Visualization of peroxisomes in living plant cells reveals acto-myosin-dependent cytoplasmic streaming and peroxisome budding

Here we examine peroxisomes in living plant cells using transgenic Arabidopsis thaliana plants expressing the green fluorescent protein (GFP) fused to the peroxisome targeting signal 1 (PTS1). Using time-lapse laser scanning confocal microscopy we find that plant peroxisomes exhibit fast directional movement with peak velocities approaching 10 microm s(-1). Unlike mammalian peroxisomes which move on microtubules, plant peroxisome movement is dependent on actin microfilaments and myosin motors, since it is blocked by treatment with latrunculin B and butanedione monoxime, respectively. In contrast, microtubule-disrupting drugs have no effect on peroxisome streaming. Peroxisomes were further shown to associate with the actin cytoskeleton by the simultaneous visualization of actin filaments and peroxisomes in living cells using GFP-talin and GFP-PTS1 fusion proteins, respectively. In addition, peroxisome budding was observed, suggesting a possible mechanism of plant peroxisome proliferation. The strong signal associated with the GFP-PTS1 marker also allowed us to survey cytoplasmic streaming in different cell types. Peroxisome movement is most intense in elongated cells and those involved in long distance transport, suggesting that higher plants use cytoplasmic streaming to help transport vesicles and organelles over long distances.     

Peroxisomal localization of a myosin XI isoform in Arabidopsis thaliana

The genome of Arabidopsis thaliana contains 13 myosin XI isoforms. Here we prepared a specific antibody against a peptide that mimics a unique C-terminal region from the myosin XI isoform, MYA2. The resulting antibody was used to demonstrate that MYA2 in Arabidopsis protein extracts co-sedimented with actin filaments and dissociated from the filaments with ATP treatment. Immunolocalization studies showed that MYA2 co-localized predominantly with actin filaments in clustered punctuate dots in leaf epidermal cells, root hair cells and suspension-cultured cells. In a transgenic plant in which peroxisomes are labeled with green fluorescent protein, some MYA2 signals were localized on peroxisomes in an actin-dependent manner. We propose that the peroxisome is one of the cargos translocated by MYA2 on actin filaments.     

Distribution and characterization of peroxisomes in Arabidopsis by visualization with GFP: dynamic morphology and actin-dependent movement

Peroxisomes were visualized in living cells of various tissues in transgenic Arabidopsis by green fluorescent protein (GFP) through the addition of the peroxisomal targeting signal 1 (PTS1) or PTS2. The observation using confocal laser scanning microscopy revealed that the GFP fluorescence signals were detected as spherical spots in all cells of two kinds of transgenic plants. Immunoelectron microscopic analysis using antibodies against the peroxisomal marker protein, catalase, showed the presence of GFP in peroxisomes, confirming that GFP was correctly transported into peroxisomes by PTS1 or PTS2 pathways. It has been also revealed that peroxisomes are motile organelles whose movement might be caused by cytoplasmic flow. The movement of peroxisomes was more prominent in root cells than that in leaves, and divided into two categories: a relatively slow, random, vibrational movement and a rapid movement. Treatment with anti-actin and anti-tubulin drugs revealed that actin filaments involve in the rapid movement of peroxisomes. Moreover, abnormal large peroxisomes are present as clusters at the onset of germination, and these clusters disappear in a few days. Interestingly, tubular peroxisomes were also observed in the hypocotyl. These findings indicate that the shape, size, number and movement of peroxisomes in living cells are dynamic and changeable rather than uniform.     

The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin

We have investigated changes in the distribution of peroxisomes through the cell cycle in onion (Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek (Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the -oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.Abbreviations BDM 2,3-butanedione monoxime - DAPI 4,6-diamidino-2-phenylindole - ER endoplasmic reticulum - GFP green fluorescent protein     

RNA processing bodies, peroxisomes, Golgi bodies, mitochondria, and endoplasmic reticulum tubule junctions frequently pause at cortical microtubules

Organelle motility, essential for cellular function, is driven by the cytoskeleton. In plants, actin filaments sustain the long-distance transport of many types of organelles, and microtubules typically fine-tune the motile behavior. In shoot epidermal cells of Arabidopsis thaliana seedlings, we show here that a type of RNA granule, the RNA processing body (P-body), is transported by actin filaments and pauses at cortical microtubules. Interestingly, removal of microtubules does not change the frequency of P-body pausing. Similarly, we show that Golgi bodies, peroxisomes, and mitochondria all pause at microtubules, and again the frequency of pauses is not appreciably changed after microtubules are depolymerized. To understand the basis for pausing, we examined the endoplasmic reticulum (ER), whose overall architecture depends on actin filaments. By the dual observation of ER and microtubules, we find that stable junctions of tubular ER occur mainly at microtubules. Removal of microtubules reduces the number of stable ER tubule junctions, but those remaining are maintained without microtubules. The results indicate that pausing on microtubules is a common attribute of motile organelles but that microtubules are not required for pausing. We suggest that pausing on microtubules facilitates interactions between the ER and otherwise translocating organelles in the cell cortex.     

