Actin-endoplasmic reticulum complexes inDrosera

In epidermal cells ofDrosera tentacles that have been preserved for ultrastructural analysis through high pressure freeze fixation and freeze substitution we describe the frequent occurrence of microfilament (MF)-endoplasmic reticulum (ER) complexes. These are found throughout the cytoplasm where they are observed in close association with the plasmalemma (PL), the tonoplast, nuclei, mitochondria, chloroplasts, and microbodies. The MF component of the complexes is identified as actin based on immunogold labelling with actin antibodies. The actin-ER complexes are prominent in the cortical cytoplasm. In this region a network of predominantly tubular ER occupies an intermediary position in which it associates closely with both the PL and the actin MFs. We suggest that the ER, especially those elements adjacent to the PL in the cortical cytoplasm, stabilizes the actin MFs and provides the necessary anchor against which the forces for cytoplasmic streaming are generated.Abbreviations CF chemical fixation - ER endoplasmic reticulum - FS freeze substitution - HPF high pressure freezing - MF microfilaments - MT microtubules - PL plasmalemma     

Intercellular cytoplasm transport during oogenesis of the moth midge, Tinearia alternata Say (Diptera: Psychodidae)

Polytrophic ovaries of the nematocerous dipteran, Tinearia alternata Say consists of several developmentally synchronized ovarioles each housing only one functional egg chamber with 15 nurse cells and an oocyte. At the early stages of previtellogenesis the nurse cells become polyploid and synthetically active. Their nuclei contain polytene chromosomes and prominent nucleoli. With the advance of previtellogenic growth the nurse cell cytoplasm is loaded with the growing number of ribosomes and contain perinuclear nuage material, mitochondria, electron dense bodies and aggregations of endoplasmic reticulum. All these organelles are transported into the oocyte thanks to the massive and rapid flow of the nurse cell cytoplasmic contents. Nurse cell-oocyte transport is mediated by actin cytoskeleton. Prior to the rapid cytoplasm transfer, F-actin network is associated with the nurse cell membranes while tiny bundles of microfilaments form actin baskets connected with ring canals. Nurse cells in Tinearia lack an extensive scaffold of radially oriented, F-actin bundles (cables) that would tether their nuclei in place, thus preventing ring canals from plugging. The way the nuclei are anchored to their central positions within the cells remains unclear. Towards the final stages of oogenesis nurse cells are almost devoid of cytoplasm and degenerate. Although their nuclei undergo dramatic morphological transformations, typical hallmarks of apoptotic pathway could not be clearly observed. Rapid ooplasmic streaming does not occur.     

Structural basis of cytoplasmic streaming in Characean internodal cells. A hydrodynamic analysis

Summary Hydrodynamic equations were derived which relate the velocity profile of endoplasmic streaming with the motive force generated by active sliding of endoplasmic organelles in Characean internodal cells, under two implicit assumptions that (1) the sliding velocity of putative organelles is comparable to the streaming velocity of endoplasm, and (2) subcortical endoplasm is far less viscous than bulk endoplasm.The equations were extended so as to calculate the velocity profile in flattened or perfused internodal cells. Calculated profiles were basically consistent with reported patterns of streaming under these conditions.Utilizing published data, we deduce some hydrodynamic parameters of streaming, and predict the dimensions of putative organelles expected to drive entire cytoplasm. A revision for published values of the motive force of streaming is proposed.Hydrodynamic analyses made earlier on the spherical organelles are repeated. The results show that the organelles may generate streaming, depending on the configurationin vivo of fine filaments protruding from the body of the organelles.     

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.     

Hydrodynamic models of viscous coupling between motile myosin and endoplasm in characean algae

Cytoplasmic streaming in characean algae is thought to be driven by interaction between stationary subcortical actin bundles and motile endoplasmic myosin. Implicit in this mechanism is a requirement for some form of coupling to transfer motive force from the moving myosin to the endoplasm. Three models of viscous coupling between myosin and endoplasm are presented here, and the hydrodynamic feasibility of each model is analyzed. The results show that individual myosinlike molecules moving along the actin bundles at reasonable velocities cannot exert enough viscous pull on the endoplasm to account for the observed streaming. Attachment of myosin to small spherical organelles improves viscous coupling to the endoplasm, but results for this model show that streaming can be generated only if the myosin-spheres move along the actin bundles in a virtual solid line at about twice the streaming velocity. In the third model, myosin is incorporated into a fibrous or membranous network or gel extending into the endoplasm. This network is pulled forward as the attached myosin slides along the actin bundles. Using network dimensions estimated from published micrographs of characean endoplasm, the results show that this system can easily generate the observed cytoplasmic streaming.     

