Polarized pigment granule transport occurs in the absence of
microtubules in squirrelfish erythrophores: studies of the effects of
estramustine
MARK E. STEARNS and MIN WANG
Department of Pharmacology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA
Summary
We have re-examined the involvement of microtubules in the process of pigment granule transport in squirrelfish erythrophores in situ (i.e. on
scales). Light-microscopic studies revealed that
following exposure to 5 jUM-nocodazole for 1 h at
4°C erythrophores retained an ability to aggregate and disperse their pigment uniformly,
though at reduced rates. Serial thick-section
stereo high-voltage electron-microscopic studies
showed that the entire microtubule population
was removed by drug treatment and that the
microtubules were not reassembled as a result of
pigment translocation processes in the presence
of reduced levels of nocodazole (0-4/iM).
Immunofluorescence microscopic studies confirmed that nocodazole (0-5-1 uM) produced
rapid disassembly of the microtubules. Wholemount electron-microscopic studies showed that
the pigment granules were suspended in a crosslinking network of 3-10nm filaments, which
appeared to support ordered pigment transport
in situ in the absence of microtubules. Drug
inhibition studies showed that micromolar levels
of estramustine, a novel anti-MAPs (microtubuleassociated proteins) drug, reversibly inhibited
pigment transport. The results suggest that an
estramustine-sensitive cytomatrix component
might produce polarized pigment transport in
intact erythrophores.
Introduction
the microtubules disassemble, and then reassemble
during some stage of pigment dispersion. Unfortunately, the importance of such dramatic changes in
microtubule number is equivocal, since the author's
sample size was small, and studies of melanophores
from other fish (e.g. Fundulus), erythrophores and
chromatophores, in general (Murphy & Tilney, 1974;
Porter et al. 1983), have not demonstrated any measurable fluctuations in microtubule numbers during pigment translocations.
Other attempts to unravel how granule transport is
controlled have relied heavily on studies of the experimental effects of anti-cytoskeletal drugs (e.g. cytochalasins, DNase I, nocodazole, colchicine). Studies
of colchicine-treated chromatophores in situ have
suggested that microtubules might not be essential for
pigment granule transport. However, in these in situ
studies it was unclear if colchicine uniformly or consistently removed the entire microtubule population from
cells (Junqueira et al. 1974, 1977; Murphy & Tilney,
A rather remarkable though largely uncharacterized
form of microtubule-directed fast organelle transport is
termed 'resolute' transport. On the basis of lightmicroscopic studies, Porter et al. (1983) defined 'resolute' motion as the non-saltatory, unidirectional and
uniform transport of a select group of organelles
(i.e. chromosomes, pigment granules). An excellent
example is evident in the uniform aggregation and
dispersion of pigment from the centrally located microtubule-organizing centre (MTOC) in chromatophores
(Junqueiraei al. 1974, 1977; Murphy & Tilney, 1974;
Wisko & Novales, 1969).
We do not understand the basic mechanochemical
mechanisms regulating resolute motion. Thin-section
studies of cultured angelfish melanophores have indicated that dynamic fluctuations in microtubule number
might contribute to resolute motion (Schliwa & Bereiter-Hahn, 1973). During aggregation about 55% of
Journal of Cell Science 87, 565-580 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
Key words: pigment transport, erythrophores,
estramustine.
565
1974; Wisko & Novaks, 1969), making it difficult to
assess the results. Several high-voltage electron-microscopic (HVEM) and immunofluorescence studies of
cultured chromatophores have revealed that colchicine
and nocodazole produce disassembly of the microtubule structures in angelfish melanophores (Schliwa,
1979; Schliwa & Euteneuer, 1978) and in squirrelfish
erythrophores (Beckerle & Porter, 1983). The drugs
also completely disrupt 'saltatory' and 'resolute' transport, so that agents (epinephrine) that normally produced complete pigment aggregation now induced an
incomplete clumping (beading) of the granules at the
cell periphery. These latter results showed that microtubules were essential and played a pivotal role in
guiding saltatory and resolute motion.
Interestingly, the HVEM studies of cultured chromatophores revealed that pigment was suspended in a
microtubule-associated cytomatrix of 3-10 nm filaments (Byers & Porter, 1977; Luby & Porter, 1980;
Luby-Phelps & Porter, 1982; Porter et al. 1983;
Schliwa, 1979). Porter et al. (1983) further reported
that part of the cytomatrix, termed the 'ar-cytomatrix',
coaggregated with the pigment while a second component, termed the '/J-cytomatrix', remained immobile
in association with the surfaces of microtubules,
smooth endoplasmic reticulum (SER) and mitochondria in quick-frozen erythrophores in culture. They
suggested that the cytomatrix might power transport
and possibly regulate the spatial distribution of
individual granules, since the granules consistently
returned to their original position after each cycle of
aggregation and dispersion (McNiven & Porter, 1984).
Their results are controversial, however, as Ip et al.
(1984) examined quick-frozen, deep-etched erythrophores in situ and found little evidence for a mobile
tf-cytomatrix component. Further studies are warranted to resolve this important issue.
The role of a distinct cytomatrix component in
organelle transport could be clarified if its protein
composition was determined and if a class of drugs was
identified that specifically binds a cytomatrix protein
and/or inhibited one or more transport events. Recently, we have shown that a microtubule-associated
protein, MAP-2, was a component of the ar-cytomatrix
(Stearns & Binder, 1987) and that MAP-2 antibodies
inhibited pigment transport (unpublished data), indicating that MAP-2 might somehow regulate transport.
