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Results for “Cytoplasmic Streaming”

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PubMed1997

Secretion of slime, the extracellular matrix of the plasmodium, as visualized with a fluorescent probe and its correlation with locomotion on the substratum.

Slime, the extracellular matrix of Physarum plasmodium, is secreted by the exocytosis of a vesicles that contain a slime precursor. Using an antibody raised against biochemically purified slime, we detected the intracellular localization of the slime vesicle. Slime vesicles are abundant in the advancing front of the plasmodium, as confirmed by electron microscopic observation in two different cross-sectional angles. Screening various reagents, we found that rhodamine-phosphatidylethanolamine (Rh-PE) binds specifically to slime in both its intravesicular and extracellular forms, as confirmed by immunoelectron microscopy using an antibody against fluorochrome rhodamine. The plasmodia vitally stained with Rh-PE exhibited dynamic fluorescent patterns during the course of locomotion. The fluorescence was conspicuous at the periphery of the leading pseudopods and oscillated according to the shuttle streaming that accompanied the relaxation and contraction of the periphery; it was intense in the relaxation phase when pseudopods extended, and became weak in the contraction phase when pseudopods contracted. The results collectively mean that the slime vesicles carried by the cytoplasmic streaming accumulated prior to secretion at the advancing margin of the plasmodium.

Animals↗

PubMed2003

Cellular organelle transport and positioning by plasma streaming.

Our analysis of known data reveals that translocations of passively movable cellular organelles from tiny granules up to large cell nuclei can be ascribed to transport by streaming cytoplasm. The various behaviours, such as velocity changes during more or less interrupted movements, forth and back shuttling and particle rotation result from different types of plasma circulation. Fast movements over long distances, as observed in the large characean internodial cells occur in strong streams generated by myosin in bundles of actin filaments in the direction of the barbed filament ends. Slow movements with frequent reversions of the direction are typical for neuronal axons, in which an anterograde plasma flow, produced in a thin layer of membrane-attached actin filaments, is compensated by a retrograde stream, produced by dynein activity in the central bundle of microtubules. Here particle rotation is due to steep flow velocity gradients, and frequent changes of particle movements result from minor particle displacements in radial directions. Similar shuttling of pigment granules in the lobes of epidermal chromatophores results from the same mechanism, whereby the centrifugal movement along astral microtubules is due to flow generated by excess of kinesin activity and the centripetal movement to the plasma recycling through the intermicrotubular space. If the streaming pattern is reversed by switching to excess dynein activity, the moving granules are trapped in the high microtubule density at the aster center. The presence of larger bodies in asters disturbs the regular, kinesin-dependent microtubule distribution in such a way that a superimposed centrifugal plasma flow develops in the microtubule-dense layer along them, which is recycled in the microtubule-free space, created by their presence. Consequently, at excess kinesin activity, nuclei, mitochondria as well as chromosome fragments move towards the aster center until they reach a dynamically stabilized position that depends on the local microtubule density. These various behaviours are not rationally explainable by models based on a mechanical stepping along microtubules or actin filaments.

Animals↗

PubMed1980

Formation of multinuclear cells induced by dimethyl sulfoxide: inhibition of cytokinesis and occurrence of novel nuclear division in Dictyostelium cells.

Our previous studies showed that 10 percent dimethyl sulfoxide (DMSO) induces the formation of actin microfilament bundles in the cell nucleus together with the dislocation of cortical microfilaments from the plasma membrane. The present study investigated the effects of DMSO on diverse activities mediated by cellular microfilaments as the second step toward assessing potential differences between nuclear and cytoplasmic actins of dictyostelium mucoroides. DMSO was found to reversibly inhibit cell-to- glass as well as cell-to-cell adhesion, cell locomotion, and cell multiplication, whereas cytoplasmic streaming and phagocytosis were not obviously inhibited. Also, 5 percent DMSO inhibited cytokinesis but did not totally inhibit cell growth thus leading to the development of giant cells more than 10 times larger than normal cells. Transmission electron microscopy using serial thin sections showed the occurrence of multinucleation in the DMSO- induced giant cells. After the removal of DMSO, the giant multinuclear cells underwent multiple cytoplasmic cleavage producing normal-sized mononuclear cells. The nuclear division in the DMSO-induced giant cells was unique in that no spindle microtubules were formed, and vesicles appeared inside the nucleus forming a transverse partition of the nuclear envelope. The presence of actin filaments in those nuclei was demonstrated by a binding study with skeletal muscle myosin subfragment-1, and their possible involvement in this mode of nuclear division is discussed.

