The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1

We have examined the cytoskeletal architecture and its relationship with synaptic vesicles in synapses by quick-freeze deep-etch electron microscopy (QF.DE). The main cytoskeletal elements in the presynaptic terminals (neuromuscular junction, electric organ, and cerebellar cortex) were actin filaments and microtubules. The actin filaments formed a network and frequently were associated closely with the presynaptic plasma membranes and active zones. Short, linking strands approximately 30 nm long were found between actin and synaptic vesicles, between microtubules and synaptic vesicles. Fine strands (30-60 nm) were also found between synaptic vesicles. Frequently spherical structures existed in the middle of the strands between synaptic vesicles. Another kind of strand (approximately 100 nm long, thinner than the actin filaments) between synaptic vesicles and plasma membranes was also observed. We have examined the molecular structure of synapsin 1 and its relationship with actin filaments, microtubules, and synaptic vesicles in vitro using the low angle rotary shadowing technique and QF.DE. The synapsin 1, approximately 47 nm long, was composed of a head (approximately 14 nm diam) and a tail (approximately 33 nm long), having a tadpole-like appearance. The high resolution provided by QF.DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles, forming approximately 30-nm short strands. The head was on the actin and the tail was attached to the synaptic vesicle or actin filament. Microtubules were also cross-linked by a single synapsin 1, which also connected a microtubule to synaptic vesicles, forming approximately 30 nm strands. The spherical head was on the microtubules and the tail was attached to the synaptic vesicles or to microtubules. Synaptic vesicles incubated with synapsin 1 were linked with each other via fine short fibrils and frequently we identified spherical structures from which two or three fibril radiated and cross-linked synaptic vesicles. We have examined the localization of synapsin 1 using ultracryomicrotomy and colloidal gold-immunocytochemistry of anti-synapsin 1 IgG. Synapsin 1 was exclusively localized in the regions occupied by synaptic vesicles. Statistical analyses indicated that synapsin 1 is located mostly at least approximately 30 nm away from the presynaptic membrane. These data derived via three different approaches suggest that synapsin 1 could be a main element of short linkages between actin filaments and synaptic vesicles, and between microtubules and synaptic vesicles, and between synaptic vesicles in the nerve terminals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Attachment sites and interconnections of the microtubular system in pigment-containing epidermal cells of Carausius morosus (Insecta,Phasmida)

Summary The apical plasma membranes of epidermal cells in the stick insect Carausius morosus Brunner, 1908 forms deep invaginations, the inner surfaces of which are covered with a zone of fine filamentous, electron-dense material. These and similar electron-dense zones, forming circumscribed areas of the basal membrane, are for the attachment of microtubules. These structures may be microtubuleintiating sites, where microtubules start to polymerize. Microtubules have sidearms that project freely or are attached to the stem, to sidearms of other microtubules or to filaments 4–6 nm in diameter and to a fine filamentous electron-dense meshwork. Their attachment to the apical and basal plasma membranes and to each other make them a firmly anchored cytoskeleton, an important prerequisite for pigment movement.     

Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein

We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three- dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button- like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule- associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75- kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic immunocytochemistry with a monoclonal antibody (D57) against sea urchin sperm flagellar 21S dynein and colloidal gold-labeled second antibody. Immunogold particles were closely associated with spindle microtubules. 76% of these were within 50 nm and 55% were within 20 nm from the surface of the microtubules. These gold particles were sporadically found on both polar and kinetochore microtubules of half-spindles at both metaphase and anaphase. They localized also on the microtubules between sister chromatids in late anaphase. These data indicate that cytoplasmic dynein is attached to the microtubules in sea urchin mitotic spindles.(ABSTRACT TRUNCATED AT 400 WORDS)     

The fine structure of the axostyle and its associations with organelles in Trichomonads

The fine structure of the axostyle in the protists Tritrichomonas foetus and Monocercomonas sp is described using transmission electron microscopy after quick-freezing techniques and immunocytochemistry. The axostyle microtubules presents a lateral projection formed by two protofilaments in addition to the 13 protofilaments normally found in microtubules. The axostyle is associated with other cell structures such as hydrogenosomes, endoplasmic reticulum, sigmoid filaments and glycogen particles. The microtubules of the pelta-axostylar system are connected to each other by bridges regularly spaced with an interval of 9 nm. Labeling of the axostyle was observed after cell incubation with monoclonal antibodies recognizing alpha-tubulin and acetylated-tubulin.     

