219

J. Anat. (1986), 147, pp. 219-233

With 7 figures
Printed in Great Britain

Mitochondria in living cells cultured from human

chorionic villi: the effects of colchicine on numbers
and distribution
FREDERICK KWAKU ADDAI* AND
COLIN DOUGLAS OCKLEFORD
Department of Anatomy, University of Leicester Medical School,
University Road, Leicester LE1 7RH

(Accepted 7 November 1985)

INTRODUCTION

The study of mitochondria in living cells has been undertaken using vital dyes and
bright field microscopy, phase contrast optics, video-enhanced differential interference contrast microscopy, and cine micrography (Michaelis, 1900; Lewis & Lewis,
1914; Frederic, 1958; Kacher, Brigman & Arnheiter, 1984) but there has only been
generalised study of placental cells in culture (Lueck & Aladjem, 1980). These
methods have provided interesting information about the distribution and behaviour
of m tochondria in cells (reviewed by Lehninger, 1964; Ernster & Schatz, 1981).
Effective research recently has been directed towards study of the association of
mitochondria with the microtubular cytoskeleton (Ball & Singer, 1982). This progress
has identified a bidirectional mobility of mitochondria that is facilitated by microtubules (Koonce & Schliwa, 1985).
Mitochondrial study has also progressed as a result of the use of the vital
fluorescent dye Rhodamine 123 (Johnson, Walsh & Chen, 1980; Johnson, Walsh,
Bockus & Chen, 1981; Kazuyuki & Murakami, 1984). This permits high definition
microscopy of mitochondria in living cells.
The present experiments were designed to estimate approximate length, distribution and number of mitochondria in cell cultures derived from human placental
tissue in the absence and presence of colchicine, a drug well known for its ability to
cause depolymerisation of cytoplasmic microtubules.
MATERIALS AND METHODS

Isolation and culture of cells from first trimester placenta

First trimester placenta was obtained from Leicester Royal Infirmary and transported in Hank's balanced salt solution (HBSS, Flow) containing 5 % Pen-Strep
solution at 4 'C. The villi were dissected from decidua (white fibrous maternal tissue)
in a disposable petri dish (Sterilin) using sterile surgical blades (Swan-Morton Ltd).
By means of a pair of flamed dissecting scissors, the villi were minced into minute
pieces. While mincing the villi, the bathing solution of HBSS was changed 4-6 times
to wash out blood and contaminating bacteria or other micro-organisms. The minced
villi were trypsinised for 5 minutes at room temperature using 10 ml 0-1 % trypsin*

Permanent address: Department of Anatomy, University of Ghana Medical School, PO Box 4236,

Accra, Ghana.
8-2

F. K. ADDAI AND C. D. OCKLEFORD

220
EDTA solution, in a universal bottle. The bottle and its contents were agitated by
swirling, the tissue allowed to settle and the supernatant (trypsinate) was discarded.
Fresh trypsin was added and the procedure repeated three times. Three further
trypsminsations were carried out in which the trypsinates were saved and diluted by
adding 5 ml growth medium (Ham's F-12K medium, 3 % Pen-Strep solution, 1 %
L-glutamine, and 30 % fetal calf serum). Cell suspensions obtained from the trypsinates were centrifuged at 1200 rev/min for 5 minutes; the resultant cell pellet was
resuspended in growth medium to rinse cells free from trypsin, and again centrifuged
as above. The final cell pellet was gently resuspended in 2 ml growth medium,
transferred into a 25 ml culture flask and placed in an incubator at 37 C in 5 %
carbon dioxide. About three hours later when some cells had attached to the bottom
of the culture flask, 5 ml more of the medium was added to the culture which was
then loosely closed and incubated as previously. The culture was examined daily
using an inverted microscope to assess cell growth. The growth medium was replaced
every three days. The principles but not the details of this method are based on those
first published by Loke & Borland (1970) and Loke (1983). All the results shown in
this paper were derived from cells cultured from a single placenta. However, the
widespread applicability of the conclusions is indicated by experiments using cells
derived from 8 placentae.

Culture of choriocarcinoma cells (Be Wo)

The BeWo culture was obtained from the American Type Culture Collection
(ATCC, CCL98) and stored in Ham's medium with 45 % fetal calf serum, and 10 %
dimethylsulphoxide, under liquid nitrogen. The cells were grown by conventional
cell culture techniques in medium containing 81 % Ham's F-12K medium (Flow
laboratories), 15 % fetal calf serum (Sera-Lab), 1 % L-glutamine (Flow), and 3 %
Pen-Strep solution containing 5000 iu/ml penicillin, 5000 ,g/ml streptomycin (Flow).
Stock cultures were maintained in 50 ml Nunc flasks (Inter-Med) with 7 ml of
medium. Cells were prepared for experiments by subculturing on glass coverslips
(Chance Propper) in repli-dish wells and fed with 1.5 ml medium per well.