Actins↗

PubMed1980

Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin.

An active fluorescent derivative of the actin-binding mushroom toxin phallacidin has been synthesized. Convenient methods were developed to stain actin cytoskeletal structures in living and fixed cultured animal cells and actively streaming algal cells. Actin binding specificity was demonstrated by competitive binding experiments and comparative staining of well-known structures. Large populations of living animal cells in culture were readily stained by using a relatively mild lysolecithin permeabilization procedure facilitated by the small molecular size of the label. Actin in animal cells was stained stress fibers, ruffles, the cellular geodome, and in diffuse appearing distributions apparently associated with the plasma membrane. Staining of actin cables in algae with nitrobenzoxadiazole (NBD)-phallacidin did not inhibit cytoplasmic streaming. NBD-phallacidin provides a convenient actin-specific fluorescent label for cellular cytoskeletal structures with promise for use in studies of actin dynamics in living systems.

Actins↗

PubMed2000

Plant nuclei can contain extensive grooves and invaginations.

Plant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus.

Cell Nucleus↗

PubMed1973

Mechanism of inhibition of translocation by localized chilling.

Arrhenius plots of translocation velocity as a function of petiole temperature show a marked increase in temperature dependence below 10 C in bean (a chilling-sensitive species) but not in sugar beet (chilling-resistant). The increased temperature dependence below 10 C was not observed for cytoplasmic streaming or oxygen uptake in bean. Bean petioles were served to release pressure in order to determine whether sieve tubes are obstructed in cold-treated petioles. The resulting pressure release caused serious displacement of the crystalline protein bodies in the sieve tubes of petioles at 25 C, but in those locally cooled to 0 C for 30 minutes little displacement occurred, indicating obstruction in the latter. An ultrastructural study of sieve tubes in tissue frozen rapidly in situ and dehydrated by freeze substitution revealed that treatment at 0 C for 30 minutes caused structural alteration and displacement of the cytoplasmic material lining the sieve tube wall resulting in occlusion of sieve plates. The sieve plates of the control petioles at 25 C were generally clear of obstructions. The results indicate that inhibition of translocation by chilling in chilling-sensitive plants results from physical blockage of sieve plates rather than from direct inhibition of a metabolic process which drives translocation.

Journal Article↗

PubMed1985

Preprophase bands, phragmoplasts, and spatial control of cytokinesis.

Features of preprophase bands (PPBs) of microtubules (MTs), and the spatial relationship between phragmosomes, PPB sites, and developing phragmoplasts during cytokinesis, are reviewed, setting new observations in the context of current knowledge. PPBs in onion root tip cells are present by the beginning of the G2 period of the cell cycle. They are at first wide, but later become more compact, narrower bands. MTs traverse the cytoplasm between the band at the cell cortex and the nuclear envelope. This whole assemblage of nucleus, PPB and intervening MTs remains together when the cell is ruptured during preparation for examination by immunofluorescence microscopy. Double bands are occasionally seen in early stages of PPB development, perhaps as a consequence of double induction from neighbouring cells. Calmodulin is not present in PPBs at a higher concentration than in the general cytoplasm, but it is more abundant in parts of the spindle and in the phragmoplast. The PPB MTs disappear at prophase, but nevertheless the new cell plate fuses with the parental cell walls at the PPB site. This spatial relationship can be disrupted by treatment with CIPC. Another experimental disruption of the relationship, accomplished by making minute wounds in the PPB site of mitotic cells in Tradescantia stamen hairs, is described. In other experiments on these cells the phragmoplast is shown to become tethered to the PPB site when the cell plate is half to three-quarters developed, although the telophase nuclei are free to move. Rhodamine-labelled phalloidin reveals putative F-actin in the phragmoplast of Tradescantia, but not in the gap between the extending phragmoplast and the PPB site. Rhodamine-labelled phalloidin also stains cytoplasmic strands that exist when cytoplasmic streaming occurs before and after (but not during) mitosis. Cytochalasin B treatment blocks incorporation of actin into the phragmoplast, which, however, can still develop, though usually abnormally. The F-actin of the phragmoplast may function in consolidation of the cell plate, rather than in spatial guidance of its growth toward the PPB site at the cell surface.

