Correlation between chloroplast motility and elastic properties of tobacco mesophyll protoplasts

Translocations of chloroplasts induced by blue light were investigated in both leaves and protoplasts isolated from leaf mesophyll of Nicotiana tabacum. In the leaf tissue, the responses of chloroplasts were similar to those observed in other, higher and lower plant species. Weak and strong light induced movements of chloroplasts towards cell walls perpendicular and parallel to the light direction, respectively. Treatment with cytochalasin D, an actin-disturbing agent, blocked the movements. This shows that actin is involved in the motile system of chloroplast translocation in tobacco. By monitoring the response of chloroplasts to light in isolated protoplasts, we addressed the question whether the presence of the cell wall is necessary for the translocations of chloroplasts to occur. In control protoplasts (isolated at room temperature from unstressed leaves), no clear light intensity-dependent changes were observed in chloroplast distribution pattern. In contrast, in protoplasts obtained from plants treated with 4 °C for 8 h the chloroplasts maintained their responsiveness to light. Atomic Force Microscopy was used to measure elastic properties of the protoplasts. Young’s modulus, which reflects rigidity of the material, was 10 times higher for protoplasts of the coldstressed plants as compared to those isolated from the control plants. The rigidity of protoplasts isolated from the plants treated with low temperature was reduced four-fold by exposure to cytochalasin D. It appears that the status of protoplast actin is a factor responsible for elasticity of protoplasts. We speculate that unknown, cold stress-induced factors, maintain the orientational movements due to anchorage of the actin cytoskeleton in the plasma membrane despite the cell wall removal.     

Phototropin Mediated Relocation of Myosins in Arabidopsis thaliana

Background

The mechanism of the light-dependent movements of chloroplasts is based on actin and myosin but its details are largely unknown. The movements are activated by blue light in terrestrial angiosperms. The aim of the present study was to determine the role of myosin associated with the chloroplast surface in the light-induced chloroplast responses in Arabidopsis thaliana. The localization of myosins was investigated under blue light intensities generating avoidance and accumulation responses of chloroplasts. The localization was compared in wild type plants and in phot2 mutant lacking the avoidance response.

Results

Wild type and phot2 mutant plants were irradiated with strong (36 µEm⁻²s⁻¹) and/or weak (0.8 µEm⁻²s⁻¹) blue light. The leaf tissue was immunolabeled with antimyosin antibodies. Different arrangements of myosins were observed in the mesophyll depending on the fluence rate in wild type plants. In tissue irradiated with weak blue light myosins were associated with chloroplast envelopes. In contrast, in tissue irradiated with strong blue light chloroplasts were almost myosin-free. The effect did not occur in red light and in the phot2 mutant.

Conclusions

Myosin displacement is blue light specific, i.e., it is associated with the activation of a specific blue-light photoreceptor. We suggest that the reorganization of myosins is essential for chloroplast movement. Myosins appear to be the final step of the signal transduction pathway starting with phototropin2 and leading to chloroplast movements.Key Words: Arabidopsis, blue light, chloroplast movements, myosins, phototropins     

Actin-based mechanisms for light-dependent intracellular positioning of nuclei and chloroplasts in Arabidopsis

The plant organelles, chloroplast and nucleus, change their position in response to light. In Arabidopsis thaliana leaf cells, chloroplasts and nuclei are distributed along the inner periclinal wall in darkness. In strong blue light, they become positioned along the anticlinal wall, while in weak blue light, only chloroplasts are accumulated along the inner and outer periclinal walls. Blue-light dependent positioning of both organelles is mediated by the blue-light receptor phototropin and controlled by the actin cytoskeleton. Interestingly, however, it seems that chloroplast movement requires short, fine actin filaments organized at the chloroplast edge, whereas nuclear movement does cytoplasmic, thick actin bundles intimately associated with the nucleus. Although there are many similarities between photo-relocation movements of chloroplasts and nuclei, plant cells appear to have evolved distinct mechanisms to regulate actin organization required for driving the movements of these organelles.Key words: actin, Arabidopsis, blue light, chloroplast positioning, phototropin, nuclear positioning     

Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.     

