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PubMed1987

Microfilaments: dynamic arrays in higher plant cells.

By using fluorescently labeled phalloidin we have examined, at the light microscope level, the three-dimensional distribution and reorganization of actin-like microfilaments (mfs) during plant cell cycle and differentiation. At interphase, mfs are organized into three distinct yet interconnected arrays: fine peripheral networks close to the plasma membrane; large axially oriented cables in the subcortical region; a nuclear "basket" of mfs extending into the transvacuolar strands. All these arrays, beginning with the peripheral network, disappear at the onset of mitosis and reappear, beginning with the nuclear basket, after cytokinesis. During mitotic and cytokinetic events, mfs are associated with the spindle and phragmoplast. Actin staining in the spindle is localized between the chromosomes and the spindle poles and changes in a functionally specific manner. The nuclear region appears to be the center for mf organization and/or initiation. During differentiation from rapid cell division to cell elongation, mf arrays switch from an axial to a transverse orientation, thus paralleling the microtubules. This change in orientation reflects a shift in the direction of cytoplasmic streaming. These observations show for the first time that actin-like mfs form intricate and dynamic arrays in plant cells which may be involved in many as yet undescribed cell functions.

Actin Cytoskeleton↗

PubMed1970

A direct light effect on maintaining photosynthetic activity of Nitella chloroplasts.

The chloroplasts of internodal cells of Nitella are fixed to a stationary layer of cytoplasm whereas the nuclei and most of the cytoplasm stream along the longitudinal axis. Isolated internodal cells were maintained for several days with half the cell kept in the dark, the other half kept under continuous light. Photosynthetic activity of the cells was checked by placing the cell evenly illuminated in a (14)CO(2) atmosphere. Chloroplasts of the previously dark half of the cell were found to fix only half as much CO(2) as the chloroplasts which were continuously illuminated. These results are discussed in relation to the possible direct effect of light on biosynthetic reactions of mature chloroplasts.

Carbon Isotopes↗

PubMed1975

Identification of actin in situ at the ectoplasm-endoplasm interface of Nitella. Microfilament-chloroplast association.

Using a glycerination procedure designed to avoid excessive plasmolysis or disruption of the ectoplasm, microfilaments in bundles at the ectoplasm-endoplasm interface of Nitella internode cell segments were found to bind rabbit heavy meromyosin (HMM) in situ. All HMM arrowheads in a bundle seem to have the same polarity and many lie in register as judged from the electron micrographs; the arrowhead periodicity is approximately 380 . The decorated microfilaments are thus similar to those seen in negatively stained cytoplasmic suspensions of internode cells. In glycerinated material, as well as in suspensions, the microfilaments are closely associated with chloroplasts. The microfilaments lie adjacent to or are attached to the chloroplast envelope. The results provide further evidence that the microfilaments thought to play a role in cytoplasmic streaming in vivo in Nitella consist of actin and suggest that they may be anchored to the chloroplasts.

Actins↗

PubMed1980

A morphological study of plasma and phagosome membranes during endocytosis in Acanthamoeba.

Particle ingestion by Acanthamoeba is rapid. Within 40 s bound particles can be surrounded by pseudopods, brought into the cytoplasm, and released as phagosomes into the cytoplasmic stream. In electron micrographs the phagosome appears as a flasklike invagination of the surface. Separation from the surface occurs by fragmentation of the attenuated "neck+ of the invagination. The separated phagosome membrane has a three- to fourfold greater density of intramembrane particles than the plasma membrane from which it derives. This change is evident within 15 min of ingestion and is detectable while the membrane is still tightly apposed to the particle. There is no direct evidence for the mechanism of this increase; no increase in particle density was seen in the membrane at an early stage in the forming phagosomes still connected to the surface. These morphological observations are consistent with chemical analyses, to be reported in a separate communication, that show that the phagosome membrane has a higher protein to phospholipid ratio and a higher glycosphingolipid content than the plasma membrane. Enlarged phagosomes (presumptive phagolysosomes) show multiple small vesiculations of characteristic morphology. The small vesicles are postulated to be the major route of membrane return to the cell surface.

Amoeba↗

PubMed2000

Cloning and characterization of a myosin from characean alga, the fastest motor protein in the world.

