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Journal of Experimental Botany, Vol. 53, No. 369, pp. 659–667, April 2002 Plant mitochondria move on F-actin, but their positioning in the cortical cytoplasm depends on both F-actin and microtubules K. Van Gestel1, R.H. Köhler 2 and J-P. Verbelen1,3 1 2 Department of Biology, University of Antwerp UIA, Universiteitsplein 1, 2610 Wilrijk, Belgium Lion Bioscience Research Inc., Cambridge, MA 02139, USA Received 27 August 2001; Accepted 26 November 2001 Abstract Introduction Mitochondrion movement and positioning was studied in elongating cultured cells of tobacco (Nicotiana tabacum L.), containing mitochondrialocalized green fluorescent protein. In these cells mitochondria are either actively moving in strands of cytoplasm transversing or bordering the vacuole, or immobile positioned in the cortical layer of cytoplasm. Depletion of the cell’s ATP stock with the uncoupling agent DNP shows that the movement is much more energy demanding than the positioning. The active movement is F-actin based. It is inhibited by the actin filament disrupting drug latrunculin B, the myosin ATPase inhibitor 2,3-butanedione 2-monoxime and the sulphydryl-modifying agent N-ethylmaleimide. The microtubule disrupting drug oryzalin did not affect the movement of mitochondria itself, but it slightly stimulated the recruitment of cytoplasmic strands, along which mitochondria travel. The immobile mitochondria are often positioned along parallel lines, transverse or oblique to the cell axis, in the cortical cytoplasm of elongated cells. This positioning is mainly microtubule based. After complete disruption of the F-actin, the mitochondria parked themselves into conspicuous parallel arrays transverse or oblique to the cell axis or clustered around chloroplasts and around patches and strands of endoplasmic reticulum. Oryzalin inhibited all positioning of the mitochondria in parallel arrays. Displacements and changes in shape of mitochondria are thought to depend on driving cytoskeletal forces as well as on intrinsic factors (Bereiter-Hahn and Vöth, 1994). In animal cells mitochondrion movement is mainly associated with microtubules (MTs) (Heggeness et al., 1987; Hirokawa, 1998). Only in neuronal axons (Kuznetsov et al., 1992) and in specific insect cells (Sturmer et al., 1995) do actin filaments (AFs) serve as a track for mitochondrial transport. In yeasts, the MTcytoskeleton is indispensable for mitochondrial distribution in Schizosaccharomyces pombe (Yaffe et al., 1996), while in Saccharomyces cerevisiae only AFs and intermediate filaments have been implicated in mitochondrial translocations (reviewed by Hermann and Shaw, 1998). For plant cells, the basic model for AF-based cytoplasmic movement of organelles (Williamson, 1993) is still valid, also for mitochondria. Structural associations of organelles with AFs have been described in fixed material (Menzel, 1987; Lichtscheidl et al., 1990), while AF-based movement of GFP-labelled Golgi stacks has been demonstrated in vivo in tobacco leaf epidermis (Boevink et al., 1998). Evidence for the mechanism of mitochondrion movement and positioning is, however, scarce and mostly based on comparison with other organelles. In onion (Allium cepa L.) epidermal cells using the fluorochrome DiOC6(3), mitochondria and spherosomes were found to follow the same pathway as the ER-strands, whose movement was shown to be dependent on F-actin (Quader et al., 1987, 1989; Allen and Brown, 1988). In the same tissue DiOC6(3)-stained mitochondria were shown to co-localize with rhodamine–phalloidin labelled AFs (Olyslaegers and Verbelen, 1998). In tobacco BY-2 cells Nebenführ et al. observed GFP-labelled Key words: Actin, green fluorescent protein, mitochondrion, Nicotiana tabacum L., tubulin. 