Microfilaments and microtubules control the shape ... - Springer Link

Protoplasma (2004) 224: 145–157 DOI 10.1007/s00709-004-0075-1

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Microfilaments and microtubules control the shape, motility, and subcellular distribution of cortical mitochondria in characean internodal cells I. Foissner* Fachbereich Zellbiologie, Universität Salzburg, Salzburg Received May 14, 2004; accepted June 17, 2004; published online December 22, 2004 © Springer-Verlag 2004

Summary. The shape, motility, and subcellular distribution of mitochondria in characean internodal cells were studied by visualizing fluorescent dyes with confocal laser scanning microscopy and conducting drug-inhibitor experiments. Shape, size, number, and distribution of mitochondria varied according to the growth status and the metabolic activity within the cell. Vermiform (sausage-shaped), disc-, or amoebalike mitochondria were present in elongating internodes, whereas very young cells and older cells that had completed growth contained short, rodlike organelles only. Mitochondria were evenly distributed and passively transported in the streaming endoplasm. In the cortex, mitochondria were sandwiched between the plasma membrane and the stationary chloroplast files and distributed in relation to the pattern of pH banding. Highest mitochondrial densities were found at the acid, photosynthetically more active regions, whereas the alkaline sites contained fewer and smaller mitochondria. In the cortex of elongating cells, small mitochondria moved slowly along microtubules or actin filaments. The shape and motility of giant mitochondria depended on the simultaneous interaction with both cytoskeletal systems. There was no microtubule-dependent motility in the cortex of nonelongating mature cells and mitochondria only occasionally travelled along actin filaments. These observations suggest that mitochondria of characean internodes possess motor proteins for microtubules and actin filaments, both of which can be used either as tracks for migration or for immobilization. The cortical cytoskeleton probably controls the spatiotemporal distribution of mitochondria within the cell and promotes their association with chloroplasts, which is necessary for exchange of metabolites during photosynthesis and detoxification. Keywords: Chloroplast-mitochondrion association; Mitochondrial motility; Mitochondrial shape; Cytoskeleton; Giant mitochondrion; Characeae. Abbreviations: AFW artificial fresh water; CD cytochalasin D; DASPMI 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide; DiOC6 3,3-dihexyloxacarbocyanine iodide; ICPC isopropyl N-(3-chlorophenyl)carbamate.

* Correspondence and reprints: Arbeitsgruppe Pflanzenphysiologie, Fachbereich Zellbiologie, Universität Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria. E-mail: ilse.foissner@sbg.ac.at

Introduction In many cells, mitochondria are highly dynamic and frequently change their shape and position (e.g., Bereiter-Hahn and Vöth 1994, Gunning 2004). Their motility guarantees the transport to specific regions of the cell and is a prerequisite for fusion of widely spaced organelles. Mitochondrial movement is also frequently associated with fission, although the division machinery does not require an external cytoskeleton (e.g., Westermann and Prokisch 2002, Logan 2003). The movement of higher-plant mitochondria has been shown to depend on actin filaments, whereas their positioning (immobilization) in the cortex additionally requires microtubules (Van Gestel et al. 2002). Translocation of mitochondria along actin filaments has also been described in the filamentous green alga Spirogyra crassa (Grolig 1990). In other lower eukaryotes and animal cells, however, mitochondrial motility predominantly relies on kinesin– microtubule interactions (see references in Steinberg 2000, Westermann and Prokisch 2002, Logan 2003). It is therefore of great interest to compare the mitochondria of higher plants with those of the members of the family Characeae, assumed to be close relatives of their common green algal ancestors (Turmel et al. 2003). The thallus of these multicellular algae resembles that of the primitive land plants known as horsetails (equisetums). Both the main axis and lateral branches consist of a regular alternation of groups of small, roundish nodal cells and cylindrical internodes that may attain a length of up to several centimeters (see Fig.1a). The cytoplasm of the internodes contains a stationary cortical layer including helically arranged files of chloroplasts and the streaming endoplasm (Foissner and Wasteneys 2000). Endoplasmic bulk streaming is generated

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through the interaction of myosin-coated cisternae of the endoplasmic reticulum with subcortical actin bundles attached to the inner surface of the chloroplasts and may reach a velocity of up to 80 m/s (Kachar and Reese 1988; reviewed by Grolig and Pierson 2000). The majority of other organelles, including the endoplasmic mitochondria, are passively transported. Occasional contact of endoplasmic mitochondria with the subcortical bundles results in active translocation at maximal rates of about 10 m/s (Kachar 1985, Foissner et al. 1996). Mitochondria are also present near the plasma membrane. These cortical mitochondria are shielded from cytoplasmic bulk streaming by the stationary chloroplasts and have previously been considered to be immobile (Foissner 1983). In this paper, I describe their morphology, dynamics (including shape changes and motility), and distribution by incorporating fluorescent dyes into living cells and observing their behavior with confocal laser scanning microscopy. Time-lapse studies reveal that these organelles are able to move actively, although at low velocities, and inhibitor experiments suggest the involvement of both actin filaments and microtubules in this migration, positioning, and immobilization. The previously unknown dynamic interaction of plant mitochondria with microtubules is shared by the cortical endoplasmic reticulum, which likewise uses microtubules as tracks for reorganization in elongating internodes (I. Foissner and G. O. Wasteneys unpubl.). The motile properties of cortical mitochondria probably ensure that these organelles are transported to and retained at regions with high metabolic activity and guarantee close contact with chloroplasts, a prerequisite for interorganellar exchange of metabolites during photosynthesis (Padmasree et al. 2002). Material and methods Plant material and culture conditions Chara corallina Klein ex Willd., em. R.D.W., Nitella translucens var. axillaris (A.Br.) R.D.W., and Nitella flexilis (L.) Ag. were cultured at room temperature in 10- to 100-liter aquaria filled with peat, sand, and distilled water. Fluorescent lamps (Gro-lux; Silvana, Erlangen, Germany) provided a photoperiod of 14 h light and 10 h dark. Thalli were grown under low light conditions to induce the formation of giant mitochondria (Foissner 1981). The light intensity, measured with an LI-189 quantum photometer (LI-COR, Lincoln, Nebr., U.S.A.), inside the culture vessels and between the thalli was about 1 mol/m2 s, a value similar to that occurring in natural habitats of characean algae (Foissner 1981). Internodal cells were trimmed of neighboring internodes and left in artificial fresh water (AFW) (0.1 mM NaCl, 0.1 mM KCl, 1 mM CaCl2) until use. Unless otherwise stated, experiments described in this study were performed with internodal cells of the lateral branchlets of Chara corallina. Staining methods and inhibitor treatments Cells were stained with 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI) (Sigma, Deisenhofen, Federal Republic of Germany),

