Cyclosis-mediated intercellular transmission of photosynthetic metabolites in Chara revealed with chlorophyll microfluorometry.

Symplastic interconnections of plant cells via perforations in adjoining cell walls (plasmodesmata) enable long-distance transport of photoassimilates and signaling substances required for growth and development. The pathways and features of intercellular movement of assimilates are often examined with fluorescent tracers whose molecular dimensions are similar to natural metabolites produced in photosynthesis. Chlorophyll fluorescence was recently found to be a sensitive noninvasive indicator of long-distance intracellular transport of physiologically produced photometabolites in characean internodes. The present work shows that the chlorophyll microfluorometry has a potential for studying the cell-to-cell transport of reducing substances released by local illumination of one internode and detected as the fluorescence increase in the neighbor internode. The method provides temporal resolution in the time frame of seconds and can be used to evaluate permeability of plasmodesmata to natural components released by illuminated chloroplasts. The results show that approximately one third of the amount of photometabolites released into the streaming cytoplasm during a 30-s pulse of local light permeates across the nodal complex with the characteristic time of ~ 10 s. The intercellular transport was highly sensitive to moderate elevations of osmolarity in the bath solution (150 mM sorbitol), which contrasts to the view that only transnodal gradients in osmolarity (and internal hydrostatic pressure) have an appreciable influence on plasmodesmal conductance. The inhibition of cell-to-cell transport was reversible and specific; the sorbitol addition had no influence on photosynthetic electron transport and the velocity of cytoplasmic streaming. The conductance of transcellular pores increased in the presence of the actin inhibitor cytochalasin D but the cell-to-cell transport was eventually suppressed due to the deceleration and cessation of cytoplasmic streaming. The results show that the permeability of plasmodesmata to low-molecular photometabolites is subject to upregulation and downregulation.

Adenylyl cyclase mRNA localizes to the posterior of polarized DICTYOSTELIUM cells during chemotaxis.

BACKGROUND: In Dictyostelium discoideum, vesicular transport of the adenylyl cyclase A (ACA) to the posterior of polarized cells is essential to relay exogenous 3',5'-cyclic adenosine monophosphate (cAMP) signals during chemotaxis and for the collective migration of cells in head-to-tail arrangements called streams. RESULTS: Using fluorescence in situ hybridization (FISH), we discovered that the ACA mRNA is asymmetrically distributed at the posterior of polarized cells. Using both standard estimators and Monte Carlo simulation methods, we found that the ACA mRNA enrichment depends on the position of the cell within a stream, with the posterior localization of ACA mRNA being strongest for cells at the end of a stream. By monitoring the recovery of ACA-YFP after cycloheximide (CHX) treatment, we observed that ACA mRNA and newly synthesized ACA-YFP first emerge as fluorescent punctae that later accumulate to the posterior of cells. We also found that the ACA mRNA localization requires 3' ACA cis-acting elements. CONCLUSIONS: Together, our findings suggest that the asymmetric distribution of ACA mRNA allows the local translation and accumulation of ACA protein at the posterior of cells. These data represent a novel functional role for localized translation in the relay of chemotactic signal during chemotaxis.

Active diffusion positions the nucleus in mouse oocytes.

In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function.

Changes in actin dynamics are involved in salicylic acid signaling pathway.

Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented.

Chitosan surface enhances the mobility, cytoplasm spreading, and phagocytosis of macrophages.

A chitosan micropattern was prepared on glass by inkjet printing to visualize and compare in real-time macrophage developments on chitosan versus glass during microfluidic culture. The mobility of macrophages on chitosan was significantly higher, since the cells on glass were anchored by the development of podosomes whereas those on chitosan did not form podosomes. The phagocytosis of bacteria by macrophages was considerably more effective on chitosan because of: (1) the macrophages' higher mobility to scavenge nearby bacteria and (2) their cyotoplasm's ability to spread, re-distribute, and recover more freely to engulf the bacteria. Consequently, bacteria growth on chitosan surface was significantly reduced in the presence of macrophages in comparison to that on glass surface, as measured by surface bacteria density and effluent bacteria concentration. These findings suggest the synergistic effect of chitosan as a potential coating material on biomedical implants in promoting macrophage response upon the arrival of opportunistic bacteria.

