J. Cell Sd. 61, 87-105 (1983) 87Printed 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 CHENDana-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 theobservation of changes in mitochondrial distribution in single cells under a variety of experimentalconditions. Visualization of mitochondria in a number of cell lines followed by processing of thesecells to study different cytoskeletal elements by indirect immunofluorescence, revealed good but notabsolute correlation between mitochondria and microtubules or intermediate filaments. Mitochon-dria and microfilament distribution within the same cell did not show such a correlation. On thebasis of observations made by various experimental approaches, we suggest that mitochondrialdistribution 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 absenceof the other.

INTRODUCTION

Mitochondria are some of the most extensively studied organelles and have beencharacterized biochemically; their major role being in energy production essential forcell survival and proliferation (Lehninger, 1964; Tandler & Hoppel, 1972; Racker,1976; Hinckle & McCarty, 1978). Earlier investigations have described theintracellular 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 andmovement 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 ofmicrotubules has been demonstrated in close proximity to the organelle of interest,and movement of such intracellular packages has been observed parallel to the longaxis of the microtubules. Physical connections between organelles and microtubuleshave been resolved in a few instances; for example, that of chromosome kinetochoreswith the spindle apparatus (Roth, Wilson & Chakraborty, 1966; Barnicot, 1966) andthat 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 concentrationof 1 mg/ml and subsequently diluted to 10/xg/ml in Dulbecco's modified Eagle's medium. Cells oncoverslips were incubated with rhodamine 123 (10^g/ml) for lOminat 37 °C, rinsed in medium andmounted in medium supplemented with 5 % calf serum on a live-cell observation chamber (Johnsonet al. 1980). Stained cells were viewed by epifluorescent illumination at 485 nm on a Zeissphotomicroscope III, and photographs were taken with a 40X Planapo objective lens using Tri-Xfilm.

Immunofluorescence staining

Three different preparative fixation techniques were used, depending on the filament system tobe stained.

Microtubules. Cells growing on coverslips were washed for 30 s at room temperature withstabilization buffer (01 M-piperazine-N,./V'-bis-2-ethanesulphonic acid, sodium salt adjusted topH6-9 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 thesame buffer containing 0 5 % Triton X-100 (Sigma). After this treatment cells were washed twicewith 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 for5 min in cold methanol ( — 20°C), then transferred back to PBS and washed in three changes duringa 10 min period.

Microfilaments. Cells were fixed in 3-7 % formaldehyde for 20 min, washed in PBS for 2-5 minand then permeabilized in cold acetone ( — 20°C) for 5 min. Cells were rinsed thoroughly in PBSbefore processing for immunofluorescence.

After fixation cells were processed in the same manner. All cells were washed thoroughly in PBSafter fixation, drained, overlaid with the appropriate cytoskeletal antiserum and incubated for30 min at 37 °C in a humidified chamber. Coverslips were then rinsed thoroughly in PBS andoverlaid with rhodamine-conjugated goat anti-rabbit immunoglobulin G (IgG; Miles) at a dilutionof 1/10 and incubated for a further 30min at 37°C. After rinsing again, coverslips were mountedin 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).

MicroinjectionMouse 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 glasscapillary 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 andpressure device was used for micromanipulation. A Leitz Diavert phase-contrast microscopeequipped with an RCA Newvicon camera, a Panasonic television monitor and a Panasonic videorecorder was used for visualization of cells during the course of the microinjection process. Allprocedures were essentially as described by Graessman & Graessman (1976), Feramisco (1979) andJohnson et al. (1982). Before fixation for immunofluorescence, microinjected cells were stainedwith rhodamine 123 and photographed as described above.

RESULTS

Live gerbil fibroma cells (CCL146) incubated with rhodamine 123 (lO^ig/ml) for10 min and viewed using epifluorescent microscopy, display a discontinuous filamen-tous array of cytoplasmic structures previously shown to be mitochondria (Johnsonet al. 1980, 1981). All mitochondria of the cell take up the dye and show uniformfluorescent intensity within a single cell. Observation of the mitochondria in a par-ticular cell and relocation of the same cell after processing for immunofluorescencewas used for the study of mitochondrial distribution and its correlation with differentcytoskeletal 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 anddistribution within and between cells. The non-uniform distribution of this organellethroughout the cytoplasm, where mitochondria often appeared in parallel array (Figs1A, 2A, 3A), suggested a directive element present within the cellular environment.Photographic visualization of mitochondria followed by processing of the cells forindirect immunofluorescence, using antibodies to different cytoskeletal elements,revealed a close correlation between the mitochondrial distribution and microtubuleswithin the same cell (Fig. 1A, B), as has been previously described by Heggeness etal. (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, withmitochondria distributed along major intermediate filament pathways within the cell(Fig. 2A, B). In both cases the distribution and orientation of mitochondria within asingle cell corresponded with the major filamentous networks visible after stainingwith tubulin or vimentin antibody, suggesting a possible channelling of mitochondriaalong these filaments. In contrast to these observations, processing of cells to visualizemitochondria and the cytoplasmic microfilament system recognized by actin antibodyrevealed no consistent correlation with respect to distribution. Major mitochondrialchannels often displayed actin filaments running along their length; however, other