I. Foissner: Dynamics of cortical mitochondria in characean internodal cells 50 M diluted in AFW from a 100 mM stock solution in dimethylsulfoxide (DMSO), or with 3,3-dihexyloxacarbocyanine iodide (DiOC6) (Molecular Probes, Leiden, the Netherlands), 2–4 M diluted from a 10 mM stock solution in DMSO. DASPMI labelled mitochondria after 30 min incubation (Bereiter-Hahn 1976). Staining was reversible, could be repeated several times, and allowed combined treatment with inhibitors. DiOC6 labelled the mitochondria and – at the higher concentration – also the endoplasmic reticulum (Terasaki et al. 1984, Quader and Schnepf 1986, Grolig 1990). DiOC6 staining up to 2 h had no effect on the shape and motility of mitochondria and the endoplasmic reticulum, but longer incubation times or simultaneous treatment with inhibitors was detrimental. Before observation, cells were briefly washed in AFW (with or without inhibitor, see below), mounted on the coverslip bottom of perfusion chambers and topped with a coverslip fragment supported by petrolatum-lanolin as described (Foissner et al. 2002). Perfusion of internodal cells, as described by Williamson et al. (1989), was used to visualize the cytoskeleton. Briefly, an internodal cell was placed on the bottom of a perfusion chamber and pressed lightly into vacuum grease lines positioned several millimeters away from the cell ends. Small reservoirs with grooves were carefully placed over the grease lines and pressed down firmly without damaging the cells. The central portion of the cell between the reservoirs was then covered with silicon fluid (Wacker, Burghausen, Federal Republic of Germany) in order to prevent desiccation. The ends of the cells within the reservoirs were bathed in isotonic perfusion solution: 200 mM sucrose, 70 mM KCl, 4.5 mM MgCl2, 5 mM ethyleneglycoltetraacetic acid (EGTA), 1.48 mM CaCl2, 10 mM piperazine-N,N-bis(2-ethanesulfonic acid) (PIPES), pH 7.0. Following reduction of turgor, cells were cut with small scissors and a small difference in solution levels between the two reservoirs ensured a gentle flow of perfusion solution through the cell. This treatment removed the cell sap and most of the endoplasm and washed out actin and tubulin monomers which could otherwise polymerize during the subsequent staining step. After about 5 min, the perfusion solution was replaced with perfusion solution containing fluorescent stains. Actin filaments were visualized by 0.32 M rhodamine or Alexa phalloidin (Molecular Probes), 6.6 M stock solution in methanol. Microtubules were stained by perfusion with 1–2 M Flutax-2 (Calbiochem, Darmstadt, Federal Republic of Germany) freshly prepared from frozen aliquots of a 0.5 mM stock solution in DMSO. At the higher concentration, Flutax-2 also labelled the mitochondria and eventually the endoplasmic reticulum (I. Foissner and G. O. Wasteneys unpubl.). Images of actin filaments and microtubules obtained by this method were similar to those seen after immunolabelling. Giant mitochondria, however, fragmented into smaller organelles. In order to perturb the cytoskeleton, cells were treated with 40 M cytochalasin D (CD) (Sigma, Deisenhofen, Federal Republic of Germany), stock solution 10 mM in DMSO; 10 M oryzalin (Riedel-de Haen, Seelze, Federal Republic of Germany), stock solution 10 mM in acetone; 5 M isopropyl N-(3-chlorophenyl)-carbamate (ICPC) (Sigma), stock solution 10 mM in ethanol; or 10 M taxol (Molecular Probes), stock solution 2 mM in DMSO. Stock solutions were diluted with AFW. DMSO, ethanol, or acetone at equivalent concentrations were included in the medium of control cells and had no effect on the shape or behavior of mitochondria. Confocal laser scanning and video-enhanced contrast microscopy The confocal laser scanning microscope used in this study was a Zeiss LSM 510 coupled to a Zeiss Axiovert inverted microscope. Projections of optical-section series (Z-series) were produced by the LSM 510 software and images were further processed by Adobe Photoshop. Organelle dynamics were studied by analysing time series taken at minimum laser intensity and pixel time in order to avoid photobleaching and irreversible fragmentation of giant mitochondria (Bereiter-Hahn et al. 1983, Dixit and Cyr 2003). Series of 10–50 images taken at 2–15 s intervals revealed the dynamics of the cortical mitochondria. For the faster moving endoplasmic mitochondria continuous, fast scanning without interval was necessary.

