"Cap" on the tip of Salmonella flagella

Flagellar filaments isolated intact from a Salmonella short-flagella mutant are unable to serve as nuclei for flagellin polymerization in vitro, whereas the filaments reconstructed in vitro from the mutant flagellin are able to do so. The inability of intact flagella to nucleate flagellin polymerization appears to be common to wild-type bacteria and thus suggests that the tip of intact flagella are generally inactivated or capped in vivo. Careful observations of the tips of intact flagella and reconstructed flagellar filaments of a wild-type species have revealed marked difference between them: the intact flagella usually have blunt ends, whereas reconstructed filaments have concave, "fish-tail" ends. Moreover, a thin structure is often observed attaching to the very end of the intact flagella. We suspect that this "capping" structure is essential to the elongation mechanism of flagellar filaments.     

Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear

Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed.     

辛德毕斯病毒装配及其6K蛋白与中间纤维的关系

用温和的选择性抽提方法与整装细胞电镜技术,DGD包埋-去包埋剂电镜技术相结合,对辛德毕斯病毒的装配与宿主细胞中间纤维的关系进行了探讨。电镜观察清晰地显示了病毒“装配中心”被中间纤维所网络,病毒的装配过程显然是以中间纤维为支架;正在装配的核壳体与已装配的核壳体紧密结合在中间纤维丝上。根据电镜照片分析,核壳体可能是沿中间纤维由装配中心向外扩散。应用人工合成6K蛋白所得抗体进行胶体金标记,证明辛德毕斯病毒非结构性6K蛋白也与中间纤维紧密结合。     

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan muscles: evidence that twitchin interacts with myosin, myorod, and paramyosin core and affects properties of actomyosin

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan smooth muscles postulates in vivo existence of twitchin links between thin and thick filaments that arise in a phosphorylation-dependent manner [N.S. Shelud'ko, G.G. Matusovskaya, T.V. Permyakova, O.S. Matusovsky, Arch. Biochem. Biophys. 432 (2004) 269-277]. In this paper, we proposed a scheme for a possible catch mechanism involving twitchin links and regulated thin filaments. The experimental evidence in support of the scheme is provided. It was found that twitchin can interact not only with mussel myosin and rabbit F-actin but also with the paramyosin core of thick filaments, myorod, mussel thin filaments, "natural" F-actin from mussel, and skeletal myosin from rabbit. No difference was revealed in binding of twitchin with mussel and rabbit myosin. The capability of twitchin to interact with all thick filament proteins suggests that putative twitchin links can be attached to any site of thick filaments. Addition of twitchin to a mixture of actin and paramyosin filaments, or to a mixture of Ca(2+)-regulated actin and myosin filaments under relaxing conditions caused in both cases similar changes in the optical properties of suspensions, indicating an interaction and aggregation of the filaments. The interaction of actin and myosin filaments in the presence of twitchin under relaxing conditions was not accompanied by an appreciable increase in the MgATPase activity. We suggest that in both cases aggregation of filaments was caused by formation of twitchin links between the filaments. We also demonstrate that native thin filaments from the catch muscle of the mussel Crenomytilus grayanus are Ca(2+)-regulated. Twitchin inhibits the ability of thin filaments to activate myosin MgATPase in the presence of Ca(2+). We suggest that twitchin inhibition of the actin-myosin interaction is due to twitchin-induced switching of the thin filaments to the inactive state.     

Structure of multinucleated smooth muscle cells of the ascidian Halocynthia roretzi

Summary Cells isolated from ascidian smooth muscle were about 1.5–2 mm in length. Each contained 20–40 nucle in proportion to cell length. The cytoplasm was characterized by the presence of an enormous quantity of glycogen particles, tubular elements of sarcoplasmic reticulum coupled to the cell membrane, and conspicuous contractile elements. Thick and thin filaments had diameters of about 14–16 nm and 6–7 nm, respectively. The population density of the thick filaments was much higher (mean 270/m² filament area) than in vertebrate smooth muscles. The ratio of thick to thin filaments was about 16. All the thick filaments were surrounded by a single row of 5–9 thin filaments forming a rosette, and cross-bridges with periodicities of 14.5 and 29 nm were found between them. The contractile apparatus consisted of numerous myofibrils which were arranged nearly along the cell axis and were separated from each other by a network of 10-nm filaments. The myofibrils further consisted of many irregularly arranged sarcomerelike structures, each of which was comprised of a small group of thick and thin filaments with attached dense bodies.     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