Staining of living cultured cells with Rhodamine 123 for fluorescent

localisation of mitochondria (Johnson et a!. 1980)
The purified laser dye Rhodamine 123 (Sigma) was dissolved in double distilled
water to a concentration of 1 mg/ml and subsequently diluted to 10 jug/ml in Ham's
F-12K medium supplemented with fetal calf serum. BeWo cells on coverslips were
incubated with 1.5 ml Rhodamine 123 (10 jug/ml) for 20 minutes in a 5 % carbon
dioxide incubator at 37 'C. First trimester placental cells were incubated in Rhodamine 123 for 10 minutes, otherwise using the same conditions. The coverslips were
rinsed through three 5 minutes changes of medium (I 5 ml per rinse) and inverted
on to growth medium making a live cell observation chamber. The chamber was
limited by drawing a circular ring of silicon grease on a standard 25 x 75 mm microscope slide. Stained cells were examined under epifluorescent illumination using the
dichroic filter set fitted to a Zeiss photomicroscope equipped with a neofluar/phasecontrast objective lens ( x 100, oil immersion). Photographs were taken using Kodak
Ektachrome 400 film (ASA 400).

Mitochondria in placental cells

221

Treatment of living cultured placental cells with colchicine and

subsequent staining for mitochondria
Placental cells subcultured on glass coverslips (Chance Propper No. 1j; 13 mm
diameter) were used. Colchicine (Sigma) was dissolved in growth medium to a
concentration of 10,ug/ml. The cells on coverslips were placed in repli-dish wells
containing 1*5 ml of the colchicine solution (10 jug/ml) per well and incubated at
37 C in 5 % carbon dioxide for three hours. Coverslips were transferred to fresh
wells which contained 15 ml Rhodamine 123 (10 jug/ml in growth medium) and
incubated as above for 10 minutes. Procedures for rinsing of coverslips, mounting,
examination under the fluorescence microscope, and photomicrography were as
described earlier. In control experiments, cells were passed simultaneously through
the various stages of the experiment, but colchicine-free medium was used when the
experimental cells were treated with colchicine.
Estimation of lengths of mitochondrial strands
The focal depth of the x 100 objective permitted in-focus imaging of most of the
mitochondria in these flattened cells. In a few cases careful adjustment of the fine
focus allowed mitochondria above or below the nucleus to be imaged when they
were not seen clearly at optimum focus.
For the quantitative study, focussing at different depths in different cells permitted
the choice of cells for measurement in which the majority of the mitochondria were
in focus at the optimum position.
The length of the two dimensional photographic projection of the mitochondria
was used as a basis for measurement. Measurement was undertaken using an
electronic planimeter pen which left an ink trace indicating which mitochondria had
already been measured. In cases of apparently crossed or branching mitochondria,
an arbitrary decision was taken as to which strand to follow at junction points,
usually avoiding crossing over another strand in the image. The remaining strands
were measured independently and entered into the data with the status of separate
mitochondria.
Monochromatic prints (5 x 7' or 5 x 4') of micrographs taken under the Zeiss
photomicroscope were used in which Rhodamine-stained mitochondria showed as
white strands. A set of data processing instruments employed consisted of a MOP
(Kontron) electronic planimeter, APPLE II microcomputer, a Hitachi monitor and
a printer. The photomicrographs were placed on the MOP graphics tablet and the
strands individually traced as the computer recorded individual strand length. Data
collected included the number of strands measured, mean apparent length of these
strands, standard error and deviation, as well as Student's t value when experimental
results were compared with controls.
RESULTS

Light microscopic observations of Rhodamine 123 labelled mitochondria

Living cultured placental cells
Using the appropriate dichroic filter set, structures stained with Rhodamine 123
appeared filamentous when examined by fluorescence microscopy; the fluorescence
was prone to fade when illuminated for more than one minute. The fluorescent

222

F. K. ADDAI AND C. D. OCKLEFORD

Fig. I (-b). Livingculturedhumanfirst trimesterplacentalcellvital-stainedwithRhodamine 123.