3 To whom correspondence should be addressed. Fax: q32 3 8202271. E-mail: [email protected] ß Society for Experimental Biology 2002 660 Van Gestel et al. Golgi stacks and fluorochrome-labelled mitochondria following the same travelling paths and showing similar stop-and-go-movements (Nebenführ et al., 1999). Stably transformed tobacco (Köhler et al., 1997) and Arabidopsis (Logan and Leaver, 2000) plant lines with GFP targeted to the mitochondria were developed. In Arabidopsis the heterogeneity in morphology and dynamics of mitochondria was described. Using the transformed tobacco plants (Köhler et al., 1997), mitochondrion movement and positioning in cells was analysed by means of different inhibitor treatments. In these cells it is demonstrated that fast directional movement of mitochondria is dependent on an intact F-actin–myosin system while the positioning of immobile mitochondria in the cortical cytoplasm is both AF- and MT-based. in DMSO, 2,3-butanedione 2-monoxime (BDM) (Sigma) was prepared as a 1 M stock in distilled H2O (50 8C). Appropriate amounts of stock solutions were dissolved in K3A-culture medium (Potrykus and Shillito, 1986) to reach the following concentrations: 1.25 mM latrunculin B (Gibbon et al., 1999), 10 mM oryzalin (Hugdahl and Morejohn, 1993), 50 mM NEM (Liebe and Quader, 1994), 40 mM DNP (Markova et al., 1990), and 20 mM BDM (May et al., 1998). Incubation times were 1–5 h for latrunculin B and oryzalin, 1–2 h for NEM, BDM and DNP. Microscopy Fluorescence of GFP, DiOC6(3), and Alexa-phalloidin were imaged using the 488 nm laser line of a Bio-Rad MRC 600 confocal system mounted on a Zeiss Axioskop microscope, equipped with a 40 3 (NA 0.9) water-immersion objective. Part of the micrographs are z-series projections of optical sections, made with the standard COMOS software (Bio-Rad Microscience Ltd.). Brightfield and phase-contrast images were acquired using the transmission mode of the confocal system. Materials and methods Plant material and labelling Elongated tobacco cells were regenerated from protoplasts (Verbelen et al., 1992) which were isolated (Potrykus and Shillito, 1986) from leaves of sterile-grown, transgenic plants of Nicotiana tabacum L. cv. Petite Havana expressing mitochondrion targeted GFP (Köhler et al., 1997). This targeting is mediated by the yeast cytochrome oxidase subunit IV (coxIV) transit peptide, which is removed upon import of the GFP molecule into the mitochondrion. The specificity and the non-toxicity of the GFP targeting have been demonstrated (Köhler et al., 1997). For visualization of the ER, AFs and MTs, tobacco cells without mitochondrion targeted GFP were used. The ER, together with the mitochondria, was stained with 0.87 mM 3,39-dihexyloxa-carbocyanine iodide (DiOC6(3)) (Molecular Probes). Alexa-phalloidin (Molecular Probes) was used to label AFs after permeabilization of the cells with detergents, and MTs were labelled by immunocytochemistry (Vissenberg et al., 2000). Drug treatments Stock concentrations of 2.5 mM latrunculin B (Calbiochem), 100 mM oryzalin (Merck), 50 mM N-ethylmaleimide (NEM) (Sigma), and 40 mM dinitrophenol (DNP) (Merck) were made Results The protoplast derived cells used in these experiments are a model system for cell elongation (Vissenberg et al., 2000). After the regeneration of a wall, cells elongate and thereby increase in volume 10–15 times. Regarding mitochondrion movement, the cytoplasm of elongating tobacco cells can be divided in two domains: fast and directional movement is concentrated in conspicuous strands of cytoplasm, bordering or transversing the vacuole, while the more static mitochondria reside in the very thin cortical layer of cytoplasm appressed against the plasma membrane. Mitochondrion movement in cytoplasmic strands Mitochondria move vigorously through transvacuolar and vacuole bordering cytoplasmic strands and they often stop, change direction, fuse or divide. This activity is most easily illustrated with pictures of the transvacuolar strands. In Fig. 1, a sequence of four fluorescence images, taken 3 s apart, demonstrates the directional movement Fig. 1. Sequence of four confocal images, taken 3 s apart, demonstrating the directional movement of GFP-tagged mitochondria in a transvacuolar cytoplasmic strand of a tobacco cell. Arrowheads indicate the fast moving mitochondria while the arrow points to immobile mitochondria