Calmodulin↗

PubMed1986

Function of Rhizodermal Transfer Cells in the Fe Stress Response Mechanism of Capsicum annuum L.

A variety of red pepper (Capsicum annuum L., cv Yaglik) responds to Fe deficiency stress with simultaneously enhanced H(+) extrusion, reduction of ferric ions and synthesis of malic and citric acid in a swollen subapical root zone densely covered with root hairs. It is demonstrated that these stress responses temporally coincide with the development of rhizodermal and hypodermal transfer cells in this root zone. During stress response the transfer cells show a marked autofluorescence which could arise from endogenous iron chelators of the phenolic acid type. The presence of organelle-rich cytoplasm which often exhibits rotational cytoplasmic streaming points to high physiological activity and makes these cells, with their increased plasmalemma surface, particularly well suited for the entire stress response mechanism. Since Fe stress-induced acidification is diminished by vanadate and erythrosin B, both specific inhibitors of plasmalemma ATPases, it seems reasonable to suppose that H(+) pumping from transfer cells is activated by an ATPase located in their plasmamembrane. H(+) extrusion is also shown to be inhibited by abscisic acid. Raised phosphoenolpyruvate carboxylase activity and simultaneous accumulation of malate in the swollen root zone point to the action of a pH stat preventing a detrimental rise in cytoplasmic pH of transfer cells during enhanced H(+) extrusion. The simultaneous increase in citric acid concentration favors chelation of iron at the site of its uptake and thus ensures long distance transport to the areas of metabolic demand. A direct link between citrate accumulation and ferric ion reduction as proposed in recent literature further supports the crucial role of transfer cells in the response to Fe deficiency stress.

Journal Article↗

PubMed2005

Acto-myosin cytoskeleton dependent viscosity and shear-thinning behavior of the amoeba cytoplasm.

The mechanical behavior of the human parasite Entamoeba histolytica plays a major role in the invasive process of host tissues and vessels. In this study, we set up an intracellular rheological technique derived from magnetic tweezers to measure the viscoelastic properties within living amoebae. The experimental setup combines two magnetic fields at 90 degrees from each other and is adapted to an inverted microscope, which allows monitoring of the rotation of pairs of magnetic phagosomes. We observe either the response of the phagosome pair to an instantaneous 45 degrees rotation of the magnetic field or the response to a permanent uniform rotation of the field at a given frequency. By the first method, we concluded that the phagosome pairs experience a soft viscoelastic medium, represented by the same mechanical model previously described for the cytoplasm of Dictyostelium discoideum [Feneberg et al. in Eur Biophys J 30(4):284-294 2001]. By the second method, the permanent rotation of a pair allowed us to apply a constant shear rate and to calculate the apparent viscosity of the cytoplasm. As found for entangled polymers, the viscosity decreases with the shear rate applied (shear-thinning behavior) and exhibits a power-law-type thinning, with a corresponding exponent of 0.65. Treatment of amoeba with drugs that affect the actin polymer content demonstrated that the shear-thinning behavior of the cytoplasm depends on the presence of an intact actin cytoskeleton. These data present a physiologic relevance for Entamoeba histolytica virulence. The shear-thinning behavior could facilitate cytoplasm streamings during cell movement and cell deformation, under important shear experienced by the amoeba during the invasion of human tissues. In this study, we also investigated the role of the actin-based motor myosin II and concluded that myosin II stiffens the F-actin gel in living parasites likely by its cross-linking activity.