I. Foissner: Dynamics of cortical mitochondria in characean internodal cells The shape and behavior of mitochondria were also studied by videoenhanced contrast microscopy with the Argus 20 system (Hamamatsu, Herrsching, Federal Republic of Germany) in order to rule out potential staining artefacts. All images included in this study are positioned with vertical sides parallel to the long axes of the cells. Statistical analysis Mitochondrial area, mitochondrial number per unit cortical area, and mitochondrial density (percentage of cortical area occupied by mitochondria) were measured with an imaging system (Lucia, London, U.K.). Data were derived from at least 4 cells or cell regions and analysed by t-test or Dixon and Mood sign test (Sachs 1984). Differences were considered to be significant when P 0.05.

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Results Shape and size of mitochondria depend on growth status of the cell The observations and experiments described in this study were mostly made on internodal cells of the branchlets of Chara corallina (Fig.1a). During their development, characean internodal cells undergo periods with different elongation rates (Wasteneys and Williamson 1994). Initially they grow slowly, reach their maximum relative elongation rate after about 10 days, and then elongate at continuously

Fig. 1 a–h. Growth-dependent morphology and distribution of cortical mitochondria in characean internodal cells. a Schematic representation of a thallus of Chara corallina. Huge internodes (I) alternate with groups of small nodal cells (nodes, N ) from which whorls of lateral branchlets arise. The branchlets likewise consist of 2–3 internodes, separated by nodes, and are terminated by a small, cone-shaped cell. b and c DiOC6-stained internodes of branchlets from the first, upper whorl. The cortex of the two young cells contains fine-meshed endoplasmic reticulum but only a few mitochondria (arrows). d–f DASPMI-stained cortical mitochondria in rapidly growing internodes from branchlets of the second and third whorl. In addition to small mitochondria, giant vermiform or amoeba-like organelles are present. Acid regions (d and lower part of f) contain significantly more and larger mitochondria than alkaline sites (e and upper part of f). The border between the acid and alkaline region in f is indicated by arrows. g and h Cortical mitochondria in an acid (g) and alkaline region (h) of a slowly elongating internode from branchlets of the third whorl. i In nongrowing mature cells (whorl number 4 and higher), cortical mitochondria are small and distributed between the autofluorescent chloroplasts. Bar in a: for a, 1 cm; for b, 7 m; for c, 5 m; for d–h, 10 m; for i, 15 m

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I. Foissner: Dynamics of cortical mitochondria in characean internodal cells

Table 1. Characteristics of mitochondria at acid and alkaline regions of Chara corallina internodal cells Characteristic

Length (m) Width (m) Area (m2) Nr./100 m2 Density b

Value for mitochondria a amoeba-like, vermiform, and short in whorl-2-cell region

vermiform and short in whorl-3-cell region

short in whorl-4-cell region

acid

alkaline

acid

alkaline

acid

alkaline

7.9 9.3* 1.2 0.6* 9.6 11.9* 2.1 0.4 17 5*

4.3 4.0 0.9 0.5 4.0 4.9 1.9 0.4 72

10.2 36.7* 0.9 0.2 8.8 33.3* 4.9 1.0* 18 9*

2.3 2.0 0.8 0.2 1.8 1.6 3.4 0.6 62

4.4 5.3* 0.8 0.2 3.2 3.9* 7.1 1.0* 15 6*

1.6 1.3 0.8 0.2 1.2 0.7 3.4 0.8 42

a

Data are means with standard deviations from at least 4 cells; significant differences between acid and alkaline regions of the same cell type are marked by asterisks (t-test) or plus symbols (Dixon and Mood sign test) b Percentage of cortical area occupied by mitochondria

lower rates until they have reached their final length at about day 30 of their development. The youngest, slowly growing internodes investigated, harvested from the upper part of the thallus, were slightly elongate, with a length not exceeding twice the diameter. Their cytoplasm was already differentiated into a stationary cortex and a streaming endoplasm. The cortex contained small-meshed endoplasmic reticulum, sandwiched between the plasma membrane and chloroplast files (Fig.1b), but mitochondria were mostly restricted to the endoplasm. During further growth, an increasing number of mitochondria appeared near the plasma membrane (Fig.1c). Longer cells from whorl 2, i.e., internodes extending from the second node, were rapidly elongating and also contained amoeba-like mitochondria with diameters of up to 20 m and/or vermiform organelles up to 30 m long (Fig.1d–f). Similar elongate mitochondria, which occasionally had platelike inflations, were present in the endoplasm (compare Fig. 4p). The mean size of the mitochondria decreased during further growth. Those of slowly elongating cells from whorl 3 were either vermiform or short (Fig.1g, h). Those from mature cells (whorl number 4 and higher) had a maximum length of 2 m, and their number in the cortex, and probably also in the endoplasm, decreased steadily during ageing (Fig.1i and Table 1). Giant mitochondria disappeared within one day after cells were isolated and, judging by the fact that the area fraction occupied by cortical mitochondria remained approximately the same over this time period under a photoperiod (Table 2), this change was probably accomplished through division into shorter organelles. This mitochondrial division also occurred when cells were incubated in darkness and probably reflected the fact that internodal cells stop growing when detached from the thallus (my own unpubl. obs.).