In characean algae, very rapid cytoplasmic streaming is generated by sliding movement of an unconventional myosin on fixed actin cables. The speed of this sliding movement is the fastest among many molecular motors known so far. We have cloned a set of overlapping cDNAs encoding the heavy chain of this myosin by immunoscreening with antibody raised against characean myosin. The molecular mass of this heavy chain is 248 kDa, and the protein has a conserved motor domain, six IQ motifs, an extensive alpha-helical coiled-coil domain, and a C-terminal globular domain. Phylogenetic analysis suggested that this myosin belongs to class XI.

Amino Acid Motifs↗

PubMed1986

The inhibitory Ca2+-regulation of the actin-activated Mg-ATPase activity of myosin from Physarum polycephalum plasmodia.

Myosin was rapidly prepared from the slime mould, Physarum polycephalum to a high level of homogeneity (greater than 95%), in a high yield (about 10 mg/100 g tissue) and in a phosphorylated state (about 5 mol phosphate/mol of 500,000 Mr myosin). Actin activated the Mg-ATPase activity of this myosin in the absence of Ca2+ about 30-fold, and this actin-activated ATPase activity was reduced to about 20% of the original activity when Ca2+ concentration was increased to 50 microM, i.e., the actin-myosin-ATP interactions show Ca-inhibition. The Ca2+ concentration giving half-maximum inhibition was 1-3 microM. The Ca-inhibition was clearly observed at physiological concentrations of Mg2+ but was obscured at both lower and higher concentrations of Mg2+. The Ca-inhibitory effect on ATP hydrolysis by actomyosin reconstituted from skeletal actin and Physarum myosin was quick and reversible. Ca-binding measurement showed that myosin bound Ca2+ with half-maximal binding at 2 microM Ca2+ and maximum binding of 2 mol per mol myosin, indicating that Ca2+ may inhibit the ATPase activity by binding to myosin. The involvement of this myosin-linked regulatory system in the Ca2+ -control of cytoplasmic streaming is discussed.

Actins↗

PubMed2007

Microtubule- and actin filament-dependent motors are distributed on pollen tube mitochondria and contribute differently to their movement.

The pollen tube exhibits cytoplasmic streaming of organelles, which is dependent on the actin-myosin system. Although microtubule-based motors have also been identified in the pollen tube, many uncertainties exist regarding their role in organelle transport. As part of our attempt to understand the role of microtubule-based movement in the pollen tube of tobacco, we investigated the cooperation between microtubules and actin filaments in the transport of mitochondria and Golgi vesicles, which are distributed differently in the growing pollen tube. The analysis was performed using in vitro motility assays in which organelles move along both microtubules and actin filaments. The results indicated that the movement of mitochondria and Golgi vesicles is slow and continuous along microtubules but fast and irregular along actin filaments. In addition, the presence of microtubules in the motility assays forces organelles to use lower velocities. Actin- and tubulin-binding tests, immunoblotting and immunogold labeling indicated that different organelles bind to identical myosins but associate with specific kinesins. We found that a 90 kDa kinesin (previously known as 90 kDa ATP-MAP) is associated with mitochondria but not with Golgi vesicles, whereas a 170 kDa myosin is distributed on mitochondria and other organelle classes. In vitro and in vivo motility assays indicate that microtubules and kinesins decrease the speed of mitochondria, thus contributing to their positioning in the pollen tube.

Actins↗

PubMed1999

Changes in the diffusion of water and intracellular metabolites after excitotoxic injury and global ischemia in neonatal rat brain.