ULTRASTRUCTURE,DEVELOPMENT AND CYTOPLASMIC ROTATION OF SETA-BEARING CELLS OF COLEOCHAETE SCUTATA (CHLOROPHYCEAE)1

Part of the cytoplasm, which always contains the plastid, of seta-bearing cells of Coleochaete scutata Bréb. rotates clockwise about the base of the seta. Many golgi bodies, vesicles and much endoplasmic reticulum occupy the bridges between the rotating central core of cytoplasm and the stationary peripheral layer of these cells. The setae, which grow from their base, are devoid of organelles other than vesicles and elongate mitochondria. At irregular intervals along the thin seta wall are annular thickenings containing callose. Microtubules which encircle the base of the seta disappear on treatment with colchicine. This drug had no effect on the speed of rotational streaming or the growth rate of existing hairs but did inhibit the development of new setae. Cytochalasin B slowed, but did not stop, streaming after 3 h exposure. However caffeine, but not EDTA, EGTA or the Ca ionophore A23187, reversibly inhibited cyclosis. The mechanism of cytoplasmic rotation is discussed in the light of these drug treatments and the presence of actin in the alga.     

Immunolocalization of myosin in rhizoids ofChara globularis Thuill

Summary Myosin-related proteins have been localized immunocytochemically in gravity-sensing rhizoids of the green algaChara globularis using a monoclonal antibody against the heavy chain of myosin from mouse 3T3 cells and a polyclonal antibody to bovine skeletal and smooth muscle myosin. In the basal zone of the rhizoids which contain a large vacuole, streaming endoplasm and stationary cortical cytoplasm, the monoclonal antibody stained myosin-related proteins as diffusely fluorescing endoplasmic strands. This pattern is similar to the arrangement of subcortical actin filament bundles. In the apical zone which contains an aggregation of ER membranes and secretory vesicles for tip growth, diffuse immunofluorescence was detected; the intensity of the signal increasing towards the apical cell wall. The most prominent myosin-staining was associated with the surface of statoliths in the apical zone. The polyclonal antibody produced a punctate staining pattern in the basal zone, caused by myosin-related proteins associated with the surface of drganelles in the streaming endoplasm and the periphery of the nucleus. In the apical zone, this antibody revealed myosin-immunofluorescence on the surface of statoliths in methacrylate-embedded rhizoids. Neither antibody revealed myosin-immunofluorescence on the surface of organelles and vesicles in the relatively stationary cytoplasm of the subapical zone. These results indicate (i) that different classes of myosin are involved in the various transport processes inChara rhizoids; (ii) that cytoplasmic streaming in rhizoids is driven by actomyosin, corresponding to the findings onChara internodal cells; (iii) that actindependent control of statolith position and active movement is mediated by myosin-related proteins associated with the statolith surfaces; and (iv) that myosin-related proteins are involved in the process of tip growth.     

The sliding theory of cytoplasmic streaming: fifty years of progress

Fifty years ago, an important paper appeared in Botanical Magazine Tokyo. Kamiya and Kuroda proposed a sliding theory for the mechanism of cytoplasmic streaming. This pioneering study laid the basis for elucidation of the molecular mechanism of cytoplasmic streaming—the motive force is generated by the sliding of myosin XI associated with organelles along actin filaments, using the hydrolysis energy of ATP. The role of the actin–myosin system in various plant cell functions is becoming evident. The present article reviews progress in studies on cytoplasmic streaming over the past 50 years.     

Alternative modes of organellar segregation during sporulation in Saccharomyces cerevisiae

Formation of ascospores in the yeast Saccharomyces cerevisiae is driven by an unusual cell division in which daughter nuclei are encapsulated within de novo-formed plasma membranes, termed prospore membranes. Generation of viable spores requires that cytoplasmic organelles also be captured along with nuclei. In mitotic cells segregation of mitochondria into the bud requires a polarized actin cytoskeleton. In contrast, genes involved in actin-mediated transport are not essential for sporulation. Instead, efficient segregation of mitochondria into spores requires Ady3p, a component of a protein coat found at the leading edge of the prospore membrane. Other organelles whose mitotic segregation is promoted by actin, such as the vacuole and the cortical endoplasmic reticulum, are not actively segregated during sporulation but are regenerated within spores. These results reveal that organellar segregation into spores is achieved by mechanisms distinct from those in mitotic cells.     