[Spectral analysis of self-oscillating motility in isolated plasmodial strand of Physarum polycephalum].

In this study the experimental dependencies of the velocity of shuttle endoplasmic motion in the isolated plasmodial strand of Physarum polycephalum obtained by laser Doppler microscopy are presented. The spectral analysis of the time dependencies of the endoplasm allows obtaining two distinct harmonic components. Influence of KCN and SHAM--inhibitors of cellular respiration--leads to a complete cessation of endoplasmic motion in the strand. After removal of the inhibitors the respiratory system becomes normal, gradually restoring the activity of both harmonic oscillation sources. Based on the spectral analysis the simulated time-dependent velocity of the endoplasmic motion is rather good consistent with experimental data.

Cyclosis-mediated transfer of H2O 2 elicited by localized illumination of Chara cells and its relevance to the formation of pH bands.

Cytoplasmic streaming occurs in most plant cells and is vitally important for large cells as a means of long-distance intracellular transport of metabolites and messengers. In internodal cells of characean algae, cyclosis participates in formation of light-dependent patterns of surface pH and photosynthetic activity, but lateral transport of regulatory metabolites has not been visualized yet. Hydrogen peroxide, being a signaling molecule and a stress factor, is known to accumulate under excessive irradiance. This study was aimed to examine whether H2O2 produced in chloroplasts under high light conditions is released into streaming fluid and transported downstream by cytoplasmic flow. To this end, internodes of Chara corallina were loaded with the fluorogenic probe dihydrodichlorofluorescein diacetate and illuminated locally by a narrow light beam through a thin optic fiber. Fluorescence of dihydrodichlorofluorescein (DCF), produced upon oxidation of the probe by H2O2, was measured within and around the illuminated cell region. In cells exhibiting active streaming, H2O2 first accumulated in the illuminated region and then entered into the streaming cytoplasm, giving rise to the expansion of DCF fluorescence downstream of the illuminated area. Inhibition of cyclosis by cytochalasin B prevented the spreading of DCF fluorescence along the internode. The results suggest that H2O2 released from chloroplasts under high light is transported along the cell with the cytoplasmic flow. It is proposed that the shift of cytoplasmic redox poise and light-induced elevation of cytoplasmic pH facilitate the opening of H(+)/OH(-)-permeable channels in the plasma membrane.

Inhibition of cytoplasmic streaming by cytochalasin D is superior to paraformaldehyde fixation for measuring FRET between fluorescent protein-tagged Golgi components.

Protein-protein interaction at the organelle level can be analyzed by using tagged proteins and assessing Förster resonance energy transfer (FRET) between fluorescent donor and acceptor proteins. Such studies are able to uncover partners in the regulation of proteins and enzymes. However, any organelle movement is an issue for live FRET microscopy, as the observed organelle must not change position during measurement. One of the mobile organelles in plants is the Golgi apparatus following cytoplasmic streaming. It is involved in the decoration of proteins and processing of complex glycan structures for the cell wall. Understanding of these processes is still limited, but evidence is emerging that protein-protein interaction plays a key role in the function of this organelle. In the past, mobile organelles were usually immobilized with paraformaldehyde (PFA) for FRET-based interaction studies. Here, we show that the actin inhibitor Cytochalasin D (CytD) is superior to PFA for immobilization of Golgi stacks in plant cells. Two glycosyltransferases known to interact were tagged with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, coexpressed in Nicotiana benthamiana leaves and analyzed using confocal microscopy and spectral imaging. Fixation with PFA leads to reduced emission intensity when compared to CytD treatment. Furthermore, the calculated FRET efficiency was significantly higher with CytD than with PFA. The documented improvements are beneficial for all methods measuring FRET, where immobilization of the investigated molecules is necessary. It can be expected that FRET measurement in organelles of animal cells will also benefit from the use of inhibitors acting on the cytoskeleton.

Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes.

Mature mammalian oocytes are poised for completing meiosis II (MII) on fertilization by positioning the spindle close to an actomyosin-rich cortical cap. Here, we show that the Arp2/3 complex localizes to the cortical cap in a Ran-GTPase-dependent manner and nucleates actin filaments in the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 activity leads to rapid dissociation of the spindle from the cortex. Live-cell imaging and spatiotemporal image correlation spectroscopy analysis reveal that actin filaments flow continuously away from the Arp2/3-rich cortex, driving a cytoplasmic streaming expected to exert a net pushing force on the spindle towards the cortex. Arp2/3 inhibition not only diminishes this actin flow and cytoplasmic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, moving the spindle away from the cortex. Thus, the asymmetric MII spindle position is dynamically maintained as a result of balanced forces governed by the Arp2/3 complex.

Rapid mass movement of chloroplasts during segment formation of the calcifying siphonalean green alga, Halimeda macroloba.

BACKGROUND: The calcifying siphonalean green alga, Halimeda macroloba is abundant on coral reefs and is important in the production of calcium carbonate sediments. The process by which new green segments are formed over-night is revealed here for the first time. METHODOLOGY/PRINCIPAL FINDINGS: Growth of new segments was visualised by epifluorescence and confocal microscopy and by pulse amplitude modulation (PAM) fluorimetry. Apical colourless proto-segments were initiated on day 1, and formed a loose network of non-calcified, non-septate filaments, containing no chloroplasts. Rapid greening was initiated at dusk by i) the mass movement of chloroplasts into these filaments from the parent segment and ii) the growth of new filaments containing chloroplasts. Greening was usually complete in 3-5 h and certainly before dawn on day 2 when the first signs of calcification were apparent. Mass chloroplast movement took place at a rate of ∼0.65 µm/s. Photosynthetic yield and rate remained low for a period of 1 to several hours, indicating that the chloroplasts were made de novo. Use of the inhibitors colchicine and cytochalasin d indicated that the movement process is dependent on both microtubules and microfilaments. SIGNIFICANCE: This unusual process involves the mass movement of chloroplasts at a high rate into new segments during the night and rapid calcification on the following day and may be an adaptation to minimise the impact of herbivorous activity.

Cyclosis-related asymmetry of chloroplast-plasma membrane interactions at the margins of illuminated area in Chara corallina cells.

Cytoplasmic streaming in plant cells is an effective means of intracellular transport. The cycling of ions and metabolites between the cytosol and chloroplasts in illuminated cell regions may alter the cytoplasm composition, while directional flow of this modified cytoplasm may affect the plasma membrane and chloroplast activities in cell regions residing downstream of the illumination area. The impact of local illumination is predicted to be asymmetric because the cell regions located downstream and upstream in the cytoplasmic flow with respect to illumination area would be exposed to flowing cytoplasm whose solute composition was influenced by photosynthetic or dark metabolism. This hypothesis was checked by measuring H(+)-transporting activity of plasmalemma and chlorophyll fluorescence of chloroplasts in shaded regions of Chara corallina internodal cells near opposite borders of illuminated region (white light, beam width 2 mm). Both the apoplastic pH and chlorophyll fluorescence, recorded in shade regions at equal distances from illuminated area, exhibited asymmetric light-on responses depending on orientation of cytoplasmic streaming at the light-shade boundary. In the region where the cytoplasm flowed from illuminated area to the measurement area, the alkaline zone (a zone with high plasma membrane conductance) was formed within 4-min illumination, whereas no alkaline zone was observed in the area where cytoplasm approached the boundary from darkened regions. The results emphasize significance of cyclosis in lateral distribution of a functionally active intermediate capable of affecting the membrane transport across the plasmalemma, the functional activity of chloroplasts, and pattern formation in the plant cell.

Effects of lanthanum on calcium and magnesium contents and cytoplasmic streaming of internodal cells of Chara corallina.