Alignment, zippering and splaying of microtubules during axogenesis

Neurodegenerative diseases may result in part from defects in motor-driven vesicle transport in neuronal cells. Myosin-V, an actin-based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin-V interacts with the microtubule-based motor, kinesin, to form a 'hetero-motor' complex on vesicles. The complex of these two motors, one microtubule-based and the other actin-based, facilitates the movement of vesicles from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons, we hypothesize that one of the functions of the tail–tail interaction is to provide feedback between the two proteins to allow seamless transition of vesicles from microtubules to actin filaments. To study the interactions of the globular tail domain of myosin-V to kinesin and to neuronal vesicles, we used a GST-tagged globular tail fragment in motility assays. The MyoV tail fragment inhibited vesicle transport by 81–91% and thereby exhibited a dominant negative effect. These data show that the recombinant protein blocked the activity of native myosin-V presumably by binding to vesicles and competing away the native myosin-V motors. The GST-MyoV-tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates and therefore it exhibited binding properties of native myosin-V. These data show that the headless myosin-V fragment is an effective inhibitor of vesicle transport in cell extracts. These studies support the hypothesis that tail–tail interactions may be a mechanism for feedback between myosin-V and kinesin to allow transition of vesicles from microtubules to actin filaments. Acknowledgements: Supported by NSF grant MCB9974709.     

角蛋白纤维的新功能--在爪蟾卵提取物核膜重组装中的作用

激活的非洲爪蟾卵提取物温育过程中,直径２００ｎｍ的膜泡附着在一种直径１０ｎｍ纤维上,形成“珠链”结构。用透射电镜整装制样技术观察了温育中“珠链”结构的形成过程,发现１０ｎｍ纤维可抗Ｔｒｉｔｏｎ抽提,免疫荧光和蛋白免疫印迹试验表明１０ｎｍ纤维可能是由５６ｋＤ的碱性角蛋白与４２ｋＤ的酸性角蛋白构成。向提取物中加入碱性角蛋白抗体ＡＥ３则可抑制环状片层的形成,而核膜的组装也受到很大影响。这些结果显示角蛋白     

Flagellar elongation and shortening in Chlamydomonas. III. structures attached to the tips of flagellar microtubules and their relationship to the directionality of flagellar microtubule assembly

Two structures on the distal ends of Chlamydomonas flagellar microtubules are described. One of these, the central microbutule cap, attaches the distal ends of the central pair microtubules to the tip of the flagellar membrane. In addition, filaments, called distal filaments, are observed attached to the ends of the A-tubules of the outer doublet microtubules. Inasmuch as earlier studies suggested that flagellar elongation in vivo occurs principally by the distal addition of sublnits and because it has been shown that brain tubulin assembles in vitro primarily onto the distal ends of both central and outer doublet microtubules, the presence of the cap and distal filaments was quantitated during flagellar resorption and elongation. The results showed that the cap remains attached to the central microtubules throughout flagellar resorption and elongation. The cap was also found to block the in vitro assembly of neurotubules onto the distal ends of the central microtubules. Conversely, the distal filaments apparently do not block the assembly of neurotubules onto the ends of the outer doublets. During flagellar elongation, the distal ends of the outer doublets are often found to form sheets of protofilaments similar to those observed on the elongating ends of neurotubules being assembled in vitro. These results suggest that the outer doublet microtubules elongate by the distal addition of subunits, whereas the two central microtubules assemble by the addition of subunits to the proximal ends.     