Cytoplasmic bags: Containers for discarded paraflagellar membranes in spermiogenesis of graphosome lineatum (Pentatomidae,Hemiptera, Insecta)

The process of cytoplasmic sloughing is described in spermiogenesis of a stink bug, Graphosoma lineatum, using transmission electron microscopy of ultrathin sections. Tails of young spermatids possess a wide cytoplasmic layer lateral to the axoneme and the nenbenkern derivatives. Membranous sheets, comprised of cisternae of endoplasmic reticulum with very narrow lumina, are arranged parallel to these organelles. More advanced spermatids show only a thin cytoplasmic layer largely devoid of membranes. At this stage, large evaginations of the flagellar membrane, termed cytoplasmic bags, are found in association with the spermatid tails. The most prominent elements within these bags are concentric layers of endoplasmic reticulum of the type previously found in spermatid tails. This relationship suggests that the cells rid themselves of cytoplasmic membranes throughout spermiogenesis via inclusion into cytoplasmic bags. Upon release from the nucleate cytoplasm, the cytoplasmic bags become more and more electron-dense and degenerate. © 1995 Wiley-Liss, Inc.     

Cytoplasmic streaming in Chara: a cell model activated by ATP and inhibited by cytochalasin B.

After vacuolar perfusion of Chara internode cells, the cytoplasm remaining in situ can be reactivated by ATP to give full rates of streaming. Observations during both perfusion and reactivation indicated that the generation of the motive force was associated with fibres consisting of bundles of microfilaments. In the absence of ATP, the remaining endoplasmic organelles were immobilized along such fibres. When ATP was introduced, organelles moved along the fibres at speeds up to 50 mum S minus 1, but but were progressively released from contact to leave the fibres in a conspicuously clean state. Inorganic pyrophosphate freed the organelles from the fibres without supporting movements. Motility required millimolar Mg2nlevels, free Ca2nat 10 minus 7 M or less and was inhibited by high levels of Clminus and by pH's on either side of 7.0. The reactivated movements were rapidly and completely inhibited by 25mug ml minus 1 cytochalasin B. The results are interpreted in terms of actin filaments in the stationary cortex interacting with a myosin-like protein which is able to link to endoplasmic organelles. Movement results from an active shear type of mechanism.     

The ultrastructure of the sea urchin egg cortex isolated before and after fertilization

The three-dimensional organization of cortices isolated from unfertilized and fertilized Strongylocentrotus purpuratus eggs has been examined by several techniques of light and electron microscopy. It has been found that when moderate shear forces are used, the isolated unfertilized egg cortex, in addition to cortical granules, contains acidic vesicles and an elaborate network of rough endoplasmic reticulum. This network provides a physical link between the cell surface and several kinds of cytoplasmic organelles (mitochondria, yolk granules, acidic vesicles) which are retained as part of the isolated cortex when gentle shear forces are applied. Furthermore a good visualization of actin in the cortex is provided: it is present as short filaments and mostly within the stubby microvilli of the egg. Finally, it has been noted that plaques exist on the inside face of the plasma membrane ready to assemble into typical clathrin coats that prefigure the burst of coated vesicle endocytosis that takes place after fertilization. The cortex isolated soon after fertilization is shown to contain coated pits and a scaffolding of filaments (mostly actin) in which many acidic vesicles are embedded.     

