Ritsu Kamiya

Formation of spherical and rod-like micelles of cetyltrimethylammonium bromide in aqueous NaBr solutions

Abstract Static light scattering from aqueous NaBr solutions of cetyltrimethylammonium bromide (CTAB) has been measured at 35°C with 488-nm laser light, and the molecular weight, radius of gyration, and the other parameters of its micelles have been evaluated. In water, spherical micelles are formed above a critical micelle concentration, 0.04 × 10 −2 g cm −3 , and each micelle consists of 91 monomers of CTAB. In aqueous solutions having NaBr concentrations higher than 0.06 M , rod-like micelles are formed, besides spherical micelles. The molecular weight of rod-like micelles increases rapidly with increasing NaBr concentration. The rod-like micelle formed in 0.5 M NaBr has molecular weight as high as 3,470,000, and is considerably flexible. The persistence length of micelle is evaluated to be 44 nm. Electron micrographic observations have been carried out for specimens prepared from CTAB solutions in water and in 0.5 M NaBr at 35°C. A specimen prepared from aqueous solution of CTAB and negatively stained with uranyl acetate displays many globular images, which have a uniform diameter around 5–6 nm and can be assigned to spherical micelles. A specimen from 0.5 M NaBr solution shows tortuous thread-like images with a uniform diameter of 4.5–6 nm, which can be regarded as flexible rod-like micelles. Thus, the size and shape of spherical and rod-like micelles of CTAB observed by the light-scattering measurement are confirmed by electron microscopy.

Helical transformations of Salmonella flagella in vitro.

Abstract Helical transformations of reconstituted Salmonella flagella were visualized by dark-field light microscopy. Flagella from SJ670 strain were lefthanded helices with a pitch of 2.3 μm at neutral pH. When, however, the pH of the solution was lowered to 4.7, they were discontinouously transformed into close-coils with a pitch of 0.5 μm and a diameter of 1.2 μm, and a further lowering of the pH converted these coiled flagella into so-called curly ones, righthanded helices with a pitch of 1.1 μm. The transformation was rapid and reversible. Two other kinds of flagella (SJ25 and SJ30) also underwent such polymorphic conversions. Thus pH is an important factor in the control of flagellar transformation. As a result of the transformation, the degree of flow birefringence of a flagellar solution depends strongly on pH. Measurements of this parameter were useful in the study of the effects on the transformation of salt concentration and temperature.

Microtubule translocation caused by three subspecies of inner-arm dynein from Chlamydomonas flagella

To help understand the function of inner‐arm dynein in nagellar motility, dynein samples from an outer arm‐missing mutant of Chlamydomonas (odal) were examined for the ability to translocate microtubules in vitro. High‐salt extract ofaxonemes containing inner‐arm dynein was separated by ion‐exchange chromatography into 7 peak fractions with ATPase activities. Of these, three fractions containing different sets of dynein heavy chains translocated microtubules. The maximal velocities were all between 3 and 5 , which were comparable to the microtubule sliding rate in disintegrating oda axonemes.

Elastic properties of bacterial flagellar filaments: II. Determination of the modulus of rigidity

Elongation of a helical bacterial flagellar filament subjected to fluid flow was calculated on the assumption that one end of the filament is firmly attached to a substratum. It was found that the quantity [E(d/2 pi r)2 + 2 mu] could be determined by measuring the elongation at various flow rates, where E is Youngs modulus, mu the modulus of rigidity, r the radius of the helix, and d the helical pitch. Experiments were carried out to determine the above quantity for Salmonella flagellar filaments assuming a close-coil form. Because the above quantity is almost equal to 2 mu for a helical form with a large radius/pitch ratio, we were able to determine the modulus of rigidity for this kind of flagellar filament from plots of elongation vs. flow rates. The modulus of rigidity was determined to be about 1 X 10(11) dyn/cm2, i.e., 2 orders of magnitude larger than the previously estimated value.

Shape of microtubules in solutions.

Abstract Although several authors have presented dark-field micrographs of axonemes or of outer doublet microtubules from sperm tails, singlet microtubules have not been observed individually by light microscopy. This study demonstrates the technical possibility of observing individual microtubules by dark-field microscopy. While polymerized brain microtubules were always observed to be straight, the outer doublet microtubules from sperm tails assumed a coiled form, as demonstrated by Summers & Gibbons [1] and Zobel [2]. The coiled conformation of the outer doublets was found to be a left-handed helix, with a nearly uniform diameter.

