5. From these experimental results, it was concluded that the primary structure of flagellin remains unchanged after polymerization into P-filament.

Micro-video study of discontinuous growth of bacterial flagellar filaments in vitro.

Abstract In a microscope slide preparation, monomeric flagellins were found to polymerize into flagellar filaments spontaneously, without addition of seeds. Dynamic images of individual growing filaments in a dark-field light microscope were recorded throughout their growth by an ultrasensitive video camera. Each filament had its own unique growth curve. The growth curves consisted of two kinds of discrete phase; namely, the elongation and the rest phase. In the former, a filament elongates at a constant rate, fairly similar among all filaments. In the latter, elongation stops completely. Each filament exists in either of the two phases and alternates between them in a stochastic manner. A mean elongation rate of 89 + 15 nm per minute was obtained at the flagellin concentration of 2 mg/ml, for filaments in the elongation phase.

Unidirectional melting of Salmonella flagella in vitro

Abstract Asakura et al . (1968) have shown that when short fragments of Salmonella flagella (seeds) are added, at room tempetature, to a solution containing monomeric flagellin at physiological ionic strength and pH, each fragment grows longer at one of the two ends. This end and the opposite end were named T and H, respectively. In the present study we investigated the direction of the reversed process, melting, which may be brought about by simple heating. By the method of cross-mixing of monomers and seeds derived from strains SJ25 and SJ670, two types of block copolymers were prepared: the first type, ( n-i ) † , was made by mixing n -seed and i -monomer at a ratio of 1:6, and the second type, ( i-n ), was made by mixing i -seed and i -monomer first and secondly n -monomer at a ratio of 1:5:1. Both preparations were heated at 50 °C for four minutes for partial melting, and the products were labelled with antibody against n -flagella and observed by electron microscopy. It was found that the proportion of block copolymers in ( n-i ) was changed little by heating, while in ( i-n ), the proportion of block copolymers decreased considerably and that of homogeneous i -polymers increased to a great extent. Before and after heating, we measured lengths of the n and i -blocks of copolymers and homogeneous n and i -polymers, which were contained in the preparations. On the basis of the statistical analyses of the data with the aid of computer simulations, it was concluded that, upon heating, flagellar polymers melt almost exclusively at their T-ends. Using 35 S-labelled n -flagella ( n ∗ -flagella), we prepared homopolymers ( n ∗ -n ) and ( n-n ∗ ) which are analogous to ( n-i ) and ( i-n ). Both homopolymers were heated at 50 °C and the release of n ∗ -monomer from each homopolymer was followed. The results obtained supported the above conclusion.