The effect of calcium activation of skinned fiber bundles on the structure of Limulus thick filaments

Here we present evidence that strongly suggests that the well-documented phenomenon of A-band shortening in Limulus telson muscle is activation dependent and reflects fragmentation of thick filaments at their ends. Calcium activation of detergent-skinned fiber bundles of Limulus telson muscle results in large decreases in A-band (from 5.1 to 3.3 microns) and thick filament (from 4.1 to 3.3 microns) lengths and the release of filament end fragments. In activated fibers, maintained stretched beyond overlap of thick and thin filaments, these end fragments are translocated to varying depths within the I-bands. Here they are closely associated with fine filamentous structures that also span the gap between A- and I-bands and attach to the distal one-third of the thick filaments. End-fragments are rarely, if ever, present in similarly stretched and skinned, but unstimulated fibers, although fine "gap filaments" persist. Negatively stained thick filaments, separated from skinned, calcium-activated, fiber bundles, allowed to shorten freely, are significantly shorter than those obtained from unstimulated fibers, but are identical to the latter with respect to both the surface helical array of myosin heads and diameters. Many end-fragments are present on grids containing thick filaments from activated fibers; few, if any, on those from unstimulated fibers. SDS-PAGE shows no evidence of proteolysis due to activation and demonstrates the presence of polypeptides with very high molecular weights in the preparations. We suggest that thick filament shortening is a direct result of activation in Limulus telson muscle and that it occurs largely by breakage within a defined distal region of each polar half of the filament. It is possible that at least some of the fine "gap filaments" are composed of a titin-like protein. They may move the activation-produced, fragmented ends of thick filaments to which they attach, into the I-bands by elastic recoil, in highly stretched fibers.     

Intermediate (skeletin) filaments in heart Purkinje fibers. A correlative morphological and biochemical identification with evidence of a cytoskeletal function

Cow Purkinje fibers contain a population of free cytoplasmic filaments which consistently differ in ultrastructural appearance from actin and myosin filaments, irrespective of preparation technique. The fixation and staining techniques, however, influenced the filament diameter, which was found to be 7.4--9.5 nm for filaments in plastic-embedded material, and 7.0 nm in cryo-sectioned material, thus intermediate as compared to actin and myosin filaments. Cross-sectional profiles suggested that the intermediate-sized filaments are composed of four subfilaments. To provide a basis for further biochemical investigations on the filaments, extraction procedures were carried out to remove other cell organelles. Electron microscopy showed that undulating bundles of intermediate filaments converging towards desmosomes still remained, after the extractions, together with Z-disk material. In spite of the extensive extraction, the shape of the individual cells and the assemblies of cell bundles remained intact. This confirms that the intermediate filaments of cow Purkinje fibers together with desmosomes do in fact have a cytoskeletal function. On account of (a) the cytoskeletal function of the filaments, (b) the similarities to the smooth muscle "100-A filament" protein subunit skeletin, and (c) the inadequate and confusing existing terminology, we suggest that the filaments be named "skeletin filaments."     

Environmentally induced formation of plasmatic filaments in the protonema ofCeratodon purpureus

Summary Incubation of spores ofCeratodon in 0.1% sucrose solution in darkness causes the formation of abundant filamentous bundles with an average width of 0.39±0.02 . The center-to-center distance of adjacent filaments is 216±1.4 Å. The individual filaments have an electron-dense tubular part with a diameter of 108±0.6 Å. Dictyosome-derived coated vesicles, which enlarge to ER-like coated tubuli, are always associated with the filaments. In younger cultures these electron-dense coated tubuli seem to spin filaments from their sides.     