Photoorientation of chloroplasts in protonemal cells of the fernAdiantum as analyzed by use of a video-tracking system

Photoorientation of chloroplasts mediated by phytochrome and blue light-absorbing pigment in protonemal cells of the fernAdiantum was studied by use of inhibitors of the cytoskeleton and was analyzed with a video-tracking system. The photoorientation responses were inhibited by cytochalasin B and by N-ethylmaleimide (NEM) but not by colchicine, suggesting that the photomovement depends on the actomyosin system. In the dark, chloroplasts moved randomly, being independent of one another. After induction of photoorientation by polarized red light, most chloroplasts that had been located at the margin of cells moved almost perpendicularly to the cell axis toward the site of photoorientation. This type of movement was hardly ever observed in the dark. Under polarized blue light, such specific movements were less evident but were still observed in the case of a few chloroplasts. After photoorientation was complete, chloroplasts still moved in random directions but their mobility was lower than that in the dark, indicating the presence of some anchoring mechanism. When EGTA was applied, photoorientation was inhibited but this inhibition was overcome by the addition of CaCl₂. Video-tracking of chloroplasts in the dark revealed that the mobility of chloroplasts was higher in medium with EGTA than in medium with EGTA plus CaCl₂ and that many of the chloroplasts moved jerkily in the medium with EGTA. This change in the nature of movements was also seen under polarized light, resulting in the disturbance of photoorientation. These results indicate that the inhibition of photoorientation at low concentrations of Ca²⁺ ions may be due to change in the nature of chloroplast movement.     

Plastid movement impaired 2, a new gene involved in normal blue-light-induced chloroplast movements in Arabidopsis

Chloroplasts move in a light-dependent manner that can modulate the photosynthetic potential of plant cells. Identification of genes required for light-induced chloroplast movement is beginning to define the molecular machinery that controls these movements. In this work, we describe plastid movement impaired 2 (pmi2), a mutant in Arabidopsis (Arabidopsis thaliana) that displays attenuated chloroplast movements under intermediate and high light intensities while maintaining a normal movement response under low light intensities. In wild-type plants, fluence rates below 20 micromol m(-2) s(-1) of blue light lead to chloroplast accumulation on the periclinal cell walls, whereas light intensities over 20 micromol m(-2) s(-1) caused chloroplasts to move toward the anticlinal cell walls (avoidance response). However, at light intensities below 75 micromol m(-2) s(-1), chloroplasts in pmi2 leaves move to the periclinal walls; 100 micromol m(-2) s(-1) of blue light is required for chloroplasts in pmi2 to move to the anticlinal cell walls, indicating a shift in the light threshold for the avoidance response in the mutant. The pmi2 mutation has been mapped to a gene that encodes a protein of unknown function with a large coiled-coil domain in the N terminus and a putative P loop. PMI2 shares sequence and structural similarity with PMI15, another unknown protein in Arabidopsis that, when mutated, causes a defect in chloroplast avoidance under high-light intensities.     

Actin-based chloroplast rearrangements in the cortex of the giant coenocytic green algaCaulerpa

Summary The giant coenocytic green algaCaulerpa is well known for its large scale amyloplast transport. The majority of chloroplasts, however, is immobilized in the cortex of the cell. By applying UV-irradiation to localized areas of the cortex chloroplasts can be induced to slowly move towards and aggregate around the irradiated spot. Chloroplast movement is blocked by cytochalasin D, but not by colchicine or the microtubule herbicide cremart. The dynein inhibitor erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA) also has no effect on chloroplast movement. However, both microtubule- and dynein-specific inhibitors block movement of amyloplasts. Using the previously developed technique of microdissection followed by immunofluorescence microscopy it can be shown that, concomitant with changes in motile behavior of chloroplasts upon irradiation, actin filaments form and rearrange around the irradiation spot. It is concluded that in contrast to amyloplast movement, immobilization and movement of chloroplasts are dependent on actin but not on microtubules. Therefore, two individual motile mechanisms appear to have evolved for independent positioning and motility of the two populations of plastids in the giant coenocyteCaulerpa.Abbreviations EHNA erythro-9-[3-(2-hydroxynonyl)] adenine - DMSO dimethylsulfoxide - MT microtubule - NEM N-ethylmaleimide     

