Unidirectional sliding of myosin filaments along the bundle of F-actin filaments spontaneously formed during superprecipitation

I reported previously (Higashi-Fujime, S., 1982, Cold Spring Harbor Symp. Quant. Biol., 46:69-75) that active movements of fibrils composed of F-actin and myosin filaments occurred after superprecipitation in the presence of ATP at low ionic strengths. When the concentration of MgCl2 in the medium used in the above experiment was raised to 20-26 mM, bundles of F-actin filaments, in addition to large precipitates, were formed spontaneously both during and after superprecipitation. Along these bundles, many myosin filaments were observed to slide unidirectionally and successively through the bundle, from one end to the other. The sliding of myosin filaments continued for approximately 1 h at room temperature at a mean rate of 6.0 micron/s, as long as ATP remained in the medium. By electron microscopy, it was found that most F-actin filaments decorated with heavy meromyosin pointed to the same direction in the bundle. Myosin filaments moved actively not only along the F-actin bundle but also in the medium. Such movement probably occurred along F-actin filaments that did not form the bundle but were dispersed in the medium, although dispersed F-actin filaments were not visible under the microscope. In this case, myosin filament could have moved in a reverse direction, changing from one F-actin filament to the other. These results suggested that the direction of movement of myosin filament, which has a bipolar structure and the potentiality to move in both directions, was determined by the polarity of F-actin filament in action.     

Aluminum toxicity studies in Vaucheria longicaulis var. macounii (Xanthophyta, Tribophyceae). II. Effects on the F-actin array

In this study, we test the hypothesis that exposure to environmentally significant concentrations of aluminum (Al, 80 μM) causes the microfilament array of Vaucheria longicaulis var. macounii vegetative filaments to become fragmented and disorganized. Changes in F-actin organization following treatment of vegetative filaments by Al are examined using vital staining with fluorescein phalloidin. In the cortical cytoplasm of the apical zone of pH 7.5 and pH 4.5 control cells, axially aligned bundles of F-actin lead to a region of diffuse, brightly stained material. Dimly stained focal masses are noted deeper in the cytoplasm of the apical zone whereas they are absent from the zone of vacuolation. The F-actin array is visualized in the cortical cytoplasm of the region of the cell, distal to the apical tip, which exhibits vigorous cytoplasmic streaming (zone of vacuolation) as long, axially aligned bundles with which chloroplasts and mitochondria associate. Thirty minutes following treatment with aluminum, and for the next 8-16 h, the F-actin array is progressively disorganized. The longitudinally aligned F-actin array becomes fragmented. Aggregates of F-actin, such as short rods, amorphous and stellate F-actin focal masses, curved F-actin bundles and F-actin rings replace the control array. Each of these structures may occur in association with chloroplasts or independently with no apparent association with organelles. Images are recorded which indicate that F-actin rings not associated with organelles may self-assemble by successive bundling of F-actin fragments. The fragmentation and bundling of F-actin in cells of V. longicaulis upon treatment with aluminum resembles those reported after diverse forms of cell disturbance and supports the hypothesis that aluminum-induced changes in the F-actin array may be a calcium-mediated response to stress.     

Protein phosphorylation regulates actomyosin-driven vesicle movement in cell extracts isolated from the green algae,Chara corallina