Symposium 4: Dynamics of the Neuronal Cytoskeleton. Actin‐based vesicle transport in nerve cell extracts: implications for neurodegenerative diseases

Neurodegenerative diseases may result in part from defects in motor‐driven vesicle transport in neuronal cells. Myosin‐V, an actin‐based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin‐V interacts with the microtubule‐based motor, kinesin, to form a ‘hetero‐motor’ complex on vesicles. The complex of these two motors, one microtubule‐based and the other actin‐based, facilitates the movement of vesicles from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons, we hypothesize that one of the functions of the tail–tail interaction is to provide feedback between the two proteins to allow seamless transition of vesicles from microtubules to actin filaments. To study the interactions of the globular tail domain of myosin‐V to kinesin and to neuronal vesicles, we used a GST‐tagged globular tail fragment in motility assays. The MyoV tail fragment inhibited vesicle transport by 81–91% and thereby exhibited a dominant negative effect. These data show that the recombinant protein blocked the activity of native myosin‐V presumably by binding to vesicles and competing away the native myosin‐V motors. The GST‐MyoV‐tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates and therefore it exhibited binding properties of native myosin‐V. These data show that the headless myosin‐V fragment is an effective inhibitor of vesicle transport in cell extracts. These studies support the hypothesis that tail–tail interactions may be a mechanism for feedback between myosin‐V and kinesin to allow transition of vesicles from microtubules to actin filaments. Acknowledgements: Supported by NSF grant MCB9974709.     

Microtubules with different diameter, protofilament number and protofilament spacing in Ornithogalum umbellatum ovary epidermis cells

Microtubules present in the epidermis of Ornithogalum umbellatum ovary in the area of lipotubuloids (i.e. aggregates of lipid bodies surrounded by microtubules) are 25-51 nm in diameter. They consist mainly of 10 and 11, sometimes 9 and 12 protofilaments. An average diameter of microtubule consisting of 9 subunits is about 32 nm, of 10-35 nm, of 11-38 nm and of 12-43 nm, however, individual microtubules in each category significantly vary in size. These differences result from varying distance between protofilaments in microtubule walls and diameters of protofilaments: in thin microtubules they are densely packed and smaller while in thicker ones they are loosely arranged and bigger. A hypothesis has been put forward that changes in microtubule diameter depend on structural changes associated with their functional status and are executed by modifications of protofilament arrangement density and their diameters in microtubule wall. The above hypothesis seems to be in agreement with the opinion formed on the basis of in vitro image of microtubules, that lateral contact between tubulin subunits in neighboring protofilaments indicates some flexibility and changeability during microtubule function.     

Cytoskeleton and vesicle mobility in astrocytes

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.     

Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution

Summary The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4–6 nm in diameter. Fine filaments 8–10 nm in diameter and 30–50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3–5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.     

Rigor crossbridges are double-headed in fast muscle from crayfish

The structure of rigor crossbridges was examined by comparing rigor crossbridges in fast muscle fibers from glycerol-extracted abdominal flexor muscle of crayfish with those in "natively decorated" thin filaments from the same muscle. Natively decorated thin filaments were obtained by dissociating the backbone of the myosin filaments of rigor myofibrils in 0.6 M KCl. Intact fibers were freeze-fractured, deep-etched, and rotary shadowed; isolated filaments were either negatively stained or freeze dried and rotary shadowed. The crossbridges on the natively decorated actin maintain the original spacing and the disposition in chevrons and double chevrons for several hours, indicating that no rearrangement of the actomyosin interactions occurs. Thus the crossbridges of the natively decorated filaments were formed within the geometrical constraints of the intact myofibril. The majority of crossbridges in the intact muscle have a triangular shape indicative of double-headed crossbridge. The triangular shape is maintained in the isolated filaments and negative staining resolves two heads in a single crossbridge. In the isolated filaments, crossbridges are attached at uniform acute angles. Unlike those in insect flight muscle (Taylor et al., 1984), lead and rear elements of the double chevron may be both double-headed. Deep-etched images reveal a twisted arrangement of subfilaments in the backbone of the thick filament.     