We have also discovered that micromolar levels of a
novel drug termed estramustine (17^3-estradiol-3-iVbis(2-chloroethyl)carbamate; EM), will rapidly diffuse
into cells and inhibit pigment granule transport in
cultured erythrophores. More significantly, the drug
exhibited a high affinity for several high molecular
weight microtubule-associated proteins (MAPs 1 and
2) in vitro (Wallin et al. 1986), inhibited microtubule
566
M. E. Stearns and M. Wang
assembly, and produced rapid microtubule disassembly in vitro and in vivo (Stearns & Tew, 1985;
Kanje et al. 1985; Wallin et al. 1985). The effects of
EM on MAPs and microtubules were reversible and
reproducible, making EM a useful compound for
studies of the potential role of MAPs in cells.
In this paper, we examined pigment transport in
squirrelfish erythrophores from scales (in situ).
Exhaustive serial-section electron-microscopic studies
clearly showed that microtubules were not essential
for 'resolute' transport. Estramustine, at micromolar
levels, was found to inhibit pigment aggregation and
dispersion rapidly and reversibly in both intact and
nocodazole-treated erythrophores. The possible role
of the cytomatrix in regulating pigment transport is
discussed.
Materials and methods
Cultured cell studies
Scales were plucked from the squirrelfish Holocentrus
ascensionus and erythrophores were cultured from the scales
using modified procedures of Stearns & Ochs (1982). Erythrophores were plated on carbon-coated coverslips or on
Formvar-coated gold grids, then allowed to attach overnight
and used in experimental studies.
In situ studies of cells
Scales were washed three times with teleost phosphate
solution (TPS) at room temperature and then incubated with
5/iM-nocodazole at 4°C for 1 h. Scales were subsequently
transferred to TPS containing 0'4^lM-nocodazole and
warmed to 32 °C with an air-current incubator for studies of
pigment transport. To stimulate transport, the nocodazoletreated cells were exposed to solutions that stimulated
pigment aggregation, washed with TPS and transferred to
pigment-dispersing solutions.
At each stage of pigment transport studied the erythrophores from three different experimental batches were fixed
and processed for electron-microscopic studies. Scales were
fixed for 30min with 025 % glutaraldehyde, 2% paraformaldehyde and 15% picric acid in phosphate buffer containing
20/iM-CaCl2, l-5niM-MgCl2, pH7-4. The scales were cut
into 1 mm X 2 mm pieces and fixed for 3 h, washed three times
with TPS, postfixed with 1 % OsC>4 in distilled water for
15 min, washed three times with distilled water, then stained
with 0-2% tannic acid for 30min, washed three times with
distilled water, stained with 2 % uranyl acetate for 1 h,
washed three times with distilled water, then dehydrated with
ethanol and embedded in Spurr's plastic for sectioning.
Sections were stained with 5 % uranyl acetate in 70 %
ethanol for lOmin for thin sections (0-1 ^m), or 40min for
thick sections (0-25 fim), washed with distilled water, and
rinsed twice with 100% methanol. Sections were stained with
a triple lead stain (Sato, 1968) for 2min for thin sections; or
10 min for thick sections, washed with distilled water and
rinsed three times with 100% methanol. Sections were air
dried, carbon coated and examined with a JEOL 100S at
100 kV or photographed in stereo with a 1000 kV HVEM at
the national facility in Boulder, Colorado. The triple lead
stain was made with 1 g lead citrate, lead nitrate, lead acetate
and 2g sodium citrate dissolved in 82 ml distilled water and
18ml of lM-NaOH.
Experimental solutions
Teleost phosphate solution (TPS) contained 169mM-NaCl,
5-4mM-KCl, l-8mM-CaCl2, 1-3 mM-MgCl2, 5-6mM-glucose,
5 mM-Tris • HC1, pH 7-2. A 1 mM stock solution of nocodazole
(Sigma), cytochalasins B and D (Sigma) was made in 100%
dimethyl sulphoxide and frozen. Thawed samples of the
stock were added to TPS. Sucrose solutions were made using
a 60% RNase-free sucrose stock solution and a 10 x TPS
stock solution to obtain a l x T P S at 660mosM as measured
with an Osmette-S osmometer. Calcium/EGTA solutions
containing known amounts of free Ca 2+ were made up
according to calculations described by Stearns & Ochs
(1982). Epinephrine and caffeine stock solutions were used to
obtain final concentrations in TPS of lXlO~7M-epinephrine
and 5 mM-caffeine. Estramustine was a gift from A. B. Leo
Research Laboratories, S25109, Helsingborg, Sweden, and
was solubilized in 100 % ethanol to make fresh stock solutions
for each experiment. Samples of about 10^1 were added to
1 ml TPS for experimental studies. Control studies were
carried out with 10 % ethanol in TPS and known amounts of
estradiol (Sigma) testosterone and nor-nitrogen mustard
(Sigma).
Results
Light-microscopic studies of pigment transport in situ
Light-microscopic studies of scales freshly plucked
from fish revealed that the pigment granules extend
outwards from the cell centre as a uniformly distributed and concentrically arranged array of granules
(Figs 1-3). Transport rates ranged from 10-20 jUms"1
during aggregation to 5-10/ims" 1 during dispersion at
temperatures of 32°C. The aggregation event was
produced by washing scales with TPS containing one
of the following agents: either 10~7M-epinephrine
(Fig. 2), 5xl0~ s M-calcium, 80mM-K + , or 660mosMsucrose. The aggregation was complete by about 5 min
and reversed (dispersion) by washing the scales with
TPS containing reduced calcium levels ( 5 X 1 0 ~ 7 M ) .