Light-induced rapid absorption changes during photosynthesis. VII. Some reactions in sonicated chloroplasts

1. Spinach chloroplasts subjected to sonication show light-induced absorption changes at 700 mμ characteristic of the photooxidation of the chlorophyll component P700. The appearance of P700 absorption changes probably resulted from the release of plastocyanin thus interrupting the electron flow between pigment systems 1 and 2. The general features of the absorption-change transients are similar to those observed previously with digitonin-treated chloroplasts. The addition of 2 mM ascorbate or 10 μM 3-(3,4-dichlorophenyl)-1, 1-dimethylurea had practically no effect on either the magnitude or the dark decay of the transient absorption change.

2. Phenazine methosulfate (PMS) (in the presence or in the absence of ascorbate) reduction appeared to be coupled to P700 photooxidation, as shown by the corresponding transients at 430 and 388 mμ. The absorbance changes at these two wavelengths indicate that the amount of PMS photoreduced was equivalent to that of P700 photooxidized. Higher PMS concentrations accelerate the dark decay of the P700 signal. When PMS alone is present, anaerobiosis caused the dark decay to become more rapid than in the presence of ascorbate.

3. Unlike PMS, other redox agents such as 2,6-dichlorophenolindophenol, N,N,N′,N′-tetramethyl-p-phenylenediamine or diaminodurol in the presence of excess ascorbate, did not noticeably affect the kinetics of the dark decay at 430 or 703 mμ, suggesting that these reduced species are not efficiently coupled to photooxidized P700.

4. The onset and decay rates of the P700 transient in the presence of PMS and excess ascorbate was insensitive to temperature between 25° and o°. However, when the chloroplast sample was frozen at temperatures ranging from −5° to −196°, all reactions ceased. When the frozen (−196°) sample was brought back to the room temperature, the reaction was restored completely. Fresh broken chloroplasts behave similarly. Digitonin-treated chloroplasts persisted down to about −25° but with diminishing magnitude and slower decay.     

Distribution pattern changes of actin filaments during chloroplast movement in Adiantum capillus-veneris

Chloroplasts change their positions in a cell in response to light intensities. The photoreceptors involved in chloroplast photo-relocation movements and the behavior of chloroplasts during their migration were identified in our previous studies, but the mechanism of movement has yet to be clarified. In this study, the behavior of actin filaments under various light conditions was observed in Adiantum capillus-veneris gametophytes. In chloroplasts staying in one place under a weak light condition and not moving, circular structures composed of actin filaments were observed around the chloroplast periphery. In contrast, short actin filaments were observed at the leading edge of moving chloroplasts induced by partial cell irradiation. In the dark, the circular structures found under the weak light condition disappeared and then reappeared around the moving chloroplasts. Mutant analyses revealed that the disappearance of the circular actin structure was mediated by the blue light photoreceptor, phototropin2.     

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.     

Pb-Induced Avoidance-Like Chloroplast Movements in Fronds of Lemna trisulca L.