Arabidopsis Formin3 Directs the Formation of Actin Cables and Polarized Growth in Pollen Tubes

Cytoplasmic actin cables are the most prominent actin structures in plant cells, but the molecular mechanism underlying their formation is unknown. The function of these actin cables, which are proposed to modulate cytoplasmic streaming and intracellular movement of many organelles in plants, has not been studied by genetic means. Here, we show that Arabidopsis thaliana formin3 (AFH3) is an actin nucleation factor responsible for the formation of longitudinal actin cables in pollen tubes. The Arabidopsis AFH3 gene encodes a 785–amino acid polypeptide, which contains a formin homology 1 (FH1) and a FH2 domain. In vitro analysis revealed that the AFH3 FH1FH2 domains interact with the barbed end of actin filaments and have actin nucleation activity in the presence of G-actin or G actin-profilin. Overexpression of AFH3 in tobacco (Nicotiana tabacum) pollen tubes induced excessive actin cables, which extended into the tubes'' apices. Specific downregulation of AFH3 eliminated actin cables in Arabidopsis pollen tubes and reduced the level of actin polymers in pollen grains. This led to the disruption of the reverse fountain streaming pattern in pollen tubes, confirming a role for actin cables in the regulation of cytoplasmic streaming. Furthermore, these tubes became wide and short and swelled at their tips, suggesting that actin cables may regulate growth polarity in pollen tubes. Thus, AFH3 regulates the formation of actin cables, which are important for cytoplasmic streaming and polarized growth in pollen tubes.     

Myosin in onion (Allium cepa) bulb scale epidermal cells: involvement in dynamics of organelles and endoplasmic reticulum

Studied with the fluorochrome 3,3-dihexyloxacarbocyanine iodide [(DIOC₆(3)], the dynamic system of the endoplasmic reticulum (ER) in epidermal cells of onion bulb scales consists of long, tubular strands moving together with organelles in the deeper cytoplasm, and of a less mobile network composed of tubular and lamellar elements at the cell periphery. Treatment with the sulfhydryl-reagent N-ethylmaleimide (NEM) inhibited organelle and ER movement, and caused the fusion of ER-tubules into flat sheets. Fixed, long, tubular ER strands were formed by lowering the cytosolic pH of NEM-treated cells. Both these observations indicate the involvement of myosin in the dynamics of organelles and ER. Using a monoclonal antibody against murine skeletal muscle myosin (known to cross-react with plant myosin; Tang et al. 1989, J. Cell Sci. 92: 569–574), myosin was identified by immunofluorescence microscopy. Mapping the distribution of myosin, actin filaments, ER, and organelles in different phases of recovery after centrifugation of epidermal cells, co-localization of myosin with ER and organelles but not with actin filaments was observed, supporting the hypothesis that a membrane bound motor protein exists in onion epidermal cells, which translocates organelles and the endoplasmic reticulum along actin filaments.     

Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system

The Golgi apparatus in plant cells consists of a large number of independent Golgi stack/trans-Golgi network/Golgi matrix units that appear to be randomly distributed throughout the cytoplasm. To study the dynamic behavior of these Golgi units in living plant cells, we have cloned a cDNA from soybean (Glycine max), GmMan1, encoding the resident Golgi protein alpha-1,2 mannosidase I. The predicted protein of approximately 65 kD shows similarity of general structure and sequence (45% identity) to class I animal and fungal alpha-1,2 mannosidases. Expression of a GmMan1::green fluorescent protein fusion construct in tobacco (Nicotiana tabacum) Bright Yellow 2 suspension-cultured cells revealed the presence of several hundred to thousands of fluorescent spots. Immuno-electron microscopy demonstrates that these spots correspond to individual Golgi stacks and that the fusion protein is largely confined to the cis-side of the stacks. In living cells, the stacks carry out stop-and-go movements, oscillating rapidly between directed movement and random "wiggling." Directed movement (maximal velocity 4.2 microm/s) is related to cytoplasmic streaming, occurs along straight trajectories, and is dependent upon intact actin microfilaments and myosin motors, since treatment with cytochalasin D or butanedione monoxime blocks the streaming motion. In contrast, microtubule-disrupting drugs appear to have a small but reproducible stimulatory effect on streaming behavior. We present a model that postulates that the stop-and-go motion of Golgi-trans-Golgi network units is regulated by "stop signals" produced by endoplasmic reticulum export sites and locally expanding cell wall domains to optimize endoplasmic reticulum to Golgi and Golgi to cell wall trafficking.     