Peroxisomal Delta(3),Delta(2)-enoyl CoA isomerases and evolution of cytosolic paralogues in embryophytes

Delta(3),Delta(2)-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the beta-oxidation cycle. Three genes encoding Delta(3),Delta(2)-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Deltaeci1 mutant to degrade 10Z-heptadecenoic acid, demonstrating Delta(3),Delta(2)-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Delta(3),Delta(2)-enoyl CoA isomerase, indicating that evolution of cytosolic Delta(3),Delta(2)-enoyl CoA isomerases is restricted to the plant kingdom.     

Improved prediction of peroxisomal PTS1 proteins from genome sequences based on experimental subcellular targeting analyses as exemplified for protein kinases from Arabidopsis

Due to current experimental limitations in peroxisome proteome research, the identification of low-abundance regulatory proteins such as protein kinases largely relies on computational protein prediction. To test and improve the identification of regulatory proteins by such a prediction-based approach, the Arabidopsis genome was screened for genes that encode protein kinases with predicted type 1 or type 2 peroxisome targeting signals (PTS1 or PTS2). Upon transient expression in onion epidermal cells, the predicted PTS1 domains of four of the seven protein kinases re-directed the reporter protein, enhanced yellow green fluorescent (EYFP), to peroxisomes and were thus verified as functional PTS1 domains. The full-length fusions, however, remained cytosolic, suggesting that PTS1 exposure is induced by specific signals. To investigate why peroxisome targeting of three other kinases was incorrectly predicted and ultimately to improve the prediction algorithms, selected amino acid residues located upstream of PTS1 tripeptides were mutated and the effect on subcellular targeting of the reporter protein was analysed. Acidic residues in close proximity to major PTS1 tripeptides were demonstrated to inhibit protein targeting to plant peroxisomes even in the case of the prototypical PTS1 tripeptide SKL>, whereas basic residues function as essential auxiliary targeting elements in front of weak PTS1 tripeptides such as SHL>. The functional characterization of these inhibitory and essential enhancer-targeting elements allows their consideration in predictive algorithms to improve the prediction accuracy of PTS1 proteins from genome sequences.     

A class I ADP-ribosylation factor GTPase-activating protein is critical for maintaining directional root hair growth in Arabidopsis

Membrane trafficking and cytoskeletal dynamics are important cellular processes that drive tip growth in root hairs. These processes interact with a multitude of signaling pathways that allow for the efficient transfer of information to specify the direction in which tip growth occurs. Here, we show that AGD1, a class I ADP ribosylation factor GTPase-activating protein, is important for maintaining straight growth in Arabidopsis (Arabidopsis thaliana) root hairs, since mutations in the AGD1 gene resulted in wavy root hair growth. Live cell imaging of growing agd1 root hairs revealed bundles of endoplasmic microtubules and actin filaments extending into the extreme tip. The wavy phenotype and pattern of cytoskeletal distribution in root hairs of agd1 partially resembled that of mutants in an armadillo repeat-containing kinesin (ARK1). Root hairs of double agd1 ark1 mutants were more severely deformed compared with single mutants. Organelle trafficking as revealed by a fluorescent Golgi marker was slightly inhibited, and Golgi stacks frequently protruded into the extreme root hair apex of agd1 mutants. Transient expression of green fluorescent protein-AGD1 in tobacco (Nicotiana tabacum) epidermal cells labeled punctate bodies that partially colocalized with the endocytic marker FM4-64, while ARK1-yellow fluorescent protein associated with microtubules. Brefeldin A rescued the phenotype of agd1, indicating that the altered activity of an AGD1-dependent ADP ribosylation factor contributes to the defective growth, organelle trafficking, and cytoskeletal organization of agd1 root hairs. We propose that AGD1, a regulator of membrane trafficking, and ARK1, a microtubule motor, are components of converging signaling pathways that affect cytoskeletal organization to specify growth orientation in Arabidopsis root hairs.     

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.     