in the cortical cytoplasm layer. Scale bar ¼ 10 mm. Movement of plant mitochondria 661 Fig. 2. The effect of latrunculin B on cytoplasmic architecture. The figure represents a bright-field image (left side) and a single plane confocal image (right side) of two tobacco cells. (A) In a control cell cytoplasmic strands containing mitochondria radiate from the nucleus (n) towards the cortical layer. (B) Latrunculin B (1 h) has a disruptive effect on the cytoplasmic strands. Scale bar ¼ 20 mm. of mitochondria in a transvacuolar strand from top right to bottom left of the image. Note also the immobile mitochondria in the cortical layer of cytoplasm at the right side of the images. The speed of this directional movement is similar to that of cytoplasmic streaming and can reach up to 10 mm s1. A movie of this dynamic transvacuolar mitochondrion movement can be seen at http:uubio-www.uia.ac.beubioufymoukvg. In Fig. 2A, the multiple transvacuolar strands radiating from the nucleus to the cortical layer (brightfield image) contain many moving mitochondria (fluorescence image). Treatment of the cells with 1.25 mM latrunculin B causes an abrupt cessation of directional mitochondrion movement and a breakdown of most cytoplasmic strands within 1 h (Fig. 2B). Mitochondria from the broken transvacuolar strands become incorporated in the cortical cytoplasm. Both NEM (50 mM) and BDM (20 mM) cause a cessation of mitochondrial tracking accompanied by a disturbance of the cytoplasmic strands (results not shown). Recovery of structure and movement after removal of the drugs only occurred in the case of latrunculin B and BDM. Oryzalin (10 mM) did not affect the movement of the individual mitochondria. On the contrary, after several hours of incubation in oryzalin, a stimulation of the mobility was noticed in the population of mitochondria due to an increase in the number of cytoplasmic strands within the cells. In a sample of 50 cells, oryzalin-treated cells contain about 9.2 transvacuolar strands against 7.5 in non-treated control cells (P-0.01, Student’s t-test). Mitochondria in the cortical layer of cytoplasm The proportion of mitochondria being immobile depends on the developmental stage of the cells. In freshly isolated protoplasts (Fig. 3A) and young (2–5 d) cells in culture (Fig. 3B) most mitochondria move actively throughout the cortical cytoplasm and very few are immobile (see http:uubio-www.uia.ac.beubioufymoukvg for a movie). No particular positioning of these cortical mitochondria was observed besides an association with chloroplasts. In the protoplasts, latrunculin B causes all mitochondria to cluster around chloroplasts and small disc-shaped structures (Fig. 3F). In the young cells part of the mitochondria form additional aggregates, showing no particular orientation (Fig. 3G). From the onset of elongation on, cells contain many more mitochondria (Fig. 3C). Active movement in the cortical cytoplasm is restricted to vacuole bordering cytoplasmic strands and in the areas which are devoid of these strands, numerous mitochondria are immobile. The position of the immobile mitochondria in the thin cortical layer is not immediately affected by latrunculin B. After sustained incubation (1–2 h) these mitochondria cluster around chloroplasts, disc-shaped and strand-like structures, but it is their positioning in parallel transverse or oblique arrays (Fig. 3H) that is conspicious. In older well-elongated cells, part of the immobile mitochondria is already arranged along parallel lines transverse or oblique to the cell axis (Fig. 3D). In these cells too, latrunculin B causes a redistribution of mitochondria to the circumference of the chloroplasts, the disc-shaped and the strand-like structures. But it is again their positioning into clear parallel arrays, transverse or oblique to the cell axis which is the most striking feature (Fig. 3I). In cells treated with oryzalin, all transverse arrays of mitochondria are lost (Fig. 3E) and mitochondria are positioned evenly throughout the cortical cytoplasm. A simultaneous incubation in latrunculin B and