Biological and environmental effects of lanthanide series of elements have received much attention recently due to their wide applications. In this study, effects of La(3+) treatments on calcium and magnesium concentrations as well as cytoplasmic streaming of internodal cells of Chara corallina were investigated. At all treatment concentrations (10, 100, and 1,000 µM), La(3+) significantly decreased calcium concentrations in the cell-wall fractions after 5-h treatments. Calcium concentrations in the cell contents and magnesium concentrations in the cell-wall fractions were reduced by 100 and 1,000 µM La(3+) treatments. However, cytoplasmic streaming as an indicator of [Ca(2+)](cyt) was only inhibited at the highest La(3+) concentration (1,000 µM). The results suggest that La(3+) may affect cellular calcium homeostasis by actions other than as a simple Ca(2+) antagonist. La(3+) could partially compensate for calcium deficiency at certain concentrations.

Speckle fluctuation spectroscopy of intracellular motion in living tissue using coherence-domain digital holography.

Dynamic speckle from 3-D coherence-gated optical sections provides a sensitive label-free measure of cellular activity up to 1 mm deep in living tissue. However, specificity to cellular functionality has not previously been demonstrated. In this work, we perform fluctuation spectroscopy on dynamic light scattering captured using coherence-domain digital holography to obtain the spectral response of tissue that is perturbed by temperature, osmolarity, and antimitotic cytoskeletal drugs. Different perturbations induce specific spectrogram response signatures that can show simultaneous enhancement and suppression in different spectral ranges.

Isolation and characterization of a large soluble form of fibronectin that stimulates adhesion, spreading, and alignment of mouse erythroleukemia cells.

Fibronectin (FN) is a major component of the extracellular matrix which plays important roles in a variety of cellular processes including cell adhesion, and migration. The soluble cellular form of FN has a monomer molecular weight of approximately 250 kDa, and generally exists as a dimer of 500 kDa. We have isolated a different form of soluble FN from mouse breast cancer cell line SC115 conditioned medium (CM) and purified it to homogeneity as evidenced by both native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE. It still exhibits a monomeric form of about 250 kDa while its form in the CM is stable and soluble with an apparent tetrameric molecular weight in the range of 800-1000 kDa. This form of FN is a potent cell adhesion factor (AF) that induces adhesion to polystyrene, elongation, spreading, alignment or "track" formation, and migration of mouse erythroleukemia cells. Column fractions homogeneous for AF protein were able to stimulate 10% cell adhesion at concentrations of 23 ng/ml and 1.9 ng/cm(2). Purified AF induced 50% cell adhesion at 94 ng/ml and 7.5 ng/cm(2). AF also increased the migration of human aortic smooth muscle and vascular endothelial cells. However, this form of FN differs from other forms as it does not bind tightly to either gelatin or heparin. Studies of this AF should shed light on adhesion of cells to extracellular matrix molecules and on cell migration, both of which are critical in several biological processes such as wound healing, metastasis, matrix formation and structure, and organ development.

HGF-induced invasion by prostate tumor cells requires anterograde lysosome trafficking and activity of Na+-H+ exchangers.

Hepatocyte growth factor (HGF) is found in tumor microenvironments, and interaction with its tyrosine kinase receptor Met triggers cell invasion and metastasis. It was previously shown that acidic extracellular pH stimulated peripheral lysosome trafficking, resulting in increased cathepsin B secretion and tumor cell invasion, which was dependent upon sodium-proton exchanger (NHE) activity. We now demonstrate that HGF induced the trafficking of lysosomes to the cell periphery, independent of HGF-induced epithelial-mesenchymal transition. HGF-induced anterograde lysosome trafficking depended upon the PI3K pathway, microtubules and RhoA, resulting in increased cathepsin B secretion and invasion by the cells. HGF-induced NHE activity via increased net acid production, and inhibition of NHE activity with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), or a combination of the NHE1-specific drug cariporide and the NHE3-specific drug s3226 prevented HGF-induced anterograde trafficking and induced retrograde trafficking in HGF-overexpressing cells. EIPA treatment reduced cathepsin B secretion and HGF-induced invasion by the tumor cells. Lysosomes were located more peripherally in Rab7-shRNA-expressing cells and these cells were more invasive than control cells. Overexpression of the Rab7 effector protein, RILP, resulted in a juxtanuclear location of lysosomes and reduced HGF-induced invasion. Together, these results suggest that the location of lysosomes is an inherently important aspect of invasion by tumor cells.