Complete pigment dispersion occurred by about 5 min
at32°C (Fig. 3).
We examined the effects of the anti-tubulin drug
nocodazole on pigment transport and microtubule
structure to determine if microtubules were essential
for the orderly transport of pigment granules. Scales
were exposed to 5/iM-nocodazole for 1 h at 4°C and
then transferred to TPS containing 0-4/iM-nocodazole
at 32°C to 37°C. Fig. 4 shows that the pigment
remained dispersed in all the erythrophores and numerous clear 'vacuolar-like' areas appeared throughout
the cytoplasm. The erythrophores retained this appearance even after several hours at 32CC or 37°C in TPS
containing 0-4^tM-nocodazole. We observed that all
saltatory motion was inhibited by nocodazole and was
not reinitiated when the cells were warmed to 32°C in
the presence of 0-4^iM-nocodazole. When the scales
were transferred to TPS containing 0'4^iM-nocodazole
plus agents known to induce pigment aggregation in
intact cells, the granules slowly aggregated, albeit at
reduced rates of 3-6(ims" 1 . The rate and extent of
aggregation was identical (in all the erythrophores)
for several different aggregating agents tested, including 2xlO~ 7 M-epinephrine, 5X 10~~5M-Ca2+, and
660mosM-sucrose. This first aggregation event was
unique and differed from that observed in intact
erythrophores. It involved the initial formation of
numerous small patches or clumps of pigment in the
peripheral margins of the cell by about 5 min. After
about 15 min single granules from these mini-aggregates migrated in.a 'stream-like' fashion to the cell
centre. Once most of the granules were aggregated
around the cell centre, by about 30 min, the granules
began to move (pulse) in a uniform manner as a
concentrically displayed array. That is, they pulsed in
and out from the cell centre for distances of about onethird of the cell's diameter at rates of 10-20 ^irns" 1 .
The cells continued the incomplete cyclic dispersion
and aggregation of their pigment for about 5 min, at
which point the granules remained aggregated in a
compact mass at the cell centre (Fig. 5). We found that
erythrophores that were exposed to hyperosmolar sucrose solutions (660mosM) were the most active and
exhibited an extensive amount of 'pulsatory action'
during the process of pigment aggregation. By comparison, erythrophores stimulated with 10~6M-calcium
solutions and epinephrine exhibited considerably less
pulsatory action as the pigment accumulated at the cell
centre.
We investigated the reverse process of pigment
dispersion in the nocodazole-treated erythrophores. In
erythrophores stimulated to aggregate with high calcium and hyperosmolar sucrose solutions, dispersion
occurred by 30min at 32°C in isotonic TPS or TPS
containing either 5 mM-caffeine,
lXl0~ 7 M-Ca 2 +
(Fig. 6), or 0-2M-NaCl. After about 5 min the granules
usually dispersed in uniform concentric arrays at rates
of about 6 ± 2s.D. j i m s " ' . The granules were evenly
distributed throughout the cytoplasm and most of the
clear vacuoles observed upon initial exposure to nocodazole (Fig. 5) did not reappear (Fig. 6). We found that
the pigment granules did not spontaneously pulsate but
uniformly aggregated and dispersed as a concentrically
distributed array at rates of 5-6finis" 1 when erythrophores were exposed to the agents that produced
pigment aggregation and dispersion. The cyclic translocations of pigment closely resembled that which we
observed in intact erythrophores, except that transport
was slower and increased amounts of the pigment
Pigment transport without microtubules
567
remained dispersed after two or three cycles of aggregation.
We found that transport returned to normal when
the cells were washed and incubated in fresh TPS for
1 h at 32°C, the time needed for microtubule regrowth
to occur. With recovery, all the immobilized granules
were reincorporated into the motile granule population
and saltatory motion was reinitiated. The spontaneous
pulsation of granules was now observed.
Tables 1-4 summarize the data from ten exper-.
iments on nocodazole-treated cells. The results show
that the nocodazole-treated erythrophores were competent to aggregate their pigment in response to
1X10 S M-Ca 2+ , 660 mosM-hyperosmolar sucrose, and
0-08 M - K + in isotonic TPS. The data further show that
the drug-treated erythrophores dispersed their pigment
in response to threshold levels of known dispersing
agents, including lXlO~ 7 M-Ca 2+ , 0-2M-NaCl and isotonic TPS. Usually, we used reduced calcium levels to
counter the effects of high calcium; isotonic solutions
to reverse the effects of hyperosmolar sucrose; 0-2 MNaCl to reverse the effects of 0-08 M-K + ; and caffeine
to reverse the effects of epinephrine. Note that in Table
2, cells were first exposed to 5xl0~ 6 M-Ca 2 + then
transferred to solutions containing 1, 5 or 7xlO~ 5 MCa 2+ .
Figs 1-3. Phase images showing dispersed erythrophores (Fig. 1) during one complete cycle of pigment aggregation
(Fig. 2) and dispersion (Fig. 3) in situ. Note the uniform distribution of granules in Fig. 3. X 1300.
Figs 4—6. Erythrophores and a melanophore (m) that were exposed to 5 jiM-nocodazole for 1 h at 4°C and transferred to
0'4/iM-nocodazole solutions at 32°C. The granules remained dispersed during drug treatment and large vacuolar spaces
appear in the cytoplasm (Fig. 4). Aggregation (Fig. 5) was induced after about 30min at 32°C using TPS containing
1X1O~5M-Caz+ and 10 jiM-calcium ionophore (A23187). Redispersion (Fig. 6) was induced with TPS containing 10~7Mcalcium and ionophore. Note that the granules recovered a normal uniform distribution following redispersion (compare
Figs 3, 4 and 6). X1300.