Actins↗

PubMed1994

Human neutrophil motility: time-dependent three-dimensional shape and granule diffusion.

The locomotion of human polymorphonuclear leukocytes (PMNs) was studied with two complementary methods: Three-dimensional shapes were reconstructed from time series of optical sectioning microscopy using differential interference contrast (DIC) optics, and the diffusion of cytoplasm granules within individual cells was measured using quasielastic laser light scattering (QELS). The three-dimensional cell edges outlined in the optical sections were analyzed qualitatively in time-lapse film strips and quantitatively from morphometry. The fastest locomotion occurred in chemotactic gradient with cell velocity that oscillated between 10 and 30 microns/min with a period of 50-55 seconds. Within the periodic bursts of speed, a fibroblast-like locomotory cycle was observed, with leading lamella extended and contacts formed with the substrate surface, followed by rapid motion of the cell body and nucleus over the immobile contacts. Consistent with this apparent staged motion, correlation analysis revealed a phase lag of 2-3 seconds in velocities between the bottom (ventral) and the top layers of the cell. In addition there was a tendency to a lower cell profile at times of higher velocity. The diffusion of natural cytoplasmic granules within resting PMNs was not affected by cytoskeleton disrupting drugs. During the stage of most rapid motion, when cytoplasmic streaming could be seen, diffusion of the granules decreased two- to 2.5-fold, and then returned to resting levels. These observations suggest that PMN locomotion consists of extensions near the surface to form forward contacts and then stiffening or possibly contraction of the cytoskeleton when the body of the cell is moved forward. Three-dimensional movies of PMN cells are included in the video supplement.

Cell Movement↗

PubMed2001

A Drosophila model of HIV-Tat-related pathogenicity.

To analyze the mechanism of Tat-mediated HIV pathogenicity, we produced a Drosophila melanogaster strain transgenic for HIV-tat gene and induced the expression of the protein during Drosophila development. By in vitro and in vivo experiments, we demonstrated that Tat specifically binds to tubulin via the MAP-binding domain of tubulin, and that this interaction delays the polymerization of tubulin and induces a premature stop to microtubule-dependent cytoplasmic streaming. The delay in the polymerization of microtubules, the tracks for the transport of the axes determinants, alters the positioning of the dorso-ventral axis as shown by the mislocalization of Gurken and Kinesin in oocyte of Drosophila after Tat induction. These results validate the use of Drosophila as a tool to study the molecular mechanism of viral gene products and suggest that Tat-tubulin interaction is responsible for neurodegenerative diseases associated with AIDS.

Animals↗

PubMed1985

Mini-review. On the possible importance of an intracellular circulation.

Since ultrastructural, biophysical and other studies continue to demonstrate that the internum of the cell is highly structured, the question raised and discussed in this review is whether an intracellular circulatory system is essential for the maintenance of active metabolism. Although cytoplasmic streaming is evident in large animal and plant cells, it is argued that it probably occurs in all cells irrespective of size, and is of particular importance in bringing together interacting molecules fast enough for metabolic processes to occur which would otherwise be far too slow if diffusion were the only form of motion. A common intracellular system would suffice for most metabolic processes and would also help to dissipate waste products. Interruption of this internal circulation would result in the inhibition of metabolic functioning, including for example protein turnover, for which evidence is presented to substantiate this hypothesis.

Animals↗

PubMed1994

Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media.