/. C. Summerhayes, D. Wong and L. B. Chen

Fig. 1. Mitochondrial staining in live gerbil fibroma cell (A) and the correspondingmicrotubule (tubulin) staining in the same cell (B). Bar, 25 ̂ m.

Mitochondrial distribution and cytoskeleton 91

Fig. 2. Mitochondrial staining in live gerbil fibroma cell (A) and the corresponding inter-mediate filament (vimentin) staining in the same cell (B). Bar, 30/im.

Fig. 3. Mitochondrial staining in a portion of live gerbil fibroma cell (A) and the corres-ponding microfilament (actin) staining in the same cell (B). Bar,

Mitochondrial distribution and cytoskeleton 93

areas within the same cell showed mitochondria and microfilaments orientated at 90 °to one another (Fig. 3A, B), suggesting the absence of actin involvement in mitochon-drial distribution.

Mitochondrial distribution and the effects of colchicine and eolcemid

Colchicine (10//g/ml) and eolcemid (1 ^zg/ml), which depolymerize microtubulesbut not intermediate filaments (Goldman, 1971; Goldman & Knipe, 1972), enabledus to study the relative importance of microtubules and intermediate filaments inmitochondrial distribution. Staining of cells before and after a 3 h incubation periodwith colchicine, when microtubules were shown to be absent, demonstrates the effectof microtubule disruption on mitochondrial distribution. At this time in all cellsstudied mitochondria lost their extended filamentous form, appearing more wavy incontour. Staining of cells with tubulin antibody, after exposure to colchicine, revealeda complete absence of filamentous staining in most cells. Vimentin staining in cellsafter colchicine treatment for 3 h showed retraction of intermediate filaments fromperipheral cell regions with a bundling of fibres (Fig. 4B) (Goldman, 1971; Goldman& Knipe, 1972) en route to the perinuclear region to form the characteristic nuclearwhorl. Superimposing negatives of mitochondrial distribution and vimentin fila-ments in the same cell after colchicine treatment showed a strong correlation betweenthe distribution of these two cytoplasmic elements (Fig. 4A, B), although themitochondrial orientation was not always parallel to the intermediate filaments as wasobserved prior to drug treatment. Prolonged exposure of cells to colchicine (24-48 h)evoked a gradual retraction of vimentin filaments from peripheral regions of the cell,resulting in a coiled bundle of filaments surrounding the nucleus (Fig. 4D). Studiesof mitochondrial distribution during this period revealed a temporally similar with-drawal (Fig. 4c) with mitochondria and vimentin filaments displaying a close correla-tion in distribution (Fig. 4c, D). Similar events were observed with cells exposed toeolcemid. However, none of the above effects were detected in lumicolchicine-treatedcells.

Mitochondrial distribution after the removal of eolcemid

Since interpretation of the previous data could possibly be accounted for by coin-cident distribution of vimentin and mitochondria due to entrapment in cytoplasmicareas after the removal of microtubules, we studied the distribution of mitochondriaafter the removal of eolcemid before extensive regrowth of microtubules. Cells ex-posed to eolcemid for 90min were stained with rhodamine 123, photographed, andthen returned to culture in the absence of the drug. Coverslips were restained with therhodamine dye at different times after eolcemid removal and then processed forindirect immunofluorescent studies. As before, the same cell was relocated at each ofthe different stages. In all colcemid-treated cells mitochondria appeared more com-pacted and closer to the nucleus (Figs 5A, B, 6A), as seen previously in colchicine-exposed cells. Staining of cells 30min after recovery from eolcemid showed fewchanges in mitochondrial distribution as assessed by fluorescence microscopy. Sixtyminutes after removal of the drug, mitochondria had begun to be relocated in the cell

/. C. Summerhayes, D. Wong and L. B. Chen

Fig. 4. Mitochondrial staining in live gerbil fibroma cells exposed to colchicine for 3 h(A) and 24 h (c). B and D. Vimentin staining of cells in A and c, respectively. Bar, 30 fim.