Table 2. Effects of several days light or dark treatment on mitochondrial density in the cortex of Chara corallina internodes a Time of determination

After isolation After 4 days % decrease

Mitochondrial density at: b Photoperiod

Darkness

13.4 5.4 11.7 4.1 6.3 2.4

15.4 5.2 9.4 2.6* 35.4 8.6

a

Internodes of the branchlets were isolated and incubated in AFW, either under a photoperiod of 16 h light and 8 h dark or in complete darkness b Percentage of cortical area occupied by mitochondria; data are means with standard deviations from at least 9 cells scanned over their whole surface. The value of mitochondrial density after 4 days dark treatment is significantly different from that after isolation (*0.05 P 0.01, t-test)

Distribution of cortical mitochondria in elongating or mature cells correlates with pattern of photosynthesis-dependent pH banding Apart from the neutral lines, which separate up- from downstreaming regions and are mostly devoid of larger organelles, cortical mitochondria in the youngest but still slowly growing internodes were evenly distributed. Their distribution in longer, rapidly elongating or mature internodes that had ceased to grow, however, was not so uniform. Regions with high numbers of larger organelles alternated with domains in which cortical mitochondria were smaller and scarce, resulting in highly significant differences in mitochondrial density, defined as the percentage of cortical area occupied by mitochondria (Fig.1d–f and Table 1). During photosynthesis, the surface of characean internodes develops alternating bands of acid and alkaline pH (Lucas and Smith 1973). Visualization of pH banding by phenol red and comparison with the pattern of mitochondrial distribution revealed that mitochondrial


number and size were highest in the acid regions, whereas sites with fewer and smaller mitochondria were found at the alkaline bands (Fig. 2a; see also arrows in Fig.1f which indicate the border between an alkaline and an acid region). In mature cells, this pattern corresponded to that of the cortical microtubules which are longer and more abundant at acid

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bands (Wasteneys and Williamson 1992). In noncalcified, elongating cells, pH bands migrate slowly along the cell surface (Lucas and Smith 1973) and, accordingly, mitochondria changed their distribution over the course of a few days, while the overall density of cortical mitochondria remained more or less the same (Fig. 2b, c). Both pH banding and mitochondrial distribution remained stable in internodes where CaCO3 accumulated at the walls of alkaline bands (data not shown). Inhibition of photosynthesis and pH banding by several days of dark treatment caused a significant decrease in the density of cortical mitochondria and mostly a more uniform distribution (Fig. 2d and Table 2). Since chloroplast size and number in slowly elongating or mature internodes were relatively homogeneous, the heterogeneous distribution of cortical mitochondria caused significant differences in the proportion of chloroplasts to mitochondria. In acid regions of mature cells, up to 8 small mitochondria were present per chloroplast and these occupied an area equivalent to about 20% of the chloroplast area (Fig.1g and Table 1). In contrast, only up to 5 mitochondria per chloroplast, occupying an area of about 5% of the chloroplast area, were observed at alkaline bands (Fig.1h and Table 1). pH-dependent differences in the distribution of cortical mitochondria and microtubules were seen in all Chara and Nitella species investigated and are, therefore, independent of charasomes, plasma membrane invaginations which occur only in members of the genus Chara and whose positions have likewise been correlated with the pH pattern (Price et al. 1985, but see Bisson et al. 1991). Cortical mitochondria move along transverse tracks in rapidly elongating internodes

Fig. 2 a–d. pH-dependent distribution of cortical mitochondria in characean internodes. a Correlation between the positions of alkaline bands and the positions of cell regions with low densities of cortical mitochondria (correlation coefficient, 0.98). Total cell length was taken as 100%. b and c Relative mitochondrial densities in the cortex of an internodal cell immediately after isolation from the thallus (b) and after 4 days of light-dark treatment (c). Peaks correlate with the changing pH pattern visualized by staining with phenol red (alkaline regions in grey). d Inhibition of photosynthesis (and pH banding) by 3 days of dark treatment causes significantly lower mitochondrial densities and homogeneous distribution of mitochondria

The small mitochondria of very young cells moved back and forth between the streaming endoplasm and stationary cortex through the clefts between the chloroplast files. These mitochondria were observed to oscillate, apparently using Brownian movement to cross the chloroplast layer, or else to migrate more or less continuously along straight or curved tracks (Fig. 3a–c), consistent with the subcortical actin bundles extending towards or originating from the cell periphery at these locations (Fig. 3d) (Wasteneys et al. 1996). These directed movements continued at the plasma membrane but were frequently punctuated by periods of relative immobility or random, oscillating motion. The orientation of the mitochondria and the direction of cortical mitochondrial motion were generally transverse to the longitudinal axis of the internode and independent of the variable direction of the helical chloroplast files.

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Fig. 3 a–n. Dynamics of cortical mitochondria and cortical cytoskeleton in untreated internodes. a–c Time series (5 and 30 s intervals) showing two mitochondria (arrows) moving towards and along the plasma membrane in a young, slowly elongating internode. d Cortical actin filaments (and microtubules, compare with l) have a predominantly transverse orientation (arrows indicate extensions of subcortical actin bundles). e–j Two time series (10 to 50 s intervals) of giant mitochondria in the cortex of rapidly elongating cells. White arrows in e indicate fusing and dividing vermiform mitochondria. The black arrow indicates a mitochondrion that undergoes stretching transversely to the long axis of the cell. Amoeboid mitochondria (arrow and asterisk in h) form and retract branches in various directions. k The cortical actin cytoskeleton of these cells consists of a random meshwork or of transverse strands (compare with d; arrows indicate extensions of subcortical actin bundles). l Microtubules are always more or less transversely arranged and associated with mitochondrial fragments (dots). m and n Cortical microtubules have a slightly longitudinal orientation in a cell nearing growth completion (m) and a random orientation in a nonelongating internode (n). Only few mitochondrial fragments are associated with the microtubules. Bar in a: for a–c and e–j, 5 m; for d and k–n, 10 m