Lead ions are particularly dangerous to the photosynthetic apparatus, but little is known about the effects of trace metals, including Pb, on regulation of chloroplast redistribution. In this study a new effect of lead on chloroplast distribution patterns and movements was demonstrated in mesophyll cells of a small-sized aquatic angiosperm Lemna trisulca L. (star duckweed). An analysis of confocal microscopy images of L. trisulca fronds treated with lead (15 μM Pb²⁺, 24 h) in darkness or in weak white light revealed an enhanced accumulation of chloroplasts in the profile position along the anticlinal cell walls, in comparison to untreated plants. The rearrangement of chloroplasts in their response to lead ions in darkness was similar to the avoidance response of chloroplasts in plants treated with strong white light. Transmission electron microscopy X-ray microanalysis showed that intracellular chloroplast arrangement was independent of the location of Pb deposits, suggesting that lead causes redistribution of chloroplasts, which looks like a light-induced avoidance response, but is not a real avoidance response to the metal. Furthermore, a similar redistribution of chloroplasts in L. trisulca cells in darkness was observed also under the influence of exogenously applied hydrogen peroxide (H₂O₂). In addition, we detected an enhanced accumulation of endogenous H₂O₂ after treatment of plants with lead. Interestingly, H₂O₂-specific scavenger catalase partly abolished the Pb-induced chloroplast response. These results suggest that H₂O₂ can be involved in the avoidance-like movement of chloroplasts induced by lead. Analysis of photometric measurements revealed also strong inhibition (but not complete) of blue-light-induced chloroplast movements by lead. This inhibition may result from disturbances in the actin cytoskeleton, as we observed fragmentation and disappearance of actin filaments around chloroplasts. Results of this study show that the mechanisms of the toxic effect of lead on chloroplasts can include disturbances in their movement and distribution pattern.     

The role of solation-contraction coupling in regulating stress fiber dynamics in nonmuscle cells

Serum-deprived Swiss 3T3 fibroblasts constitutively form stress fibers at their edges. These fibers move centripetally towards the perinuclear region where they disassemble. Serum stimulation causes shortening of fibers in a manner suggesting active actin-myosin-based contraction (Giuliano, K.A. and D.L. Taylor. 1990. Cell Motil. and Cytoskeleton. 16:14-21). To elucidated the role of actin-based gel structure in these movements, we examined the effects of disrupting actin organization with cytochalasin. Serum-deprived fibroblasts were microinjected with rhodamine analogs of actin or myosin II and fiber dynamics were monitored with a multimode light microscope workstation using video-enhanced contrast and fluorescence modes. When cells were perfused with greater than or equal to 3 microM cytochalasin B or 0.5 microM cytochalasin D, formation and transport of stress fibers were reversibly inhibited, and rapid and immediate shortening of existing fibers was induced. Quantification of actin and myosin II fluorescence associated with individual shortening fibers demonstrated that fluorescence per length of fiber increased for both components, suggesting sliding filament contraction. However, there was also a net loss of both actin and myosin II from fibers as they shortened, indicating a self-destructive process. Loss of material from fibers coupled with increased overlap of actin and myosin II remaining in the fibers suggested that contraction could be induced not only by increasing the force exerted by contractile motors, but also by decreasing gel structure through partial solation. Finally, cytochalasin accelerated contraction of actin-myosin-based gels reconstituted from purified proteins in the absence of myosin-based regulation, further supporting solation-contraction coupling as a possible mechanism for modulating cytoplasmic contractility (Taylor, D.L. and M. Fechheimer. 1982. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 299:185-197).     

Contractile elements of Lemna trisulca L. glycerinated cell models during chloroplast translocations.

Electron microscopy of Lemna glycerinated cell models depicts contractile elements during chloroplast translocations. One contractile element, the thin ectoplasmic layer, is < or = 0.4 microm thick, pressed against plasma membrane-cell wall. Thin ectoplasmic layer contains numerous oriented filaments and some appear to be actin and myosin. Another contractile element is the outer chloroplast membrane which envelops each chloroplast and joins or fuses with the thin ectoplasmic layer. Chloroplast interconnections are formed between two or more chloroplasts by outer chloroplast membranes; they enhance chloroplast communications, translocations, and molecular exchanges.     