The reduction of the apparent diffusion coefficient (ADC) of brain tissue water in acute cerebral ischemia, as measured by diffusion-weighted magnetic resonance imaging, is generally associated with the development of cytotoxic edema. However, the underlying mechanism is still unknown. Our aim was to elucidate diffusion changes in the intracellular environment in cytotoxic edematous tissue. The ADC of intracellular metabolites was measured by use of diffusion-weighted 1H-magnetic resonance spectroscopy after (1) unilateral N-methyl-D-aspartate (NMDA) injection and (2) cardiac arrest-induced global ischemia in neonatal rat brain. The distinct water ADC drop early after global ischemia was accompanied by a significant reduction of the ADC of all measured metabolites (P < 0.01, n = 8). In the first hours after excitotoxic injury, the ADC of water and the metabolites taurine and N-acetylaspartate dropped significantly (P < 0.05, n = 8). At 24 and 72 hours after NMDA injection brain metabolite levels were diminished and metabolite ADC approached contralateral values. Administration of the NMDA-antagonist MK-801 1.5 hours after NMDA injection completely normalized the water ADC but not the metabolite ADC after 1 to 2 hours (n = 8). No damage was detected 72 hours later and, water and metabolite ADC had normal values (n = 8). The contribution of brain temperature changes (calculated from the chemical shift between the water and N-acetylaspartate signals) and tissue deoxygenation to ischemia-induced intracellular ADC changes was minor. These data lend support to previous suggestions that the ischemia-induced brain water ADC drop may partly be caused by reduced diffusional displacement of intracellular water, possibly involving early alterations in intracellular tortuosity, cytoplasmic streaming, or intracellular molecular interactions.

Animals↗

PubMed2002

Actin-binding proteins in the Arabidopsis genome database: properties of functionally distinct plant actin-depolymerizing factors/cofilins.

The plant actin cytoskeleton is a highly dynamic, fibrous structure essential in many cellular processes including cell division and cytoplasmic streaming. This structure is stimulus responsive, being affected by internal stimuli, by biotic and abiotic stresses mediated in signal transduction pathways by actin-binding proteins. The completion of the Arabidopsis genome sequence has allowed a comparative identification of many actin-binding proteins. However, not all are conserved in plants, which possibly reflects the differences in the processes involved in morphogenesis between plant and other cells. Here we have searched for the Arabidopsis equivalents of 67 animal/fungal actin-binding proteins and show that 36 are not conserved in plants. One protein that is conserved across phylogeny is actin-depolymerizing factor or cofilin and we describe our work on the activity of vegetative tissue and pollen-specific isoforms of this protein in plant cells, concluding that they are functionally distinct.

Actins↗

PubMed1985

Recurrent senescence in axenic cultures of Physarum polycephalum.

When subcultures of the aux-2 and aux-4 strains of Physarum polycephalum, which had been grown for more than four years in axenic shake culture, were transferred to non-axenic surface culture they displayed progressively shorter lifespans (older axenic surface cultures yield shorter lived non-axenic cultures). Similar subcultures transferred to axenic agar medium also underwent senescent-like events. These subcultures, after a period of vigorous growth, displayed a slower growth rate, reduced cytoplasmic streaming, loss of yellow pigment, and eventually they fragmented into a number of small spherical structures with the concomitant lysis of most of the plasmodium. In non-axenic culture these structures quickly degenerated (and disappeared from the culture); however, in axenic culture they revived and after several days produced new vigorous plasmodia. Following a period of vigorous growth the plasmodium again underwent senescent-like events. This cycle of senescence and growth was repeated a number of times before death finally occurred.

Cell Cycle↗

PubMed1994

The response to gravity is correlated with the number of statoliths in Chara rhizoids.

In contrast to higher plants, Chara rhizoids have single membrane-bound compartments that appear to function as statoliths. Rhizoids were generated by germinating zygotes of Chara in either soil water (SW) medium or artificial pond water (APW) medium. Differential-interference-contrast microscopy demonstrated that rhizoids form SW-grown plants typically contain 50 to 60 statoliths per cell, whereas rhizoids from APW-grown plants contain 5 to 10 statoliths per cell. Rhizoids from SW are more responsive to gravity than rhizoids from APW because (a) SW rhizoids were oriented to gravity during vertical growth, whereas APW rhizoids were relatively disoriented, and (b) curvature of SW rhizoids was 3 to 4 times greater throughout the time course of curvature. The growth rate of APW rhizoids was significantly greater than that of SW-grown rhizoids. This latter result suggests that APW rhizoids are not limited in their ability for gravitropic curvature by growth and that these rhizoids are impaired in the early stages of gravitropism (i.e. gravity perception). Plants grown in APW appeared to be healthy because of their growth rate and the vigorous cytoplasmic streaming observed in the rhizoids. This study is comparable to earlier studies of gravitropism in starch-deficient mutants of higher plants and provides support for the role of statoliths in gravity perception.

