MICROSCOPY RESEARCH AND TECHNIQUE 58:470 – 480 (2002)

Morphological Studies on the Mechanisms of

Pigmentary Organelle Transport in Fish Xanthophores
and Melanophores
VICTORIA A. KIMLER1* AND JOHN D. TAYLOR2,3
1
Department of Basic Clinical Sciences, University of Detroit Mercy; Detroit, Michigan 48219
2
Departments of Biological Sciences and Radiation Oncology, Wayne State University, Detroit, Michigan 48202
3
Gershenson Radiation Oncology Center, Harper Hospital, Detroit, Michigan 48201

KEY WORDS chromatophore; cytoskeleton; organelle transport; carotenoid droplets; melano-

somes
ABSTRACT Pigmentary organelle translocations within fish chromatophores undergo physio-
logical color changes when exposed to external signals. Chromatophores can be isolated in high
yields, and their pigmentary organelles can be tracked readily by microscopy. The combined efforts
of morphology and biomolecular chemistry have led to the identification of and determination of the
interrelationships between cytoskeletal elements and accessory proteins, motor molecules, cytoma-
trix, and pigmentary organelles of various sizes. Fish chromatophores have been classified as fast,
intermediate, and slow translocators, based on the relative numbers of microtubules. Studies on
cultured goldfish (Carassius auratus L.) xanthophores for over 20 years have demonstrated that in
this slow translocator, tubulovesicular structures of the smooth endoplasmic reticular (SER)
cisternae are involved in the disperson and aggregation of associated carotenoid droplets (CD) with
some involvement of cytoskeletal elements. Killifish (Fundulus heteroclitus L.) melanophore, a fast
translocator, was also examined. Recent work demonstrates a bright fluorescent “starburst”-like
spot that we call an actin filament-organizing center (AFOC) with radiating microfilaments, akin to
the microtubule-organizing center (MTOC) with radiating microtubules. Melanosomes translocate
single-file on microtubules and are not associated with SER cisternae. Slower CD dispersion or
aggregation in goldfish xanthophores seems to be predominantly microfilament-based transport, or
microfilament- and microtubule-based transport, respectively. Faster melanosome translocations
in killifish melanophores are based on microtubules, with our evidence indicating microfilament
involvement. Neural crest-derived chromatophores are models for vesicular transport in axons, and
immunocytochemical and imaging technologies may help to elucidate the cellular transport mech-
anisms. Microsc. Res. Tech. 58:470 – 480, 2002. © 2002 Wiley-Liss, Inc.

OVERVIEW stages of secretion (Lo et al., 1980). The stages of dis-

Neural crest-derived fish chromatophores, situated
within the dermis, may be convenient models for cy-
toskeleton-dependent anterograde and retrograde
transport in neurons, as well as for early stages of
secretion in irritable cells. The mechanisms of pigmen-
tary organelle dispersion and aggregation in the gold-
fish xanthophore have been studied in relation to cy-
toskeletal structure in the killifish melanophore. Early
work on the goldfish xanthophore, what was then
called the erythrophore, began with the characteriza-
tion of the pigmentary organelles (Matsumoto, 1965;
Matsumoto and Obika, 1968), before xanthophore stud-
ies were extended from Japan to Detroit, Michigan.
Whole fish scale tissues and cultured individual chro-
matophores have been examined by thin-section and
whole-cell (WC)TEM, immuno/fluorescence, and confo-
cal microscopy in the Taylor and Tchen laboratory. The
dispersion of CD in the xanthophore was found to be
concomitant with the dispersion of smooth endoplasmic
reticulum (Winchester et al., 1976), and actin filaments
in cultured xanthophores were involved in filopodial
formation and in CD associations, implicating early
persion or aggregation of CD involve rearrangements
and interrelationships of specific cytoskeletal elements
(Palazzo et al., 1989a; Walker et al., 1984). Later work
by WCTEM revealed that CD are attached to or are
part of elongating and retracting tubulovesicular struc-
tures of SER cisternae by WCTEM (Kimler et al.,
1993), supported by work with cytoskeletal inhibitors
and immunocytochemical identification (Kimler and
Taylor, 1995).
Four physiological states for pigmentary organelle
distribution can be generalized to all chromatophores:
dispersing, dispersed, aggregating, and aggregated
states, which are induced by hormones, such as ACTH,
for dispersion and epinephrine, for aggregation and
other agonists, such as caffeine or camp, for dispersion.
*Correspondence to: Dr. Victoria A. Kimler, Department of Basic Clinical
Sciences, College of Health Professions, University of Detroit Mercy; Detroit, MI
48219. E-mail: kimlerva@udmercy.edu
Received 11 December 2001; accepted in revised form 15 January 2002
DOI 10.1002/jemt.10165
Published online in Wiley InterScience (www.interscience.wiley.com).

© 2002 WILEY-LISS, INC.