The fine structure of chromatin in Paranosema grylli (Microsporida)

Nucleosomes were found for the first time in the nuclear chromatin of Microsporida--organisms known among the smallest eukaryotes on Earth. Chromatin of Paranosema grylli sporoplasm was studied by Miller's technique. On low ionic-strength cell spreads, this chromatin was represented by 10 nm nucleosome filaments, 20 nm filaments, and "smooth" (nucleosome-free) filaments of 3-4 nm in diameter. Nucleosome filaments display structural heterogeneity seen as irregular arrangement of nucleosome particles along the filament length. Different nucleosome filaments show 13-30 nucleosomes per 1 microm with the length of linker DNA ranging from 10 to 45 nm. The present results suggest that microsporidian chromatin is weakly condensed. Only lower-order chromatin packaging levels displayed some structural peculiarities.     

Kinetics of the formation and dissociation of actin filament branches mediated by Arp2/3 complex

The actin filament network at the leading edge of motile cells relies on localized branching by Arp2/3 complex from "mother" filaments growing near the plasma membrane. The nucleotide bound to the mother filaments (ATP, ADP and phosphate, or ADP) may influence the branch dynamics. To determine the effect of the nucleotide bound to the subunits of the mother filament on the formation and stability of branches, we compared the time courses of actin polymerization in bulk samples measured using the fluorescence of pyrene actin with observations of single filaments by total internal reflection fluorescence microscopy. Although the branch nucleation rate in bulk samples was nearly the same regardless of the nucleotide on the mother filaments, we observed fewer branches by microscopy on ADP-bound filaments than on ADP-P(i)-bound filaments. Observation of branches in the microscope depends on their binding to the slide. Since the probability that a branch binds to the slide is directly related to its lifetime, we used counts of branches to infer their rates of dissociation from mother filaments. We conclude that the nucleotide on the mother filament does not affect the initial branching event but that branches are an order of magnitude more stable on the sides of new ATP- or ADP-P(i) filaments than on ADP-actin filaments.     

Actin filaments in sensory hairs of inner ear receptor cells

Receptor cells in the ear are excited through the bending of sensory hairs which project in a bundle from their surface. The individual stereocilia of a bundle contain filaments about 5 nm in diameter. The identity of these filaments has been investigated in the crista ampullaris of the frog and guinea pig by a technique of decoration with subfragment-1 of myosin (S-1). After demembranation with Triton X-100 and incubation with S-1, "arrowhead" formation was observed along the filaments of the stereocilia and their rootlets and also along filaments in the cuticular plate inside the receptor cell. The distance between attached S-1 was 35 nm and arrowheads pointed in towards the cell soma. It is concluded that the filaments of stereocilia are composed of actin.     

THE ORGANIZATION OF CONTRACTILE FILAMENTS IN A MAMMALIAN SMOOTH MUSCLE

Ordered arrays of thin filaments (65 A diameter) along with other apparently random arrangements of thin and thick filaments (100–200 A diameter) are observed in contracted guinea pig taenia coli rapidly fixed in glutaraldehyde. The thin-filament arrays vary from a few to more than 100 filaments in each array. The arrays are scattered among isolated thin and thick filaments. Some arrays are regular such as hexagonal; other arrays tend to be circular. However, few examples of rosettes with regular arrangements of thin filaments surrounding thick filaments are seen. Optical transforms of electron micrographs of thin-filament arrays give a nearest-neighbor spacing of the thin filaments in agreement with the "actin" filament spacing from x-ray diffraction experiments. Many thick filaments are closely associated with thin-filament arrays. Some thick filaments are hollow circles, although triangular shapes are also found. Thin-filament arrays and thick filaments extend into the cell for distances of at least a micron. Partially relaxed taenia coli shows thin-filament arrays but few thick filaments. The suggestion that thick filaments aggregate prior to contraction and disaggregate during relaxation is promoted by these observations. The results suggest that a sliding filament mechanism operates in smooth muscle as well as in striated muscle.     