Both cortical actin filaments (Fig. 3d) and microtubules (compare Fig. 3l) in such cells had a distinctly transverse orientation. Transverse alignment and trajectories were also typical for the vermiform cortical mitochondria observed in rapidly elongating internodes. These mitochondria often migrated over considerable distances, just as the smaller mitochondria, or stopped movement after a few micrometers and then returned to their original position. Some of the vermiform mitochondria appeared to undergo transient stretching into longer and thinner shapes (Fig. 3e–g). Amoeba-like mitochondria were more randomly oriented and their motility was intimately related to changes in organelle shape (Fig. 3h–j). They mostly remained in place but slowly formed and retracted branches in different directions. The motility of giant mitochondria was frequently associated with fission activity, and the resultant fragments usually moved several micrometers before fusing with another mito-

chondrion. Concerted movements of organelles were often observed, even if they were widely spaced, and different mitochondria appeared to travel in line on the same track (compare Fig. 3a–c) (Grolig 1990, Lichtscheidl 1995, Lichtscheidl and Url 1995). Giant mitochondria frequently migrated through the chloroplast layer towards the endoplasm but not in the reverse direction, unlike small mitochondria which, as described above, moved in both directions. The cortical actin cytoskeleton of rapidly elongating cells consisted either of transverse strands (Fig. 3d) or of a random meshwork (Fig. 3k). Cortical microtubules, whose orientation is also transverse to the long axis, were associated with mitochondrial fragments originating from longer organelles that were not preserved during perfusion (Fig. 3l). Slowly elongating cells often contained vermiform or short mitochondria that were preferentially localized parallel to and between the chloroplast files. The orientation and motility of these mitochondria corresponded to that of the longitudinally


or slightly helically arranged cortical microtubules (Fig. 3m) or to the extensions of subcortical actin bundles (not shown). In other cells, however, cortical mitochondria were randomly arranged and more or less immobile, even if transverse or longitudinal microtubules were still present. The small mitochondria of nongrowing cells were exclusively localized between or at the periphery of chloroplasts, i.e., at those regions where the chloroplast surface was not tightly associated with the plasma membrane and created a pattern (Fig.1g–i) which was independent of the mostly randomly oriented actin filaments or microtubules (Fig. 3n). Motility events were exclusively Brownian or else directed movements across the chloroplast layer. The velocity of migrating cortical mitochondria and the speed at which branches were formed or retracted varied but never exceeded 1 m/s. Analysis of frequency distribution plots of maximum velocities measured for different mitochondria or trajectories revealed no distinct peaks, which would be indicative of alternative use of motor proteins working at different velocities (Kachar 1985). The slow motility of the cortical mitochondria contrasted with that of the fast but passively moving endoplasmic organelles, which reached rates of up to 80 m/s. The orientation of these endoplasmic mitochondria was more or less parallel to the direction of cytoplasmic streaming or that of the subcortical actin bundles, respectively (Fig. 4p). Giant vermiform mitochondria, if present, often became deformed and underwent snakelike turning and folding movements. Branched organelles were rarely observed. Actin filaments and microtubules control shape and motility of cortical mitochondria in elongating cells The comparison of mitochondrial trajectories with the orientation of cytoskeletal elements suggested that cortical mitochondria may interact both with microfilaments and microtubules. In order to distinguish between actin- and microtubule-dependent movements, inhibitor experiments were performed. Because of the great variations in shape, size, motility, and distribution of cortical mitochondria (outlined in the previous sections), the effects of drugs had to be analyzed in the same cell regions from which the control images had been made. As expected, the effects of cytoskeleton-disturbing drugs were most severe in the young, rapidly elongating internodes in which cortical mitochondrial dynamics were greatest. Treatment with CD at a concentration of 40 M arrested cytoplasmic streaming within 30 min, after which endoplasmic organelles only oscillated. Directed movement of mitochondria along linear tracks across the

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chloroplast layer stopped, but close to the cell membrane, small and vermiform organelles continued to move along transverse tracks, although at lower frequency. The most significant changes were observed with amoeba-like and disc-shaped mitochondria. These organelles became vermiform and some of them started to move transverse to the long axis of the cell (Fig. 4a–g) even when they had been immobile before treatment. The morphology of the subcortical actin bundles and that of cortical microtubules (Fig. 4h) remained unchanged upon treatment with CD, but the cortical actin filament meshwork reorganized into strands of distinct transverse orientation (Fig. 4i). Treatment with microtubule-depolymerizing drugs generated opposite changes in the morphology of giant cortical mitochondria. After 1 h of incubation in 10 M oryzalin or 50 M ICPC, the amoeboid mitochondria became more or less disc-shaped and relatively immobile (Fig. 4 j, k, o). Vermiform mitochondria of transverse orientation (compare Fig. 3e–g) became amoeboid, and these occasionally formed and retracted branches in various directions (Fig. 4l, m). The number of motility events (translocation and shape changes) decreased, but directed movements of small and vermiform mitochondria across the chloroplast layer and along the plasma membrane and slight changes in the morphology of amoeboid mitochondria (see above) were not completely inhibited. Cortical microtubules were absent (Fig. 4n) and the small-meshed, transverse cortical endoplasmic reticulum of untreated cells (compare Fig.1b, c) reorganized into a network with wide, random meshes (Fig. 4o). Neither the morphology of the actin cytoskeleton nor the cytoplasmic bulk streaming were affected, and elongate mitochondria were present in the streaming endoplasm, just as prior to treatment (Fig. 4 p). The microtubule-stabilizing drug taxol had no effect on the motility or shape of mitochondria (data not shown). Combined treatment with oryzalin and CD for 1 h eliminated all microtubules but preserved actin filaments in transverse orientation. This combined treatment inhibited all longdistance movements across the chloroplast layer and near the cell membrane. The giant mitochondria either retained their shape or became discoid. The CD- or oryzalin-induced effects on the cytoskeleton and the cortical mitochondria were reversible after 6 h recovery in AFW. Cells treated with oryzalin and CD at the same time could not recover (not shown). In mature cells, cytoskeletal inhibitors did not change the morphology of the small mitochondria and had only minor effects on their (reduced) motility. CD inhibited all long-distance movements along the plasma membrane and across the chloroplast layer. Treatment with oryzalin had no effect on mitochondrial motility.