 PIGMENTARY ORGANELLE TRANSLOCATIONS
The rates of these pigmentary organelle translocations
will vary among chromatophores: the goldfish xan-
thophore is one of the slowest translocators, while the
killifish melanophore is one of the most rapid, and the
increased density of microtubules seems to relate to the
increased rates of pigmentary organelle translocations
(Junqueira et al., 1974; Schliwa, 1982). Slower trans-
locators, such as the goldfish xanthophore (Palazzo,
1984; Palazzo et al., 1989a; Winchester et al., 1976)
appear to rely on the elongation and retraction of tu-
bulovesicular SER, which carry the pigmentary or-
ganelles. The cisternal elongation and retraction re-
quires microfilaments, microtubules, and intermediate
filaments in different phases of CD translocations
(Kimler et al., 1993). The faster translocators, such as
the killifish melanophore (Bickle et al., 1966; Jun-
quieira and Porter, 1971) use radial microtubules as a
means for pigmentary organelle transport. Recently,
microfilaments were found to emanate from the cyto-
center, which followed the radial pattern of the micro-
tubules in the killifish melanophore. We are calling
this central region of actin labeling the AFOC (Haddad
et al., 1995; Kimler and Vallarapu, 1997; Kimler et al.,
2002), and melanosomes may also translocate upon its
radial microfilament tracks. Fluorescence and confocal
microscopy indicated that the AFOC is close to the
MTOC, and that the radial distribution of microfila-
ments remains similar in three of the four states of
pigmentary distribution. The fully pigment-aggregated
state blocks AFOC viewing. The structural continuum
of aligned melanosomes within the killifish melano-
phore (Green, 1968) supports this observation of both
radial and parallel microtubules and microfilaments. It
would be interesting to determine if the AFOC is
involved in organizing and trafficking pigmentary
organelles from this central “hub” in the fast translo-
cators, and, also if the AFOC exists in the slow trans-
locators, where the SER may need to be compartmen-
talized because of its size. The ultrastructural observa-
tions of chromatophores may provide insights into the
stages of a vesicle’s path of transport in several excit-
able cells, through a physiological as well as a devel-
opmental time-course. It is interesting to note that the
slow migration of pigmentary organelles in the goldfish
xanthophore is still faster than what is considered to be
fast vesicle transport of both the anterograde and the
retrograde directions in the neuron’s axon (Baitinger et
al., 1982; Hammerschlag and Brady, 1989; Lasek and
Brady, 1982; Ochs, 1981b; Tytell et al., 1981). Micros-
copy in the twenty-first century is a powerful tool that
can aid molecular biology to answer how structure and
function can complement each other in these compar-
ative organelle transport systems.
GOLDFISH XANTHOPHORES
Early Studies: Thin-Section TEM Work
The goldfish xanthophore was first investigated us-
ing tissue sections of the scale (Lo, 1979; Winchester et
al., 1976) or cell suspensions for thin-section TEM. In
consequence of the development of culturing tech-
niques, the cells could be viewed by WCTEM (Lo et al.,
1979, 1980). Initial WCTEM work of air-dried xan-
thophores demonstrated the formation of pigmented
filopodia when cells were stimulated with ACTH, while
thin-section TEM showed heavy meromyosin labeling
471
of F-actin within filopodia. WCTEM showed inhibition
of filopodial formation by cytochalasin B destabiliza-
tion, suggesting that CD are associated with microfila-
ments during pigment dispersion (Lo et al., 1980).
Thin-section TEM experiments on the goldfish xan-
thophore indicated that there is melanocyte-stimulat-
ing hormone (MSH)- or cAMP-induced dispersion of
carotenoid-containing SER, which is inhibited by cy-
tochalasin B, an actin destabilizer; and epinephrine-
induced aggregation of this SER by colchicine, a micro-
tubule destabilizer. It was also suggested that the dis-
persion of carotenoids does not involve the release of
pigment from, but rather the movement of, the SER
(Winchester et al., 1976), yet lipid monolayers sur-
rounding carotenoid droplets were found within such
SER cisternae (Lo et al., 1979). Thin-section TEM re-
sults also suggested that carotenoids might be trans-
ported within the agranular reticulum in goldfish
erythrophores (Matsumoto and Obika, 1968). Hor-
mone-induced dispersion of CD and SER was inhibited
by cytochalasin B in primary cultures of xanthophores,
as well (Obika et al., 1978b).
SUBSEQUENT STUDIES: PERMEABILIZED
AND CELL-FREE SYSTEMS
Xanthophores were now recoverable in high yields
with purity for cell culture (Lo et al., 1982), and exper-
iments on the signaling mechanisms for CD transport
began. Molecular players in dispersion and aggrega-
tion were eludicated in permeabilized cell and cell-free
systems (Lynch et al., 1981, 1986a, b; Palazzo et al.,
1989b; Tchen et al., 1988; Wu et al., 1990; Yu et al.,
1989, 1990; Zeng et al., 1989) along with the immuno-
logic characterization of intermediate filaments
(Walker et al., 1989) and the CD phosphoprotein, p57
(Gu, 1991). In order to carefully study the cytoskeleton,
xanthophores were extracted and fixed after inducing
different states of pigment distribution. Early to mid-
dispersion of CD in xanthophores demonstrated micro-
filament involvement, while final dispersion repre-
sented a steady state between microfilament and inter-
mediate filament involvement. Early aggregation was
shown to involve intermediate filaments, while micro-
tubules were suggested to help form and hold together
the final state of pigment aggregation (Palazzo et al.,
1989a; Tchen et al., 1986). Intermediate filaments were
immunolabeled with monoclonal antibodies generated
against goldfish erythrophoroma cell intermediate fil-
ament proteins. The filaments were seen as a perinu-
clear band around aggregated CD and as loosely ar-
ranged when the pigment was dispersed (Walker et al.,
1989). Furthermore, the destabilization of intermedi-
ate filaments by acrylamide treatment inhibited the
aggregation of CD (Tchen et al., 1988). High-resolution
scanning electron microscopy, however, demonstrated
that CD are attached to intermediate filaments in frac-
tured xanthophores (Lim et al., 1987). This intermedi-
ate filament system showed unique reorganizing char-
acteristics in response to pigment aggregation (Wang
et al., 1997), with microtubule involvement in CD
translocations (Chen and Wang, 1993). In the squir-
relfish erythrophore, a fast translocator with numerous
microtubules and large CD, intermediate filaments