In Characean cells endoplasmic streaming stops upon membrane depolarization accompanied by Ca(2+) entry. We investigated the mechanism of this cessation of endoplasmic streaming by reconstituting the vesicle movement in vitro. In a living cell of Chara corallina, there are a number of vesicles moving along actin cables. Vesicles in the endoplasm squeezed out of the cell into a medium containing Mg-ATP showed directional movements under a dark field microscope. When the extracted endoplasm was treated with 20 nM okadaic acid, vesicles showed only movements like the Brownian motion. When it was treated with 50 nM staurosporine, directional movements of vesicles were activated. These movements were analyzed by image processing of videomicroscopic records. Vesicle movements along F-actin filaments were also observed by merging both images of the same field by dark field microscopy and fluorescence microscopy, indicating that myosin on the vesicle surface was responsible for vesicle movements. We also examined the effects of okadaic acid and staurosporine on in vitro sliding of F-actin on Chara myosin. When Chara myosin was treated with 20 nM okadaic acid in the cell extract, the number of sliding F-actin filaments was greatly reduced. In contrast, it increased when Chara myosin was treated with 50 nM staurosporine. In addition, Chara myosin treated with protein kinase C greatly diminished its motility. These results suggest that inactivation of Chara myosin via its phosphorylation is responsible for cessation of endoplasmic streaming.     

Filaments associated with the endoplasmic reticulum in the streaming cytoplasm of Chara corallina

Perfused Chara cells capable of resuming ATP-dependent cytoplasmic streaming in low free Ca++ solutions have been examined by electron microscopy for myosin-like filaments. Filaments 44 nm in diameter and up to 3 micron in length have been found associated with the endoplasmic reticulum that along with mitochondria, microbodies and dictyosomes from the endoplasm becomes immobilised around the sub-cortical actin bundles when ATP is depleted. Such endoplasmic filaments have not been detected in association with mitochondria or microbodies and they have not been found in the stationary cortex. These filaments are extracted from the perfused cell by ATP unless motility-inhibiting levels of cytochalasin B are present. The filaments are not detectable in cells inactivated in solutions containing high (10(-4) M) Ca++ concentrations even when the Ca++ level is subsequently lowered. Consistent with their being required for motility, cytoplasmic streaming cannot be effeiciently reactivated by ATP in such filament-depleted cells. The possibility is discussed that the filaments contain myosin and that the endoplasmic reticulum with which they are associated has a major role in generating and transmitting the motive force for streaming.     

Studies on the motility of the foraminifera. I. Ultrastructure of the reticulopodial network of Allogromia laticollaris (Arnold)

Allogromia laticollaris, a benthic marine foraminifer, extends numerous trunk filopodia that repeatedly branch, anastomose, and fuse again to form the reticulopodial network (RPN), within which an incessant streaming of cytoplasmic particles occurs. The motion of the particles is saltatory and bidirectional, even in the thinnest filopodia detected by optical microscopy. Fibrils are visible by differential interference microscopy, and the PRN displays positive birefringence in polarized light. These fibrils remain intact after lysis and extraction of the RPN in solutions that stabilize microtubules (MTs). Electron micrographs of thin sections through these lysed and stabilized cytoskeletal models reveal bundles of MTs. The RPNs of living Allogromia may be preserved by standard EM fixatives only after acclimatization to calcium-free seawater, in which the streaming is normal. The MTs in the RPN are typically arranged in bundles that generally lie parallel to the long axis of the trunk and branch filopodia. Stereo electron micrographs of whole-mount, fixed, and critical-point-dried organisms show that the complex pattern of MT deployment reflects the pattern of particle motion in both flattened and highly branched portions of the RPN. Cytoplasmic particles, some of which have a fuzzy coat, are closely associated with, and preferentially oriented along, either single MTs or MT bundles. Thin filaments (approximately 5 nm) are also observed within the network, lying parallel to and interdigitating with the MTs, and in flattened terminal areas of the filopodia. These filaments do not bind skeletal muscle myosin S1 under conditions that heavily decorate actin filaments in controls (human blood platelets), and are approximately 20% too thin to be identified ultrastructurally as F-actin.     