568
M. E. Stearns and M. Wang
Interestingly, we found that the nocodazole-treated
erythrophores (5 [IM for 1 h) were incapable of translocating their pigment if they were exposed to reactivation solutions that contained more than 1 fiMnocodazole. Thus, the experimental solutions always
contained reduced levels of nocodazole of about 0-4fiM.
At this concentration of nocodazole the microtubules
did not regrow and pigment transport was not inhibited
by nocodazole.
Agents that inhibit transport in nocodazole-treated
erythrophores
In intact cells, pigment transport was completely
inhibited after 2 h exposure to the metabolic inhibitors,
0-01% sodium azide and 2xl0~ 6 M-dinitrophenol
(DNP). Similarly, transport was inhibited after 30min
exposure to 0-01 % sodium azide or 2x 10~6M-DNP in
erythrophores previously exposed to 5 jiM-nocodazole
for 1 h at 4°C and in TPS containing 0-4 /JM-nocodazole. For example, erythrophores exposed to metabolic inhibitors (i.e. 2xlO~ 6 M-DNP) and stimulated
with 2xlO~ 7 M-epinephrine did not aggregate their
pigment or even form patches in the peripheral margins
of the cell. The inhibitory effects of the DNP were
reversed following washes with fresh TPS and incubation in TPS containing 0-4 jXM-nocodazole for 60min
at 37°C. Following recovery the cells aggregated their
pigment in the presence of epinephrine and redispersed
their pigment in caffeine solutions.
We investigated the effects of anti-actin drugs and a
novel anti-mitotic drug, termed estramustine, which
Table 1. Effects of caffeine and epinephrine on dispersed cells
Dispersion
Reagent
Aggregation
Pulsation
Saltation
Reversal
7
Caffeine
(1X1O"5M)
1X10 M-epinephrinc
660mo8M hyper TPS
0-08 M-K+, TPS
Epinephnne
lx!0~ 5 M-caffeine
7
(1X10~ M)
All of cells were prctreated with 5 /iM-nocodazole at 4°C for 1 h and then exposed to the reagent for 30min at 32°C in the presence of
0'4/JM-nocodazole.
Table 2. Effect of calcium on dispersed cells
Dispersion
Aggregation
Pulsation
Saltation
Reversal
5xlO" 6 M*
+
-
-
-
lxlO~ 7 M-epinephrine
5xlO" 5 M»
lXlO~ 5 Mt
+
+
+
+
5X10~5M|
+
+
+
7xlO" 5 Mf
+
+
+
Calcium
-
Cells were treated with 5/iM-nocodazole at 4°C for 1 h and exposed to calcium/EGTA solutions containing 10/iM calcium ionophore
(A23187) and known amounts of calcium. The effects of calcium were tested with (•) or without (f) 0'4/iM-nocodazole.
Table 3. Effect of hyperosmolarity on dispersed cells
Osmolarity
Dispersion
Aggregation
660
-
+
Pulsation
Saltation
-
-
Reversal
+
•
Cells were treated with 5 /JM-nocodazole at 4°C for 1 h and then with a hypertonic solution containing 660 mosM-sucrose in TPS at 32°C
for 15-20 min.
•With a calcium/EGTA solutions (5X 10~6M-Ca2+) containing 10/JM calcium ionophore (A23187), or with 0-2M-Na+ in TPS.
Table 4. Effect of high levels of potassium or sodium on dispersed cells
Solution
Dispersion
+
0-08 M-K in TPS
0-2M-Na + inTPS
Aggregation
Pulsation
Saltation
Reversal
1X 10~5 M-caffeine
Hyperosmolar solution
(600 mosM)
Cells were treated with 5/iM-nocodazole at 4°C for 1 h and at 32°C with K + or Na + , TPS solutions containing 0-4/iM-nocodazolc.
Pigment transport without microtubules
569
has been found to bind high molecular weight MAPs in
vitro (Stearns & Tew, unpublished data; Wallin et al.
1986). We found that cytochalasins B and D at 10/ZM
levels for 1 h at 32 °C did not inhibit or disrupt pigment
aggregation and dispersion in intact erythrophores or in
nocodazole-treated erythrophores (5 jiM for 1 h at 4°C)
that were transferred to 0-4 /iM, or 0-8 /iM-nocodazole at
32°C. In comparison, estramustine used at 30 ytA levels
for 30min at 32°C inhibited pigment aggregation and
pigment dispersion in the nocodazole-treated erythrophores. When the erythrophores were permitted to
regrow their microtubules in fresh TPS at 37°C for
60min, higher dosages of estramustine (60/ZM-EM for
60min at 32°C) were required to inhibit transport. If
the drug(s) was removed with washes of TPS transport
was restored by about 5 min at 32°C.
In control studies the effects of 10% ethanol, 60 fiMestradiol, 60/iM-testosterone and 60 /ZM-nor-nitrogen
mustard were monitored for 1 h. We found that when
erythrophores were pretreated with 5//M-nocodazole
they responded to epinephrine and aggregated their
pigment in the presence of these agents. The nitrogenmustard-treated cells eventually died (after a day),
presumably as a result of toxic alkylating effects of the
drug, effects that were not displayed by estramustine,
testosterone or estradiol.