Chlorophyta↗

PubMed1999

Inhibitory regulation of higher-plant myosin by Ca2+ ions

Myosin isolated from the pollen tubes of lily (Lilium longiflorum) is composed of a 170-kD heavy chain (E. Yokota and T. Shimmen [1994] Protoplasma 177: 153-162). Both the motile activity in vitro and the F-actin-stimulated ATPase activity of this myosin were inhibited by Ca2+ at concentrations higher than 10(-6) M. In the Ca2+ range between 10(-6) and 10(-5) M, inhibition of the motile activity was reversible. In contrast, inhibition by more than 10(-5) M Ca2+ was not reversible upon Ca2+ removal. An 18-kD polypeptide that showed the same mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that of spinach calmodulin (CaM) was present in this myosin fraction. This polypeptide showed a mobility shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a Ca2+-dependent manner. Furthermore, this polypeptide was recognized by antiserum against spinach CaM. By immunoprecipitation using antiserum against the 170-kD heavy chain, the 18-kD polypeptide was coprecipitated with the 170-kD heavy chain, provided that the Ca2+ concentration was low, indicating that this 18-kD polypeptide is bound to the 170-kD myosin heavy chain. However, the 18-kD polypeptide was dissociated from the 170-kD heavy chain at high Ca2+ concentrations, which irreversibly inhibited the motile activity of this myosin. From these results, it is suggested that the 18-kD polypeptide, which is likely to be CaM, is associated with the 170-kD heavy chain as a light chain. It is also suggested that this polypeptide is involved in the regulation of this myosin by Ca2+. This is the first biochemical basis, to our knowledge, for Ca2+ regulation of cytoplasmic streaming in higher plants.

Journal Article↗

PubMed1999

Tracheary element differentiation uses a novel mechanism coordinating programmed cell death and secondary cell wall synthesis

Tracheary element differentiation requires strict coordination of secondary cell wall synthesis and programmed cell death (PCD) to produce a functional cell corpse. The execution of cell death involves an influx of Ca2+ into the cell and is manifested by rapid collapse of the large hydrolytic vacuole and cessation of cytoplasmic streaming. This precise means of effecting cell death is a prerequisite for postmortem developmental events, including autolysis and chromatin degradation. A 40-kD serine protease is secreted during secondary cell wall synthesis, which may be the coordinating factor between secondary cell wall synthesis and PCD. Specific proteolysis of the extracellular matrix is necessary and sufficient to trigger Ca2+ influx, vacuole collapse, cell death, and chromatin degradation, suggesting that extracellular proteolysis plays a key regulatory role during PCD. We propose a model in which secondary cell wall synthesis and cell death are coordinated by the concomitant secretion of the 40-kD protease and secondary cell wall precursors. Subsequent cell death is triggered by a critical activity of protease or the arrival of substrate signal precursor corresponding with the completion of a functional secondary cell wall.

Journal Article↗

PubMed1999

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.

Actomyosin↗

PubMed1977

Freezing injury in onion bulb cells: I. Evaluation of the conductivity method and analysis of ion and sugar efflux from injured cells.

Onion (Allium cepa L.) bulbs were frozen to -4 and -11 C and kept frozen for up to 12 days. After slow thawing, a 2.5-cm square from a bulb scale was transferred to 25 ml deionized H(2)O. After shaking for standard times, measurements were made on the effusate and on the effused cells. The results obtained were as follows.Even when the scale tissue was completely infiltrated, and when up to 85% of the ions had diffused out, all of the cells were still alive, as revealed by cytoplasmic streaming and ability to plasmolyze. The osmotic concentration of the cell sap, as measured plasmolytically, decreased in parallel to the rise in conductivity of the effusate. The K(+) content of the effusate, plus its assumed counterion, accounted for only 20% of the total solutes, but for 100% of the conductivity. A large part of the nonelectrolytes in the remaining 80% of the solutes was sugars.The increased cell injury and infiltration in the -11 C treatment, relative to the -4 C and control (unfrozen) treatments, were paralleled by increases in conductivity, K(+) content, sugar content, and pH of the effusate. In spite of the 100% infiltration of the tissue and the large increase in conductivity of the effusate following freezing, no increase in permeability of the cells to water could be detected.The above observations may indicate that freezing or thawing involves a disruption of the active transport system before the cells reveal any injury microscopically.