Cytochalasin B-induced depolymerization of F-actin in chick embryo fibroblasts is dependent on cell density and anchorage to substratum

The effect of cytochalasin B on F-actin amount and organization was measured in chick embryo fibroblasts (CEF) grown on solid substratum at low density, at high density, and suspended in a fluid medium. It was found that: 1) Cytochalasin B induced decrease in F-actin content only in cells growing at low density, in density-inhibited or suspended cells cytochalasin B had no effect on F-actin amount. 2) In cells grown at low density F-actin filaments organized in stress fibers are more resistant to cytochalasin B than F-actin which is not organized in fibrils. In cell density-inhibited or suspended in a fluid medium F-actin filaments are insensitive to the action of cytochalasin B, although they are not organized in stress fibers. These results are interpreted to reflect the influence of contact reactions on treadmilling in F-actin filaments.     

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501- 520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.     

Actin-organelle interaction: association with chloroplast in arabidopsis leaf mesophyll cells.

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.     

Localization of actin filaments in internodal cells of characean algae. A scanning and transmission electron microscope study

New methods of visualizing subcortical actin filament bundles, or fibrils, in Characean internodes confirm that they are associated with chloroplasts at the surface facing the streaming endoplasm, and reveal that they are continuous over long distances. With the scanning electron microscope, an average of four to six fibrils are seen bridging a file of chloroplasts. The same configuration appears in negatively stained preparations of large blocks of chloroplast files connected by actin fibrils. Few branches of the subcortical fibrils are evident. These findings are discussed with respect to the mechanism of cytoplasmic streaming in Characeae.     

The 135-kDa actin-bundling protein from lily pollen tubes arranges F-actin into bundles with uniform polarity

 A plant 135-kDa actin-bundling protein (P-135-ABP) isolated from pollen tubes of Lilium longiflorum (Thunb.) binds stoichiometrically to F-actin filaments and bundles them in vitro (E. Yokota et al., 1998, Plant Physiol. 116: 1421–1429). To further understand the mechanism of actin-filament bundle formation by P-135-ABP, the polarity of each F-actin filament in bundles was examined using myosin subfragment 1 (S-1). Dissociation of F-actin filaments from bundles organized by P-135-ABP was induced by S-1. However, F-actin filaments that remained in a bundle and decorated by S-1 showed uniform polarity. These results indicate that P-135-ABP arranges F-actin filaments into bundles with uniform polarity and consequently plays a key role in the orientation of cytoplasmic streaming in pollen tubes. Received: 23 February 1999 / Accepted: 22 April 1999     

Motile Fibrils and Cytoplasmic Streaming in Germinated Pollen

The highly vigrous subprotoplasts were prepared from the germinated pollen of Lilium. As the protoplasm mass contracted, many cytoplasmic fibrils with free-ends which moved like animal sperm tails appeared at the surface of the mass. The winding movement of the fibril could tow the free-end's cytoplasm mass, but did not affect the particles moving along the fibril. Only when the fibril free-end adhered to the inner side of the cell membrane, could the, particle movement along the fibril occur, with the disappearance of the fibril’s winding movement. In vitro, the fibril contraction could make both cytoplasmic particles and subprotoplast move in unidirection, and the fibrils could specifically bind fluorescent beads coated with rabbit myosin. This indicates that the fibrils were composed of F-actin. We think that the cytoplasmic streaming may be based on the contraction of F-actin which must adhere to some points of the inner side of the cell membrane, and the contraction of F-actin drives the membrane-bound organells to move, at the same time, propels the sol cytoplasm thus forming the cytoplasmic streaming observed by light microscopy.     

Filamentous actin in paramecium cells: functional and structural changes correlated with phalloidin affinity labeling in vivo