Substructure of sidearms on squid axoplasmic vesicles and microtubules visualized by negative contrast electron microscopy

We present a high-resolution electron microscopic study of the sidearms on microtubules and vesicles that are suggested to form the crossbridges which produce the microtubule-based vesicle transport in squid axoplasm. The sidearms were found attached to the surfaces of the anterogradely transported vesicles in the presence of ATP. These sidearms were made of one to three filaments of uniform diameter. Each filament measured 5-6 nm in width and 30-35 nm in length. The filaments in some of the sidearms had splayed apart by pivoting at their base, thereby assuming a "V" shape. The spread configuration illustrated the independence of the individual filaments. The filaments in other sidearms were closely spaced and oriented parallel to each other, a pattern called the compact configuration. In axoplasmic buffer containing AMP-PNP, structures indistinguishable from the filaments of the sidearms on the vesicles were observed attached to microtubules. Pairs of filaments, thought to represent the basic functional unit, were observed attached to adjacent protofilaments of the microtubules by their distal tips. These data support a model of vesicle movement in which a pair of filaments within a sidearm forms two crossbridges and moves a vesicle by "walking" along the protofilaments of the microtubule.     

Electron microscopical observations on the brush border of proximal tubule cells of mammalian kidney

Summary The ultrastructure of the plasma membrane and the core of microvilli of proximal tubule cells has been investigated by electron microscopy using sectioned and negatively stained material. By the technique of negative staining, a particulated coat is disclosed on the outside of the plasma membrane of microvilli of brush borders isolated from rat, rabbit and ox. This coat is composed of 30 to 60 Å particles and is 150 to 300 Å thick and appears to be a distinguishing feature for the luminal plasma membrane (brush border) of proximal tubule cells. The plasma membrane of the basal part of tubule cells is found to be smooth. By thin sectioning, an axial bundle of 50 to 70 Å diameter filaments regularly arranged in an 1+6 configuration, one axially located filament being surrounded by a ring of six, is disclosed. The distance from the ring of filaments to the inner surface of the plasma membrane is 250–300 Å, the diameter of the ring 300 Å and the center-to-center distance between filaments 120 Å. Negative staining also discloses 60 Å filaments in microvilli of isolated brush borders. Broken off, single microvilli (fingerstalls) are observed with thin filaments projecting from their broken ends. Filaments up to 1 in length are seen. At high magnification, the filaments appear beaded and show strong resemblance with actin filaments isolated from skeletal muscle. Based on present evidence, it is postulated that microvilli constituting renal brush borders possess contractile properties, which may play a role in the absorption process operating at the luminal part of the cells.The authors are indebted to Miss Kirsten Sjöberg for skilled technical assistance, and to the Danish State Research Foundation and the Tuborg Foundation for financial support.     

Assembly of smooth muscle myosin into side-polar filaments

The in vitro assembly of myosin purified from calf aorta muscle has been studied by electron microscopy. Two types of filament are formed: short bipolar filament similar to those formed from skeletal muscle myosin, and longer "side-polar" filaments having cross bridges with a single polarity along the entire length of one side and the opposite polarity along the other side. Unlike the case with skeletal myosin filaments, antiparallel interactions between myosin molecules occur along the whole length of side-polar filaments. The side-polar structure may be related to the in vivo form of myosin in vertebrate smooth muscle.     

Intermediate-sized filaments present in Sertoli cells are of the vimentin type.

The cytoplasmic structure of Sertoli cells of rat testes has been studied by electron microscopy of ultrathin sections. Sertoli cells contain numerous intermediate-sized (7-11 nm) filaments which form a meshwork extending throughout the whole cytoplasm. Often the frequency of such filaments appears especially high in juxtanuclear and cortical regions, including the apical recesses containing the spermatids. Examination of frozen sections of testes by indirect immunofluorescence microscopy using guinea pig antibodies to prekeratin and vimentin has shown the absence of intermediate-sized filaments of the cytokeratin type in all cells of the testes but the presence of filaments of the vimentin type in Sertoli cells as well as in cells of the interstitial space. These results show that the intermediate-sized filaments, abundant in Sertoli cells, are of the vimentin type. In addition we conclude that the "germ epithelium" differs from others true epithelia by the absence of cytokeratin filaments and typical desmosomes and, in Sertoli cells, the presence of vimentin filaments, suggestive of a mesenchymal character or derivation.     