(a) Phase-contrast image. Phase-dense mitochondria are visible in this micrograph where the focal
plane is somewhat above the nucleus. (b) The same area showing the fluorescence emitted by the
mitochondria. A few mitochondria appear to overlie the nucleus (arrows).

strands were superimposable in location with phase-dense structures identified in the

corresponding phase contrast image of the same cells. Johnson et al. (1980) have
identified these as mitochondria in similar cells. The nuclei of cells did not stain but
were represented by ellipsoidal, oval, or spherical dark spaces (Fig. 1). A few instances
of fluorescent strands apparently occurring in the region of the nucleus were shown
by careful focussing to be in cytoplasm either above or below the nucleus. Along the
horizontal axis of the cells, however, there was usually a high density of labelled
mitochondria around the nucleus, such that the fluorescence tended to be brighter
(Fig. 2). This perinuclear assemblage of strands exhibited two peculiar features:

Mito'chondria in placental cells

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Fig. 2(a-b). Perinuclear dipolar (*) assemblage of mitochondria in a Rhodamine 123-stained

cell. Living cultured human first trimester placenta. (a) Phase contrast. (b) Fluorescence.

(a) Polarity; the assemblage of strands concentrated at opposite ends ofthe nucleus
(dipolar perinuclear aggregation: Fig. 2), or on one side of the nucleus (unipolar
perinuclear aggregation: Fig. 3).
(b) Parallelism; the strands immediately adjacent to the nuclear envelope usually
lay parallel to it (Fig. 3).
Mitochondria appeared to radiate from the perinuclear aggregates towards the
cell periphery in a tangle of filaments. Hence, the topography of mitochondrial
arrangement was usually ill-defined near the nucleus, but was better revealed in the

224

F. K. ADDAI AND C. D. OCKLEFORD

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Fig. 3(a-b). Unipolar (*) arrangement of mitochondria in a Rhodamine 123-stained cell. Note
the loop of a mitochondrion in an adjacent cell (arrow) and the apposition of mitochondria to
the surface of the nucleus. Living cultured human first trimester placenta cells. (a) Phase
contrast. (b) Fluorescence.

peripheral regions of the cells where the mitochondria were less densely packed.
Here mitochondria were generally elongated but were also branched (Fig. 4), curved
(Fig. 4), looped (Fig. 3) and possessed swellings (Fig. 4). Mitochondria commonly
terminated with free endings in the vicinity of the cell membrane (Fig. 5).
Living cultured choriocarcinoma (Be Wo) cells
The distribution of mitochondria in BeWo cells will be described in relation to
that in normal placental cells. A difference in the general organisation of mitochondria was very evident. Particularly noteworthy was the small size and close

Mitochondria in placental cells

225

Fig. 4(a-b). The peripheral regions of two adjoining cells showing branching, curving and
complex topography of mitochondria in a vital dye preparation. The arrowheads indicate
varicosities in the mitochondria. Living cultured human first trimester placenta cells. (a) Phase
contrast. (b) Fluorescence.

packing of these cells compared with the normal ones. The mitochondria, were
visually more slender and less prominent than in cultured placental cells. They were
also apparently shorter. The perinuclear aggregates described in normal cells were
not obvious in BeWo cells. Nuclei of choriocarcinoma cells, like those oftheir normal
relatives, did not stain with the Rhodamine dye. Similarly, they infrequently displayed
strands which lay above or below the nucleus. The mitochondria were diffusely
distributed in BeWo cytoplasm. Superpositioning of phase-dense structures in phase
contrast micrographs and fluorescent strands in epifluorescence micrographs of the
same cell was less striking in choriocarcinoma cultures (Fig. 7). A remarkable
finding was that the tumour cells required twice the exposure time needed by their
normal counterparts for sufficiently bright labelling of mitochondria by Rhodamine
123.

226

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Fig. 5(a-b). The periphery of this vital stained cell is marked by the dotted line. Note the close
approximation of the distal ends of some mitochondria to the cell surface. Varicosities and
branching of mitochondria are apparent. Living cultured human first trimester placenta cells.
(a) Phase contrast. (b) Fluorescence.