Electron-microscope studies
Thin and thick serial-section electron-microscopic
studies showed that erythrophores normally contain
hundreds of radially arrayed microtubules, which emanate from a centrally located centrosome and extend
continuously to the cell periphery (Figs 7-9). Pieces of
other filaments of variable diameter (3-10 nm) were
visible in cross-sectional view (Fig. 8, double arrowhead) and in cross-sectional views (Fig. 9, arrowheads). Treatment of erythrophores with nocodazole
(5 /ZM for 1 h at 4°C) followed by incubation in 0-4 fJMnocodazole for 2h at 32°C resulted in the complete
disappearance of the microtubules (Figs 10-14). The
thin-section images in Figs 10—14 show that the microtubules were completely removed at the cell periphery
and in the vicinity of the centrosome. Other radially
aligned filaments such as 'intermediate filaments' or
microfilament bundles per se were not observed,
although radially arrayed and easily identified intermediate filaments were readily visible in nearby iridophores and melanophores. Short pieces of filament of
3-10 nm diameter (double arrowheads, arrowheads)
were evident throughout the cytoplasm. Also, a coarse
or fuzzy 'fibrillar-like' cytoplasmic ground substance
was evident throughout the cell, which appeared to
increase in density towards the cell centre.
Thin and thick stereo HVEM serial-section studies
of erythrophores fixed in the aggregated state, and in
the dispersed state showed that the microtubules were
570
M. E. Stearns and M. Wang
not reassembled during one or more phases of pigment
transport in the presence of 0-4^M-nocodazole and/or
30fiM-estramustine. The thin-section images in Figs
12-14 show regions of a nocodazole-treated erythrophore that had completed two cycles of pigment
aggregation and dispersion prior to fixation in the
aggregated state. Nocodazole did not have an effect on
cell shape or the spatial distribution of granules. The
large vacuolar-like spaces that appeared during the
initial exposure to the drug did not reappear after one
or two cycles of pigment transport (Fig. 12). Figs 13
and 14 demonstrate that the microtubules were completely absent at the cell centre (Fig. 13) and at the cell
margins (Fig. 14). The 10 nm filaments (arrowheads)
and the coarse fibrillar, granular material were the
principle cytoplasmic material that was evident. We
also examined thick sections in stereo to determine if
additional filamentous structures might show up that
were not detected in thin sections. Fig. 15 shows one
stereo pair of six through the cell centre in a dispersed
erythrophore that was embedded and examined by
serial thick-section HVEM (Figs 15, 16). In all our
studies, neither microtubules nor microtubule fragments were observed. Because of the reliable nature of
such an assay, serial sections were routinely done to
confirm the absence of microtubules in the experiments
described in Tables 1-4. In each of these studies, the
only structural element evident was the coarse granular
material, and some 3-10 nm filaments.
When nocodazole was removed by washing the fish
scales with TPS and incubation in fresh TPS for 1 h,
microtubules were easily detected throughout the cells,
even at relatively low magnifications and without the
benefit of stereo viewing techniques. In other words,
the microtubules were not obscured by the plastic
embedment in thin and thick sections (Figs 17, 18).
Figs 17 and 18 show a region of the cell margin and the
cell centre in a cell rescued from the effects of
nocodazole and embedded for thick-section studies.
Microtubules were easily detected in all regions of the
cell. As in untreated cells, the microtubule surfaces
were cross-linked by 4nm filaments (double arrowheads). Intermediate filaments and microfilament
bundles per se were not evident, although pieces of the
3-10nm filaments were readily visible (arrows). In
general, the 3-10 nm filaments were more easily visualized in thin sections (Figs 10—14) and difficult to detect
in thick sections (Figs 15-18), presumably as a result of
increased electron scattering by the Spurr's embedding
plastic in thick sections. However, the serial thicksection studies provided definitive proof that microtubules were absent in the nocodazole-treated cells.
Whole-mount studies of cultured erythrophores
Whole-mount electron-microscopic studies were carried out to determine the nature of the 'wispy' 3-10 nm
filaments observed by thin- or thick-section microscopy, and to assess further the effects of nocodazole
and estramustine.
For these studies, the erythrophores were removed
from scales by collagenase digestion and plated on
Formvar-coated gold grids in preparation for wholemount electron-microscopic analysis. Following
nocodazole treatment (0-5)JM for 1 h at 4°C), we
attempted to stimulate pigment aggregation with
2x10 7M-epinephrine and with 5x10 6M-calcium
plus 10/XM-calcium ionophore (A23187). We observed
by light microscopy that localized pigment patches
formed in the cell periphery after 5-10 min, but the
pigment granules were unable to aggregate to the cell
centre. Some redispersion of these 'mini-aggregates'
was observed if the calcium-treated cells were exposed
to reduced calcium levels (1X 10~7 M-Caz+ for 1 h) or to
5 mM-caffeine. Whole-mount HVEM studies showed
Fig8 7-9. Thin-section images of intact erythrophores (e) in situ. A longitudinal view (Fig. 7) and cross-sectional views of
the centrosomal complex (Fig. 8) and the cell margin (Fig. 9) show numerous microtubules (mt) emanating from the cell
centre. Pigment granules (/>), centrosomal complex (cc), membrane (m), collagen fibres (co), iridophore (/), guanine
crystals (g). Fig. 7, x 12400; Fig. 8, X47 4O0; Fig. 9, X41700.