Rapid Severing and Motility of Chloroplast-Actin Filaments Are Required for the Chloroplast Avoidance Response in Arabidopsis

Phototropins (phot1 and phot2 in Arabidopsis thaliana) relay blue light intensity information to the chloroplasts, which move toward weak light (the accumulation response) and away from strong light (the avoidance response). Chloroplast-actin (cp-actin) filaments are vital for mediating these chloroplast photorelocation movements. In this report, we examine in detail the cp-actin filament dynamics by which the chloroplast avoidance response is regulated. Although stochastic dynamics of cortical actin fragments are observed on the chloroplasts, the basic mechanisms underlying the disappearance (including severing and turnover) of the cp-actin filaments are regulated differently from those of cortical actin filaments. phot2 plays a pivotal role in the strong blue light–induced severing and random motility of cp-actin filaments, processes that are therefore essential for asymmetric cp-actin formation for the avoidance response. In addition, phot2 functions in the bundling of cp-actin filaments that is induced by dark incubation. By contrast, the function of phot1 is dispensable for these responses. Our findings suggest that phot2 is the primary photoreceptor involved in the rapid reorganization of cp-actin filaments that allows chloroplasts to change direction rapidly and control the velocity of the avoidance movement according to the light’s intensity and position.     

Polytene chromosome structure at the submicroscopic level

Nuclear DNA obtained by SDS treatment or phenol extraction of isolated polytene salivary gland nuclei of D. melanogaster and D. hydei was investigated electron-microscopically. All preparations contained only linear doublestranded DNA filaments of various length. The mean length of a sample of 52 DNA filaments of D. melanogaster produced by SDS treatment was 37.3 . For D. hydei a mean length of 24.2 was established on account of a sample of 51 filaments obtained by SDS treatment. In samples obtained by phenol extraction a mean length of 23.8 (26 filaments) was found. Pronase digestion following SDS treatment gave a mean length of 29.1 for D. melanogaster (46 filaments) and of 17.1 for D. hydei (57 filaments). — The mean length of DNA filaments from D. hydei sperm was 21.5 on the basis of 25 filaments measured. The length distribution of the DNA of the samples of filaments measured varied. Preparations of single-stranded DNA obtained by heat denaturation of samples of D. hydei nuclear DNA revealed very long filaments. An obvious increase in the number of filaments shorter than 30 as compared with double-stranded DNA could not be established.     

The pattern of organization of intermediate filaments and their asymmetrical association with dense bodies in smooth muscle of an ascidian Halocynthia roretzi

Summary An extensive network of intermediate filaments that interconnected cytoplasmic dense bodies and connected the dense bodies to the cell surface was revealed in double-fixed, tannic acid-stained preparations of ascidian smooth muscle. The filament network ran through spaces in the continuous network of myofibrils, connecting them longitudinally, obliquely and transversely to form an intimately associated, dual network. In their transverse passage, the intermediate filaments ran across myofibrils along I-zones exclusively, interconnecting successive dense bodies.The pattern of attachment of intermediate filaments to dense bodies was predominantly one-sided. The filaments, which themselves were not incorporated into the contractile apparatus, remained folded or unfolded between myofibrils and between sarcomere-like structures in synchrony with the contraction-relaxation cycles.These results suggest that the intermediate filaments mechanically maintain the organization and arrangement of myofibrils via an intimate association with the myofibrils in the regions of the dense bodies, in such a way that the filaments do not impede muscle function.Based on these observations, a new model for the network of intermediate filaments in smooth muscle cells is proposed.