J. Cell Sd. 61, 87-105 (1983) 87 Printed in Great Britain © The Company of Biologists Limited 1983 EFFECT OF MICROTUBULES AND INTERMEDIATE FILAMENTS ON MITOCHONDRIAL DISTRIBUTION IAN C. SUMMERHAYES, DAVID WONG AND LAN BO CHEN Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, 44 Binney Street, Boston, MA 02115, U.SA. SUMMARY The laser dye rhodamine 123 specifically stains mitochondria in living cells and facilitates the observation of changes in mitochondrial distribution in single cells under a variety of experimental conditions. Visualization of mitochondria in a number of cell lines followed by processing of these cells to study different cytoskeletal elements by indirect immunofluorescence, revealed good but not absolute correlation between mitochondria and microtubules or intermediate filaments. Mitochon- dria and microfilament distribution within the same cell did not show such a correlation. On the basis of observations made by various experimental approaches, we suggest that mitochondrial distribution is under the strong influence of the two systems, microtubules and intermediate fila- ments. Neither plays an absolute role but one seems able to play a more dominant role in the absence of the other. INTRODUCTION Mitochondria are some of the most extensively studied organelles and have been characterized biochemically; their major role being in energy production essential for cell survival and proliferation (Lehninger, 1964; Tandler & Hoppel, 1972; Racker, 1976; Hinckle & McCarty, 1978). Earlier investigations have described the intracellular distribution of mitochondria and observed the high motility and con- siderable morphological heterogeneity displayed by these organelles (Lewis & Lewis, 1914; Palade, 1953; Gey, Shapres & Borysko, 1954; Tobioka & Biesele, 1956; Bier- ling, 1954; Mann, 1975); however, the mechanisms that determine the location and movement of such organelles are little understood. Electron-microscopic studies in various organisms, involving different organelles, have implicated microtubules as the directing influence in both cytoplasmic organiza- tion (Tilney & Porter, 1965), and organelle movement and distribution (Ledbetter & Porter, 1963; Whaley & Mollenhauer, 1963; Rudzinska, 1965; Bikle, Tilney & Por- ter, 1966; Holmes & Choppin, 1968; Murphy & Tilney, 1974; Smith, Jarlfors & Cameron, 1975; Smith, Jarlfors & Cayer, 1977). In all these studies the presence of microtubules has been demonstrated in close proximity to the organelle of interest, and movement of such intracellular packages has been observed parallel to the long axis of the microtubules. Physical connections between organelles and microtubules have been resolved in a few instances; for example, that of chromosome kinetochores with the spindle apparatus (Roth, Wilson & Chakraborty, 1966; Barnicot, 1966) and that of mitochondria in neuronal axons, by electron-dense cross-bridges between the 88 /. C. Summerhayes, D. Wong and L. B. Chen structures (Smith et al. 1975, 1977). Using anti-mitotic drugs such as colchicine, disassembly of microtubules can be shown to have profound effects on organelle movement (Holmes & Choppin, 1968) and distribution (Heggeness, Simon & Singer, 1978; Johnson, Walsh & Chen, 1980) within different cell types, providing strong evidence for the influence of microtubules on organelle distribution. With the recent discovery that rhodamine 123 and other permeant cationic fluores- cent probes can be utilized for staining mitochondria in living cells (Johnson et al. 1980; Johnson, Walsh, Bockus & Chen, 1981; Johnson, Summerhayes & Chen, 1982), we have a unique opportunity to observe the distribution of this organelle without complicating fixation factors. Since the laser dye rhodamine 123 is found to be non-toxic to fibroblasts at low concentrations, we have been able to observe the mitochondrial distribution in a single cell prior to, during and after recovery from different drug treatments. In this paper we describe a possible involvement of inter- mediate filaments (see review by Lazarides, 1980) and microtubules in mitochondrial distribution. MATERIALS AND METHODS Mitochondrial staining The laser dye rhodamine 123 (Eastman) was dissolved in dimethyl sulphoxide at a concentration of 1 mg/ml and subsequently diluted to 10/xg/ml in Dulbecco's modified Eagle's medium. Cells on coverslips were incubated with rhodamine 123 (10^g/ml) for lOminat 37 °C, rinsed in medium and mounted in medium supplemented with 5 % calf serum on a live-cell observation chamber (Johnson et al. 1980). Stained cells were viewed by epifluorescent illumination at 485 nm on a Zeiss photomicroscope III, and photographs were taken with a 40X Planapo objective lens using Tri-X film. Immunofluorescence staining Three different