were implicated to hold CD in position and coordinate
their movements during transport (Murphy and
472 V.A. KIMLER AND J.D. TAYLOR
Grasser, 1984). These bands were confirmed by fluo-
rescence studies to be vimentin, circumscribing the CD
aggregate (Wang and Stearns, 1986), but not associ-
ated with CD (Stearns and Wang, 1987).
LATEST STUDIES: DEVELOPMENT OF THE
WCTEM TECHNIQUE FOR HIGHLY
FLATTENED XANTHOPHORES
The ability to view ultrastructural features of the
different stages of pigmentary organelle dispersion or
aggregation in cultured xanthophores was now en-
hanced with the use of cytoskeletal stabilizing agents,
phalloidin and taxol, tannic acid with the second glu-
taraldehyde fixation, staining with uranyl acetate, de-
hydration and critical-point drying with freon, as the
WCTEM technique development evolved into its recent
stage. Sharper resolution without the requirement to
extract or permeabilize cells was noted (Kimler et al.,
1993). One of the other utilities developed for optimum
viewing of a xanthophore in culture was to permit cell
process spreading as a function of time in culture.
Individual xanthophores became maximally flattened
until they could not become any thinner and broader
(Flattening Index; F.I. ⫽ 5). The various degrees of
dispersion or aggregation of CD were scored by light
microscopy (Taylor et al., 1991). Cells could be cultured
at flattening indices from 1 to 5, with correlative pig-
mentary organelle indices (P.O.I.) from 1 (fully aggre-
gated) to 5 (fully dispersed). This light microscopic
index could be partially applied to WCTEM, because
the viewing area of a F.I. ⫽ 5 xanthophore was approx-
imately 40% of the entire cell, with the exception of the
cytocenter and the nucleus, where the region was too
thick to accommodate penetrating electrons (Kimler et
al., 1993). The older 5-stage index of pigmentary or-
ganelle dispersion based on in situ melanophores of
amphibian skin (Hogben and Slome, 1931), demon-
strated the positive interdependency of enhanced cel-
lular flattening and also of pigmentary organelle dis-
persion, without demonstrating evidence of highly flat-
tened cells possessing fully aggregated pigment and
highly rounded cells having fully dispersed pigment.
NATURE OF THE CAROTENOID DROPLET
ASSOCIATIONS TO THE TUBULOVESICULAR
STRUCTURES BY WCTEM
By WCTEM, CD were seen in association with the
SER, except in rare cases at the periphery of the cell
where they seemed to be clustered on cortical micro-
filaments. These dynamic cisternae elongated with
ACTH stimulation and dispersed the CD, while they
retracted upon removal of ACTH and consequently ag-
gregated the CD (Kimler et al., 1993; Taylor, 1992).
Tubulovesicular cisternae emerged from and retracted
into carotenoid vesicles, which are smooth surfaced,
electron dense and presumably filled with carotenoid
pigments. Tubulovesicular SER with associated CD
(Fig. 1) is seen in a cultured xanthophore (F.I. ⫽ 5;
P.O.I. ⫽ 5) treated with ACTH for CD dispersion by
WCTEM (for review see Kimler et al., 1993). The
greater electron density of the carotenoid vesicle sug-
gested that this was the origin of the CD aneurysms
found on electron-lucent membranous protrusions. The
cisternae appeared to serve as a ferry to transport CD
(Kimler et al., 1993; Taylor, 1992). Although the outer
leaflet of the lipid bi-layer of the electron-lucent cister-
num was shown to be continuous with the single mem-
brane of the more electron-dense CD, the inner mem-
brane is absent. The electron-lucent short stem that
attaches CD to the cisternum has a lipid bilayer. This
supports earlier observations of lipid monolayers
around CD in xanthophores by thin-section TEM (Lo et
al., 1979), and in squirrelfish erythrophores where CD
lack a limiting membrane by WCTEM (Porter et al.,
1983). There may be hydrophobic interactions between
the diglycerides and the carotenoids, causing the col-
lapse of the inner membrane into the pigment (Kimler
et al., 1993). Thin-section TEM of shrimp chromato-
phores demonstrates that monolayer membrane lipo-
carotenoid granules are attached to lipid bilayers of the
SER, suggesting that these lipocarotenoids are derived
from the external surfaces of the SER cisternae and not
from the Golgi apparatus (McNamara and Sesso,
1982). Microlipid droplets appear to originate from ER
membranes of milk-secreting mammary gland epithe-
lial cells, as well (Deeney et al., 1985; Zaczek and
Keenan, 1990). Even the melanosomes of the xanthic
sailfin molly (Poecilia latipinna Lesueur) develop in
part from an ER-derived vesicle that develops an in-
ternal lamellar framework (Blanchard et al., 1991).
Radioactive tracer and cytochemical studies for TEM
demonstrated SER movements within neuronal axo-
plasm without evidence of cytoskeletal dependency
(Tsukita and Ishikawa, 1979). However, the entire con-
tinuous network of SER probably does not move but
rather small vesicles pinch off and refuse at another
aspect of the cisternum. This may be paralleled with
the elongation and retraction of cisternae from station-
ary SER. Dense-cored vesicles are shown to be attached
to tubulovesicular cisternae in neurons (Droz et al.,
1975); in PC-12 cells by thin-section TEM (Luckenbill-
Edds et al., 1979), and also by WCTEM (Kimler et al.,
1991b). Vesicles were also seen as attached to a retic-
ulum in central nervous system growth cones by
WCTEM (Tsui et al., 1983).
COMPOSITION OF THE TUBULOVESICULAR
STRUCTURES
The next attempt was to determine if the tubulove-
sicular structures were actually SER-derived cisternae
within the xanthophore. Cultured xanthophores were
stained for fluorescence microscopy with a vital dye,
DiOC6(3), originally used to identify SER in CV-1 ep-
ithelial cells (Terasaki, 1990). This staining was found
to be positive for xanthophore SER cisternae, and
ACTH dependent elongation of the cisternae from ca-
rotenoid vesicles (Figs. 2a– c) was demonstrated with
time course fluorescence images of xanthophores (ini-
tial F.I. ⫽ 5; P.O.I. ⫽ 4) treated with ACTH for disper-
sion of CD (for review see Kimler et al., 1993). SER
tubular membranes can be found parallel along the
length of and in association with microtubules. These
membranes change their course of translocation, alter
their shapes from slender to bulbous, and sprout new
termini in time-course studies within several cell types
and in cell-free systems (Dabora and Sheetz, 1988b;
Lee et al., 1989; Terasaki, 1990; Terasaki et al., 1990).
Individual SER cisternal termini have also been
tracked by fast axonal anterograde and retrograde
transport in neurons (Gallant et al., 1992). In the xan-
 PIGMENTARY ORGANELLE TRANSLOCATIONS 473