Colchicine-treated (10 ,ug/ml, 3 hours) living cultured placental cells

The most obvious observable feature was a disorganisation of the mitochondrial
distribution. On average, strands were diffusely distributed closer to the nucleus.
Some groups of mitochondria were located towards the outer reaches of the cell,
usually on one side of the nucleus only and with an irregular outline (Fig. 6). The
complexity of the arrangement of mitochondrial filaments was markedly reduced, a
numerical increase in the strands and more open spacing in their distribution was
apparent. The dispersion observed using fluorescence microscopy was not clearly
detectable using the phase contrast microscope.
Analyses of data on estimates of mitochondrial lengths
A summary of results obtained from measurements and calculations is presented
in Table 1. The lengths of all the mitochondria in each of fifteen living cultured
placental cells and in an equal number of cells treated with 10 sg-ml colchicine
(3 hours) were measured. The mean length of strands ranged from a maximum of

Mitochondria in placental cells

227

Fig. 6. Fluorescence micrograph of a living cultured first trimester human placental cell treated
with colchicine (10 sgm/ml) for 3 hours and stained with Rhodamine 123. The positions of
cell nuclei are indicated by an area of absence of stain (n).

15-67 to 8-23 ,um for normal (untreated) cells. These data were obtained from cells
containing 32 and 23 mitochondria respectively. In the colchicine-treated cells, the
mean strand length per cell varied from 9171 ,um (22 mitochondria) to 4 69 #m
(44 mitochondria). Prior to making measurements, the colchicine-treated cells were
paired arbitrarily with untreated (normal) controls to facilitate statistical analyses.
Table 1 shows that in the paired cells examined, the mean lengths of mitochondrial
strands were perceptibly greater in the control cells than in their colchicine-treated
counterparts. In addition, the cell to cell variation in mean strand length was
more marked in normal cells compared with treated pairs. The dispersion of individual strand measurements from the mean length in each cell, as indicated by the
standard deviation (Table 1), showed higher values for controls. This confirmed
subjective observations that mitochondrial filaments were more uniform in length
in colchicine-treated than in normal placental cells. When the colchicine-treated and
untreated cells were compared in terms of the total number of mitochondrial strands
present in each cell (Table 1), appreciably higher numbers were found in treated cells
than in their untreated partners.
Table 1 also shows Student's t values resulting from a statistical comparison of
mean lengths in the random pairs of colchicine-treated and untreated cells. In fifteen
pairs, twelve values were significant at P < 0 01, one at P < 0 05, and only two were
not significant at P < 0 05. An analysis based on the total numbers of mitochondrial
strands per cell yielded a Student's t value of 2-49 with 28 D.F. which was significant
at P < 0-02. Thus the number of strands occurring in each treated cell was statistically greater than in untreated controls. However, a similar comparison of the total
length of mitochondria per cell (mean length x number of strands) gave a t value of
1I 74 that was not significant. This indicated that there was no statistically significant
difference between the total length of mitochondria in colchicine-treated cells and
their normal untreated controls.

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Fig. 7(a-b). Choriocarcinoma (BeWo) cells vital stained with Rhodamine 123 show less
organisation of mitochondria than the first trimester human placental cells. (a) Phase contrast.
(b) Fluorescence.
DISCUSSION

Mitochondrial distribution
The results show that disruption of the mic-rotubular cytoskeleton leads to reorganisation of the mitochondria, indicating that mitochondria are associated with

microtubules.

The action of colchicine is interpreted as being mediated by its effect on microtubules (Dustin, 1978). Colchicine, however, is also known to affect other cellular
activities (Weatherbee, 1981) such as inhibition of nucleoside transport (Mizel &
Wilson, 1972), partial inhibition of protein synthesis (Creasy, Bensch & Malawista,
1971) and inhibition of fluid transport by membranes (Beebe, Feagans, BlanchetteMackie & Nau, 1979). There is a variety of reasons why these latter effects are not
likely to be important in the present context; perhaps the most convincing is the