Direct observation of microtubules with the scanning tunneling microscope

To observe surface topography of microtubules, we have applied scanning tunneling microscopy (STM), which can image metal and semiconductive surfaces with atomic resolution. Isolated microtubules fixed in 0.1% glutaraldehyde in reassembly buffer containing 0.8 M glycerol were imaged in air on a graphite substrate. The presence of microtubules in solution was verified by electron microscopy. At atmospheric pressure and room temperature, STM probing of both freeze-dried and hydrated microtubules reveals structures approximately 25 nm in width, consisting of longitudinal filaments about 4 nm in width. These structures match electron microscopy images of microtubules and their component protofilaments. Microtubules imaged by STM frequently appear buckled and semiflattened. Top-view shaded scans show what appear to be individual tubulin subunits within protofilaments. We believe these results represent the first direct STM observation of protein assemblies in which components can be identified. Although the microtubule image resolution described here is no better than that presently obtainable by other techniques (e.g., electron microscopy with freeze-drying, shadowing, and/or negative staining), it is significant that suitably prepared biomolecules may be sufficiently conductive and stable for STM imaging, which is ultimately capable of atomic resolution. Further development of STM technology, computer-enhanced image processing, and elucidation of optimal STM sample preparation indicate that STM and related applications will offer unique opportunities for the study of biomolecular surfaces.     

Spermatogenesis in anthozoa: Differentiation of the spermatid

The fine structure of spermatids has been examined in Calliactis, Protanthea, Gonactinia and Parazoanthus (Cnidaria, Anthozoa). The sperm cells are relatively simple and lack distinct acrosomes. Their nuclei, spherical in the zoanthid, in the actinians are slendertipped cones. Condensation of the chromatin is interpreted in terms of progressive coiling of densely-stained filaments and the elimination of nucleoplasm. Nuclear elongation occurs in the absence of microtubules. A well-developed centriolar complex is attached to the nuclear envelope by fibres and in this area (that of a shallow fossa in actinian spern) the nuclear membranes seem to be thickened. The centrioles are surrounded by a mitochondrial collar, especially pronounced in Calliactis. In contact with the mitochondria and nucleus is a ring of lipid-containing vesicles 300-700 nm in diameter. A system of densely-staining vesicles 150-300 nm in size corresponds to the "pro-acrosomal vesicles" described for other coelenterates. They are scattered in the peripheral cytoplasm and are regarded as derivatives of the endoplasmic reticulum. Problems of organelle function and of differentiation during spermiogenesis are discussed.     

Synthetic lipid (DOPG) vesicles accumulate in the cell plate region but do not fuse

The cell plate is the new cell wall, with bordering plasma membrane, that is formed between two daughter cells in plants, and it is formed by fusion of vesicles (approximately 60 nm). To start to determine physical properties of cell plate forming vesicles for their transport through the phragmoplast, and fusion with each other, we microinjected fluorescent synthetic lipid vesicles that were made of 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) into Tradescantia virginiana stamen hair cells. During interphase, the 60-nm wide DOPG vesicles moved inside the cytoplasm comparably to organelles. During cytokinesis, they were transported through the phragmoplast and accumulated in the cell plate region together with the endogenous vesicles, even inside the central cell plate region. Because at this stage microtubules are virtually absent from that region, while actin filaments are present, actin filaments may have a role in the transport of vesicles toward the cell plate. Unlike the endogenous vesicles, the synthetic DOPG vesicles did not fuse with the developing cell plate. Instead, they redistributed into the cytoplasm of the daughter cells upon completion of cytokinesis. Because the redistribution of the vesicles occurs when actin filaments disappear from the phragmoplast, actin filaments may be involved in keeping the vesicles inside the developing cell plate region.     

Short-range axonal/dendritic transport by myosin-V: A model for vesicle delivery to the synapse

Myosin-V is a versatile motor involved in short-range axonal/dendritic transport of vesicles in the actin-rich cortex and synaptic regions of nerve cells. It binds to several different kinds of neuronal vesicles by its globular tail domain but the mechanism by which it is recruited to these vesicles is not known. In this study, we used an in vitro motility assay derived from axoplasm of the squid giant axon to study the effects of the globular tail domain on the transport of neuronal vesicles. We found that the globular tail fragment of myosin-V inhibited actin-based vesicle transport by displacing native myosin-V and binding to vesicles. The globular tail domain pulled down kinesin, a known binding partner of myosin-V, in affinity isolation experiments. These data confirmed earlier evidence that kinesin and myosin-V interact to form a hetero-motor complex. The formation of a kinesin/myosin-V hetero-motor complex on vesicles is thought to facilitate the coordination of long-range movement on microtubules and short-range movement on actin filaments. The direct interaction of motors from both filament systems may represent the mechanism by which the transition of vesicles from microtubules to actin filaments is regulated. These results are the first demonstration that the recombinant tail of myosin-V inhibits vesicle transport in an in vitro motility assay. Future experiments are designed to determine the functional significance of the interaction between myosin-V and kinesin and to identify other proteins that bind to the globular tail domain of myosin-V.     