Fig. 1. The elongated electron-lucent lumen of the tubulovesicular carotenoid vesicles (v). Trilaminar membranes (arrowheads) are
SER (er) and the electron dense CD (cd), which are associated with the found on the cisternae. The CD are part of the cisternae where the
cisternae in a xanthophore (F.I. ⫽ 5: P.O.I. ⫽ 5) treated with ACTH outer layer of the trilaminar membrane is continuous with the CD
for dispersion. The cisternae appear to emerge from electron-dense monolayer membrane (m) by WCTEM. 69,505⫻.

Fig. 2. Fluorescence micrographs showing that a xanthophore, maintained in CD-aggregation me-

dium, and then stimulated with ACTH– containing dispersion medium for 40 minutes, followed by
staining with DiOC6(3) in the dispersion medium, while alive, and photographed at 2-minute intervals,
exhibits the emergence of protrusions (arrowheads) from carotenoid vesicles (v). 1,508⫻. Modified from
Kimler et al., 1993.

thophore, carotenoid vesicles with protrusions could CYTOSKELETAL ELEMENTS-SER CISTERNAL

only be found between the cell center and the periph-
ery, because of the central thickness by WCTEM. At
this level of resolution, protrusions had associated CD.
Carotenoid vesicles with protrusions of different
lengths were seen throughout the cell by DiOC6(3)
staining for fluorescence microscopy, a level of micro-
scopic resolution not able to detect individual CD (Kim-
ler, 1993; Kimler et al., 1993).
INTERACTIONS BY WCTEM
The next stage of experiments was carried out by
WCTEM to determine what cytoskeletal elements are
in proximity to, and which cytoskeletal elements asso-
ciate with CD and/or the tubulovesicular cisternae in
the different phases of dispersion and aggregation. In
the xanthophore, microfilaments and intermediate fil-
474 V.A. KIMLER AND J.D. TAYLOR
aments were in found in bundles and as individual
filaments in proximity to the CD-studded tubulovesicu-
lar structures. Microfilaments were seen in bundles in
the cortical regions and as stress fibers, in proximity to
the tubulovesicular structures in the cell peripheries.
It appeared that elongated cisternae were actually be-
ing pulled into filopodia during lamellar formation
(Kimler, 1993; Taylor, 1992). This supported earlier
observations of dynamic process formation (Lo et al.,
1980; Obika et al., 1978b) as dependent upon a pro-
posed microfilament nucleation center on the plasma
membrane (Lo et al., 1979). Bundles of intermediate
filaments in proximity to cisternae were seen during
pigment aggregation (Kimler et al., 1993), supporting
the fluorescence data of perinuclear band formation by
intermediate filament immunolabeling during aggre-
gation (Walker et al., 1989). Microtubules were seen
parallel or perpendicular to cisternae. Not all of the
tubulovesicular cisternae had microtubules adjacent to
them; however, some cisternae could be found without
adjacent microtubules. Very few of the microtubules
were found without proximal cisternae; most had a
parallel cisternum. Since xanthophore microtubules
emanate from within the cytocenter (Palazzo et al.,
1989a), they may be involved in the retraction of cis-
ternae with consequent aggregation of CD. Meshwork-
like arrangements of microfilaments and intermediate
filaments appeared to make direct contact with CD-
studded cisternae, sometimes appearing as cross
bridges (Kimler, 1993). Recent various microscopic
techniques demonstrated that individual microfila-
ments support vesicular and entire ER translocations
in several plant and animal cells (reviewed in DePina
and Langford, 1999).
CYTOSKELETAL INHIBITOR STUDIES
In organelle transport studies, cytoskeletal inhibi-
tors are used to determine: (1) the presence of a cy-
toskeletal element; (2) how that element interrelates
with other elements; (3) whether cisternae were seen
adjacent to cytoskeletal elements; (4) how cytoskeletal
destabilization may affect the integrity and movement
of organelles; and finally, (5) which cytoskeletal ele-
ments are not involved in maintaining organelle integ-
rity or translocations. Diameter measurements are not
adequate to ensure the identity of cytoskeletal ele-
ments, and cytoskeletal destabilization and immunola-
beling studies are required. The effect of microtubule
stabilization by taxol, or destabilization by nocodazole,
on tubulovesicular cisternae by DiOC(6)3 fluorescence
staining, has been investigated (Lee et al., 1989; Te-
rasaki et al., 1990). In studies by WCTEM, the depoly-
merization of microfilaments or microtubules by re-
spective cytoskeletal destabilizers cytochalasin B and
D or nocodazole revealed different patterns of pertur-
bance of the CD-studded tubulovesicular structures,
indicating that these cytoskeletal elements are proba-
bly necessary to maintain the integrity and the elon-
gation/retraction of the cisternae. By treatment with
the cytochalasin B and D congeners, tubulovesicular
structures became constricted along their length; CD
appeared as if they were being pulled from these SER