(Fig. 5B), with little microtubule regrowth (Fig. 5c). The small number of micro-tubules shown in Fig. 5c appear to be colcemid-resistant. Mitochondria were oftenlocalized in areas where no microtubules were found. At 90 min after the removal ofcolcemid, similar results were obtained (Fig. 5D, E, F). Similar studies 90 min afterrecovery from colcemid (Fig. 6A, B), followed by immunofluorescent staining tovisualize vimentin filaments, revealed a good correlation between the redistributedmitochondrial pattern and vimentin filaments (Fig. 6B, C). However, mitochondriaat this stage still appeared wavy in contour, lacking the more extended appearance ofmitochondria in untreated cells. It appears that full recovery of the microtubulenetwork is essential for the reappearance of normal mitochondrial morphology.

Mitochondrial distribution and cytoskeleton 95

Fig. 5. Mitochondrial staining in live colchicine-treated gerbil fibroma cells (A, D) and thesame cells 60 min (B) and 90 min (E) after the removal of colchicine. Microtubule stainingof cells in B and E is shown in c and F, respectively. Bar, 30jUm.

96 /. C. Summerhayes, D. Wong and L. B. Chen

Fig. 6. Mitochondrial staining in live colchicine-treated gerbil fibroma cell (A) and thesame cell 90 min after the removal of colchicine (B). Vimentin staining of the same cell isshown in c. Bar, 30 pirn.

Mitochondrial distribution and cytoskeleton 97

Mitochondrial distribution and disruption of vimentin filaments

Although the above experiments suggest that, in the absence of an intact micro-tubule system, mitochondrial distribution seems to be correlated with vimentin fila-ment distribution, it is still not known how mitochondria would behave if micro-tubules were kept intact but vimentin distribution were altered. Until recently it hasnot been possible to alter vimentin organization without affecting microtubules. Arecent report by Sharpe, Chen, Murphy & Fields (1980) describes one of the methodsin monkey kidney CV-1 cells, where vimentin filament distribution was disruptedafter protein synthesis was inhibited by cycloheximide treatment. The other method,involving microinjection of antibody against vimentin-filament-associated protein hasbeen described by Lin & Feramisco (1981). Microtubule and microfilament arrange-ments appeared unaffected by either treatment, as assessed by indirect im-munofluorescence.

CV-1 cells show the same correlation of mitochondrial distribution with micro-tubules or intermediate filaments as observed in the gerbil fibroma cell line. Exposureof these cells to cycloheximide (lOjUg/ml) blocks 80% of protein synthesis within5 min and results in disruption of the mitochondrial distribution after 3—5 h, when thevimentin filament pattern appears disorganized. Mitochondria in the cycloheximide-treated cells aggregate around the nucleus and often appear isolated at the cell peri-phery (Fig. 7B), lacking their usual orientation (Fig. 7A). Staining of vimentin in thesame cell after cycloheximide treatment reveals disruption of intermediate filamentsinto disorganized arrays (Fig. 7c), but not depolymerization of the vimentin fila-ments. Staining of cells with tubulin antibodies, after the same period of cyclo-heximide exposure, shows a complete, extended, microtubular network apparentlyunaffected by drug treatment and similar to those reported by Sharpe et al. (1980).Recovery from these treatments results in reorganization of both mitochondria andvimentin filaments after 24 h.

Fig. 7. Mitochondrial staining in live CV-1 cell before (A) and after cycloheximide treat-ment (B). Vimentin staining of the same cell is shown in c. Bar, 30|Um.

98 /. C. Summerhayes, D. Wong and L. B. Chen

Gerbil fibroma cells (CCL146) were microinjected with monoclonal antibody(JLB7) against vimentin-filament-associated protein (Lin, 1981). Fig. 8 shows thatvimentin filaments gradually retract toward the perinuclear region upon micro-injection, as described by Lin & Feramisco (1981). However, mitochondrialdistribution is clearly unaffected by the altered vimentin organization. Unlike thosein the cycloheximide experiment, these results suggest that an intact intermediatefilament system is not an absolute requirement for normal mitochondrial distribution.

Fig. 8. Microinjection of gerbil fibroma cell with monoclonal antibody JBL7. Mitochon-dria staining (A) and vimentin staining of the same cell (B). Bar, 30/xm.