Dynamin-dependent biogenesis, cell cycle regulation and mitochondrial association of peroxisomes in fission yeast

Peroxisomes were visualized for the first time in living fission yeast cells. In small, newly divided cells, the number of peroxisomes was low but increased in parallel with the increase in cell length/volume that accompanies cell cycle progression. In cells grown in oleic acid, both the size and the number of peroxisomes increased. The peroxisomal inventory of cells lacking the dynamin-related proteins Dnm1 or Vps1 was similar to that in wild type. By contrast, cells of the double mutant dnm1Delta vps1Delta contained either no peroxisomes at all or a small number of morphologically aberrant organelles. Peroxisomes exhibited either local Brownian movement or longer-range linear displacements, which continued in the absence of either microtubules or actin filaments. On the contrary, directed peroxisome motility appeared to occur in association with mitochondria and may be an indirect function of intrinsic mitochondrial dynamics. We conclude that peroxisomes are present in fission yeast and that Dnm1 and Vps1 act redundantly in peroxisome biogenesis, which is under cell cycle control. Peroxisome movement is independent of the cytoskeleton but is coupled to mitochondrial dynamics.     

A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae.

In vivo time-lapse microscopy reveals that the number of peroxisomes in Saccharomyces cerevisiae cells is fairly constant and that a subset of the organelles are targeted and segregated to the bud in a highly ordered, vectorial process. The dynamin-like protein Vps1p controls the number of peroxisomes, since in a vps1Delta mutant only one or two giant peroxisomes remain. Analogous to the function of other dynamin-related proteins, Vps1p may be involved in a membrane fission event that is required for the regulation of peroxisome abundance. We found that efficient segregation of peroxisomes from mother to bud is dependent on the actin cytoskeleton, and active movement of peroxisomes along actin filaments is driven by the class V myosin motor protein, Myo2p: (a) peroxisomal dynamics always paralleled the polarity of the actin cytoskeleton, (b) double labeling of peroxisomes and actin cables revealed a close association between both, (c) depolymerization of the actin cytoskeleton abolished all peroxisomal movements, and (d) in cells containing thermosensitive alleles of MYO2, all peroxisome movement immediately stopped at the nonpermissive temperature. In addition, time-lapse videos showing peroxisome movement in wild-type and vps1Delta cells suggest the existence of various levels of control involved in the partitioning of peroxisomes.     

Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins.     

Cortical fine actin filaments in higher plant cells visualized by rhodamine-phalloidin after pretreatment with m-maleimidobenzoyl N-hydroxysuccinimide ester

Summary Actin filaments in cultured tobacco cells were stained by rhodamine-phalloidin after pretreatment with 100 M m-maleidobenzoyl N-hydroxysuccinimide ester (MBS) followed by formaldehyde fixation. The use of MBS prior to formaldehyde fixation enabled us to visualize fine, transversely arranged cortical actin filaments in a majority of interphase tobacco cells. It also enabled us to double-stain fine actin filaments and microtubules in the same cells. The pattern of actin filaments and that of microtubules in the cortical region of a single tobacco cell bore a close resemblance to each other. The method which employed MBS was found to be useful also in visualizing fine cortical actin filaments in inner epidermal cells of onion bulbs.Rhodamine-phalloidin seemed to induce the bundling of actin filaments both tobacco cells and in onion cells when it was applied to the cells which had not been subjected to fixation, indicating that the application of fluorescent-dye-labeled phallotoxins to unfixed cells involves the risk of observing artifically bundled actin filaments.     

New guidelines for fluorophore application in peroxisome targeting analyses in transient plant expression systems

Peroxisome research has been revolutionized by proteome studies combined with in vivo subcellular targeting analyses. Yellow and cyan fluorescent protein(YFP and CFP) are the classical fluorophores of plant peroxisome research. In the new transient expression system of Arabidopsis seedlings co-cultivated with Agrobacterium we detected the YFP fusion of one candidate protein in peroxisomes, but only upon co-transformation with the peroxisome marker, CFP-PTS_1. The data suggested that the YFP fusion was directed to peroxisomes due to its weak heterodimerization ability with CFP-PTS_1,allowing piggy-back import into peroxisomes. Indeed, if co-expressed with monomeric Cerulean-PTS_1(mCer-PTS_1),the YFP fusion was no longer matrix localized. We systematically investigated the occurrence and extent of dimerization-based piggy-back import for different fluorophore combinations in five major transient plant expression systems. In Arabidopsis seedlings and tobacco leaves both untagged YFP and monomeric Venus were imported into peroxisomes if co-expressed with CFP-PTS_1 but not with mCer-PTS_1. By contrast, piggy-back import of cytosolic proteins was not observed in Arabidopsis and tobacco protoplasts or in onion epidermal cells for any fluorophore combination at any time point. Based on these important results we formulate new guidelines for fluorophore usage and experimental design to guarantee reliable identification of novel plant peroxisomal proteins.     

