Supramolecular structures in mycoplasmas

Mycoplasma pneumoniae cells treated with Triton X-100 showed a detergent-resistant cytoskeleton. This cytoskeleton consists of microfilaments which seem related to eukaryotic actin filaments, both morphologically and in some chemical properties, including specific staining by anti-actin antibodies and rhodamine-labeled phalloidin. The degree of homology, however, is still unclear. In motile cells the filaments form an irregular network in the cytoplasm of the cell body and a bundle in the frontal projection corresponding to the leading edge of the gliding cells. This particular arrangement may reflect different functions. The microfilaments could be isolated by differential centrifugation. Analysis of the microfilament fraction by SDS gel electrophoresis revealed five major polypeptide bands. One of these proteins, with a molecular weight of 42.5 kd, co-migrated with rabbit muscle actin. No filaments could be found in a nonmotile mutant, M-22.     

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara

Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin. To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB. Use of two fluorescent actin probes, fluorescein, isothiocyanate-heavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phallacidin (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB. Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment, NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions.     

Sarcomere structures in the rabbit psoas muscle as revealed by fluorescent analogs of phalloidin

Summary The fluorescent analogs of phalloidin (rhodamine-and fluorescein-phalloidin) bind tightly to the skinned fibres of rabbit psoas muscle at essentially the same sites as phalloidin and mainly stain the known regions of actin localization in the sarcomere: the thin filaments and Z bands. On both sides of the Z bands, unstained zones were observed, suggesting the presence of proteins tightly bound to the thin filaments. In myofibrils which are stretched to such an extent that the actin and myosin filaments do not overlap, stained bands could also be seen at the myosin-band border, which suggests the localization of actin at these sites.     

Noncooperative stabilization effect of phalloidin on ADP.BeFx- and ADP.AlF4-actin filaments

Actin plays important roles in eukaryotic cell motility. During actin polymerization, the actin-bound ATP is hydrolyzed to ADP and P i. We carried out differential scanning calorimetry experiments to characterize the cooperativity of the stabilizing effect of phalloidin on actin filaments in their ADP.P i state. The ADP.P i state was mimicked by using ADP.BeF x or ADP.AlF 4. The results showed that the binding of the nucleotide analogues or phalloidin stabilized the actin filaments to a similar extent when added separately. Phalloidin binding to ADP.BeF x- or ADP.AlF 4-actin filaments further stabilized them, indicating that the mechanism by which phalloidin and the nucleotide analogues affect the filament structure was different. The results also showed that the stabilization effect of phalloidin binding to ADP.BeF x or ADP.AlF 4-bound actin filaments was not cooperative. Since the effect of phalloidin binding was cooperative in the absence of these nucleotide analogues, these results suggest that the binding of ADP.BeF x or ADP.AlF 4 to the actin modified the protomer-protomer interactions along the actin filaments.     

Cytoplasmic actin and cochlear outer hair cell motility

Summary Isolated outer hair cells of the guinea pig lacking a cuticular plate and its associated infracuticular network retain the ability to shorten longitudinally and become thinner. Membrane ghosts lacking cytoplasm retain the cylindrical shape of the hair-cell, and although they do not shorten, they retain the ability to constrict and become thinner. These data suggest that cytoplasmic components are associated with outer hair-cell longitudinal shortening and that the lateral wall is responsible for maintaing cell shape and for constriction. Actin, a protein associated with the cytoskeleton and cell motility, is thought to be involved in outer hair-cell motility. To study its role, actin was localized in isolated outer hair cells by use of phalloidin labeled with fluorescein and antibodies against actin coupled to colloidal gold. In permeabilized guinea-pig hair cells stained with phalloidin, actin filaments are found along the lateral wall. In frozen-fixed hair cells actin filaments are distributed uniformly throughout the cytoplasm. Electron-microscopic studies show that antibodies label actin throughout the outer hair-cell body. Thus cytoplasmic actin filaments may provide the structural basis for the contraction-like events.     