Mitochondria in placental cells

231

finding that rotenone, also a microtubule inhibitor (Brinkley, Barham, Barranco &
Fuller, 1974; Ketelbant-Balasse & Neve, 1976; Dustin, 1978), but apparently without
the other effects on cells which colchicine might inflict, also changes mitochondrial
distribution in a similar fashion (Johnson et al. 1981). Further evidence is that
lumicolchicine, an ultraviolet light derivative of colchicine, which is inactive in
preventing microtubule assembly yet as active as colchicine in inhibition of nucleoside transport (Dustin, 1978), has been found by Johnson et al. (1980) to have no
effect on mitochondria revealed by Rhodamine 123 staining. Recent experiments
(F. K. Addai, unpublished observations), have indicated similar conclusions in the
chorionic villus culture system.
Prior to treatment, at least some of the mitochondria extend out to the periphery
of the cells where they may be associated with the cell surface. This type of distribution is also seen in microtubules (Ockleford, Dearden & Badley, 1984). The pattern
of disassembly of microtubules in cultured cells usually leads to shorter microtubules
based on more centrally localised microtubule organising centres from which they
grow out afresh during recovery. It is, therefore, very interesting to note that after
treatment of cells with antimicrotubular drugs the location of the mitochondria is
more central. Here, it may be of interest also to point out the possibility that the
uni/dipolar patterns of mitochondria observed in untreated human placental cells
may reflect the presence of either one or two organising centres (centrioles and
associated materials) which replicate as the cell cycle proceeds. Again on this point,
it is known that there are multiple microtubule organising centres in BeWo cells
(Ockleford et al. 1984) and in this location mitochondrial unipolar and dipolar
patterns are not distinguishable. There is, therefore, in the results of these experiments further confirmation of the suggested structural link between microtubules and
mitochondria.
The close association noted above (Figs. 2-4, 7) between mitochondria and nuclear
envelope is an intriguing one. Questions arise as to what are the functional significances of this association. Is the mitochondrion, which is a genetically inadequate
organelle (Tribe, Morgan & Whittaker, 1981), receiving informational macromolecules from the nucleus? Or amongst other possibilities, are these associated
mitochondria providing energy-rich substrates required for trans-nuclear envelope
transport processes? Another possibility suggested by Lehninger (1964) is that the
association reflects a biogenetic link between mitochondria and the nuclear envelope.
It is remarkable that the intracellular origin of mitochondria is still not understood.

Length control of mitochondria

The fact that the average length of mitochondria is reduced after colchicine treatment but that the total length of all the mitochondrial strands within a cell is maintained, invites the conclusion that shortening results from separation of strands into
tandem fragments. This is an important conclusion because it appears to exclude
the possibility that much of the shortening is elastic in nature. The appearance of
varicosities and gently curving loops of mitochondrial strands in untreated cells is
also consistent with the view that the majority of mitochondria are not under tension
to the extent of stretching as a result of their interaction with the forces transmitted
by the microtubular cytoskeleton.
The number of mitochondria may change with time in cells (James & Bohman,
1981) and it has been suggested that fusion and separation occur at right angles to
the long axis of the mitochondrion (Lehninger, 1964). The motile force required to

232

F. K. ADDAI AND C. D. OCKLEFORD

effect separation may be generated in association with microtubules. Conversely, the

attachment of short mitochondrial strands to a microtubular track may facilitate
their approximation and terminal mutual contact prior to membrane fusion.

Mitochondria in transformed cells

The fact that the exposure time to Rhodamine 123 required to achieve equivalent
mitochondrial staining in BeWo cells is double that for first trimester human placental
cells in culture, indicates a significant but not unexpected difference between the
carcinoma cell line and a non-malignant cell culture derived from its tissue of origin.
It is intriguing in this context that Rhodamine 123 has differential anti-tumour
effects (Bernal, Lampidis, Summerhayes & Chen, 1982; Bernal, Lampidis, McIsaac &
Chen, 1983; Lampidis, Bernal, Summerhayes & Chen, 1983). The ability of mitochondria to maintain a high internal concentration of Rhodamine 123 is known to
be affected by metabolic poisons (Johnson et al. 1981 ; Tanphaichitr, Chen & Bellve,
1984) and their physiological state (Summerhayes et al. 1982; Haim, Munck,
Fourcade & Lampidis, 1984). An obvious explanation for 'the different staining
reactions of the cells is altered mitochondrial function in the tumour cells. The
possibility that differences in the cell surfaces of the two types of cell are of significance in explaining this observation must be considered, but has been shown to be
unimportant in previous studies where differences in staining of this type were
observed (Johnson et al. 1981).
SUMMARY

Fluorescence microscopy of living first trimester human placental cells and

choriocarcinoma cells in cultures was carried out using the vital fluorescent dye
Rhodamine 123. The length and distribution of mitochondria and, in the normal cells,
their reaction to colchicine treatment is described.
It appears that in the presence of colchicine the distribution of mitochondria in
normal placental cells becomes more restricted to the perinuclear cytoplasm and that
the mean length of mitochondria is reduced. However, the total length of all the
mitochondria in treated cells is not significantly different from that in paired cells
which were not exposed to the drug.
It is inferred from this result that mitochondrial shortening in the presence of
colchicine is not an elastic shortening consequent on the removal of cytoskeletal
elements which promote extension of the organelle. Rather it is brought about by
fragmentation of the organelle into tandem segments.
We thank Anne Clode and Christopher Rigel-d' Lacey for technical and computing assistance and Professor McVicar and the obstetricians and gynaecologists
of the Leicester Royal Infirmary for clinical coordination. F. K. A. gratefully
acknowledges a British Council Scholarship.
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