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Fig. 4. Effect of actin- (a–i) and microtubule-disturbing drugs (j–p) on cortical mitochondria, cortical endoplasmic reticulum and cytoskeleton. a–d Amoeboid mitochondria become vermiform after 1 h of treatment with 40 M CD. The cortical endoplasmic reticulum is reorganized into a finemeshed net (d). Images a and b (DASPMI-stained) and c and d (DiOC6-stained) were taken from the same cell region before and after treatment. e–g Time series (12 s intervals) of an elongate mitochondrion probably moving along microtubules of opposite polarity in a CD-treated cell (compare with h). Note wiggling motion (f) between periods of continuous movement (e and g). h and i The orientation of microtubules is not affected by CD (h) but the cortical actin becomes reorganized into strands with a distinct transverse orientation (i). j and k Amoeboid mitochondria become discshaped and nearly immobile after depolymerization of microtubules by 10 M oryzalin. Images j and k were taken from the same cell region before and after 1 h of treatment. l and m Previously vermiform mitochondria (compare with Fig. 3e–g) become amoeboid and some of them are still able to change their shape (arrow; time interval, 7 s). n and o Cortical microtubules are absent (n) and the cortical endoplasmic reticulum is reorganized into a random, wide-meshed reticulum (o). Note also disc-shaped mitochondria in o. p In the streaming endoplasm, mitochondria are still elongate (arrows). Bar in a: for a, b, h–k, n, and o, 10 m; for c–g, l, and m, 5 m; for p, 15 m

Discussion This study shows that cortical mitochondria in characean internodes accumulate at regions with high rates of photosynthesis, suggesting an important role of the cytoskeleton in the differential positioning of mitochondria. The inhibitor experiments indicate that, in the cortex of characean internodes, mitochondria directly interact with the cytoskeleton, whereas they are passively transported in the rapidly streaming endoplasm. These mitochondria are likely to possess motor proteins for association with both microtubules and actin filaments. The motors can be used for either immobi-

lization or relocation and thereby facilitate the spatiotemporal distribution of mitochondria. Interactions between mitochondria and chloroplasts Mitochondrial metabolism is essential for photosynthetic carbon assimilation (Padmasree et al. 2002). Intimate contact between mitochondria and chloroplasts has often been described (Stickens and Verbelen 1996, Logan and Leaver 2000) and facilitates interorganellar exchange of metabolites. In characean internodes, endoplasmic mitochondria are in constant motion and mostly far away from the peripheral chloroplast layer.


Only cortical mitochondria, i.e., those located near the plasma membrane, may establish the close contact with chloroplasts required for efficient interaction. It thus appears reasonable to assume that mitochondria have different functions or metabolic activities depending on their location. In higher plants, the ratio of mitochondria to chloroplasts increases with the rate of photosynthesis (Wang et al. 2003). The present study shows a similar relationship in members of the Characeae, with the ratio of cortical mitochondrial area to chloroplast area decreasing significantly upon dark treatment, irrespective of the size of individual organelles. Reduced photosynthetic activity is probably also the reason for the low ratios observed in old internodal cells because these are usually shaded by the upper parts of the thallus. In members of the Characeae, photosynthesis-dependent variations in organelle distributions occurred not only under different light conditions but also within different regions of the same cell. In the light, internodes develop alternating bands of acid and alkaline regions (Spear et al. 1969). A feature of the acid bands is increased availability of CO2 and enhanced photosynthesis (Lucas and Smith 1973, Plieth et al. 1994, Bulychev and Vredenberg 2003). These acid regions contained not only higher numbers of mitochondria but also significantly larger organelles, irrespective of their shape (Table1). In contrast, cortical mitochondria were not only less abundant and smaller at alkaline regions but were also weakly stained, indicating lower metabolic activity (membrane potential; Bereiter-Hahn et al. 1983, Terasaki 1994). Mitochondrial metabolism is not only essential for the continuous supply of metabolites for photoassimilation, but oxidative electron transport also protects chloroplasts against the reactive oxygen species produced during photosynthesis (MacKenzie and McIntosh 1999, Padmasree et al. 2002). The differential positioning of mitochondria in the cortex could therefore also be an important strategy against photodamage. The chloroplasts of characean internodes are immobilized in the cortex and are not able to escape detrimental light intensities by relocation, as described for higher plants, ferns, mosses, and other algal cells (Wada et al. 2003). In order to compensate for the lack of chloroplast avoidance movements, the members of the Characeae likely evolved another mechanism and minimize photodamage by accumulating detoxifying mitochondria at photosynthetically active regions. The motility of cortical mitochondria described here suggests an important role for the cytoskeleton in optimizing the interaction between chloroplasts and mitochondria. Unfortunately, the experimental proof for this hypothesis requires long-term inhibitor studies which necessitate elaborate control studies to exclude nonspecific side effects.