黄蝉和姜花生殖细胞内肌动蛋白微丝的定位

用荧光标记的鬼笔碱染色,对离体的黄蝉和姜花的生殖细胞内肌动蛋白微丝的分布进行了研究,结果证明两种植物的生殖细胞内部都存在一个微丝网络,黄蝉生殖细胞的比姜花的简单,微丝束较粗。但姜花生殖细胞的网络微丝束比黄蝉的更紧密地环绕着核。用免疫荧光技术在黄蝉生殖细胞的分裂前期和中期,可以观察到一些微丝束的存在,但在分裂后期和末期细胞内的肌动蛋白则变为颗粒状。     

Molecular and biochemical characterization of a novel actin bundling protein in Acanthamoeba

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to 10 microM of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.     

Motility in Echinosphaerium nucleofilum. II. Cytoplasmic contractility and its molecular basis

Echinosphaerium nucleofilum exhibits at least three kinds of movement: locomotion by the bending and shortening of its many axopodia, feeding by means of food-cup pseudopodia formed from its cortical cytoplasm, and saltatory motion of cytoplasmic particles, especially in the cortex and axopodia. Since previously presented evidence indicated that the microtubular axoneme is not essential for particle motion, the cytoplasm was investigated for the possible existence of contractile behavior and for the possible presence of linear elements other than microtubules. Cytoplasm can be isolated in physiological media in which rigor, relaxation, and contraction can be induced, as in muscle, by manipulating the concentrations of calcium ions and magnesium-adenosine triphosphate. Contraction is initiated by calcium ions at concentrations above 2.4 times 10-minus 7 M. The rigor-to-relaxation transition occurs at subthreshold calcium concentrations on the addition of 10-minus 3 M ATP. Negatively stained preparations of isolated cytoplasm show two types of filaments: thin filaments identified as cytoplasmic actin by virtue of their binding heavy meromyosin from striated muscle in characteristic arrowhead arrays, and thicker filaments which do not strictly resemble myosin aggregates from muscle or amoeba but could conceivably by myosin aggregated in an unfamiliar form.     

Rhodamine-phalloidin and anti-tubulin antibody staining of spindle fibres that were irradiated with an ultraviolet microbeam

Summary We irradiated chromosomal spindle fibres in crane-fly spermatocytes with an ultraviolet microbeam of 270 nm wavelength light with total energies near those that cause actin filaments in myofibrils to depolymerize; after irradiation we stained the cells with rhodamine-labelled phalloidin and with anti-tubulin antibodies. In some cells, the irradiation reduced both phalloidin and tubulin staining of the chromosomal spindle fibres; in other cells, the irradiations reduced phalloidin staining but not tubulin staining; in yet other cells, the irradiations reduced tubulin staining but not phalloidin staining. In all irradiated cells in which phalloidin staining was reduced in the irradiated areas phalloidin staining also was reduced poleward from the irradiated areas. These results show that phalloidin staining of chromosomal spindle fibres is not dependent on the presence of kinetochore microtubules, and, therefore, that actin filaments are present in the spindle fibres in vivo. We suggest that actin filaments present in spindle fibres in vivo may be involved in causing chromosome movements during anaphase.     

Actin hydrophobic loop 262-274 and filament nucleation and elongation

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by ∼ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by ∼ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.     

Possible involvement of protein phosphorylation in the regulation of cytoplasmic organization and streaming in root hair cells as revealed by a protein phosphatase inhibitor, calyculin A

Summary The effects of a protein phosphatase inhibitor, calyculin A (CA), on cytoplasmic streaming and cytoplasmic organization were examined in root hair cells ofLimnobium stoloniferum. CA at concentrations higher than 50 nM inhibited cytoplasmic streaming and also induced remarkable morphological changes in the cytoplasm. The transvacuolar strands, in which actin filament bundles were oriented parallel to the long axis, disappeared and spherical cytoplasmic bodies emerged in the CA-treated cells. In these spherical bodies, actin filaments were present and the spherical bodies were connected to each other by thin strands of actin filaments. Upon CA removal, transvacuolar strands, in which actin filament bundles were aligned, and cytoplasmic streaming reappeared. A nonselective inhibitor for protein kinases, K-252a, delayed the inhibitory effect of CA on cytoplasmic streaming and suppressed the CA-induced formation of the spherical bodies. From these results, it is suggested that phosphatases sensitive to CA regulate cytoplasmic streaming and are involved in the organization of the cytoplasm in root hair cells.Abbreviations APW artificial pond water - CA calyculin A     

Phalloidin and tropomyosin do not prevent actin filament shortening by the 90 kD protein-actin complex from brain