Pigment transport without microtubules
571
that after exposure to 0-5-1/iM-nocodazole for 1 h at
4°C the microtubules were completely removed from
these cells. The only remaining structural component
was a three-dimensional cross-linking network of filaments that measured 3-10 nm in diameter. In cells
where mini-aggregates of pigment formed the granules
were cross-linked by numerous 3-10 nm filaments
(Fig. 19). Some redispersion of the mini-aggregates
was observed if the cells were exposed to reduced
calcium levels (1X 10~7 M-Caz+ for 1 h) or caffeine (not
shown).
Immunofluorescence studies with monoclonal antibodies raised against tubulin (antibody 27B) showed
that erythrophores exposed to 0-5/ZM-nocodazole for
Figs 10—11. Thin-section micrographs showing the cell margin (Fig. 10) and cell centre (Fig. 11) of an erythrophore fixed
in the dispersed state after exposure to 5/iM-nocodazole for 1 h at 4CC. The microtubules were completely removed and the
cytoplasm appeared to be occupied by granular material and filaments of varying diameters (3-10nm). The filaments of
Snm (double arrowheads) and 10nm (arrowheads) diameter are indicated. The centrosomal complex (cc) consists of
centrioles (c) and dense bodies (d). Pigment (p), membrane (m). Fig. 10, X 100000; Fig. 11, X80 000.
572
M. E. Stearns and M. Wang
1 h at 4°C were barren of any microtubules (Fig. 20).
The immunofluorescence studies further confirmed
that microtubules were not reassembled in living
erythrophores exposed to different aggregating agents,
including epinephrine (Fig. 20A), calcium (Fig. 20B)
and hyperosmolar sucrose (not shown). Time-course
studies (Fig. 20D-F) showed that the microtubules
were reassembled from the centrosome at the cell
centre following removal of nocodazole from living
cells by washing with fresh TPS. Light-microscopic
studies revealed that concomitant with microtubule
regrowth a spontaneous, cyclic pulsation of the pigment was reinitiated. Usually this occurred after about
30 min when the microtubules were almost completely
reassembled. During recovery the pigment was observed to pulsate in uniform concentrically distributed
arrays from the cell centre for distances of about half
the cell's diameter. Following longer recovery times of
60 min, the granules were observed to disperse all the
way to the cell periphery and this behaviour was
directly correlated with immunofluorescent evidence of
microtubule reassembly.
Figs 12-14. Thin sections showing that microtubules were not re-assembled either at the cell centre (cc) (Fig. 13) or at the
cell margin (Fig. 14) following pigment aggregation. Erythrophore (e), iridiphore (j), guanine crystal (g), collagen (co),
membrane (m), pigment (p), dense bodies (d), centriole (c). Fig. 12, X850O; Fig. 13, X62000; Fig. 14, X71 000.
Pigment transport without microtubules
573
15
Figs 15-16. Stereo high-voltage electron micrographs (HVEM) showing one stereo pair from serial thick sections through
the cell centre of a dispersed erythrophore (Fig. 15). Fig. 16 shows, at higher magnification, a region of the cell shown in
Fig. 15. The tannic acid stained three-dimensional images show a complete absence of microtubules in both dispersed and
aggregated cells. Only a dense granular, sometimes filamentous, material remained in the cytoplasm. Centriole (c), pigment
(/>). Fig. 15, X12000; Fig. 16, X54000.
Effects of estramustine on whole-mount erythrophores
Whole-mount electron-microscopic studies and immunofluoresence studies with tubulin antibody showed
that 60-120 ^iM-estramustine removed all the microtubules by 40-20min, respectively (Fig. 21). Estramustine did not destroy the 3-10 nm filaments or the
filamentous linkages observed between pigment granules (Fig. 21, arrowheads). In live cells washed with
fresh media and allowed to recover from the effects
of the drug for 1 h, the microtubules were rapidly
reassembled by 20 min at 37 °C and transport was
reinitiated.
To determine if EM could inhibit transport independently of its effects on microtubules, erythrophores
were exposed to 1 /ZM-taxol for 3 h in order to stabilize
the microtubules prior to exposure to EM. Electron
microscopy showed that in taxol-stabilized cells,
120^iM-estramustine did not produce microtubule disassembly or noticeable disruption of the microtubuleassociated cytomatrix, even after 2h of exposure
(Fig. 22). Light-microscopic studies of living cells
revealed that 120jUM-EM inhibited saltatory transport
after about 15 min and then subsequently inhibited
resolute motion by about 30 min in the taxol-treated
erythrophores. Following removal of EM with washes
of fresh media, a rapid recovery of saltatory and
574
M. E. Stearns and M. Wang
resolute pigment transport was observed by 15 min.
Identical results were obtained in studies of taxoltreated erythrophores in situ (on fish scales). Similar
dosages of EM ( 1 2 0 ^ for 30 min) were required to
inhibit resolute pigment transport in taxol-treated
erythrophores on scales. Saltatory motion was more
sensitive than resolute transport and was inhibited with
60/XM-EM by 15 min. Fig. 22 shows a stereo view of
part of a taxol-treated erythrophore exposed to 120 JJMEM for 30 min. Note that the microtubules and 3—
10 nm-filamentous network (arrowheads) were largely
intact.
Control studies with 120^iM-estradiol and nor-nitrogen mustard, the constituents of estramustine, and
120j!M-testosterone showed that transport was not
inhibited in intact erythrophores in situ or in culture
after about 60min of exposure to each compound.
Prolonged exposure to nor-nitrogen mustard for about
4h produced cell death, however.