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Cytoskeleton-dependent motility and morphology of cortical mitochondria A recent study demonstrated that in higher-plant cells, mitochondria move along actin filaments but that their immobilization was microtubule-dependent (Van Gestel et al. 2002). A similar use of the cytoskeleton has been reported for dictyosomes (Nebenführ et al. 1999) and chloroplasts (Kwok and Hanson 2003). In the characean cells, both cytoskeletal systems appear to be involved in mitochondrial transport and immobilization. Whether actin filaments are used as tracks for translocation or as a matrix for anchorage appears to depend on the size of the mitochondrion and on the number of motor proteins on its surface. Small mitochondria, which – because of their small size – may only possess one actinbinding motor protein (or one actin-binding motor protein which is sterically able to interact with actin), were found to use actin filaments or actin bundles for active migration through the chloroplast layer and long-distance movement parallel to the cell membrane. Giant mitochondria mostly remained in place but formed branches in different directions that disappeared upon treatment with CD. These observations suggest that larger mitochondria interact with the cortical actin filament array via several motor proteins and that a combination of forces is at least partly responsible for their amoeboid shape and motile behavior. A similar mechanism appears to determine whether interaction with microtubules results in translocation or immobilization. Small mitochondria moved over several micrometers, not only in untreated cells but also in cytochalasin-treated cells, suggesting that these mitochondria migrated along microtubules. Many of the larger mitochondria in CD-treated cells were immobilized along cortical microtubules and their vermiform shape indicated the presence of several microtubulebinding (motor) proteins, which could act to anchor the mitochondria through attachment to adjacent microtubules of opposite polarity. This elongate, unbranched morphology was also consistent with the parallel orientation of cortical microtubules. After depolymerization of microtubules by oryzalin or ICPC, amoeboid and vermiform mitochondria became immobile and nearly platelike, with few, indistinct protrusions. The results of these experiments and those of the CD treatments suggest that the branched morphology and the motility or immobilization of giant mitochondria in untreated, elongating cells are the result of simultaneous interactions of mitochondria with microtubules and actin filaments, in which microtubules exert strong forces transverse to the long axis of the cells and actin filaments “pull” in various directions. A similar balance of forces has been suggested for transverse nuclear positioning in algal (Grolig 1998) or fungal cells (Steinberg

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1998). Interaction of cortical mitochondria with both actin filaments and microtubules may also be important for their displacement parallel to the longitudinal axis of the cell, which perhaps is required for the accumulation of cortical mitochondria at the acidic regions (see Fig. 5 and below). The fact that mitochondria in characean internodes may move along microtubules as well as along actin filaments could be an ancient character, because it is shared by chloroplasts of the coenocytic alga Bryopsis spp. (Menzel and Schliwa 1986) and by moss chloroplasts which are also able to migrate both along microtubules and microfilaments (Sato et al. 2001). The identity of mitochondrial motors in the characean cells remains to be determined. There is little doubt that the molecular motor for actin-dependent mitochondrial motility is a myosin (Simon et al. 1995, Zhao et al. 1999). In the subcortex and in isolated cytoplasm, mitochondria move along actin filaments at rates of several micrometers per second. The velocity of actin-dependent motility in the cortex is less than 1 m/s, even along the thicker bundles, suggesting that the conditions in the cortex reduce the activity of the myosin motor(s). The motor protein for microtubule-dependent motility is probably a kinesin because kinesin-related proteins with a mitochondrial targeting signal have been found in plants (Itoh et al. 2001), and a kinesin-binding protein involved in the correct distribution of mitochondria in plant cells has recently been identified (Logan et al. 2003). A kinesin-like protein from characean internodes has been identified on Western blots, and immunofluorescence images indicate that it is responsible for microtubule-dependent dynamics of the cortical endoplasmic reticulum (I. Foissner and G. O. Wasteneys unpubl.). A kinesin-like calmodulin-binding protein which associates with cortical microtubules and whose level decreases during cotton fiber development has been described recently (Preuss et al. 2003). The loss of microtubule dependency of cortical endoplasmic reticulum and mitochondria in mature internodes likewise indicates depletion or inactivation of the motor protein because these cells still contain microtubules. Although there are some indications for endogenous mitochondrial actin (Lo et al. 2003), our observations suggest that the motility and part of the morphology of cortical mitochondria in characean internodes are due to the interaction of outer-membrane-bound motor proteins with “external” actin filaments and/or microtubules, in agreement with findings published by other authors (review by Grolig 2004, Steinberg 2000, Westermann and Prokisch 2002). Two observations supporting the argument for an involvement of external cytoskeletal elements in mitochondrial dynamics are the transverse orientation of mito-


chondria in elongating cells and the concerted movement of more widely spaced organelles, neither of which can be explained by the activity of intraorganellar proteins. These observations on the rather slowly migrating cortical mitochondria of characean cells, however, do not rule out a possible contribution of endogenous cytoskeletal elements in the highly dynamic mitochondria of other organisms (Jarosch 1978, Logan and Leaver 2000). Does the cytoskeleton mediate the pH-dependent distribution of cortical mitochondria? Mitochondrial densities were high at the photosynthetically more active acid bands and low at alkaline bands. This accumulation may be caused by differential growth of organelles, migration from the endoplasm and/or retention in the cortex, relocation within the cortex, or a combination of these possibilities. But even if enhanced mitochondrial growth is the reason for the higher mitochondrial densities at acid regions, it must be coupled with increased migration into the endoplasm when acid regions eventually become alkaline, and this points to a central role for the cytoskeleton in mitochondrial distribution. Active migration of mitochondria through the chloroplast layer involves the participation of actin filaments or actin bundles and, as previously discussed, immobilization may occur both along the cortical actin meshwork and the microtubules. Translocation in the longitudinal direction, i.e., along the axis in which the pH bands migrate, is less easy to explain. Neither actin filaments nor microtubules possess the required orientation except for a very short period at the end of the elongation phase, when microtubules are longitudinally arranged but probably no longer used as tracks. A characteristic feature of the acid bands is that they contain significantly more microtubules than the alkaline bands (Wasteneys and Williamson 1992). If mitochondria possess protein(s) which allow them to bind along microtubules, they will accumulate at microtubule-rich regions even if they are randomly distributed by the actin filament meshwork. A possible mechanism that explains migration of giant mitochondria perpendicular to transverse microtubules, with the aid of an actin meshwork and taking account of microtubule polymerization and depolymerization during migration of pH bands, is shown in Fig. 5a–c. It is also possible that the rapid turnover of cortical microtubules (Wasteneys et al. 1993) in combination with the appropriate motor protein (Lombillo et al. 1995) contributes to mitochondrial movement. Alternatively, giant mitochondria may not migrate as a whole but undergo fission, and their fragments could travel towards, accumulate, and fuse at a newly formed acid region.