Previously we reported the purification from bovine brain of the 90 kD protein-actin complex that shortens actin filaments. In the present work we study the effect of this complex on actin polymerized in the presence of phalloidin (PL) or tropomyosin (TM) which are known to stabilize actin filaments. The effect of the complex has been compared with that of cytochalasin D (CD), a fungal metabolite that also shortens actin filaments. Low shear viscosimetry and electron microscopy showed that PL or TM could not prevent the shortening of actin filaments in the presence of 90 kD protein-actin complex whereas they effectively protected actin filaments from shortening by CD. We conclude that the 90 kD protein-actin complex is a more potent filament-shortening factor than CD.     

Glutathionyl(cysteine-374) actin forms filaments of low mechanical stability

Rabbit muscle actin reacts with 2,4-dinitrophenylglutathionyldisulfide, forming a mixed disulfide in position 374. The product S-(cysteine-374)glutathionyl actin forms filaments which are easily disrupted under shearing stress. Even weak mechanical strain, as exerted, for example, during capillary viscometry or heating the solution to 37°C, leads to considerable breakage of these filaments. Because of spontaneous repair which consumes ATP, the mechanically broken filaments exhibit an approx. 6-fold enhanced steady-state ATPase activity as compared to normal F-actin. Monomers of glutathionyl actin have a reduced affinity for their bound nucleotide and a slightly increased critical concentration. Disruption of the filaments and enhanced ATPase activity are reversed by the addition of KCl or the mushroom toxin phalloidin. By the large stabilizing effects of KCl and phalloidin on glutathionyl actin filaments we propose glutathionyl actin as a tool for detecting filament-stabilizing agents and for studying the different mechanisms of filament stabilization     

Ultrastructural localization of RNA in the chromatoid bodies of undifferentiated cells (neoblasts) in planarians by the RNase–gold complex technique

Undifferentiated cells of planarians (Platyhelminthes, Turbellaria), also called neoblasts, are totipotent stem cells, which give rise to all differentiated cell types, while maintaining their own density by cell proliferation. Neoblasts are the only somatic cells of planarians bearing chromatoid bodies in their cytoplasm; these organelles disappear as differentiation takes place. Studies on germinal cells of several groups of organisms have shown that chromatoid bodies contain substantial amounts of RNA. To test its presence in neoblasts, we have used an RNase–gold technique. We found chromatoid bodies labeled with RNase–gold particles. Heterogeneity in the density of the label, may be correlated with the functionality and complexity of these organelles. The gold marker was also present over the nucleus and rough endoplasmic reticulum, but mitochondria, secretory granules, and the extracellular space were devoid of label. This specific localization of RNA in planarian chromatoid bodies supports earlier findings on germ cells and embryonic cells in a variety of organisms, indicating that chromatoid bodies are information-storage structures, essential during the process of cell differentiation. © 1993 Wiley-Liss, Inc.     

Organization of actin filaments in regenerating and outgrowing subprotoplasts from pollen tubes ofNicotiana tabacum L.

The dynamics of actin-filament organization in pollen-tube subprotoplasts ofNicotiana tabacum L. cv. Samsun during regeneration and outgrowth was examined using phalloidin probes and a non-fixation method. A succession of actin arrays was examined during subprotoplast regeneration that strongly resembled the actin dynamics described for developing microspores by Van Lammeren et al. (1989, Planta178, 531–539) and activated pollen by Tiwari and Polito (1988, Protoplasma147, 5–15). At the end of the succession the actin filaments often became extended between two opposite polar foci. The ordering of the cortical actin filaments reflected a polarity in the subprotoplasts which determined the plane of outgrowth. The site of outgrowth was often marked by a ring of actin filaments. As growth proceeded and tube-like structures were formed, the arrangement of cortical actin filaments was found to be transverse to the elongation axis. Since the patterns of actin distribution were identical in both caryoplasts and cytoplasts, it was concluded that the pollen-tube cytoplasm has the intrinsic capacity of reorganizing actin filaments and imposing polarity on the spherical subprotoplasts.     

Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.

We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-1 s-1) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate.     