oryzalin, causes the mitochondria to cluster around chloroplasts and around disc-shaped and strand-like structures with an obvious absence of any clear transverse orientation (Fig. 3J). The typical patterns of mitochondria localization in elongated cells, as described above, are strongly amplified by incubating the cells in the uncoupling agent 2,4-dinitrophenol (DNP). This DNP treatment results 662 Van Gestel et al. Fig. 3. Z-series projections of confocal images of GFP-tagged mitochondria in the cortical cytoplasm of tobacco cells at different stages of development (A–D) and (F–I), and after different drug treatments (E-J). The grey discs (arrow in A) represent autofluorescent chloroplasts. The sequence of development from protoplast (A), after regeneration of a cell wall (B), at the onset of elongation (C), to a well-elongated cell (D), demonstrates that mitochondria increase in number and become quantitatively less mobile and finally adopt a parallel orientation transverse to the cell axis. Latrunculin B (1 h) makes the mitochondria cluster around chloroplasts and disc-shaped ER (arrow) in protoplasts (F) and young spherical cells (G). At the onset of elongation (H) mitochondria additionally lie in parallel transverse arrays (1–2 h treatment). In fully elongated cells (I) these Movement of plant mitochondria in a situation where all cortical mitochondria lie immobile and head-to-tail on parallel transverse arrays (Fig. 4A). All mitochondria are immobile in the strands as well. This situation is, however, fully reversible as mitochondria regain their former behaviour directly after washing the cells with DNP-free culture medium. In latrunculin B-treated cells, the DNP effect is very similar, the array-like head-to-tail positioning of mitochondria is 663 accentuated (Fig. 4B). In oryzalin-treated cells, however, DNP did not induce any array-like positioning of cortical mitochondria (Fig. 4C). BDM (20 mM) and NEM (50 mM) cause the immobile mitochondria of elongated cells to aggregate along short lines, with an average transverse orientation (Fig. 5A, B). In NEM-treated cells, however, more irregular clumping of the mitochondria was observed. Fig. 4. Z-series projections of confocal images of GFP-tagged mitochondria in the cortical cytoplasm of DNP-treated (1–2 h) tobacco cells. (A) In normal cells DNP induces head-to-tail parking of immobile mitochondria on parallel arrays transverse to the cell axis. (B) In latrunculin B-treated (4 h) cells DNP has a similar effect on those mitochondria which are not clustered around chloroplasts or ER-structures. (C) In oryzalin-treated (4 h) cells DNP could not induce transverse array-like positioning of the mitochondria. Scale bar ¼ 50 mm. Fig. 5. Z-series projections of confocal images of GFP-tagged mitochondria in the cortical cytoplasm of BDM- and NEM-treated (1–2 h) tobacco cells. Both BDM (A) and NEM (B) induce clustering of mitochondria along short lines, NEM has, however, a more clumping effect. Scale bar ¼ 15 mm. transverse arrays (right side of the picture) and the clustering along ER-strands (arrowheads) are predominant (2– 4 h treatment). Oryzalin (4 h) destroys the parallel positioning of the mitochondria in elongated cells (E). Simultaneous treatment with latrunculin B and oryzalin (4 h) results in clustering around chloroplasts and disc-shaped ER, without any parallel transverse orientation (J). Scale bar ¼ 25 mm. 664 Van Gestel et al. To evaluate the proper effects of the inhibitors latrunculin B, oryzalin, NEM, and BDM on the cytoskeleton, AFs and MTs were visualized in the cells after the different treatments. AFs were unaffected by treatment with BDM and oryzalin, NEM caused a conspicuous decrease in number, intactness and thickness of the filaments and latrunculin B resulted in a complete breakdown of the AFs (data not shown). MTs were unaffected by BDM and latrunculin B, NEM had a slight disrupting effect, while oryzalin destroyed them completely (data not shown). The disc-shaped and strand-like structures around which the mitochondria cluster in cells treated with latrunculin