C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states

Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments’ interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506–793) and CaDH2 (residues 683–767) on the structure of actin–tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin–tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle ‘ghost’ fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)–phalloidin or fluorescein-5′-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Φ_A, Φ_E, Θ_1/2 and N were calculated. Φ_A and Φ_E are angles between the fiber axis and the absorption and emission dipoles, respectively; Θ_1/2 is the angle between the F-actin filament axis and the fiber axis; N is the relative number of randomly oriented fluorophores. Actin–tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the ‘on’ conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca²⁺) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca²⁺) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between ‘off’ (caldesmon and CaDH1) and ‘on’ (S-1 and CaDH2) states in a manner analogous to troponin.     

Macrophage deformability and phagocytosis

The influence of several metabolic inhibitors and pharmacologic agents on macrophage deformation (induced by fluid shear stress) was examined in relationship to changes in ATP content and phagocytosis of latex beads. Two relatively specific inhibitors of glycolysis (iodoacetate [IA], and sodium fluoride [NaF]) and a sulfhydryl-binding agent (N-ethylmaleimide [NEM] markedly inhibited phagocytosis and reduced cell deformability. A microtubule-disrupting agent (vinblastine) and a highly specific inhibitor of glycolysis (2-deoxyglucose) markedly inhibited phagocytosis without influencing cell deformability. An organomercurial sulfhydryl binding agent p-chloromercuribenzene (PCMBS) and a microfilament-disrupting agent (cytochalasin B) inhibited phagocytosis and increased cell deformability. The effects of these agents on phagocytosis and cell deformability bore no consistent relationship to alterations in cellular content of ATP. The observation that 2-deoxyglucose, the most specific inhibitor of glycolysis examined, reduced ATP content to levels far lower (15 percent of control values) than those achieved by any other agent examined and inhibited phagocytosis without altering cell deformability, suggests that alterations in cell deformability induced by NaF, IA, NEM, PCMBS, and cytochalasin B are not due to inhibition of glycolysis per se, but instead result from direct or indirect effects of these agents on cell constituents, possibly contractile proteins, which are determinants of cell deformability. The finding that cytochalasin B, NEM, PCMBS, and IA interfere with phagocytosis and alter cell deformability, together with evidence that these agents interact with isolated actin and myosin, suggests that contractile proteins are important both in phagocytosis and as determinants of cell deformability. The observation that vinblastine, colchicines, and heavy water (D(2)O) did not alter cell deformability, even though vinblastine caused formation of intracellular crystals of microtubular protein, indicates that microtubules are not major determinants of cell deformability. The observations that beads adhered normally to surfaces of cytochalasin B- and of PCMBS-treated cells and that shear-stress induced deformation was increased whereas phagocytosis was markedly inhibited, suggest that deformation of cells around beads associated with ingestion depends on some form of cellular (contractile?) activity, whereas deformation of cells by fluid shear stress is a passive phenomenon.     

Photoinduction of formation of circular structures by microfilaments on chloroplasts during intracellular orientation in protonemal cells of the fernAdiantum capillus-veneris

Summary Changes in the organization of cortical actin microfilaments during phytochrome-mediated and blue light-induced photoorientation of chloroplasts were investigated by rhodamine-phalloidin staining in protonemal cells of the fernAdiantum capillusveneris. Low- and high-fluence rate responses were induced by partial irradiation of individual cells with a microbeam of 20 m in width. In the low-fluence rate responses to red and blue light, a circular structure composed of microfilaments was induced on the chloroplast concentrated in the irradiated region, on the side facing the plasma membrane, as already reported in the case of the low-fluence rate response induced by polarized red or blue light. Such a structure was not observed on the chloroplasts located far from the microbeam. Time-course studies revealed that the structure was induced after the chloroplasts gathered in the illuminated region and that the structure disappeared before chloroplasts moved out of this region when the microbeam was turned off. In the high-fluence rate response to blue light, chloroplasts avoided the irradiated site but accumulated in the shaded area adjacent the edges of microbeam. The circular structure made of microfilaments was also observed on the chloroplasts gathered in the area and it showed the same behavior with respect to its appearance and disappearance during a light/dark regime as in the case of the low-fluence rate response. However, no such circular structure was observed in the high-fluence rate response to red light, in which case the chloroplasts also avoided the illuminated region but no accumulation in the adjacent areas was induced. These results indicate that the circular structure composed of microfilaments may play a role in the anchorage of the chloroplast during intracellular photo-orientation.     