Rhodaminylated (R)-phalloidin microinjected into Paramecium tetraurelia cells at a final concentration of greater than or equal to 20 micrograms/ml produces considerable functional and structural changes. F-actin bundles (with 20 micrograms/ml phalloidin within 15 min) are formed, which subsequently (greater than 30 min) are sequestered into autophagic vacuoles; simultaneously, the originally intense fluorescence of a narrow cortical layer becomes more and more diminished. When such microinjected cells are processed for electron microscopy, they display concomitant ultrastructural alterations, namely, the formation of transcellular bundles of 5-7 nm-thick filaments, which subsequently appear in autophagosomes, as well as a considerable reduction of filamentous materials in the cortex. This, in turn, entails a considerable restructuring of the cortex, enabling free access of various structural components to the cortex. Higher doses of R-phalloidin abolish cytoplasmic streaming (e.g., 50 micrograms/ml after 20-30 min); although the cells may survive, new secretory organelles (trichocysts) are no longer docked to the cell membrane. In contrast, exocytosis of docked trichocysts (as well as subsequent membrane resealing and retrieval) is not impaired under any conditions. Cortical F-actin may account for the cytoplasmic streaming that may normally guarantee the delivery of new trichocysts to free docking sites at the cell membrane. When docking is inhibited by high R-phalloidin doses, excess free trichocysts are sequestered into autophagosomes (crinophagy). One of the most sensitive cell functions is food vacuole formation (assayed by prelabeling with India ink), which correlates with the presence of R-phalloidin labeling in the cytostomal region and around food vacuoles. The main conclusions from this work are that filamentous actin may be involved in structuring of the cortex and in cytoplasmic streaming, and may therefore influence the formation, and possibly the transcellular transport (cyclosis), of food vacuoles, as well as the docking of trichocysts, whereas it does not play a role in exocytosis per se or in the steps immediately following.     

A cytochalasin-sensitive actin filament meshwork is a prerequisite for local wound wall deposition inNitella internodal cells

Summary Reorganization of the actin cytoskeleton following cell wall puncturing of characean internodal cells was studied by immunofluorescence and confocal laser scanning microscopy. Injury locally destroyed the parallel subcortical actin filament bundles and cortical actin strands that are characteristic of unwounded regions. At wounds, a delicate three-dimensional interlaced structure of actin strands, with meshes up to 5 m wide, formed by de novo assembly of isolated filaments and by the elongation of residual subcortical actin bundles and cortical actin strands. The actin meshwork persisted for up to 2 h, corresponding to the duration of intense wound wall secretion. Actin filament bundles continuous with the subcortical bundles outside the wound then regenerated, their parallel alignment probably assisted by endoplasmic flow. Cytochalasin D concentrations that arrested cytoplasmic streaming completely inhibited the formation of the actin meshwork, wound wall deposition and recovery of actin bundles. Concentrations that only reduced streaming velocity delayed meshwork formation and wound walls were thinner than in controls. The actual amount of F-actin within the meshwork, however, was clearly greater in the presence of low cytochalasin concentrations. In late stages of recovery, the actin bundles became very thick and intervening spaces became wider thereby forming a conspicuous, three-dimensional lattice that was continuous with interwebbing subcortical bundles and cortical actin around the periphery of the wound. Our experiments suggest that actin meshwork formation is a prerequisite for plasma membrane-directed transport of vesicles involved in wounding-induced exocytosis in characean internodes. Stabilization of the meshwork by subinhibitory concentrations of cytochalasin D is probably caused by actinbinding properties of the drug that either induce bundling or impede function of associated proteins.Abbreviations AFW artificial fresh water - BSA bovine serum albumin - CLSM confocal laser scanning microscope (microscopy) - DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - MBS m-maleimidobenzoyl N-hydroxy-succinimide ester - PBS phosphate-buffered saline - SCAB subcortical actin bundle     

The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin filaments

Electron microscopy of directly frozen giant cells of characean algae shows a continuous, tridimensional network of anastomosing tubes and cisternae of rough endoplasmic reticulum which pervade the streaming region of their cytoplasm. Portions of this endoplasmic reticulum contact the parallel bundles of actin filaments at the interface with the stationary cortical cytoplasm. Mitochondria, glycosomes, and other small cytoplasmic organelles enmeshed in the endoplasmic reticulum network display Brownian motion while streaming. The binding and sliding of endoplasmic reticulum membranes along actin cables can also be directly visualized after the cytoplasm of these cells is dissociated in a buffer containing ATP. The shear forces produced at the interface with the dissociated actin cables move large aggregates of endoplasmic reticulum and other organelles. The combination of fast-freezing electron microscopy and video microscopy of living cells and dissociated cytoplasm demonstrates that the cytoplasmic streaming depends on endoplasmic reticulum membranes sliding along the stationary actin cables. Thus, the continuous network of endoplasmic reticulum provides a means of exerting motive forces on cytoplasm deep inside the cell distant from the cortical actin cables where the motive force is generated.     