Lily pollen tubes possess a steep, tip-focused intracellular Ca2+ gradient and a tip-directed extracellular Ca2+ influx. Ratiometric ion imaging revealed that the gradient extends from above 3.0 microM at the apex to approximately 0.2 microM within 20 microns from the tip, while application of the Ca(2+)-specific vibrating electrode indicated that the extracellular influx measured between 1.4 and 14 pmol cm-2 sec-1. We examined the relationship between these phenomena and their role in tube growth by using different 1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA)-type buffers and hypertonic media. Injection of active BAPTA-type buffers or application of elevated levels of sucrose reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-focused gradient and the extracellular Ca2+ flux. Of the BAPTA-type buffers, 5,5'-dibromo-BAPTA (dissociation constant [Kd] is 1.5 microM) and 4,4'-difluoro-BAPTA (Kd of 1.7 microM) exhibited greater activity than those buffers with either a higher affinity (5,5'-dimethyl-BAPTA, Kd of 0.15 microM; BAPTA, Kd of 0.21 microM; 5,5'-difluoro-BAPTA, Kd of 0.25 microM) or lower affinity (5-methyl, 5'-nitro-BAPTA, Kd of 22 microM) for Ca2+. Our findings provide evidence that growing pollen tubes have open Ca2+ channels in their tip and that these channels become inactivated in nongrowing tubes. The studies with elevated sucrose support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca2+ signal.

Buffers↗

PubMed2003

Involvement of myosin in intracellular motility and cytomorphogenesis in Micrasterias.

Myosin was detected on Western blots of Micrasterias denticulata extracts by use of antibodies from different sources. Inhibitors with different targets of the actomyosin system, such as the myosin ATPase-blockers N-ethylmaleimide (NEM) and 2,3-butanedione monoxime (BDM), or the myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexhydro-1,4-diazapine (ML7), had similar effects on intracellular motility during cell development in the green alga Micrasterias, thus pointing towards a participation of myosin in these processes. The drugs markedly altered the mode of postmitotic nuclear migration, slowed down cytoplasmic streaming, changed cell pattern development and prevented normal chloroplast distribution and spreading into the growing semicell. In addition, an increase and dilatations in ER cisternae and marked morphological changes of the Golgi system were observed by transmission electron microscopy after exposure of growing cells to BDM. Neither BDM nor ML7 exhibited any effect on the distribution or arrangement of the cortical F-actin network nor on the F-actin basket around the nucleus, characteristic of untreated growing Micrasterias cells (J Cell Sci 107 (1994) 1929). This is particularly interesting since BDM caused disintegration of the microtubule system co-localized to the F-actin cage during normal nuclear migration. Together with the fact that other microtubules not connected to the F-actin system remained uninfluenced by BDM, this observation is evidence of an integrative function of myosin between the cytoskeleton elements.

Actins↗

PubMed2001

Amyloplast sedimentation dynamics in maize columella cells support a new model for the gravity-sensing apparatus of roots.

Quantitative analysis of statolith sedimentation behavior was accomplished using videomicroscopy of living columella cells of corn (Zea mays) roots, which displayed no systematic cytoplasmic streaming. Following 90 degrees rotation of the root, the statoliths moved downward along the distal wall and then spread out along the bottom with an average velocity of 1.7 microm min(-1). When statolith trajectories traversed the complete width or length of the cell, they initially moved horizontally toward channel-initiation sites and then moved vertically through the channels to the lower side of the reoriented cell where they again dispersed. These statoliths exhibited a significantly lower average velocity than those sedimenting on distal-to-side trajectories. In addition, although statoliths undergoing distal-to-side sedimentation began at their highest velocity and slowed monotonically as they approached the lower cell membrane, statoliths crossing the cell's central region remained slow initially and accelerated to maximum speed once they reached a channel. The statoliths accelerated sooner, and the channeling effect was less pronounced in roots treated with cytochalasin D. Parallel ultrastructural studies of high-pressure frozen-freeze-substituted columella cells suggest that the low-resistance statolith pathway in the cell periphery corresponds to the sharp interface between the endoplasmic reticulum (ER)-rich cortical and the ER-devoid central region of these cells. The central region is also shown to contain an actin-based cytoskeletal network in which the individual, straight, actin-like filaments are randomly distributed. To explain these findings as well as the results of physical simulation experiments, we have formulated a new, tensegrity-based model of gravity sensing in columella cells. This model envisages the cytoplasm as pervaded by an actin-based cytoskeletal network that is denser in the ER-devoid central region than in the ER-rich cell cortex and is linked to stretch receptors in the plasma membrane. Sedimenting statoliths are postulated to produce a directional signal by locally disrupting the network and thereby altering the balance of forces acting on the receptors in different plasma membrane regions.