B, with or without oryzalin, are very probably of ER nature. They are visible using phase-contrast optics (Fig. 6) and can be labelled with the fluorochrome DiOC6(3) (Fig. 7). These ER-structures are far more fluorescent than the cortical ER network, which is not visible in Fig. 7, but are comparable in fluorescence intensity to the mobile ER-stretches in cytoplasmic strands of control cells (data not shown). The transverse orientation of the strand-like ER bordered by the mitochondria is very conspicuous in elongated cells (Fig. 7A). Part of the mitochondria in latrunculin B-treated cells are, however, positioned in transverse arrays without association with these conspicuous ER structures (Fig. 7B). Fig. 6. The strand-like (A) and disc-shaped (B) ER-structures (arrows) along which GFP-tagged mitochondria cluster in the cortical cytoplasm after latrunculin B treatment (4 h) are visible with phase-contrast microscopy (right side). The left part of the pictures is a single plane confocal image indicating the position of the mitochondria. Scale bar ¼ 10 mm. Fig. 7. Single plane confocal images of mitochondria and ER, both stained with DiOC6(3), in latrunculin B-treated (4 h) elongated tobacco cells. (A) Mitochondria are clustered around disc-shaped and strand-like parts of the ER. (B) Part of the mitochondria in latrunculin B-treated cells are, however, positioned in transverse arrays without association with these transverse ER structures. Scale bar ¼ 10 mm. Movement of plant mitochondria Discussion In elongating tobacco cells, there is a clear distinction between mitochondria in the cytoplasmic strands— through and along the vacuole—and those in the very thin cortical layer of cytoplasm. While the former display a fast directional tracking, the latter are often hardly moving. A similar difference between the inner and the cortical cytoplasm has been described for the movement of the ER (Allen and Brown, 1988) and of the Golgi stacks (Nebenführ et al., 1999). The ratio between mobile and immobile mitochondria depends on the physiological status of the cell. In protoplasts and cells that have just generated a cell wall, very few mitochondria are immobile. When the cells elongate, the number of mitochondria increases, especially in the non-mobile part of the population. Mitochondrion movement in the cytoplasmic strands depends on F-actin–myosin interaction while the recruitment of strands is negatively controlled by MTs Mitochondria move through the cytoplasmic strands in a directional but non-uniform way. There is a great variation in mobility within a cell, between cells and in time. Similar movements of fluorochromically labelled mitochondria have been reported and called ‘stop-and-go movements’ (Quader et al., 1987; Nebenführ et al., 1999). Mitochondial mobility is a very energy-demanding process. The uncoupling agent DNP abolishes the proton gradient over the inner mitochondrial membrane, leading to a depletion of ATP in the cells (Rawn, 1989). In cells treated with DNP, movement of mitochondria was totally abolished. This movement of mitochondria involves F-actin as it was totally arrested by the F-actin disrupting drug latrunculin B. The myosin ATPase inhibitor BDM and the sulphydryl-modifying agent NEM were used to confirm the involvement of myosins in mitochondrial mobility, as reported previously for onion epidermal cells (Liebe and Quader, 1994). In the tobacco cells both drugs inhibited mitochondrial movement. Recently, it has been demonstrated that myosin is associated with the surface of mitochondria prepared from etiolated wheat (Triticum aestivum L.) seedlings (Zhao et al., 1999). The MT-depolymerizing drug oryzalin did not affect the movement of mitochondria itself, but it caused a slight increase in the frequency of cytoplasmic strands with moving mitochondria, hence leading to a slightly higher number of mobile mitochondria. This study thus confims the results obtained previously with fluorochromically labelled mitochondria (Quader et al., 1989): mitochondrion movement is dependent on an intact F-actin–myosin