cisternae. Furthermore, some CD were also seen as
dissociated from the cisternae in the peripheries of the
cell. As a result of this treatment, microtubules and
intermediate filaments changed their spatial relation-
ships. Because CD failed to aggregate and sometimes
accumulated within the distal lamellae with this treat-
ment, it is likely that microfilaments may be required
for aggregation of CD. Nocodazole treatment demon-
strated that some, but not all, cisternae became vesic-
ulated and then collapsed, suggesting that microtu-
bules are required for the integrity and for the retrac-
tion of those adjacent tubulovesicular structures. As a
result of this treatment, microfilaments and interme-
diate filaments did not appear to change their spatial
relationships, possibly due to the lower numbers of
microtubules in the xanthophore. Destabilization of
intermediate filaments with cyclohexamide resulted in
extensive alteration of the appearance of cells. The
cells appeared to be perturbed morphologically as if
they were in the process of dying. Cycloheximide de-
stabilizes intermediate filaments (Sharpe et al., 1980)
along with the cisternae in xanthophores (Kimler et al.,
1993), which is likely secondary to cycloheximide’s ef-
fect on inhibiting protein synthesis having other cell
biological effects (Lee et al., 1989). Fluorescence mi-
croscopy revealed diffuse staining of cisternae with
DiOC6(3) when xanthophores were treated with cy-
tochalasin B and D, and collapsed cisternae when the
cells were treated with nocodazole. These studies were
performed during the course of pigment aggregation by
cisternal retraction, in that WCTEM viewing was pos-
sible in such flatter peripheral processes (Kimler et al.,
1993).
IMMUNOGOLD LABELING
Immunogold labeling of cytoskeletal elements in the
xanthophore was studied by WCTEM. One cytoskeletal
element, the microtubules, labeled by polyclonal com-
mercial anti-␤ tubulin; actin microfilaments, labeled by
different monoclonal (generated in our laboratory,
Walker, 1984) and commercial polyclonal anti-actin;
and intermediate filaments, labeled by monoclonal
anti-intermediate filament domain proteins p45a and
p60, also generated in our laboratory, were observed
(Walker et al., 1989). Actin labeling was seen on indi-
vidual and in bundles of microfilaments, and associ-
ated with CD-studded tubulovesicular cisternae, indi-
cating the linkage between microfilaments and these
organelles. In this immunocytochemical technique,
monoclonal and polyclonal antibodies against various
phosphoproteins, intracellular receptors, and motor
molecules were also used (Kimler and Taylor, 1995).
CD phosphoprotein (Lynch et al., 1986a), labeled by
monoclonal anti-p57 generated in our laboratory (Gu,
1991; Wu, 1987) was situated upon the CD surface.
Cytoplasmic dynein, labeled by polyclonal anti-
MAP1C, was found within the cytomatrix and on CD.
The protein may be a motor molecule within the xan-
thophore. Furthermore, xanthophore CD appear to
have immunoreactivity to polyclonal anti-PCD6 for a
subunit of the ryanodine receptor (Takei et al., 1990).
Changes in the calcium concentration during pigmen-
tary organelle dispersion and aggregation have been
measured in squirrelfish erythrophores (Kotz and Mc-
Niven, 1992), but the question of whether the CD/
tubulovesicular structures have calcium channels
would need to be determined. The 6 –10-nm filaments,
cytomatrix elements, and cisternae/vesicles (and occa-
 PIGMENTARY ORGANELLE TRANSLOCATIONS
sionally CD) were suggested to have a microtubule-
associated protein (MAP) 2a ⫹ 2b by polyclonal anti-
MAP 2a ⫹ 2b labeling. This MAP was also bound to
intermediate filaments as well as to microtubules
(Bloom and Vallee, 1983) and found to induce actin
gelation (Caceres et al., 1984) in neurons, and was
associated with vesicles in chromaffin cells (Severin et
al., 1991). Immunogold-antibody conjugate labeling
techniques demonstrate the molecular localization of
cytoskeletal actors involved in the different stages of
CD translocations, and the identity of the accessory
proteins that propel the CD-studded cisternal machin-
ery. WCTEM of non-permeabilized cells with measure-
ments of the cytoskeletal elements, cytoskeletal inhib-
itor studies, and immunogold labeling of the cytoskel-
eton, all reveal which element is prevalent in different
stages of pigmentary organelle translocations. Micro-
filaments play a major role and microtubules a minor
role in the elongation and the integrity of the CD-
studded tubulovesicular structure. Microfilaments, in-
termediate filaments, and microtubules all play a role
in retraction and the integrity of this organelle com-
plex. Microfilaments and intermediate filaments ap-
pear to work together in early aggregation of CD. These
data infer that cytoskeletal elements interact with
each other in the course of pigment dispersion and
aggregation.
SOME RECENT STUDIES ON
XANTHOPHORES OF OTHER FISH SPECIES