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.     

Cell-type-specific disruption and recovery of the cytoskeleton in Arabidopsis thaliana epidermal root cells upon heat shock stress

Summary. The cytoskeleton in plant cells plays an important role in controlling cell shape and mediating intracellular signalling. However, almost nothing is known about the reactions of cytoskeletal elements to heat stress, which represents one of the major environmental challenges for plants. Here we show that living epidermal root cells of Arabidopsis thaliana could cope with short-term heat shock stress showing disruption and subsequent recovery of microtubules and actin microfilaments in a time-dependent manner. Time-lapse imaging revealed a very dynamic behavior of both cytoskeletal elements including transient depolymerization and disassembly upon heat shock (40–41 °C) followed by full recovery at room temperature (20 °C) within 1–3 h. Reaction of microtubules, but not actin filaments, to heat shock was dependent on cell type and developmental stage. On the other hand, recovery of actin filaments, but not microtubules, from heat shock stress was dependent on the same parameters. The relevance of this adaptive cytoskeletal behavior to intracellular signalling is discussed. Correspondence and reprints: Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Federal Republic of Germany.     

Interaction of microtubules and microfilaments in the zone of distal elongation of Arabidopsis thaliana roots

Investigations on GFP-MAP4 and GFP-ABD2 plants of Arabidopsis thaliana with the usage of inhibitors of actin and tubulin polymerization revealed that stability of cell growth in the zone of distal elongation of a root is provided by structural interactions between microtubules and actin filaments. The experiments with clinorotation and inhibition analysis enabled us to demonstrate that gravitation is a determining factor for the interaction between cytoskeleton elements in the zone of distal elongation in a root.     

An Arabidopsis pex10 null mutant is embryo lethal, implicating peroxisomes in an essential role during plant embryogenesis

Peroxisomes participate in many important functions in plants, including seed reserve mobilization, photorespiration, defense against oxidative stress, and auxin and jasmonate signaling. In mammals, defects in peroxisome biogenesis result in multiple system abnormalities, severe developmental delay, and death, whereas in unicellular yeasts, peroxisomes are dispensable unless required for growth of specific substrates. PEX10 encodes an integral membrane protein required for peroxisome biogenesis in mammals and yeast. To investigate the importance of PEX10 in plants, we characterized a Ds insertion mutant in the PEX10 gene of Arabidopsis (AtPEX10). Heterozygous AtPEX10::dissociation element mutants show normal vegetative phenotypes under optimal growth conditions, but produce about 20% abnormal seeds. The embryos in the abnormal seeds are predominantly homozygous for the disruption allele. They show retarded development and some morphological abnormalities. No viable homozygous mutant plants were obtained. AtPEX10 fused to yellow fluorescent protein colocalized with green fluorescent protein-serine-lysine-leucine, a well-documented peroxisomal marker, suggesting that AtPEX10 encodes a peroxisomal protein that is essential for normal embryo development and viability.     

Actin-organelle interaction: association with chloroplast in arabidopsis leaf mesophyll cells.

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.     

The N-terminal domain of PMTV TGB1 movement protein is required for nucleolar localization, microtubule association, and long-distance movement

The triple-gene-block (TGB)1 protein of Potato mop-top virus (PMTV) was fused to fluorescent proteins and expressed in epidermal cells of Nicotiana benthamiana under the control of the 35S promoter. TGB1 fluorescence was observed in the cytoplasm, nucleus, and nucleolus and occasionally associated with microtubules. When expressed from a modified virus (PMTV.YFP-TGB1) which formed local lesions but was not competent for systemic movement, yellow fluorescent protein (YFP)-TGB1 labeled plasmodesmata in cells at the leading edge of the lesion and plasmodesmata, microtubules, nuclei, and nucleoli in cells immediately behind the leading edge. Deletion of 84 amino acids from the N-terminus of unlabeled TGB1 within the PMTV genome abolished movement of viral RNA to noninoculated leaves. When the same deletion was introduced into PMTV.YFP-TGB1, labeling of microtubules and nucleoli was abolished. The N-terminal 84 amino acids of TGB1 were fused to green fluorescent protein (GFP) and expressed in epidermal cells where GFP localized strongly to the nucleolus (not seen with unfused GFP), indicating that these amino acids contain a nucleolar localization signal; the fusion protein did not label microtubules. This is the first report of nucleolar and microtubule association of a TGB movement protein. The results suggest that PMTV TGB1 requires interaction with nuclear components and, possibly, microtubules for long-distance movement of viral RNA.