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bands or from the extent of photosynthesis. Possible candidates for signal transduction are calcium ions or protons, whose concentrations probably differ according to the pattern of pH banding (Smith 1984) and which are taken up by chloroplasts in the light (Plieth 2001). Both ions have profound effects on the organization of the cytoskeleton and the activity of motor proteins (Staiger et al. 2000, Holdaway-Clarke and Hepler 2003, Wasteneys and Galway 2003), and the interdependence of calcium ions and protons is well known (Felle 2001). Giant mitochondria are only formed in elongating cells grown under low light intensity

Fig. 5 a–e. Schematic representation of mitochondrial shape and motility and the effects of inhibitors. a–c Proposed mechanism for migration of a giant mitochondrion perpendicular to transverse microtubules. The amoeboid shape of the mitochondrion is due to interaction with both transverse microtubules (thick lines) and randomly oriented actin filaments (thin lines). Depolymerization of microtubules (grey) in the alkaline region (upper parts of the images) results in partial loss of tension (anchorage) and net-translocation towards the acid region (lower parts of the images) because the pulling forces exerted by the actin filaments are not sufficient to keep the organelle in place. The activity of microtubuleassociated motors at acid regions makes acto-myosin-dependent backward movement towards alkaline regions where microtubules are less abundant unlikely. Mitochondria will therefore accumulate at microtubule-rich acid regions. Arrows indicate the relevant forces for the positioning and displacement of mitochondria. d Depolymerization of microtubules results in a disc-shaped organelle because the mitochondrion is isotropically stretched by randomly oriented actin filaments. e Inhibition of actin–myosin interactions by CD causes alignment of giant mitochondria along microtubules. Binding to microtubules of opposite polarity results in immobilization (upper mitochondrion), binding to a single microtubule or to several microtubules of the same polarity eventually causes translocation (lower mitochondrion)

Irrespective of how cortical mitochondria are redistributed, differences in photosynthetic activity must be “sensed” by these organelles and the cytoskeleton. They may respond to changes in the ionic environment that result from the different transport properties of acid and alkaline

The dependency of mitochondrial shape on cell status has been described for various organisms (Stickens and Verbelen 1996). In characean internodal cells, giant mitochondria have been found in thalli grown under low light intensities, and inhibitor experiments suggest that they form under conditions of low rates of photosynthesis (Jarosch 1961; Foissner 1981, 1983). In higher-plant cells, small mitochondria fuse with each other when the oxygen pressure is low, a situation comparable to conditions where the rate of photosynthesis is reduced (Vartapetian et al. 1977, Ramonell et al. 2001, Van Gestel and Verbelen 2002). In Vaucheria sp. and Oedogonium sp., however, fusion occurs under conditions of high light intensities (Jarosch 1978), and in animal cells a wide range of metabolic conditions causes the formation of giant mitochondria (Bereiter-Hahn and Vöth 1994). These observations, and the fact that giant mitochondria were present in elongating cells only, suggest a more complex situation. A more reliable “mitochondrial” indicator for the extent of photosynthesis in characean and other cells is the ratio between mitochondrial area (volume) and chloroplast area (volume) which is independent of mitochondrial shape (see above and Wang et al. 2003). Although it cannot be excluded that giant mitochondria form by inhibition of fragmentation, the frequent observation of fission and fusion events in characean internodes, as well as the results of other studies (see above and, e.g., BereiterHahn and Vöth [1994], Westermann [2002]), suggest that their formation is due to fusion of smaller organelles, and the capability for fusion is obviously higher in elongating cells. The probability of fusion should be enhanced at regions with high mitochondrial densities. This would explain the obvious discrepancy of the higher number of giant mitochondria at the photosynthetically acid bands, although giant mitochondria – at least in members of the Characeae – are formed when the rate of photosynthesis is low.

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The morphology of characean giant mitochondria appears to be primarily due to intrinsic factors, because vermiform organelles with platelike extensions or few branches were present in the flowing endoplasm and, hence, were independent of an external cytoskeleton. In the cortex of growing cells, however, the shape of these organelles seems to be additionally controlled by the action of motor proteins (Fuchs et al. 2002). While interaction with the parallel microtubules favored an elongate, vermiform shape, interaction with the actin filament meshwork resulted in a more platelike morphology (Fig. 5d, e). Highly branched amoeba-like organelles were only seen in the cortex and their shape seemed to depend on both cytoskeletal systems. The shape of the vermiform but randomly oriented mitochondria occasionally observed in cells nearing growth completion (Fig.1g) did not change upon treatment with oryzalin, suggesting that the mitochondrial shape in the mature internodes is independent of an external cytoskeleton. The identification of mitochondrial motor proteins in characean cells, their number, distribution and regulation are the subject of present studies. The coordination between these motors that interact with an “external” cytoskeleton and intraorganellar proteins that are responsible for mitochondrial fusion and fission will be a fascinating research theme for the future. Acknowledgments This work was partially supported by a grant from the OeNB (6675). I am grateful to F. Grolig (Marburg) and G. O. Wasteneys (Vancouver) for reading the manuscript and valuable discussions.

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