Identification of actin filaments in the rhabdomeral microvilli of Drosophila photoreceptors

The phototransductive microvilli of arthropod photoreceptors each contain an axial cytoskeleton. The present study shows that actin filaments are a component of this cytoskeleton in Drosophila. Firstly, actin was detected in the rhabdomeral microvilli and in the subrhabdomeral cytoplasm by immunogold labeling with antiactin. Secondly, the rhabdomeres were labeled with phalloidin, indicating the presence of filamentous actin. Finally, the actin filaments were decorated with myosin subfragment-1. The characteristic arrowhead complex formed by subfragment-1 decoration points towards the base of the microvilli, so that the fast growing end of each filament is at the distal end of the microvillus, where it is embedded in a detergent-resistant cap. Each microvillus contains more than one actin filament. Decorated filaments extend the entire length of each microvillus and project into the subrhabdomeral cytoplasm. This organization is comparable to that of the actin filaments in intestinal brush border microvilli. Similar observations were made with the photoreceptor microvilli of the crayfish, Procambarus. Our results provide an indication as to how any myosin that is associated with the rhabdomeres might function.     

Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D. At prophase, several clear spots were observed at or near the kinetochores of the chromosomes. At anaphase, the actin filaments that appeared to be pulling chromosomes toward the division poles were demonstrated. However, as there was a slight possibility that these results might have been the artifacts of cytochalasin D treatment or the phalloidin staining, we analyzed the localization of actin filaments at the mitotic apparatus immunologically. We cloned a novel BY-2 -type actin cDNA and prepared a BY-2 actin antibody. The fluorescence of the anti-BY-2 actin antibody was clearly observed at the mitotic apparatus in both non-treated and cytochalasin D-treated BY-2 cells during mitosis. The facts that similar results were obtained in both actin staining with rhodamine-phalloidin and immunostaining with actin antibody strongly indicate the participation of actin in the organization of the spindle body or in the process of chromosome segregation. Furthermore, both filamentous actin and spindle bodies disappeared in the cells treated with propyzamide, which depolymerizes microtubules, supporting the notion that actin filaments are associated with microtubules organizing the spindle body.Hiroshi Yasuda and Katsuhiro Kanda contributed equally.     

THE CONTRACTILE BASIS OF AMOEBOID MOVEMENT : I. The Chemical Control of Motility in Isolated Cytoplasm

Cytoplasm has been isolated from single amoeba (Chaos carolinensis) in physiological solutions similar to rigor, contraction, and relaxation solutions designed to control the contractile state of vertebrate striated muscle. Contractions of the isolated cytoplasm are elicited by free calcium ion concentrations above ca. 7.0 x 10^-7 M. Amoeba cytoplasmic contractility has been cycled repeatedly through stabilized (rigor), contracted, and relaxed states by manipulating the exogenous free calcium and ATP concentrations. The transition from stabilized state to relaxed state was characterized by a loss of viscoelasticity which was monitored as changes in the capacity of the cytoplasm to exhibit strain birefringence when stretched. When the stabilized cytoplasm was stretched, birefringent fibrils were observed. Thin sections of those fibrils showed thick (150–250 Å) and thin (70 Å) filaments aligned parallel to the long axis of fibrils visible with the light microscope. Negatively stained cytoplasm treated with relaxation solution showed dissociated thick and thin filaments morphologically identical with myosin aggregates and purified actin, respectively, from vertebrate striated muscle. In the presence of threshold buffered free calcium, ATP, and magnesium ions, controlled localized contractions caused membrane-less pseudopodia to extend into the solution from the cytoplasmic mass. These experiments shed new light on the contractile basis of cytoplasmic streaming and pseudopod extension, the chemical control of contractility in the amoeba cytoplasm, the site of application of the motive force for amoeboid movement, and the nature of the rheological transformations associated with the circulation of cytoplasm in intact amoeba.     

激光原子力显微镜观察肌动蛋白体外自组织纤维结构多态性的初步研究

利用原子力显微镜(atomic force microscope,AFM)技术,研究了肌动蛋白体外通过自组织过程形成的纤维结构及其多态性。肌动蛋白在体外通过自组织过程能够聚合形成离散的树状分支的纤维丛和具有不同直径的长纤维等高级纤维结构,表现出明显的结构多态性;与微丝工具药物鬼笔环肽干预下自装配形成的主要由单根微丝和微丝束等纤维成份构成的连续网络结构明显不同。