Mitochondria! distribution and cytoskeleton 99

Mitochondrial distribution and intermediate filaments in epithelial cellsSince intermediate filaments seem to be involved in mitochondrial distribution, it

is of interest to examine the established epithelial cell lines in which the presence oftwo intermediate filament systems has been shown (Franke et al. 1979a; Franke,Schmid, Weber & Osborn, 19796). Keratin has been shown to be the major inter-mediate filament system of epithelial cells in culture (Franke et al. 19786; Sun &Green, 1978), but is absent from all the mesenchymal cells studied. In contrast, theoccurrence of both the epithelial specific intermediate filament, keratin, and themesenchymal-derived filament, vimentin, has been reported in a number ofestablished epithelial cell lines. In some cases staining with antibody to either inter-mediate filament type reveals an extensive filamentous array (Franke et al. 1979a),facilitating the study of mitochondria and these cytoskeletal elements. The kangarookidney epithelial cell line, PtK2, is one such line and was used in this study.

Incubation of PtK2 cells with rhodamine 123 revealed a mitochondrial distributiondifferent from that observed with the gerbil cell line, as mitochondria were lessfilamentous, with more even distribution throughout the cytoplasm (Fig. 9A, C).Staining of cells with the mitochondrial dye, followed by processing of these cells toobserve vimentin intermediate filaments, revealed a less clear-cut correlation of thesetwo cytoplasmic elements within the same cell, compared with observations onmesenchymal cells (Fig. 9c, D). Similar experiments with these cells, to look atkeratin distribution, revealed an elaborate array of keratin filaments throughout thecell, making interpretation difficult (Fig. 9A, B). Overlay of negatives of mitochon-drial pattern and the corresponding keratin filaments in the cell showed mitochondriapresent with keratin in all regions.

Effect of colchicine on mitochondrial distribution in PtK2 cellsIn order to differentiate between the two intermediate filament systems expressed

in PtK2 cells we exposed cells to colchicine, as it has been previously reported thatvimentin will retract to the perinuclear region as in mesenchymal cells, but keratin willbe little affected (Sun & Green, 1978; Franke et al. 19796). A 24-h exposure of cellsto colchicine led to a more granular mitochondrial form accompanied by retraction ofcells. Corresponding staining of retracted PtK2 cells for keratin showed keratin fila-ments present with mitochondria in all regions. In contrast, vimentin filaments inPtK2 cells clustered in the perinuclear region after 24 h treatment with colchicine(Fig. 10B) and did not appear to be correlated with mitochondrial distribution (Fig.10A). The differential fluorescent intensities among different PtK2 cells seen in Fig.10A reflected the different dye accumulation in mitochondria among these cells at thetime of staining, as reported previously (Johnson et al. 1981, 1982).

Mitochondrial distribution in primary bladder epithelial cell cultures after colchicinetreatment

We have reported the absence of vimentin filaments in primary bladder epithelialcell cultures using immunofluorescent techniques and two-dimensional gel analysis,

100 /. C. Summerhayes, D. Wong and L. B. Chen

Fig. 9. Mitochondrial staining in live PtK-2 cells (A, C). Keratin staining of A is shownin B, and vimentin staining of c is shown in D, respectively. Bar,

Mitochondria! distribution and cytoskeleton 101

Fig. 10. Mitochondrial staining in live PtK-2 cells treated with colchicine (A) ; and vimen-tin staining of the same cells (B). Bar, 20fitn.

whereas an extensive keratin cytoskeleton is present in these cells (Summerhayes,Cheng, Sun & Chen, 1981). Keratin cytoskeletons in these cells are little affected bycolchicine, and it is of interest to discover the factor controlling mitochondrialdistribution in cells where vimentin is absent.

Staining of epithelial outgrowths with rhodamine 123 reveals mitochondriadistributed throughout the cytoplasm in filamentous array, similar to the stainingobserved in mesenchymal cells within the same culture. A 24-h exposure of cells tocolchicine resulted in little change in staining in epithelial cells, in which mitochon-dria were still filamentous and extending out to the cell periphery. In contrast, mesen-chymal cells at the edge of the same outgrowth displayed retraction of mitochondriatoward the nucleus, consistent with our previous observations. Epithelial outgrowthsdisplaying an unaffected mitochondrial distribution showed an extensive keratincytoskeleton after immunofluorescence staining, which extended to all areas of the cellwhere mitochondria were present.

DISCUSSION

The experiments reported in this paper indicate a possible role for intermediatefilaments and microtubules in determining the distribution of mitochondria in anumber of different cell types in culture. The correlation in distribution observedbetween mitochondria and different cytoskeletal elements suggests intermediate fila-ments and microtubules as possible candidates in determining mitochondrial or-ganization and indicates that there is a close distribution of both filamentous networkswithin the cytoplasm (Geiger & Singer, 1980). In contrast, microfilament organiza^tion in cells showed no consistent correlation with mitochondrial distribution, sug-gesting a lesser role for actin filaments in determining mitochondrial location. How-ever, the possibility still remained that these observations may only reflect the fact thatmicrotubules and intermediate filaments share a major subcompartment of thecytoplasm with mitochondria, without specific interaction among them.