The recent work on the histology of xanthophores of
the gilthead seabream (Sparus aurata L.) demon-
strated that the cells are the only chromatophore spe-
cies in the epidermis, although they were also present
within the dermis (Ferrer et al., 1999). Epidermal xan-
thophores, as well as dermal ones, with CD of larger
size, which are more tightly packed in the cytoplasm
than in goldfish xanthophores, were also found in the
Antarctic notothenoid teleost (Trematomous bernacchi
Boulenger). CD are the only pigmentary organelles in
both dermal and epidermal xanthophores. These CD
were found to have no limiting membrane (Obika and
Meyer-Rochow, 1990). Additionally, studies on the ty-
rosine kinase-mediated pathway through the mamma-
lian lactation hormone, prolactin, for dispersion of pig-
ment in teleost erythrophores and xanthophores
(Oshima and Goto, 2000) may be linked to carotenoid
droplet dispersion being analogous to the first stages of
secretion in the goldfish xanthophore (Tchen et al.,
1986), if one considers the mechanism of milk droplet
release from ER of mammary epithelial cells (Deeney
et al., 1985; Zaczek and Keenan, 1990). Finally, on a
molecular level, an orthologue of the kit-related gene
fms was shown to be necessary for the differentiation of
neural crest-derived xanthophores in the zebrafish
(Danio rerio, Hamilton; Parichy et al., 2000).
KILLIFISH MELANOPHORES
Early Studies: Pulsatile Translocations
of Melanosomes
Killifish melanophores have been a popular TEM
experimental model to exemplify physiological color
change by microtubule-dependent melanosome trans-
locations within melanophores (Bickle et al., 1966;
Green, 1968; Murphy and Tilney, 1974). Melanosomes
475
have been suggested to translocate within “fixed chan-
nels,” where the granules returned to the same area
from where they came. Microtubules, which did not
terminate directly on the surface of the granules, re-
tained their constant length even when pigment gran-
ules were aggregated (Bickle et al., 1966). The pro-
cesses of the melanophore would, therefore, not follow
the distribution of the moving pigment front. Early
citations have described melanosome movements in
the killifish melanophores, in situ on the scale, as pul-
satile (Bickle, 1966; Green, 1968; Marsland, 1944; Mat-
thews, 1931; Smith, 1930; Spaeth, 1916). The driving
forces behind the pulsatile mechanisms has been based
on cytoplasmic changes in that the melanophore be-
haves like a smooth muscle cell so that its arms would
contract and relax, carrying the pigmentary granules
(Spaeth, 1916). Later, the term “contraction” became
known as an aggregation of pigment. The process is not
entirely comparable to that of isotonic muscle contrac-
tion, because in pigment aggregation, the whole cell
does not contract. Early workers who studied the cyto-
plasm of melanophores believed that pigment concen-
tration involved the contraction of the plasmagel sys-
tem where the pigment granules were affixed in the
gel-like framework. The hyaline protoplasm of the mel-
anophore soma was considered to be the plasmasol,
while the protoplasm as peripheral to the pigmentary
organelles was the plasmagel. Contraction of the pig-
mentary organelles was thought to be caused by pro-
toplasm gelation, while expansion by solation (Mars-
land, 1944). Later, workers began to call this motile
cytoplasm the microtrabecular lattice, which might be
the driving force behind organelle translocations, be-
cause its conformation changed during intracellular
motility (Porter, 1984). The external factors such as pH
and electrolyte concentration changes, in addition to
the aforementioned internal factors, might stimulate
pulsations (Bickle, 1966; Marsland, 1944; Smith, 1930;
Spaeth, 1916), which could be creating voltage-gated
responses to depolarize or repolarize chromatophores
for pigment dispersion or aggregation, respectively.
Subsequent Studies: The Cytoskeleton, the
Structural Continuum, and
Other Driving Forces
This structural continuum where individual melano-
somes translocate in single file along microtubules
(Green, 1968; Murphy and Tilney, 1974) was shown by
thin-section TEM to be bounded by ER cisternae in
that membranes surrounded the granules and perme-
ated the granule mass (Green, 1968). These melano-
somes appear to be connected to microtubules by tiny
cross bridges by thin-section TEM (Murphy and Tilney,
1974), and by WCTEM (Haddad et al., 1995; Kimler
and Vallarapu, 1997; Kimler et al., 2002), supporting
the argument of the structural continuum. The identity
of the cross bridges is yet to be determined, however,
the suggestion of motor molecules is plausible. Addi-
tionally, the energy cycling in oxidative phosphoryla-
tion (Junqueira et al., 1974) along with a force-trans-
ducing ATPase (Clark and Rosenbaum, 1984) to drive
the rapid melanosome translocations, have also been
investigated within the killifish melanophores.
476 V.A. KIMLER AND J.D. TAYLOR