Organization of the actin cytoskeleton during pollen development inGasteria verrucosa (Mill.) H. Duval visualized with rhodamine-phalloidin

The three-dimensional organization of the microfilamental cytoskeleton of developingGasteria pollen was investigated by light microscopy using whole cells and fluorescently labelled phalloidin. Cells were not fixed chemically but their walls were permeabilized with dimethylsulphoxide and Nonidet P-40 at premicrospore stages or with dimethylsulphoxide, Nonidet P-40 and 4-methylmorpholinoxide-monohydrate at free-microspore and pollen stages to dissolve the intine.Four strikingly different microfilamentous configurations were distinguished. (i) Actin filaments were observed in the central cytoplasm throughout the successive stages of pollen development. The network was commonly composed of thin bundles ramifying throughout the cytoplasm at interphase stages but as thick bundles encaging the nucleus prior to the first and second meiotic division. (ii) In released microspores and pollen, F-actin filaments formed remarkably parallel arrays in the peripheral cytoplasm. (iii) In the first and second meiotic spindles there was an apparent localization of massive arrays of phalloidin-reactive material. Fluorescently labelled F-actin was present in kinetochore fibers and pole-to-pole fibers during metaphase and anaphase. (iv) At telophase, microfilaments radiated from the nuclear envelopes and after karyokinesis in the second meiotic division, F-actin was observed in phragmoplasts.We did not observe rhodamine-phalloidin-labelled filaments in the cytoplasm after cytochalasin-B treatment whereas F-actin persisted in the spindle. Incubation at 4° C did not influence the existence of cytoplasmic microfilaments whereas spindle filaments disappeared. This points to a close interdependence of spindle microfilaments and spindle tubules.Based on present data and earlier observations on the configuration of microtubules during pollen development in the same species (Van Lammeren et al., 1985, Planta165, 1-11) there appear to be apparent codistributions of F-actin and microtubules during various stages of male meiosis inGasteria verrucosa.Abbreviation DMSO dimethylsulfoxide     

Cortical actin filaments fragment and aggregate to form chloroplast-associated and free F-actin rings in mechanically isolatedZinnia mesophyll cells

Summary Changes in F-actin organization following mechanical isolation ofZinnia mesophyll cells were documented by rhodamine-phalloidin staining. Immediately after isolation, most cells contained irregular cortical actin fragments of varying lengths, and less than 5% of cells contained intact cortical filaments. During the first 8 h of culture, filament fragments were replaced by actin rings, stellate actin aggregates, and bundled filament fragments. Some of these aggregates had no association with organelles (free actin aggregates). Other aggregates were associated with chloroplasts, which changed in shape and location at the same time actin aggregates appeared. F-actin was concentrated within or around the nucleus in a small percentage of cells. After 12 h in culture, the percentage of cells with free actin rings and chloroplast-associated actin aggregates began to decline and the percentage of cells having intact cortical actin filaments increased greatly. Intermediate images were recorded that strongly indicate that free actin rings, chloroplast-associated actin rings, and other actin aggregates self-assemble by successive bundling of actin filament fragments. The fragmentation and bundling of F-actin observed in mechanically isolatedZinnia cells resembles changes in F-actin distribution reported after diverse forms of cell disturbance and appears to be an example of a generalized response of the actin cytoskeleton to cell stress.Abbreviations FITC fluorescein isothiocyanate - MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - RhPh tetramethylrhodamine isothiocyanate-phalloidin     