102 /. C. Summerhayes, D. Wong and L. B. Chen

The rearrangement of mitochondria observed in cells exposed to colchicine wascharacterized by a compacting of these organelles and their retraction from the cellperiphery. The depolymerization of microtubules did not result in a complete disor-ganization of mitochondrial distribution, but rather an apparent reorganization thatwas correlated closely with the movement of intermediate filaments toward theperinuclear region. It is not clear whether the redistribution and reorientation ofmitochondria under these conditions is a direct result of disruption of a microtubule-mitochondrion linkage or an effect mediated through the rearrangement of inter-mediate filaments associated with the disassembly of microtubules (Goldman, 1971;Goldman & Knipe, 1972).

A role for intermediate filaments in mitochondrial distribution was demonstratedby the disruption of vimentin filaments in CV-1 cells after exposure to cycloheximide.Consistent with a previous report by Sharpe et al. (1980), we observed disorganiza-tion of vimentin filaments in CV-1 cells after exposure to cycloheximide, whereimmunofluorescent staining revealed a complete loss of the organization that had beenapparent prior to drug treatment. Concomitant with the disruption of vimentin fila-ments we observed a rearrangement of mitochondrial distribution into disorganizedaggregates around the perinuclear region, despite the fact that microtubules andmicrofilaments in these cells appeared unperturbed by the drug treatment. Whereasthese results suggest that in the presence of intact microtubules in CV-1 cells it ispossible to observe alterations in distribution of both mitochondria and vimentin,microinjection of monoclonal antibody against protein associated with vimentin fila-ments shows that vimentin organization can be induced to change without alteringmitochondrial distribution. Thus, an intact vimentin filament system is not essentialfor the normal distribution of mitochondria. It appears that in a microinjected cell,as long as the microtubule system is intact, normal mitochondrial distribution can bemaintained. It is likely that mitochondrial distribution is strongly influenced by thetwo systems, microtubules and intermediate filaments. Neither plays an absolute rolebut one seems able to play a more dominant role in the absence of the other. It isimpossible to attribute mitochondrial behaviour completely to either intermediatefilaments or microtubules and it is clear that both play a major role in determiningmitochondrial distribution. It seems likely that intermediate filaments andmicrotubules represent a multicomponent system involved in mitochondrialdistribution and orientation (Wang & Goldman, 1978).

Since intermediate filaments appear to have a role in mitochondrial distribution,established epithelial cell lines that express both keratin and vimentin intermediatefilament types appear to have two possible alternative mitochondrial associations. Insuch cell lines (Franke et al. 1979a) mitochondrial distribution was not found to becorrelated with vimentin filaments, even in the presence of colchicine when vimentinfilaments had coiled to the perinuclear region. In primary epithelial cultures, wherevimentin filaments are absent (Summerhayes et al. 1981), mitochondrialdistribution seems totally insensitive to colchicine or colcemid. The maintenance ofmitochondrial distribution under these conditions is contrary to observations inmesenchymal cells, but is consistent with the maintenance of an extended keratin

Mitochondrial distribution and cytoskeleton 103

cytoskeleton under the same conditions (Sun & Green, 1978; Franke et al. 197%).Previous literature concerning organelle movement in cells of various organisms has

provided convincing morphological evidence implicating microtubules as the direct-ing influence in this phenomenon. Although this is likely to be the case in a numberof cells from lower organisms, metazoan cells present a more complex picture, sinceintermediate filaments in these cells are often closely associated with microtubules(Wang & Goldman, 1978; Franke, Grund, Osborn & Weber, 1978a) and have beenshown to have a distribution dependent on intact microtubule structures (Goldman,1971). As a result, depolymerization of microtubules by anti-mitotic drugs greatlyaffects intermediate filament distribution in these cells. The correlation observedbetween mitochondrial distribution and intermediate filaments or microtubules inuntreated cells further emphasizes the close distribution of these cytoskeletal ele-ments, but does not necessarily suggest any physical association. In fact, disruptionof vimentin filaments in CV-1 cells by cycloheximide argues strongly against anyphysical association, since microtubules remained unaffected in these cells.

We are grateful to Dr Marcia L. Walsh for her contribution to the initial phase of this project. Thiswork has been supported by grants from National Cancer Institute, American Cancer Society andMuscular Dystrophy Association to L.B.C., who is the recipient of an American Cancer SocietyFaculty Research Award.

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(Received 1 October 1982-Accepted 19 November 1982)