preparative fixation techniques were used, depending on the filament system to be stained. Microtubules. Cells growing on coverslips were washed for 30 s at room temperature with stabilization buffer (01 M-piperazine-N,./V'-bis-2-ethanesulphonic acid, sodium salt adjusted to pH6-9 with KOH, 1 mM-ethylene glycol bisQS-aminoethyl ether)-AW-tetraacetic acid, 2-5 mM- GTP and 4 % polyethylene glycol 6000) and then incubated for 5 min at room temperature in the same buffer containing 05 % Triton X-100 (Sigma). After this treatment cells were washed twice with stabilization buffer and then fixed in cold methanol (-20°C) for 5 min (Osborn & Weber, 1977). Intermediate filaments. Coverslips were rinsed in phosphate-buffered saline (PBS) and fixed for 5 min in cold methanol ( — 20°C), then transferred back to PBS and washed in three changes during a 10 min period. Microfilaments. Cells were fixed in 3-7 % formaldehyde for 20 min, washed in PBS for 2-5 min and then permeabilized in cold acetone ( — 20°C) for 5 min. Cells were rinsed thoroughly in PBS before processing for immunofluorescence. After fixation cells were processed in the same manner. All cells were washed thoroughly in PBS after fixation, drained, overlaid with the appropriate cytoskeletal antiserum and incubated for 30 min at 37 °C in a humidified chamber. Coverslips were then rinsed thoroughly in PBS and overlaid with rhodamine-conjugated goat anti-rabbit immunoglobulin G (IgG; Miles) at a dilution of 1/10 and incubated for a further 30min at 37°C. After rinsing again, coverslips were mounted in gelatin/glycerol. Antibodies to cytoskeletal elements were generous gifts from Dr T. T. Sun (keratin) (Sun & Green, 1978), Dr F. Solomon (tubulin) (Solomon, Magendantz & Salzman, Mitochondrial distribution and cytoskeleton 89 1979), Dr K. Burridge (actin) (Burridge, 1976) and Dr R. O. Hynes (vimentin) (Hynes & Destree, 1978). Microinjection Mouse monoclonal antibody (JLB-7) (Lin, 1981), generously provided by Dr Jim J. C. Lin of Cold Spring Harbor Laboratory, was microinjected into cells grown on glass coverslips with a glass capillary needle drawn out to a tip of less than 0-5 jUrn in diameter by a Narishige PN-3 puller (Narishige Scientific Instrument, Japan). A Leitz micromanipulator equipped with a vacuum and pressure device was used for micromanipulation. A Leitz Diavert phase-contrast microscope equipped with an RCA Newvicon camera, a Panasonic television monitor and a Panasonic video recorder was used for visualization of cells during the course of the microinjection process. All procedures were essentially as described by Graessman & Graessman (1976), Feramisco (1979) and Johnson et al. (1982). Before fixation for immunofluorescence, microinjected cells were stained with rhodamine 123 and photographed as described above. RESULTS Live gerbil fibroma cells (CCL146) incubated with rhodamine 123 (lO^ig/ml) for 10 min and viewed using epifluorescent microscopy, display a discontinuous filamen- tous array of cytoplasmic structures previously shown to be mitochondria (Johnson et al. 1980, 1981). All mitochondria of the cell take up the dye and show uniform fluorescent intensity within a single cell. Observation of the mitochondria in a par- ticular cell and relocation of the same cell after processing for immunofluorescence was used for the study of mitochondrial distribution and its correlation with different cytoskeletal elements. Mitochondrial distribution and cytoskeletal elements Staining of gerbil fibroma cells with rhodamine 123 revealed filamentous mitochon- dria throughout the cytoplasm (Figs 1A, 2A, 3A), with variable morphology and distribution within and between cells. The non-uniform distribution of this organelle throughout the cytoplasm, where mitochondria often appeared in parallel array (Figs 1A, 2A, 3A), suggested a directive element present within the cellular environment. Photographic visualization of mitochondria followed by processing of the cells for indirect immunofluorescence, using antibodies to different cytoskeletal elements, revealed a close correlation between the mitochondrial distribution and microtubules within the same cell (Fig. 1A, B), as has been previously described by Heggeness et al. (1978). Cells studied in the same manner and stained for vimentin (the mesen- chymal intermediate filament type) also showed a strong correlation in every cell, with mitochondria distributed along major intermediate filament pathways within the cell (Fig. 2A, B). In both cases the distribution and orientation of mitochondria within a single cell corresponded with the major filamentous networks visible after staining with tubulin or vimentin antibody, suggesting a possible channelling of mitochondria along these filaments. In contrast