Total reconstitution of Salmonella flagellar filaments from hook and purified flagellin and hook-associated proteins in vitro

Salmonella flagellar filaments comprise the following distinct parts connected in series: a curved hook composed of a single kind of subunit (hook protein); two short segments made up of hook-associated proteins (HAP1 and HAP3); a long helical filament composed of flagellin; and a cap composed of HAP2. In this study, a procedure was developed to isolate HAPs from the culture medium of a short-flagella mutant. We demonstrate that hook-filament complex can be formed in vitro by sequential addition of HAP1, HAP3 and flagellin to hook fragments.

Transformation of straight flagella and recovery of motility in a mutant Escherichia coli

Abstract The non-motile strain W3623 ha-177 of Escherichia coli (Kondoh & Ozeki, 1976) is known to produce straight flagella as a result of a mutation in the structural gene for the flagellin. Under physiological conditions, however, flagella of this mutant undergo straight-to-helical transformation with small changes of pH. Evidence for this came from dark-field light microscope observations of reconstituted flagella. At pH values lower than 6.6 in the presence of 0.1 m -NaCl, the flagella were straight. When, however, the pH was raised above 7.3, they were transformed into left-handed helices with a pitch of 2.05 μm. The transformation was rapid and reversible. In the pH range between 6.6 and 7.3, straight and transformed flagella co-existed but no stable forms other than the two were found. Bacterial motility also depended on the pH of the medium: at pH values above 7.0, bacteria swam by means of the transformed flagella. Therefore, helically transformed flagella of the mutant strain were similar in morphology and function to normal-type flagella of the parent strain. The significance of this similarity is discussed on the basis of general considerations of polymorphism in bacterial flagella.

Structural similarity between actin bundles from characean algal cells and sea urchin oocytes

Abstract The structure of actin bundles from internodal cells of Chara australis , an algal plant, was studied by electron microscopy of negatively stained specimens and optical diffraction. Gently prepared bundles revealed paracrystalline structures resembling the Mg 2+ -induced paracrystals of rabbit skeletal muscle actin (Hanson, 1968). In addition, the algal actin bundles sometimes had transverse striations at intervals of about 130 A, as has been observed in actin bundles from sea urchin eggs (DeRosier et al. , 1977; Spudich & Amos, 1979) and sea urchin coelomocytes (De Rosier & Edds, 1980; Otto & Bryan, 1981). This finding suggests that a common mechanism might be working in a variety of cells to organize actin filaments into functional bundles.

Formation of a flagella-like but straight polymer of Salmonella flagellin

Abstract Salmonella flagellin (monomer) polymerizes into flagellar filaments with the addition of (NH4)2SO4 (Ada et al., 1963; Wakabayashi et al., 1969). When, however, this process was allowed to take place in the presence of a high concentration of NaCl (about 1.5 m ), the product consisted of flagella-like but straight filaments. This phenomenon was common to four kinds of flagellins derived from strains SJ670, SJ25, SJ30 and SJ814. When the straight filament, suspended in 0.15 m -NaCl, was heated, it depolymerized to the monomer, which could in turn be polymerized into flagellar filaments by the addition of short fragments of flagella at room temperature. Nevertheless, attempts at direct transformation between the two types of filaments were unsuccessful. In 0.15 m -NaCl, straight filaments prepared from the four kinds of flagellins had markedly different heat stabilities, which were much lower than that of any kind of flagella. When monomeric flagellin dissolved in 3.5 m -NaCl was seeded with short fragments of straight filaments, the monomer polymerized onto the ends of the short fragments, which consequently grew into long straight filaments. In this type of experiment, monomers and seeds derived from the four strains were able to interact in any combination, suggesting that straight filaments consisting of the four kinds of flagellins have the same substructures. Whether the concentration of added NaCl was 0.15 m or 3.5 m , fragments of flagella (or straight filaments) were unable to act as seeds for the formation of straight filaments (or flagellar filaments). From this and other experimental results, it was concluded that in the two filamentous structures, flagellin molecules may be packed in different ways.

Actin paracrystal in the acrosome of newt sperm

Abstract When a newt sperm-head was treated with trypsin and DNase, an arrow-like thin rod was revealed. This rod presumably corresponds to the ‘perforatorium’ described by Picheral [1, 2] in Pleurodele sperm. It consisted of one apical and one caudal part. In the apical part there appeared to be an envelope with a 530 A structural repeat, inside which coursed a filament bundle, presumably identical with that in the caudal part. In the caudal part, a characteristic filament bundle, quite similar to the paracrystal of rabbit skeletal actin [3], was observed after extensive treatment with trypsin. The optical diffraction pattern of this bundle indicates that it has the same helical symmetry as that of rabbit skeletal actin [4] but slightly different from that of the acrosomal process of Limulus sperm [5]. The diffraction pattern frequently has a strong meridional reflection at about (27 A)−1, which is usually observed only with low intensity in the actin paracrystals. This fact suggests that the structural unit in the bundle has a shape considerably different from that of the usual G-actin.