Discussion
The results have consistently demonstrated that 'resolute' transport can occur without the aid of microtubules. Following removal of the microtubules, the
bidirectional transport observed was normal in most
respects, except that aggregation rates were reduced
by about one third from 10-20fiMs" 1 to about
6 ± 1 S. D. jUM s~'. Once the pigment had initially aggregated, the system regained its composure and the
granules moved to and from the cell centre in evenly
distributed arrays. Aggregation and dispersion were
both energy-dependent and did not occur at reduced
temperatures or in the presence of metabolic inhibitors.
Fig. 19. HVEM image showing part of a whole-mount
erythrophore that was exposed to nocodazole (1 //M for 1 h).
Nocodazole removed the microtubules, leaving a threedimensional network of 3-10 nm cross-linking filaments
(arrowheads). The pigment granules appeared to be
suspended in this cytomatrix and their surfaces were crosslinked by 3-10nm filaments. X77 800.
Figs 17-18. HVEM images of 0-25 /<m thick sections
showing the cell margin (Fig. 17) and the cell centre
(Fig. 18) of an erythrophore that had recovered from
nocodazole prior to fixation. The pictures demonstrate that
microtubules (mt) were visible in thick sections without the
aid of stereo imaging techniques. Compare Figs 17, 18 with
Fig. 16. Numerous 4nm filaments (double arrowheads)
cross-linked the microtubule surface. Filaments of 10 nm
diameter (arrows) were observed throughout the
cytoplasm, p, pigment; d, dense bodies. Fig. 17, X66000;
Fig. 18, X48000.
Pigment transport without microtubules
575
Moreover, drug-treated erythrophores responded to
diverse agents that normally produced pigment aggregation and dispersion, and which have been shown
previously to produce pigment translocations in different chromatophore systems (Schliwa & Euteneuer,
1978). In addition, we found that shifts in osmolarity
also produced aggregation (660mosM) and dispersion
(330mosM), presumably as a result of physical effects
on the organization of the water content of a structured
cytomatrix. Taken together, these data suggested that a
non-microtubule component might mediate pigment
transport.
In the above studies it was imperative to demonstrate
whether the microtubules were consistently absent in
reactivated erythrophores and to determine if another
filamentous system might exist to support transport.
HVEM studies of sectioned material and whole-mount
erythrophores clearly showed that the microtubules
were removed from the nocodazole-treated erythrophores. The presence of other filaments was difficult to
Fig. 20. A - F . Immunofluorescent images of whole-mount erythrophores labelled with tubulin antibody and secondary
antibody-FITC conjugates. A,B show that the microtubules were removed with nocodazole (0-5 fm for 1 h) and that
epinephrine (A) and calcium (B) did not induce reassembly of microtubules. Control cells labelled with 2° antibody
(FITC-IgG) alone were not stained (C). D - F show that, upon removal of nocodazole, erythrophores gradually regrew
their microtubules from the centrosomal complex (arrowheads). The successive stages of microtubule reassembly are shown
after lOmin (D), 20min (E) and 30min (F) recovery.
576
M. E. Stearns and M. Wang
Fig. 21. Stereo HVEM picture of a region of an erythrophore exposed to 60/iM-EM for 30min. The occasional piece of a
microtubule (mt) still remains. The pigment granules (p) were often clumped and cross-linked by numerous 3-10 nm
filaments (arrowheads). X 103 800.
Fig. 22. Stereo HVEM picture of part of an erythrophore exposed to 1 ^M-taxol for 3 h followed by EM (60 ^M for 30min)
treatment prior to fixation. Shows numerous microtubules (mt) and a fine crosslinking network of 3-10 nm filaments
(arrowheads) some of which cross-link the microtubule and pigment (p) surfaces. X80 000.
Pigment transport without microtubules
577
discern in sections, although pieces and 'wisps' of
filaments of 3-10 nm were evident. By comparison, a
highly structured network of 3-10 nm filaments was
clearly visible from stereo images of whole-mount
erythrophores, since the cells were not embedded in
plastic or stained with lead or uranyl salts. It is this
network of filaments, termed the cytomatrix, that
appeared to mediate pigment transport in intact and
drug-treated erythrophores. The actual mechanism of
transport is not understood but probably depends on
novel properties of the cytomatrix.
What we do know about the properties of the
cytomatrix has largely come from microscope studies.
Earlier electron- and light-microscopic investigations
of melanophores (Green, 1968; Junqueira et al. 1974;
Marsland, 1944; Marsland & Meisner, 1967; Schliwa &
Euteneuer, 1978) and erythrophores (Byers & Porter,
1977; Luby & Porter, 1980; Luby-Phelps & Porter,
1982) indicated that an elastic cytoplasmic component
might produce resolute motion of granules. Green
(1968) suggested that granules were embedded in an
elastic continuum that synergistically contracted during aggregation. She envisaged the cytomatrix gathering the suspended granules together in a tightly packed
concentric ring at the cell centre. Utilizing fast-freezing
and freeze-sublimation techniques Porter et al. (1983)
in fact showed that part of the cytomatrix, the acytomatrix, co-aggregated with the granules. Ip et al.
(1984) examined fast-frozen, deep-etched rotary replica shadowed erythrophores and found that the cytomatrix was composed, in part, of 2nm filaments that
cross-linked the surfaces of pigment with microtubules. In contradiction to Porter and colleagues, they
found that there was little change in the cross-linking
activity of these filaments during transport. In further
disagreement with Porter they did not observe marked
changes in the organization of the coarse filamentous
(18 nm filaments) cytoplasmic ground substance during pigment transport.