Myosin and Ca2+-sensitive streaming in the alga Chara: detection of two polypeptides reacting with a monoclonal anti-myosin and their localization in the streaming endoplasm

A monoclonal antibody to the heavy chain of myosin from mouse 3T3 cells was used to detect and localize related proteins in the green alga Chara. Proteins of 200,000 and 110,000 Mr reacted on immunoblots of proteins precipitated rapidly with trichloroacetic acid to minimize proteolysis. Immunofluorescence of whole cells localized these proteins to organelles of the streaming endoplasm, to a system of endoplasmic strands and to the subcortical actin bundles. Except that fewer endoplasmic strands and organelles were found and the strands were tangled, the localization pattern was similar in cells rapidly perfused to remove the bulk of the streaming endoplasm. Actin was confined almost entirely to the system of subcortical actin bundles in both whole and perfused cells. Myosin that was associated with the tangled endoplasmic strands but not that associated with the organelles or actin bundles was removed by concentrations of Ca2+ inhibiting ATP-dependent streaming in perfused cells. ATP extracted both organelles and endoplasmic strands but left a continuous pattern of myosin immunostaining along the actin bundles. The findings are discussed in relation to the possible existence of two forms of myosin and of separate mechanisms moving the bulk endoplasm and individual organelles.     

The Fine Structure of the Epithelial Cells of the Mouse Prostate : I. Coagulating Gland Epithelium

The fine structure of the epithelial cells of the anterior lobe, or coagulating gland, of the mouse prostate has been investigated by electron microscopy. This organ is composed of small tubules, lined by tall, simple cuboidal epithelium surrounded by connective tissue and smooth muscle. The epithelial cells are limited by a distinct plasma membrane, which covers minute projections of the cytoplasm into the lumen. The cell membranes of adjacent cells are separated by a narrow layer of structureless material of low density. The cavities of the endoplasmic reticulum are greatly dilated, and the cytoplasmic matrix is reduced to narrow strands, in which the various organelles are visible. The content of the cavities of the endoplasmic reticulum appears as structureless material of lesser density than the cytoplasmic matrix. Material which may be interpreted as secretion products can be seen in the lumina of the tubules. The possible nature of the material inside the cisternal spaces and the secretory mechanisms in these cells is discussed.     

Effects of exogenous proteins on cytoplasmic streaming in perfused Chara cells

Cytoplasmic streaming in characean algae is thought to be generated by interaction between subcortical actin bundles and endoplasmic myosin. Most of the existing evidence supporting this hypothesis is of a structural rather than functional nature. To obtain evidence bearing on the possible function of actin and myosin in streaming, we used perfusion techniques to introduce a number of contractile and related proteins into the cytoplasm of streaming Chara cells. Exogenous actin added at concentrations as low as 0.1 mg/ml is a potent inhibitor of streaming. Deoxyribonuclease I (DNase I), an inhibitor of amoeboid movement and fast axonal transport, does not inhibit streaming in Chara. Fluorescein-DNase I stains stress cables and microfilaments in mammalian cells but does not bind to Chara actin bundles, thus suggesting that the lack of effect on streaming is due to a surprising lack of DNase I affinity for Chara actin bundles. Heavy meromyosin (HMM) does not inhibit streaming, but fluorescein-HMM (FL-HMM), having a partially disabled EDTA ATPase, does. Quantitative fluorescence micrography provides evidence that inhibition of streaming by FL-HMM may be due to a tendency for FL-HMM to remain bound to Chara actin bundles even in the presence of MgATP. Perfusion with various control proteins, including tubulin, ovalbumin, bovine serum albumin, and irrelevant antibodies, does not inhibit streaming. These results support the hypothesis that actin and myosin function to generate cytoplasmic streaming in Chara.     

Relation between cell activity and the distribution of cytoplasmic actin and myosin

We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.     

Measurement of motive force for cytoplasmic streaming in characean internodes

Summary With an attempt to measure the motive force responsible for cytoplasmic streaming in characean internodal cells, the difference between densities of cytoplasm and vacuolar sap was heightened by about 10 times (density of vacuolar sap was made larger than that of cytoplasm) by replacing the natural vacuolar sap ofChara corallina with an artificial one of higher density. Endoplasmic flow contiguous to the peripheral actin cables (peripheral flow of endoplasm) in the centrifugal direction was not influenced at all by the application of centrifugal acceleration up to 1400 g. We thus concluded that the motive force for the peripheral flow should be much larger than 12dyn/cm², a figure more than 10 times larger than that for bulk endop lasmic flow so far reported.Dedicated to Emeritus Professor Noburo Kamiya on the occasion of his 80th birthday