Regeneration of actin bundles in Chara: polarized growth and orientation by endoplasmic flow

Strong irradiation of localized areas of the alga Chara produces chloroplast damage and extensive loss of the actin bundles responsible for cytoplasmic streaming. Immunofluorescence using a monoclonal antibody binding to the actin bundles has been used to follow their regrowth. Bundle regeneration is polarized so that new bundles develop from the ends of the actin bundles delivering endoplasm to the damaged area and not from bundles removing endoplasm. According to the previously established polarity of the actin filaments this growth is occurring from the "barbed" but not the "pointed" ends of the component filaments. The frequently irregular orientation of the regenerated bundles contrasts with the straight, parallel arrangement of the bundles before destruction. The arrangement of the regenerated bundles is suggested to depend on orientation by passive endoplasmic flow rather than a cortical template. As a result, bundles follow sweeping curves and can form a U-turn connecting oppositely polarized bundles normally separated by the neutral line. In addition to development in continuity with the free ends of pre-existing bundles, visualization of small, discrete fluorescent structures suggests that bundles can begin to form in isolation within the damaged areas. The results are discussed in terms of the polarized actin polymerisation seen in vitro, additional controls which may operate on bundle growth in vivo, and the ability of flow to orient F-actin. The relevance of the findings to normal cell ontogeny is assessed.     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.     

Counterion-induced actin ring formation

Actin filaments form rings and loops when > 20 mM divalent cations are added to very dilute solutions of phalloidin-stabilized filamentous actin (F-actin). Some rings consist of very long single actin filaments partially overlapping at their ends, and others are formed by small numbers of filaments associated laterally. In some cases, undulations of the rings are observed with amplitudes and dynamics similar to those of the thermal motions of single actin filaments. Lariat-shaped aggregates also co-exist with rings and rodlike bundles. These polyvalent cation-induced actin rings are analogous to the toroids of DNA formed by addition of polyvalent cations, but the much larger diameter of actin rings reflects the greater bending stiffness of F-actin. Actin rings can also be formed by addition of streptavidin to crosslink sparsely biotinylated F-actin at very low concentrations. The energy of bending in a ring, calculated from the persistence length of F-actin and the ring diameter, provides an estimate for the adhesion energy mediated by the multivalent counterions, or due to the streptavidin-biotin bonds, required to keep the ring closed.     

High-voltage electron microscopy of whole,critical-point dried plant cells

Summary The lower epidermis ofSelaginella Helvetica leaves has numerous chloroplasts. In the diffuse light of the plant's normal habitat these are distributed over the inner wall of the cell, while in bright sunlight they move to the lateral walls. High voltage electron microscopy of whole critical-point dried cells shows that in the diffuse-light position the chloroplasts are connected by bundles of tightly-packed parallel filaments; these are distinct from, but seem to interconnect with, the filaments of the cytomatrix. In thin sections these appear as conventional microfilament bundles, while staining with rhodamineconjugated phalloidin implies that they are composed of actin. In bright light, when the chloroplasts have moved to the lateral walls, these microfilament bundles completely disappear, while filaments of the cytomatrix system remain attached to the chloroplasts. These results suggest that the function of the microfilament bundles may be to anchor the chloroplasts as much as to move them, and that the cytomatrix system may play a part in the movement; it is possible that actin microfilament bundles may actually dissociate into separate filaments within the cytomatrix. Staining of cryo-sections with FITC-labelled antitubulin reveals a typical cortical pattern of microtubules which appears to play no part in chloroplast motility.Abbreviations EDTA ethylenediaminetetra-acetic acid - EM electron microscopy - FITC fluorescein-iso-thiocyanate - HVEM high voltage electron microscopy - PIPES piperazine-NN-bis-2-ethanesulphonic acid