system. The negative control by MTs resembles the observations by Nebenführ et al. who found that the AF-based mobility of Golgi stacks 665 was negatively controlled by the presence of MTs (Nebenführ et al., 1999). Examples of interactions between MTs and AFs are numerous, both in animal and in plant cells (Gavin, 1997; Blancaflor, 2000; Šamai et al., 2000). In plant cells especially, the mechanism at the base of these interactions is not yet understood. In maize (Zea mays L.) root cells, Šamai et al. found evidence for interaction between AFs and MTs via BDM-sensitive myosins (Šamai et al., 2000). It is unclear which mechanism lies at the base of the observed increase in AF-containing cytoplasmic strands upon depolymerization of the MTs. Mitochondrial positioning in the cortical cytoplasm depends on AFs and MTs The involvement of AFs is most obvious in protoplasts and young cells. MT-dependence is predominant in elongating cells. Freshly prepared protoplasts and young cells are roughly spherical and isotropic in growth and in cellular organization; their cytoskeleton contains relatively few and randomly oriented cortical MTs and AFs (Vissenberg et al., 2000). The immobile mitochondria are positioned throughout the cortical cytoplasm. After incubation in latrunculin B the majority of mitochondria clustered around chloroplasts and ER-patches. Mitochondria found in close association with chloroplasts have been reported for Vallisneria and Arabidopsis (Liebe and Menzel, 1995; Logan and Leaver, 2000). In Arabidopsis, baskets of F-actin were visualized surrounding the chloroplasts (Kandasamy and Meagher, 1999). Although no F-actin was detected around the chloroplasts of the tobacco cells after latrunculin B treatment, it is clear that mitochondria become trapped there as a result of the F-actin breakdown in the cytoplasmic strands. The disc-shaped ER-patches, around which the mitochondria clustered in AF-depleted cells, are probably derived from previously mobile tubular ER of the cytoplasmic strands, according to the fluorescence intensity when stained with DiOC6(3). Similar cortical ER‘patches’ were observed in cytochalasin D-treated onion epidermal cells (Knebel et al., 1990). Cells that elongate have very prominent transverse parallel arrays of cortical MTs, while arrays of parallel AFs are also present (Vissenberg et al., 2000). In these cells, cytoplasmic strands bordering the vacuole are scarce and most mitochondria are immobile in the very thin cortical layer of cytoplasm. Part of these mitochondria are often positioned in parallel transverse or oblique arrays. In latrunculin B-treated cells, where all the cortical AFs were lost, the mitochondria remained and even gathered on parallel transverse arrays. Part of the mitochondria clustered around the chloroplasts, ER-patches and ER-strands which act as lifebuoys. 666 Van Gestel et al. After treatment with oryzalin, however, all transverse ordering was lost. Thus the immobile part of the mitochondrion population is preferentially positioned along MTs. Clearly this positioning is less energy-demanding than actin-based mobility. DNP, which stops mobility, does not affect the positioning along microtubules. Indeed, when treated with DNP, mitochondria were frozen head-to-tail on parallel transverse or oblique arrays in the cortical cytoplasm of normal and AF-depleted cells, but failed to do so in MT-depleted cells. All these data together make it possible to conclude that, first, in elongating cells of tobacco, mitochondrion travelling depends on intact F-actin–myosin interactions whereby MTs seem to regulate the frequency of travelling tracks. Secondly, depending on the physiology of the cells, the positioning of mitochondria in the cortical cytoplasm is based on AFs or on MTs. Co-operation between MT- and AF-based motor proteins for the transport of membranous organelles has recently come into focus for animal cells. Organelles are supposed to possess both myosins and microtubule motor proteins (Allan and Schroer, 1999; Brown, 1999). 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