Biochemical information about the cytomatrix composition would help resolve how transport occurs. On
the basis of preliminary studies of cell lysates, Murphy
& Grasser (1984) observed that 10 nm intermediate
filaments might constitute part of the cytomatrix, and
they suggested a possible involvement of 10 nm filaments in transport. Our data suggest that 10 nm
filaments might be present, perhaps arranged in an
intricate three-dimensional network.
The results presented here indicate that the cytomatrix is intimately linked with the microtubules and
the centrosome and is important for transport in intact
cells. In fact, we believe that the rapid rates of pigment
aggregation (10-20jtims~') that were observed in intact cells might depend on minute interactions of the
cytomatrix with microtubule surfaces. Clearly, removal of the microtubules indirectly disrupted the
578
M. E. Stearns and M. Wang
cytomatrix organization and inhibited transport in
cultured cells. In vivo nocodazole produced clumps of
pigment and aggregation initially involved a bizarre
pattern of pigment migration, which did not occur at
normal rates or in a recognizable, reproducible pattern.
Without the microtubules, it appeared that the cytomatrix must have slowly collapsed towards the cell
centre with the pigment in tow. Since subsequent
pigment translocations were normal, albeit at reduced
rates of 6Jumols~1, it would appear that following
aggregation the cytomatrix was reorganized into a
functional entity. For these complex events to occur we
believe that the centrosome or microtubule-organizing
centre (MTOC) must serve as an organizing centre for
the cytomatrix components, facilitating their reassembly independently of any microtubules. Clearly, the
reorganization and related activities of the cytomatrix
were not fortuitous, since pigment motion involved
energy-dependent processes that were highly polarized.
We do not understand why nocodazole levels raised
above 1 [JM should inhibit pigment transport in erythrophores. In taxol-treated erythrophores, where the
microtubules and MTOC were stabilized, 1 jUM-nocodazole did not inhibit transport, indicating that the drug
did not affect the motor or exert non-specific effects on
the cytomatrix that could culminate in the inhibition of
motion. Likewise, nocodazole at 0-4— 0-8^M levels did
not exert an additive effect when estramustine was used
to inhibit transport. One possible effect of 1 ^Mnocodazole was to disrupt the MTOC and indirectly
interfere with the reorganization and maintenance of a
functional cytomatrix. In agreement with this idea we
found that 1 /zM-nocodazole did not inhibit transport in
taxol-treated erythrophores where the microtubules
were stabilized and the MTOC maintained intact. Our
hypothesis is in keeping with our premise that the
cytomatrix might be structured in a three-dimensional
radial array of filaments that originates from the
MTOC.
Estramustine studies
Our studies revealed the micromolar levels of estramustine (EM) inhibited pigment transport in erythrophores. By comparison, nocodazole treatment of
taxol-stabilized cells and anti-microfilament drugs did
not affect transport.
Estramustine is a therapeutic agent used to treat
advanced prostatic cancer (Joonsson et al. 1977).
Prostatic carcinoma cells in culture (Hartley-Asp &
Gunnarsson, 1982) and HeLa and Walker 256 carcinoma cells (Tew«( al. 1983) have shown a sensitivity to
micromolar levels of estramustine. The cytotoxic effects of EM were independent of the drug's constituent
steroid and alkylating agent species (Tew et al. 1983),
and have been linked with hydrophobic interactions
and with the drug's destructive effects on microtubules
(Stearns & Tew, 1985, unpublished data; Wallin et al.
1985). At micromolar levels, EM caused a rapid
disassembly of microtubules in fish erythrophores and
human prostatic DU145 tumour cells (Stearns & Tew,
1985) and also produced the rapid disassembly of
mitotic spindle microtubules in DU145 cells (Stearns
& Tew, unpublished data). The effects were rapidly
reversed upon washing out the drug with fresh media
and secondary effects of the drug on cell survival were
minimal, except for DU145 cells, which accumulated
the drug in vesicles and eventually died (Stearns et al.
1985). One very interesting effect of EM was rapid yet
reversible inhibition of organelle transport even before
microtubule disassembly occurred in fish erythrophores (Stearns & Tew, 1985) and in frog sciatic nerve
fibres (Kanje et al. 1985). We investigated whether EM
might disrupt transport and ultimately destroy microtubule architecture by directly binding several microtubule-associated proteins (MAPs). We found that
[ 3 H]EM and fluorescent dansylated EM bind MAPs in
vitro to inhibit their association with microtubules
(Stearns & Tew, unpublished data), and recently we
have demonstrated that [ 3 H]EM binds purified MAP-2
with a /Q) = 15 fXtA (Stearns & Tew, unpublished data).
We believe that EM might bind the MAP (MAP-2)
component of the cytomatrix to inhibit reorganizational processes essential for both aggregation and
dispersion. Recent monoclonal antibody labelling
studies in our laboratory have shown that MAP-2
composed part of the a'-cytomatrix that cross-linked
the pigment granule surfaces during pigment translocation (Stearns & Binder, 1987). Thus, in accordance
with the studies reported here EM might bind MAP-2
and inhibit MAP-2-dependent 'cross-linking' activities
between the cytomatrix and granules. In intact cells,
the effects of EM on MAP-2 and microtubule interactions would produce microtubule disassembly. In
taxol-stabilized cells, EM would not affect microtubule
assembly but could still inhibit pigment transport.
This work was supported by American Cancer Society
grant CD-136 to M.E.S. and a grant from A. B. Leo
(Sweden). Donna Platz was of invaluable help in the typing
the manuscript.
We thank George Wray heartily for his generous help and
warm hospitality while we worked at Boulder, Colorado.
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M. E. Stearns and M. Wang
(Received 24 November 1986 - Accepted 19 February 1987)