Fig. 3. Serial reconstruction of all sections, by laser scanning confocal microscopy, to create a
three-dimensional image of a melanophore (F.I. ⫽ 4; P.O.I. ⫽ 4), maintained in control culture medium,
and for fluorescent identification, subsequently labeled with rhodamine-phalloidin. Note the AFOC
(arrowhead) and radiating microfilaments (mf) and cortical microfilaments (c). 1,541⫻.

Latest Studies: phore, by labeling cells with rhodamine-phalloidin for

The Actin-Filament Organizing Center
In challenging the assumption that microtubules
serve solely as tracks for the structural continuum as
described by Green (1968), we proposed that actin po-
lymerization may represent the motile force for mela-
nosome aggregation, while depolymerization repre-
sents the relaxation leading to melanosome dispersion
(Obika et al., 1978a). Furthermore, the microtubules of
a cultured killifish melanophore were shown to emerge
from the cell center in dense and periodic radial arrays
from the microtubule-organizing center (MTOC) by flu-
orescence microscopy. The interaction of the MTOC
with melanosomes in translocations has been eluci-
dated in killifish melanophores (Rodionov et al., 1991).
Based on the evidence of the actin polymerization/de-
polymerization and the existence of the MTOC, we
sought to determine the nature of that central “star-
burst-like” region in the center of a cultured melano-
fluorescence microscopy. We proposed the existence of
an actin filament-organizing center (AFOC) by
WCTEM, fluorescence (Haddad et al., 1995; Kimler
and Vallarapu, 1997), and laser scanning confocal mi-
croscopic examination (Kimler et al., 2002). Confocal
imaging of melanophores with dispersed pigment ap-
pears to have solved the limitation of two-dimensional
viewing of the AFOC by serial optical sectioning of the
melanophores. By rhodamine-phalloidin staining, radi-
ating microfilaments were observed on most sectional
planes with the brightest fluorescence toward the cen-
ter of the cell, indicating that the AFOC may distribute
microfilaments in three dimensions. When a serial re-
construction of all sections is performed to create a
three-dimensional image, a dense radial arrangement
of microfilaments from the cell center out to the cortex,
as well as cortical microfilaments parallel to the perim-
eter of the cell’s leading edges (Fig. 3), can be noted in
 PIGMENTARY ORGANELLE TRANSLOCATIONS
Fig. 4. a– d: Two melanophores were either treated with caffeine
for dispersion (P.O.I. ⫽ 5) or epinephrine for aggregation (P.O.I. ⫽ 1),
and double-labeled with rhodamine-phalloidin and anti-␤ tubulin. a:
The caffeine-treated melanophore demonstrated a radial pattern of
microtubules (mt) at the MTOC (arrowhead) by anti-␤ tubulin label-
ing (550⫻). b: Similar “starburst-like” center of microfilaments (mf)
from the AOC (arrowhead) with rhodamine-phalloidin labeling
this melanophore (F.I. ⫽ 4; P.O.I. ⫽ 4) by confocal
microscopy (for review see Kimler and Vallarapu, 1997;
Kimler et al., 2002). When melanophores were double-
labeled with rhodamine-phalloidin and FITC-conju-
gated anti ␤-tubulin, both radial microfilaments, as
well as radial microtubules were seen, following the
same pattern in three out of the four physiologic states
of pigment distribution. This is demonstrated in two
double-labeled melanophores that were either treated
with caffeine for dispersion (F.I. ⫽ 5; P.O.I. ⫽ 5), and
labeled with anti ␤-tubulin (Fig. 4a) or rhodamine-
phalloidin (Fig. 4b); or epinephrine for aggregation
(F.I. ⫽ 5; P.O.I. ⫽ 1) and labeled with anti ␤-tubulin
(Fig. 4c) or rhodamine-phalloidin (Fig. 4d) for fluores-
cence microscopy (for review see Kimler and Vallarapu,
1997; Kimler et al., 2002). The AFOC was obscured by
the aggregated pigment mass in most cases, and punc-
tate labelling of actin was found upon the aggregate in
several small bright regions. Finally, to elucidate the
role of the microfilaments within the aggregate, we
first treated cells with cytochalasin B to destabilize
actin filaments, and then, epinephrine, to induce ag-
gregation, in this order, and as reversed. In both cases,
477
(584⫻). c: The epinephrine-treated cell demonstrates microtubules
(mt), with a slight appearance of the MTOC (arrowhead) with anti-␤
tubulin labeling (496⫻). d: Minimal “starburst-like” center of micro-
filaments (mf) from the AOC (arrowhead) with punctate labeling
(arrowhead) on top of the aggregate by rhodamine-phalloidin fluores-
cence (530⫻).
most cells appeared to have looser aggregates, with
some pigment spilling out of the chromatophore in
several cases. Cytochalasin B inhibited the stages of
early aggregation, suggesting that microfilaments may
be required to secure the melanosomes within the ag-
gregate. Based upon this observation of a radial label-
ing at the site of the centrioles with rhodamine-phal-
loidin, it is likely that the AFOC exists. Microtubule
and microfilament interaction, as well as motor mole-
cules involvement in pigmentary organelle transloca-
tions, has been well established in fish chromatophores
(Akiyama et al., 1990; Kimler et al., 1993; Kimler and
Taylor, 1995; Palazzo et al., 1989a; Rodionov et al.,
1998; Schliwa, 1982). It is possible that the MTOC has