Involvement of actin filaments in rhizoid morphogenesis of Spirogyra.

The role of actin filaments in rhizoid morphogenesis was studied in Spirogyra. When the algal filaments were severed, new terminal cells started tip growth and finally formed rhizoids. Actin inhibitors, latrunculin B and cytochalasin D, reversibly inhibited the process. A mesh-like structure of actin filaments (AFs) was formed at the tip region. Gd(3+) inhibited tip growth and decreased AFs in the tip region. Either a decrease in turgor pressure or lowering of the external Ca(2+) concentration also induced similar results. It was suggested that the mesh-like AF structure is indispensable for the elongation of rhizoids. A possible organization mechanism of the mesh-like AF structure was discussed.

Microtubule-dependent motility and orientation of the cortical endoplasmic reticulum in elongating characean internodal cells.

Motility of the endoplasmic reticulum (ER) is predominantly microtubule- dependent in animal cells but thought to be entirely actomyosin-dependent in plant cells. Using live cell imaging and transmission electron microscopy to examine ER motility and structural organization in giant internodal cells of characean algae, we discovered that at the onset of cell elongation, the cortical ER situated near the plasma membrane formed a tight meshwork of predominantly transverse ER tubules that frequently coaligned with microtubules. Microtubule depolymerization increased mesh size and decreased the dynamics of the cortical ER. In contrast, perturbing the cortical actin array with cytochalasins did not affect the transverse orientation but decreased mesh size and increased ER dynamics. Our data suggest that myosin-dependent ER motility is confined to the ER strands in the streaming endoplasm, while the more sedate cortical ER uses microtubule-based mechanisms for organization and motility during early stages of cell elongation. We show further that the ER has an inherent, NEM-sensitive dynamics which can be altered via interaction with the cytoskeleton and that tubule formation and fusion events are cytoskeleton-independent.

Involvement of membrane potential in alkaline band formation by internodal cells of Chara corallina.

Internodal cells of Chara corallina form alkaline bands on their surface upon illumination via photosynthesis. In the present study, the effect of KCl on alkaline band formation was analyzed. When the extracellular KCl concentration was increased, alkaline band formation was extensively inhibited. Electrophysiological analysis unequivocally showed the need for inner negative membrane potential for alkaline band formation.

Water diffusion in cytoplasmic streaming in Elodea internodal cells under the effect of antimitotic agents.

The translational displacement of the cytoplasmic water in Elodea stem cells resulting from protein motor activity was measured using the NMR method. A 24-h treatment with vincristine results in a reduction of the translational displacement of the cytoplasmic water. With a constant cytoplasmic streaming velocity, the dynamics of the translational displacement of the cytoplasmic water under the effect of taxol are characterized by a continuous increase at a concentration of 0.05 mM, and reaching a plateau at a concentration of 0.5 mM.

Primary effect of bromoxynil to induce plant cell death may be cytosol acidification.

Bromoxynil, 3,5-dibromo-4-hydroxybenzonitrile, is a commonly used herbicide and is also used as a tool to trigger rapid cell death in basic botany. However, the primary effect inducing cell death is not known. Bromoxynil inhibited the cytoplasmic streaming and killed cells in Chara corallina when it was applied in the acidic external medium. At higher pH, bromoxynil was inert even at high concentrations. It was speculated that bromoxynil in the protonated form enters the cell and acidifies the cytosol by releasing H(+). Experiments using analogues of bromoxynil supported this possibility. Acidification of the cytosol by bromoxynil was confirmed by experiments using pollen tubes. Based on the acidity of the apoplast, the herbicide action of bromoxynil in higher plants was discussed.