Actins↗

PubMed1999

Actin cytoskeleton in plants: from transport networks to signaling networks.

The plant actin cytoskeleton is characterized by a high diversity in regard to gene families, isoforms, and degree of polymerization. In addition to the most abundant F-actin assemblies like filaments and their bundles, G-actin obviously assembles in the form of actin oligomers composed of a few actin molecules which can be extensively cross-linked into complex dynamic meshworks. The role of the actomyosin complex as a force generating system - based on principles operating as in muscle cells - is clearly established for long-range mass transport in large algal cells and specialized cell types of higher plants. Extended F-actin networks, mainly composed of F-actin bundles, are the structural basis for this cytoplasmic streaming of high velocities On the other hand, evidence is accumulating that delicate meshworks built of short F-actin oligomers are critical for events occurring at the plasma membrane, e.g., actin interventions into activities of ion channels and hormone carriers, signaling pathways based on phospholipids, and exo- and endocytotic processes. These unique F-actin arrays, constructed by polymerization-depolymerization processes propelled via synergistic actions of actin-binding proteins such as profilin and actin depolymerizing factor (ADF)/cofilin are supposed to be engaged in diverse aspects of plant morphogenesis. Finally, rapid rearrangements of F-actin meshworks interconnecting endocellular membranes turn out to be especially important for perception-signaling purposes of plant cells, e.g., in association with guard cell movements, mechano- and gravity-sensing, plant host-pathogen interactions, and wound-healing.

Actins↗

PubMed1993

Pure thoughts with impure proteins: permeabilized cell models of organelle motility.

Permeabilized cell models provide an experimental middle ground wherein the in vitro properties of mechanochemical proteins can be reconciled with the physical and topological constraints of the intact cell. Several well-studied examples of organelle motility are described here, including the actin-based cytoplasmic streaming of Characean algae, the microtubule-based aggregation and dispersion of pigment granules in chromatophores and the saltatory movements of vesicles along microtubules in fibroblasts and macrophages. The permeabilized models developed for these systems have helped to integrate observations in vivo with in vitro assays of motor proteins.

Actins↗

PubMed1989

Fluorescence microscopic localization of actin in pollen tubes: comparison of actin antibody and phalloidin staining.

A comparison of actin localization in pollen tubes of Nicotiana has been made using a monoclonal actin antibody and rhodamine-phalloidin (RP). The monoclonal antiactin, based on Western blotting of pollen tube extract, labels a polypeptide at 45 kD that comigrates with muscle actin. A 51-kD unknown protein and three bands less than 45 kD, presumed to be proteolytic fragments of actin, are also observed. Structural observations using this antibody reveal a network of axially oriented strands of microfilaments (MFs). The MFs are distributed throughout the length of the pollen tube except at the very tip, where diffuse staining is usually observed. A similar pattern of MFs is evident after RP staining. When pollen tubes are treated with cytochalasins (CB or CD) cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the microfilament (MF) pattern is markedly altered; however, the antibody and RP produce different staining patterns. The antibody reveals many MF strands that distribute throughout the tube length and extend into the very tip. In contrast, RP shows mostly a diffuse staining pattern with only a few short clumps of filamentous material. Immunogold labelling of sections of pollen tubes prepared by rapid-freeze fixation and freeze substitution reveals that actin MF bundles are indeed present after cytochalasin treatment. Our results thus question reports in the literature, based on phalloidin staining, asserting that cytochalasin fragments or destroys actin MFs.

Actins↗