a partner structure, the AFOC, which is mutually in-
volved in melanosome translocations. In general, the
AFOC may be able to: (1) help maintain cell shape; (2)
serve as a model organizing center for the spindle fi-
bers in mitotic cells; (3) change its conformation during
mitosis and differentiation; (4) support bi-directional
vesicular transport in chromatophores as well as in
neurons, with its scaffolding formed by microtubules
and microfilaments bounded by cross bridges; (5) serve
478 V.A. KIMLER AND J.D. TAYLOR
to secure pigment during pigment aggregate formation;
and (6) play a role in exocytosis and in endocytosis.
THE GOLDFISH XANTHOPHORE: A SLOW
TRANSLOCATOR, AND THE KILLIFISH
MELANOPHORE: A FAST TRANSLOCATOR
Since there are multiple mechanisms involved in
dispersion and aggregation of pigmentary organelles, it
is likely that microfilaments as well as microtubules,
may serve as organelle translocation tracks, contribut-
ing to the total times measured for relative transloca-
tion rates. Within a slow fish chromatophore, the gold-
fish xanthophore, pigment dispersion requires approx-
imately 50 – 60 minutes (Palazzo et al., 1989a;
Winchester et al., 1976) and aggregation takes approx-
imately 60 minutes (Palazzo et al., 1989a) or from
3 hours to overnight (Kimler, 1993). Fast chromato-
phores generally involve seconds to accomplish aggre-
gation, while slow chromatophores, which include some
species of fishes, but mostly crustacean and amphibian
chromatophores, require at least several minutes for
pigmentary organelle aggregation (Schliwa, 1982). In
the killifish melanophore, translocations have been re-
ported to take 1–2 ␮m per second (Junquieira and
Porter, 1971), or dispersion and aggregation to take a
few minutes (Bickle et al., 1966). We have classified
these killifish melanophores as fast translocators com-
pared to intermediate or slow translocators. Our clas-
sification is also based upon the amount of “membra-
nous baggage” in the form of cisternae, which the cy-
toskeleton must translocate (Kimler, 1993).
DO THE XANTHOPHORE AND THE
MELANOPHORE HAVE THE SAME KINDS OF
MOTILE SYSTEMS?
As evidenced by thin-section TEM, WCTEM, and
fluorescence microscopy, the cytoskeleton as well as the
elongating and retracting tubulovesicular cisternae ap-
pear to lack a linear order in the goldfish xanthophore,
with sparse microtubules and a meshwork of microfila-
ments and intermediate filaments throughout the cell
(Kimler et al., 1993). As shown by WCTEM, fluores-
cence, and confocal microscopy, microtubules and mi-
crofilaments in the killifish melanophore appear to
“cradle” columns of pigment in a linear conformation in
translocations (Kimler et al., 2002). Additionally, the
lipid monolayer CD as associated to the lipid bilayer of
large SER cisternae has a different membranous struc-
ture than the single-file arranged melanosomes, which
have a lipid bilayer. Finally, the xanthophore is a slow
translocator, while the melanophore is a rapid translo-
cator. It is likely that different cytoskeletal arrange-
ments are required for the xanthophore’s irregularly
shaped CD-cisternal complex resting in a heteroge-
neous cytoskeletal arrangement, versus the melano-
phore’s orderly translocation, with large melanosomes
and few SER membranes, along parallel cytoskeletal
tracks. It is probably faster for melanosomes to travel
along the linear cytoskeletal tracks than for the cister-
num, which carries attached carotenoid droplets, than
to travel along a meshwork of actin filaments with few
microtubules. These are probably inherent adaptive
coloration mechanisms that evolved for each of these
respective species of fishes in their environments.
PERSPECTIVE
The pigment cell may serve as an excellent model for
understanding the mechanisms of axonal transport
and the mechanisms of endocytosis and exocytosis
within neuronal termini. The melanophore may serve
as a model for understanding the roles of the centrioles
in the formation of spindle fibers in mitosis. Actively
dividing cells may use microfilaments in the different
phases of mitosis. The fact that the melanophore has
radiating microfilaments from the cytocenter and in
the same parallel conformation as microtubules indi-
cates that microfilaments may work with microtubules
for securing and releasing pigmentary organelles in
different states of melanosome distribution. Morpho-
logical studies such as the investigations described
above on pigmentary organelle transport and cytoskel-
etal conformation in chromatophores, permits one to
visualize the phenotypes that genes code for. Research
in molecular biology is re-emphasized when we can
study the dynamics of cell structure and function with
current microscopic techniques. Eventually, the regu-
lation of many of the chromatophores’ genes will per-
mit us to present morphological, developmental, and
pathologic scenarios as models for mammalian systems
on a cellular level.
ACKNOWLEDGMENTS
The authors thank Dr. Bryan Miller for critical re-
view of the manuscript, and Mr. Eric Jacobs for assis-
tance in the electronic imaging for the figures pre-
sented in this review.
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