Journal Article↗

PubMed1978

Irradiation-enhanced Phytochrome Pelletability: Requirement for Phosphorylative Energy in Vivo.

Short, high intensity pulses of red and far red light are used to study, at room temperature, the kinetics of the in vivo dark reaction responsible for irradiation-enhanced phytochrome pelletability. The t((1/2)) for this reaction is 2 seconds at 25 C in both Avena shoots and Zea mays coleoptiles. This is the most rapid phytochrome-far red-absorbing form (Pfr)-mediated cellular response thus far reported. Anoxia, KCN, NaN(3) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone reduce the rate (but not the final extent) of the reaction by more than an order of magnitude. The rate of the reaction under these conditions is strongly correlated with the inhibitor-induced reductions in cellular ATP levels. Likewise, recovery in ATP levels upon withdrawal of the inhibitors is accompanied by a parallel recovery in the rate of the reaction. Cytochalasin B blocks cytoplasmic streaming without diminishing the pelletability response. Colchicine is likewise without effect. These data suggest a requirement for phosphorylative energy in one or more of the Pfr-dependent intracellular events leading to enhanced phytochrome pelletability. The possibility that this event might represent an ATP-dependent modification of the pigment protein itself in the Pfr form is discussed.

Journal Article↗

PubMed1991

Electrogenic transport properties of growing Arabidopsis root hairs : the plasma membrane proton pump and potassium channels.

Ion transport, measured using double-barreled micropipettes to obtain current-voltage relations, was examined in Arabidopsis thaliana root hairs that continued tip growth and cytoplasmic streaming after impalement with the micropipette. To do this required in situ measurements with no handling of the seedlings to avoid wounding responses, and conditions allowing good resolution microscopy in tandem with the electrophysiological measurements. Two ion transport processes were demonstrated. One was a tetraethylammonium-sensitive potassium ion current, inward at hyperpolarized potentials and outward at depolarized potentials. The addition of tetraethylammonium (a potassium channel blocker) caused the potential to hyperpolarize, indicating the presence of a net inward potassium current through the ion channels at the resting potential. The potassium influx was sufficient to "drive" cellular expansion based upon growth rates. Indeed, tetraethylammonium caused transient inhibition of tip growth. The other electrogenic process was the plasma membrane proton pump, measured by indirect inhibition with cyanide or direct inhibition by vanadate. The proton pump was the dominant contribution to the resting potential, with a very high current density of about 250 microamperes per square centimeter (seen only in young growing root hairs). The membrane potential generated by the proton pump presumably drives the potassium influx required for cellular expansion. The pump appears to be a constant current source over the voltage range -200 to 0 millivolts. With this system, it is now possible to study the physiology of a higher plant cell in dynamic living state using a broad range of cell biological and electrophysiological techniques.

Journal Article↗

PubMed1992

Mathematical modeling of oxygen diffusion and respiration in legume root nodules.

The O(2) permeability of legume root nodules is under physiological control; decreases in permeability are triggered by various forms of stress. Two linked mathematical models were used to explore several hypotheses concerning the physical nature of the variable diffusion barrier in nodules. Respiration and diffusion of dissolved O(2) and oxygenated leghemoglobin were simulated for the nodule cortex and the nodule interior. Measured nodule permeabilities were shown to be inconsistent with the hypothesis that large numbers of air-filled pores penetrate the diffusion barrier. Changes in the affinity of leghemoglobin for O(2) or in the rate of cytoplasmic streaming in diffusion barrier cells did not result in the large changes in O(2) permeability reported for real nodules. The presence or absence, but not the thickness, of aqueous plugs in radial pores through the cortex was found to have a large effect on permeability. Flooding of intercellular spaces, either between layers of cells in the cortex or in the nodule interior, also caused large changes in simulated permeability. The unsteady-state O(2) method for determining nodule permeability was tested using data generated by the model. The accuracy of the method was confirmed, provided that certain assumptions (full oxygenation of leghemoglobin under pure O(2) and uniform conditions in the nodule interior) are met.

Journal Article↗