Alteration of the distribution of intermediate filaments in PtK1 cells by acrylamide. II: Effect on the organization of cytoplasmic organelles

The distribution and motility of cytoplasmic particles was examined in PtK1 cells in which intermediate filament networks had been disrupted by acrylamide. In these cells, particles (mitochondria and vesicles) accumulated near the cell center although saltatory movements continued. This left a broad sheet of agranular cytoplasm at the periphery of the cell. Particles were capable of movement into this sheet. Intermediate filaments were absent in the peripheral cytoplasm although microtubules remained in a normal configuration. Particles apparently move along the microtubules. These results indicate that particle movement along microtubules is not dependent upon the normal configuration of intermediate filaments. It is suggested that intermediate filaments are necessary for normal organelle distribution and serve as a matrix with which particles can associate to maintain position.     

Cross-bridges mediate anterograde and retrograde vesicle transport along microtubules in squid axoplasm

To assay the detailed structural relationship between axonally transported vesicles and their substrate microtubules, vesicle transport was focally cold blocked in axoplasm that was extruded from the squid giant axon. A brief localized cold block concentrated anterogradely and retrogradely transported vesicles selectively on either the proximal or or distal side of the block. Normal movement of the concentrated vesicles was reactivated by rewarming the cold-blocked axoplasm. At the periphery of the axoplasm, moving vesicles were located on individual microtubules that had become separated from the other cytomatrix components. The presence of moving vesicles on isolated microtubules permitted the identification of the structural components required for vesicle transport along microtubules. The results show that 16-18-nm cross-bridges connect both anterogradely and retrogradely moving vesicles to their substrate microtubules. These observations demonstrate that cross-bridges are fundamental are fundamental components of vesicle transport along axonal microtubules. Thus, vesicle transport can now be included among those cell motile systems such as muscle and axonemes that are based on a cross-bridge-mediated mechanism.     

Assembly and structure of neurofllaments isolated from bovine spinal cord

Neurofilaments (NFs) are neuron-specific intermediate filaments. The NFs were isolated from bovine spinal cord by differential centrifugation. The NFs were detected with electron microscopy and scanning tunneling microscopy (STM). Under STM, two kinds of sidearm of NFs were revealed: one was short, the other was long. They were arrayed along the 10-nm width core filaments one by one. The intervals between two adjacent long sidearms or two short sidearms were 20—22 nm, while those between two adjacent long and short sidearms were 10—11 nm. It was proposed that the rod domain of NF triplet prnteins was 3/4-staggered. The assembly properties of NF triplet proteins were also studied. Immuno-colloidal-gold labeling assay showed that NF-M and NF-H are able to co-assemble into long filaments with NF-L. NF-M and NF-H can also co-constitute into winding filaments.     

Assembly and structure of neurofilaments isolated from bovine spinal cord

Neurofilaments (NFs) are neuron-specific intermediate filaments. The NFs were isolated from bovine spinal cord by differential centrifugation. The NFs were detected with electron microscopy and scanning tunneling microscopy (STM). Under STM, two kinds of sidearm of NFs were revealed: one was short, the other was long. They were arrayed along the 10-nm width core filaments one by one. The intervals between two adjacent long sidearms or two short sidearms were 20–22 nm, while those between two adjacent long and short sidearms were 10–11 nm. It was proposed that the rod domain of NF triplet proteins was 3/4-staggered. The assembly properties of NF triplet proteins were also studied. Immuno-colloidal-gold labeling assay showed that NF-M and NF-H are able to co-assemble into long filaments with NF-L. NF-M and NF-H can also co-constitute into winding filaments.