Changes in the organization of actin and myosin in non-muscle cells induced by N-ethylmaleimide

There is evidence from in vitro studies that the SH reagent N-ethylmaleimide (NEM) causes the formation of ATP-resistant rigor-complexes between actin and myosin, and NEM-modified heavy meromyosin has been used by Cande et al. to study the contractile process during cytokinesis. It is reported here that treatment of tissue-cultured cells with NEM causes an immediate cessation of all motile activities and a simultaneous stabilization of the ultrastructure of the cell visualized on lysis with detergent-containing buffers. After NEM treatment a 5- to 10-fold increase in the amount of myosin was found associated with the detergent-resistant cell residues. As suggested by immunoelectron microscopy, using antibodies to non-muscle myosin together with gold-labelled protein A, increasing amounts of myosin filaments became associated with the microfilament assemblies of the cell with time of NEM treatment. In addition to this there was a slow, progressive reorganization of the cortical wave of microfilaments. The structures interpreted as myosin filaments were visualized at relatively high resolution. The immunoelectron microscopy finally also indicated the presence of a non-filamentous form of myosin in agreement with the results of others.     

Light perception and the role of the xanthophyll cycle in blue-light-dependent chloroplast movements in lemna trisulca L

In most higher plants, chloroplasts move towards the periclinal cell walls in weak blue light (WBL) to increase light harvesting for photosynthesis, and towards the anticlinal walls as an escape reaction, thus avoiding photo-damage in strong blue light (SBL). The photo- receptor(s) triggering these responses have not yet been identified. In this study, the role of zeaxanthin as a blue-light photoreceptor in chloroplast movements was investigated. Time-lapse 3D confocal imaging in Lemna trisulca showed that individual chloroplasts responded to local illumination when one half of the cell was treated with light of different intensity or spectral quality to that received by the other half, or was maintained in darkness. Thus the complete signal perception, transduction and effector system has a high degree of spatial resolution and is consistent with localization of part of the transduction chain in the chloroplasts. Turnover of xanthophylls was determined using HPLC, and a parallel increase was observed between zeaxanthin and chloroplast movements in SBL. Ascorbate stimulated both a transient increase in zeaxanthin levels and chloroplast movement to profile in physiological darkness. Conversely, dithiothreitol blocked zeaxanthin production and responses to SBL and, to a lesser extent, WBL. Norflurazon preferentially inhibited SBL-dependent chloroplast movements. Increases in zeaxanthin were also observed in strong red light (SRL) when no directional chloroplast movements occurred. Thus it appears that a combination of zeaxanthin and blue light is required to trigger responses. Blue light can cause cis-trans isomerization of xanthophylls, thus photo-isomerization may be a critical link in the signal transduction pathway.     

Phototropins and neochrome1 mediate nuclear movement in the fern Adiantum capillus-veneris

In gametophytic cells (prothalli) of the fern Adiantum capillus-veneris, nuclei as well as chloroplasts change their position according to light conditions. Nuclei reside on anticlinal walls in darkness and move to periclinal or anticlinal walls under weak or strong light conditions, respectively. Here we reveal that red light-induced nuclear movement is mediated by neochrome1 (neo1), blue light-induced movement is redundantly mediated by neo1, phototropin2 (phot2) and possibly phot1, and dark positioning of both nuclei and chloroplasts is mediated by phot2. Thus, both the nuclear and chloroplast photorelocation movements share common photoreceptor systems.     

Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of Lysosomes

An earlier report suggested that actin and myosin I alpha (MMIalpha), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIalpha were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIalpha. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIalpha impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIalpha contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors.