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Cell Motility and the Cytoskeleton 38:397?409 (1997)
Evidence for Several Roles of Dynein
in Pigment Transport in Melanophores
Hele?n Nilsson and Margareta Wallin
Department of Zoophysiology, University of Go?teborg, Go?teborg, Sweden
Melanophores are specialized cells that transport pigment granules to and from the
cell center, giving animals the ability to change skin color. A kinesin-related
plus-end motor has previously been shown to be responsible for pigment granule
dispersion [V.I. Rodionov, F.K. Gyoeva, and V.I. Gelfand. Proc. Natl. Acad. Sci.
USA. 1991, 88:4956-4960]. Here, we have microinjected a dynein antibody (70.1)
into cultured cod (Gadus morhua) melanophores and used the dynein inhibitor
vanadate on permeabilized melanophores in skin pieces, to examine the role of the
microtubule minus-end motor dynein in these cells. Both pigment granule
aggregation and maintenance of the spherical central pigment mass (CPM) were
inhibited by the antibody and by vanadate. Vanadate or antibody treatment of cells
with aggregated pigment did not induce pigment dispersion. However, when the
antibody-injected cells were induced to disperse pigment, the pigment moved
farther to the cell periphery, which resulted in a depletion of pigment in the cell
center. Similar superdispersion of previously uniformly distributed pigment was
also seen when the antibody was injected in melanophores with dispersed pigment.
Our results demonstrate that both pigment aggregation and maintenance of the
CPM are dynein-dependent processes. Our data further show that dynein is
involved in the homogeneous distribution of dispersed pigment. These results
suggest that both dynein and kinesin are active in keeping pigment granules
dispersed throughout the cytoplasm, transporting pigment granules in opposite
directions. The possibility that dynein is continuously active during both aggregation and dispersion, while kinesin might be the target for regulation, is discussed.
Cell Motil. Cytoskeleton 38:397?409, 1997. r 1997 Wiley-Liss, Inc.
Key words: melanophore; chromatophore; pigment; vanadate; kinesin; dynein; fish; Gadus morhua
INTRODUCTION
Chromatophores are regarded as excellent model
systems for studies in regulation of intracellular transport
of membrane-bound organelles [Haimo and Thaler, 1994]
and in organization of microtubules [Rodionov and
Borisy, 1997a,b]. Pigment granules can aggregate to the
center of the cell to form a spherical central pigment mass
(CPM), or disperse evenly throughout the cytoplasm.
These movements are controlled by neurohumoral stimuli
and determine the pigmentation of the animal [Fujii and
Oshima, 1994]. Microtubules are important for maintaining cell morphology and for movement of pigment
granules [Schliwa and Bereiter-Hahn, 1973a,b; Murphy
and Tilney, 1974; Ochs, 1982; Obika and Negishi, 1985;
Visconti and Castrucci, 1985; Chen and Wang, 1993;
r 1997 Wiley-Liss, Inc.
Nilsson et al., 1996], and McNiven and Ward [1988]
showed the first clear evidence that pigment granules
were associated to microtubules by using scanning electron microscopy of lysed erythrophores.
We have recently shown by immunocytochemistry
that both the minus-end directed motor dynein and the
Contract grant sponsors: Swedish Natural Science Research Council;
Helge Ax:son Johnsons Stiftelse; Nordenskjo?ldska Swedenborgsfonden; Kungl. och Hvitfeldska Stipendieinra?ttningen; Magnus Bergvalls
Stiftelse; Lars Hiertas Minne; Hierta Retzius Stipendiefond
*Correspondence to: Margareta Wallin, Department of Zoophysiology,
Go?teborg University, Medicinaregatan 18, S-413 90, Go?teborg, Sweden; E-mail: m.wallin@zool.gu.se
Received 18 August 1997; accepted 19 August 1997
398
Nilsson and Wallin
plus-end motor kinesin co-localize with both aggregated
and dispersed pigment in cultured Atlantic cod (Gadus
morhua) melanophores [Nilsson et al., 1996]. Another
indication that these proteins are bound to melanosomes
is that both dynein and a kinesin-like protein are present
on Western blots of isolated melanosomes from cultured
Xenopus melanophores [Rogers et al., 1997]. Microinjection of a polyclonal kinesin antibody into black tetra
melanophores suppressed pigment dispersion [Rodionov
et al., 1991], showing that kinesin, or a kinesin-like
protein, has an important role in dispersion (for a
discussion, see Rogers et al. [1997]).
The evidence for a role of dynein in aggregation is
more indirect, however. Vanadate and EHNA (erythro-9[3-(2-hydroxynonyl)]adenin), inhibitors of cytoplasmic
dynein ATPase in vitro [Paschal and Vallee, 1987;
Shimizu et al., 1995; Niclas et al., 1996], inhibit pigment
aggregation [Beckerle and Porter, 1982; Clark and Rosenbaum, 1982]. However, caution is necessary, as these
substanses are known to inhibit cellular activities other
than based on dynein [Kobayashi et al., 1978; Pike et al.,
1978; Penningroth, 1986; Porter et al., 1987]. Many
different types of microtubule motors have been found in
recent years [Moore and Endow, 1996], and the lack of
specific inhibitors of dynein have so far made it difficult
to assess the contribution of dynein in transport processes, as the different motors cannot be pharmacologically distinguished from one another in crude systems
[see Collins, 1994, for discussion].
A monoclonal antibody (mAb) to dynein (70.1) has
recently been shown to inhibit dynein-dependent organization of microtubules [Heald et al., 1996], an antibody
that makes it possible to determine the role of dynein in
melanophores. We have microinjected 70.1 into cultured
cod melanophores to study the role of dynein in four
different processes: aggregation, maintenance of the
CPM, dispersion, and the homogeneous distribution of
melanosomes throughout the cytoplasm. We have also
used vanadate on permeabilized skin melanophores for
similar studies to be able to discuss the use of vanadate as
a chemical tool and to make comparisons with older data.
MATERIALS AND METHODS
Chemicals and Antibodies
Sodium orthovanadate and vinblastine (Sigma
Chemical Co., St. Louis, MO) were dissolved in H2O,
stored at 220蚓 in dark, and diluted 1 : 1,000 to the
experimental concentrations in buffer just before use.
Mowiol was from Hoechst (Frankfurt, Germany).
The 70.1 antibody against chick brain cytoplasmic
dynein [Steuer et al., 1990], control ascites fluid, as well
as an antibody against b-tubulin, were from Sigma.
Secondary antibodies conjugated with Texas red or
fluorescein were from Amersham (Stockholm, Sweden).
Secondary antibodies conjugated with 12 nm colloidal
gold, as well as normal serum from donkey, were from
Jacson Immuno Research Laboratories (West Grove, PA).
Goat antimouse Ig conjugated to horseradish peroxidase
(HRP) and peroxidase substrate kit were from BioRad
(Richmond, CA).
Melanophore Culture
Melanophores were cultured according to a modified procedure described by Nilsson et al. [1996]. The cod
fin explants formed a monolayer of skin cells including
melanophores in medium 199 with Hank?s salts supplemented with 10% fetal calf serum (FCS), 1% antibiotic/
antimycotic solution, and 1% L-glutamine from Life
Technologies (Renfrewshire, Scotland). Thereafter, the
cells were further cultured in serum-free CO2-independent medium supplied with 1% antibiotic/antimycotic
solution and 1% L-glutamine (Life Technologies), 5
痢/ml insulin, 5 痢/ml transferrin, and 5 ng/ml sodium
selenite from Boehringer Mannheim (Mannheim, Germany) and 1 mg/ml glucose, to isolate melanophores on
the coverslips and induce detachment of other skin cells.
Melanophores used in the experiments had been cultured
for 1?2 weeks.
Microinjection of Dynein Antibody
and Immunocytochemistry
Microinjections were performed with micropipettes
made from borosilicate glass capillar with filament OD
1.0 mm, ID 0.78 mm, and 10-cm length, from Bergstro?m
Instrument AB (Go?teborg, Sweden), with Micropipette
Puller P-87 from Sutter Instrument (Novato, USA).
Observations of the cells were done in a Nikon diaphote
microscope (Tokyo, Japan), adapted for microinjection
with Eppendorf micromanipulator 5171 and transjector
5246 (Hamburg, Germany). The 70.1 antibody against
cytoplasmic dynein and control ascites were transferred
to a microinjection buffer made according to Rodionov et
al. [1991], by repeated filling and centrifugation at
10,000g 3 3 for 10 min at 4蚓 in Microcon 50
microconcentrator tubes from Amicon (Beverly, MA) and
in a Ole Dich centrifuge (Hvidovre, Denmark). The 70.1
antibody was concentrated to an antibody concentration
of 6 mg/ml and total protein concentration of approximately 270 mg/ml, or 3 mg/ml antibody and 135 mg/ml
proteins, respectively. Both concentrations were tested
and found to give the same effects. Only the lower
concentration was further used in the experiments. The
control ascites was concentrated to a total protein concentration of approximately 140 mg/ml. Working aliquots of
the concentrated 70.1 antibody and control ascites were
Roles of Dynein in Pigment Transport in Melanophores
TABLE III. Effect of Vanadate on Pigment Dispersion
in Skin Melanophores
TABLE I. Effect of Vanadate on Rate of Pigment Aggregation
(MI/min)
Treatment
Control
Control
Saponin
Saponin
Vanadate 10 然
Vanadate 10 然
Vanadate 50 然
Vanadate 50 然
Vanadate 100 然
Vanadate 100 然
ATP
Mean 6 s.d.
Fish
(No)
Sample
(No)
n
y
n
y
n
y
n
y
n
y
1.36 6 0.30
1.49 6 0.20
1.81 6 0.30
1.53 6 0.26
0.75 6 0.10
0.75 6 0.27
0.25 6 0.18
0.23 6 0.18
0.04 6 0.08
0.00 6 0.00
2
2
2
2
2
2
2
2
2
2
8
8
8
7
8
8
8
8
8
8
Treatment
Rate
(MI/min)
Time for
onset (min)
Mean 6 SD
Mean 6 SD
Control
Saponin
Vanadate 10 然
Vanadate 50 然
Vanadate 100 然
0.59 6 0.20
0.79 6 0.21
0.96 6 0.31
0.81 6 0.15
0.82 6 0.28
TABLE II. Effects of Microinjected 70.1 Antibody
on Pigment Aggregation*
Injected substance
Control Ascites
70.1 Antibody 3 mg/ml
Control Ascites
70.1 Antibody 3 mg/ml
Complete
aggregation
Dispersion to
periphery
Total No
cells
8
0
0
13
8
14
*Melanophores with dispersed pigment were injected and thereafter
allowed to rest for 1 h before addition of noradrenaline (NA). Response
to NA was determined after 8 min. Complete aggregation was defined
as Melanophore Index 5 1 or 2. There was a slight peripheral
aggregation in 2 antibody injected cells. Dispersion to the periphery
was analysed 30 min after removal of NA. Numbers indicate No of
cells.
kept at 220蚓. All experiments were performed at 12蚓
in CO2-independent medium. Cells in every treatment
were from at least four different fishes. Control cells were
either injected with ascites fluid, microinjection buffer,
touched with a microneedle or uninjected. Touched and
uninjected control cells were observed simultaneously as
injected cells. The responses of the cells were documented by photography. To verify that the microtubule
system was unaffected by the microinjections of 70.1
antibody, ascites, or buffer, some cells were immunocytochemically stained for tubulin using methanol as fixative
(not shown), according to Osborn and Weber [1982]. To
confirm that the 70.1 antibody solution had spread
throughout the melanophores 30 min after injection,
some cells were fixed and labelled with secondary
antibody (not shown). Because the cells were of different
sizes, the protein concentration of the injected substanses
likely variated within the cells. A clearly visible swelling
of an injected cell was defined as a proper injection.
For aggregation experiments, melanophores were
injected in dispersed state of pigment distribution and
allowed to rest for 1 h. Noradrenaline (norepinephrine,
NA) was thereafter added to the medium to a final
concentration of 1 然 to induce aggregation. After 8 and
Sample
(No)
4
4
4
4
4
16
16
16
16
16
MI 5 Melanophore Index.
TABLE IV. Effects of Microinjected 70.1 Antibody
on CPM and Pigment Dispersion*
Response to NA
Fish
(No)
9.1 6 4.0
6.2 6 2.7
3.1 6 2.3
3.5 6 1.0
2.2 6 0.8
y 5 included, n 5 not included, MI 5 Melanophore Index.
Injected substance
399
Deformed
Dispersion
Total
CPM
Dispersion to periphery No cells
1
12
16
12
0
7
16
12
*Melanophores with noradrenaline (NA) induced aggregated pigment
were injected and allowed to rest in 1 然 NA for 30 min. Deformed
central pigment masses (CPM) were determined after rest. Thereafter
dispersion was induced. Cells with dispersed pigment and dispersion to
cell periphery were determined after 30 min. Numbers indicate No of
cells.
25 min in NA, the pigment distribution in the cells was
analyzed. The cells were thereafter replaced in fresh
medium without NA to initiate dispersion.
For experiments on maintenance of spherical CPM
and dispersion, cells were incubated in 1 然 NA for 15
min to obtain spheric CPMs before the cells were
injected. Injection was made in close approximity to the
CPM. After microinjection, the cells were allowed to rest
in the presence of NA for 30 min, and the amounts of
deformed or spheric CPMs were analyzed. Thereafter the
cells were replaced in fresh medium to initiate dispersion.
Cultured melanophores with dispersed or aggregated pigment were also used for immunocytochemical
staining against dynein according to Nilsson et al. [1996],
with the 70.1 antibody.
Immunoblotting
Characterization of the dynein antibody (70.1)
immunoreactivity in Atlantic cod tissue, was performed
on a vertical 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gel (LKB, Bromma, Sweden), according to
Laemmli [1970]. Cod brains were homogenized in 1 : 2
v/v cold Pipes-MgSO4-EGTA-buffer as described in
Nilsson et al. [1996], and centrifuged at 10.000g for 10
min at 4蚓. The crude supernatant was boiled in sample
buffer (BioRad) for 5 min, and 100 痞 was loaded on each
lane of the gel for electrophoresis and subsequent Western blotting as described before [Nilsson et al., 1996].
400
Nilsson and Wallin
Fig. 1. Skin melanophores were incubated in 10, 50, or 100 然
vanadate for 10 min in lysis buffer with 0.02% saponin. Controls were
incubated in lysis buffer with or without saponin. One-half of the
samples were also treated with 100 然 ATP. Noradrenaline (NA) was
thereafter added to 1 然 to induce pigment aggregation; the process
was measured as melanophore index (MI) every 30 sec. Graph shows
mean rates of aggregation (MI/min) in each treatment. Statistical
analysis on slopes of MI/min to 3 min and on three samples/fish,
showed that pigment aggregation was inhibited by 10, 50, or 100 然
vanadate treatment (ANOVA: F4,10 5 133.1, P , 0.05 and SNK-test
p , 0.05). No influence of ATP was found.
Vanadate and Vinblastine Treatment
of Skin Explants
58triphosphate (ATP) (Sigma). Thereafter, NA was added,
and the rate of aggregation was measured every 30 s for 6
min. Pigment movement was measured according to the
melanophore index (MI), where 1 5 completely aggregated and 5 5 fully dispersed [Hogben and Slome, 1931].
In order to investigate the effects of microtubule
and dynein inhibitors on the spherical CPM, melanophores on skin pieces were induced to aggregate pigment
granules completely by addition of 1 然 NA in medium.
The microtubule disrupting agent vinblastine was thereafter added for 2 h of incubation at a concentration of 3 然
in the presence of NA. Controls were incubated in 1 然
NA only. The skin explants were thereafter fixed in
ice-cold methanol for 6 min and rinsed in PBS 2 3 5 min.
The effect on maintenance of the spherical shape of
CPMs was determined as the amount of deformed CPMs
in percentage of the amount of total CPMs counted from
each well with skin explants. Cultured cells were treated
similarily, and immunocytochemically stained by antibodies against tubulin, to verify disruption of microtubules
(not shown). To reveal whether dynein is involved in
maintenance of the CPM, vanadate was added at different
concentrations. Explants were induced to aggregate melanophore pigment completely, and thereafter incubated
with 1 然 NA with 10, 50, or 100 然 vanadate for 10
min in lysis buffer with saponin. Controls were treated
with lysis buffer with or without saponin. Thereafter the
explants were fixed as previously and the amount deformed CPMs measured.
Experiments on pigment translocation and maintenance of circular CPM were carried out on melanophores
on skin pieces of about 1 mm2 prepared from the dorsal
fins using a diaphote Nikon microscope. Skin pieces were
allowed to adhere to the bottom of sterile plastic culture
dishes with four wells in 1 drop of medium for about 1 hr
before addition of medium 199 and further incubation at
12蚓. The experiments were performed the day after at
12蚓 in medium, or lysis buffer at pH 7.0 made according
to Grundstro?m et al. [1985]. For mild permeabilization of
cell membranes to vanadate, 0.02% saponin was included
in the lysis buffer. Every treatment was performed on four
fish, and from every fish measurements were done on
melanophores on four skin pieces in different wells (total
amount of measurements: 16 per treatment). Aggregation
of pigment granules was induced by addition of 1 然 NA
in the medium. Explants that did not respond by aggregation of the melanophore pigment granules within 3 min
were discharged.
For aggregation experiments, pigment granules
were allowed to redisperse in fresh medium to the next
day. Explants were thereafter incubated in 10, 50, or 100
然 vanadate for 10 min in lysis buffer with saponin. The
time of treatment was standardized to 10 min, as longer
time in 0.02% saponin was found to inhibit NA-induced
pigment aggregation (not shown). Controls were treated
with lysis buffer with or without saponin. One-half of the
samples were also incubated with 100 然 adenosine
Roles of Dynein in Pigment Transport in Melanophores
401
Fig. 2. Characterization of the 70.1 dynein antibody in cod. Immunoblot of a crude cod brain extract (a). Cod brain proteins were separated
on 7.5% SDS-Page and blotted to a nitrocellulose membrane. The
membrane was probed with 70.1 mAb against dynein three light chains
of ,70 kD from chicken. Lane 1, Coomassie blue stained gel; lane 2,
nitrocellulose membrane stained with 70.1 mAb. Distribution of 70.1
mAb immunoreactivity in cultured melanophores with dispersed (b)
and aggregated (d) pigment. c, e: Light micrographs of the two cells,
respectively. Bar 5 10 然.
For dispersion experiments, melanophores were
first induced to aggregate pigment for 4 min by addition
of 1 然 NA. They were thereafter incubated for 10 min in
10, 50, or 100 然 vanadate together with 1 然 NA in
lysis buffer with saponin, or in lysis buffer only with or
without saponin together with 1 然 NA. Measurements
of dispersion was initiated by removal of NA by rinsing
the samples with lysis buffer including only vanadate, or
lysis buffer without vanadate for the controls, and measurements were made every min for 22 min.
In experiments with vanadate and vinblastine, measurements were ??blind?? assays in which the observer was
unaware of treatment. Determination of differences in
amount of spherical shapes of CMPs and differences in
402
Nilsson and Wallin
Fig. 3. Effects of microinjection of 70.1 mAb on pigment granule
aggregation in cultured melanophores. Melanophore touched with a
microneedle (a?d), injected with ascites fluid (e?h), and injected with
70.1 mAb (i?l). a, e, i: Micrographs showing cells before injection. f, j:
At 1 h after injection. e, g, k: After 8 min in NA. d, h, l: After 30 min in
fresh medium without NA. k: Arrow, no pigment aggregation after 8
min in NA. l: Arrows, pigment translocated to the cellular periphery
after replacement of the cells into medium without NA. Bar 5 50 痠.
Roles of Dynein in Pigment Transport in Melanophores
403
rates of pigment dispersion was performed with a factorial analysis of variance (ANOVA) with treatment as
fixed factor, individual fish as a nested factor and
perecentage deformed CPMs or slope of MIs/min in the
interval of translocation with maximum 22 min as
dependent variables. Differences in rates of pigment
aggregation were after random removal of data from one
skin explant/fish, analysed with a factorial ANOVA with
vanadate and ATP treatments as fixed factors, individual
fish as a nested factor and slope of MIs/min in the interval
of translocation with a maximum of 3 min as the
dependent variable. Data from the studies of maintenance
of CPM were transformed by arcsin as recommended for
proportions [Sokal and Rohlf, 1995]. All data were tested
for homogeneity of variance with Cochran?s C test with
a 5 0.05 [Snedecor and Cochran, 1989]. Multiple
comparisons of means were made with Student?Newman
Keul?s test. Differences in time for onset of dispersion,
measured as mean time spent as MI 5 1, were examined
with nonparametric Kruskal?Wallis and Tukey-type multiple comparisons as data were noncontinuous [Zar,
1984]. Level for significance was P , 0.05.
RESULTS
Vanadate Inhibits Aggregation of Pigment
in Skin Melanophores
The dynein inhibitor vanadate was used as one tool
to investigate whether dynein is involved in the aggregation of pigment in melanophores on skin pieces from cod.
Aggregation was induced by addition of 1 然 NA in lysis
buffer, and dispersion by removal of NA by rinsing with
lysis buffer. All vanadate treatments (in the presence or
absence of ATP) decreased the aggregation rates in a doseresponse-like appearance compared to controls (Fig. 1, Table
I). In the presence of 100 然 vanadate, the aggregation was
almost completely inhibited. The same effects was seen after
3 min in the presence of 50 然 vanadate. Vanadate also
inhibited aggregation at a concentration of 10 然, but not
as efficient as the higher concentrations.
Microinjection of Dynein Antibody Inhibits
Pigment Granule Aggregation
in Cultured Melanophores
In our study, we have used a mAb against cytoplasmic chick dynein light chains (70.1), characterized by
Fig. 4. Effects of microinjection of 70.1 mAb on pigment granule
aggregation in a cultured melanophore in comparison with simultaneously observed uninjected melanophores. a: Micrograph of melanophores with dispersed pigment. b: Cells are photographed just after
injection of 70.1 mAb in the melanophore (arrow). c: Cells after 30 min
rest after injection. d: Cells at 20 min after addition of 1 然 NA. Bar 5
50 痠.
404
Nilsson and Wallin
Fig. 5. Light micrographs of representative skin explants with melanophores with normally dispersed pigment (a), and melanophores with
completely aggregated pigment after incubation in 1 然 NA in
medium, and thereafter incubated in buffer, vinblastine, or vanadate
(c?e). A representative control explant was incubated in lysis buffer
with 0.02% saponin and NA for 10 min (b). Skin explant incubated in 3
mM vinblastine in medium with NA for 2 h (c). Explants incubated in
lysis buffer with 0.02% saponin, NA, and 50 然 vanadate (d), and lysis
buffer with 0.02% saponin, NA and 100 然 vanadate (e). Note the
deformed shape of the melanophore pigment masses after treatment
with vinblastine (b) and with vanadate (d, e,), in contrast to control (b).
Bars 5 100 mm.
Steuer et al. [1990], which has recently been shown to be
a powerful tool in studies of dynein-mediated processes
[Heald et al., 1996]. To show that the 70.1 antibody reacts
with cod dynein and is not species-specific, a crude cod
brain extract was immunoblotted with the antibody. The
antibody reacted specifically with three bands of the
appropriate Mr, of which the two upper were very closely
aligned(Fig. 2a). Cultured melanophores with dispersed
or aggregated pigment were analyzed immunocytochemically with the 70.1 antibody. The antibody labelling
showed a punctate distrubution throughout cells with
dispersed granules (Fig. 2b), and an accumulated staining of
the center of the cells with aggregated pigment (Fig. 2d).
To determine whether dynein is involved in pigment granule aggregation, the 70.1 antibody was microin-
jected into cultured cod melanophores with dispersed
pigment (Table II). Aggregation was induced 1 h after
injection by addition of 1 然 NA and the melanophores
were observed during 25 min. A complete aggregation
was seen within 6 min for control cells, which either were
touched with a microneedle, injected with ascites fluid
(Fig. 3a?c,e?g), or uninjected (not shown) . These cells
aggregated their pigment slightly slower than melanophores that were untreated (not shown). Microinjection of
the dynein antibody induced a change in pigmentation of
the dispersed cells already during the 1-h rest before NA
treatment. The pigment extended farther to the periphery,
leaving a spot with a lower density of pigment in the
central part of the cell, and a higher density of pigment in
the peripheral parts (Fig. 3j). This was in clear contrast to
Roles of Dynein in Pigment Transport in Melanophores
Fig. 6. Melanophores with completely aggregated pigment were
incubated with 1 然 NA in the presence of 10, 50, or 100 然 vanadate,
respectively, for 10 min in lysis buffer with 0.02% saponin. Controls
were treated with NA and lysis buffer with or without saponin. Mean
amount of percentage deformed CPMs of total CPMs were calculated
from 16 skin samples in every treatment. Error bars are s.d. The effects
of 50 and 100 然 vanadate were significant, as indicated by stars
(ANOVA: F4,15 5 16.5, P , 0.05, F15,60 5 16.8, P , 0.05 and SNK-test
P , 0.05).
the touched control and ascites injected cells, where a
slight aggregation was induced by the treatment (Fig.
3b,f). When NA was added to the antibody-injected cell,
the aggregation was inhibited and no CPM was formed
(Fig. 3k), in contrast to control cells (Fig. 3c,g). In Figure
4, only one of several melanophores has been injected
with dynein antibody, clearly showing the difference
between uninjected control cells and dynein mAbinjected cells during rest and NA-induced aggregation.
Dispersion of pigment (Table II), which was induced by
replacing the cells in fresh medium without NA, was
unaffected in touched or ascites injected melanophores
(Fig. 3d,h), showing that these cells behaved normally. In
the dynein antibody- injected cells, which were already in
a dispersed state caused by inhibition of aggregation, a
further translocation of pigment to the peripheral parts of
the cells was seen upon removal of NA stimulation, leading to
a very unusual pigment pattern (Fig. 3l). Our results indicate
that dynein not only is responsible for aggregation, but also in
keeping the pigment uniformly dispersed.
Vinblastine and Vanadate Treatments Induce
Deformed Shapes of the CPMs
in Skin Melanophores
To determine whether microtubules are involved in
maintenance of the spherical form of CPM, melanophores on skin pieces with pigment aggregated to punctate with 1 然 NA were treated with the microtubule
405
disrupting agent vinblastine at 3 mM in medium with NA
for 2 h (Fig. 5c). The results showed that microtubule
disruption led to a significant (P , 0.05, ANOVA: F1,6 5
216.8) deformation of 65% of the spherical CPMs,
compared with 19% in control skin pieces incubated in
medium with only NA.
To investigate whether dynein is involved in maintenance of the CPM, skin melanophores with pigment
aggregated to punctate with 1 然 NA, were also treated
with 10, 50, and 100 然 vanadate in lysis buffer with
0.02% saponin and NA for 10 min. Vanadate induced a
significant deformation of the CPMs in a dose-responsedependent way, where 100 然 vanadate induced 82%
deformed CPMs, 50 然 55% and 10 然 34% (Fig. 6B) .
A significant difference in percentage deformed CPMs
was induced by 50 and 100 然 vanadate, compared to the
controls with (20%) or without saponin (22%).
Vanadate treatment inhibited only pigment aggregation, as no significant difference in dispersion rate was
found between vanadate-treated samples, and controls
with or without saponin (Fig. 7, Table III). Even if the
dispersion rate was unaffected, the time needed for
initiation of dispersion decreased significantly in the
presence of 100 然 vanadate, compared to controls in
lysis buffer (Table III).
Microinjection of Dynein Antibody Inhibits
Maintenance of Spherical CPMs
in Cultured Melanophores
To evaluate further the role of dynein in maintenance of the spherical shape of the CPM, the 70.1
antibody against dynein was microinjected into cultured
melanophores with NA-induced aggregated pigment granules. After injection, the cells were allowed to rest for at
least 30 min in presence of NA to maintain the aggregated
state of pigment. Control cells, which were either uninjected or injected with ascites fluid, maintained their
spherical CPMs after rest in NA (Fig. 8a,b,d,e). In
contrast, microinjection of the dynein antibody induced a
loosening of the spherical CPM, as observed after the rest
period (Fig. 8g,h). The results show that dynein is
important for maintenance of the spherical CPM. Dispersion of pigment was induced by replacing the cells in
fresh medium without NA for 20 min (Fig. 8c,f,i, (Table
IV)). Pigment moved to a uniformal distribution throughout the control cells (Fig. 8 c,f). In contrast, a further
translocation of pigment to the peripherial parts of the
cells was seen in the antibody injected cells (Fig. 8i). This
was even more visible after prolonged time (not shown).
DISCUSSION
In this study we have for the first time shown that
dynein is involved in three different processes in melanophores; aggregation, maintenance of the central pigment
ma ss, and homogeneous distribution of pigment granules
406
Nilsson and Wallin
Fig. 7. Skin melanophores were incubated in NA to induce pigment
aggregation. Skin explants were then incubated in 10, 50, or 100 然
vanadate with NA for 10 min in lysis buffer. The explants were
thereafter rinsed in lysis buffer with vanadate and in absence of NA to
induce pigment dispersion. Measurement of dispersion as MI was done
every min. The graph shows the mean rate of dispersion (MI/min) in
each treatment. No difference in slopes of time/MI between treatments
was found (ANOVA: F4,15 5 1.42, P . 0.05). Time for onset of
dispersion (detemined as time until MI 2 is reached) differed between
treatments (Kruskal?Wallis: P , 0.05). Multiple comparisons detected
a quicker onset of dispersion after treatment with 100 然 vanadate
compared to controls in lysis buffer (Tukey type: P , 0.05).
throughout the cells. The 70.1 antibody against chicken
dynein has proved an important tool with broad species
specifity for functional studies of dynein in both Xenopus
egg extracts [Heald et al., 1996] and cod melanophores.
Vanadate, which we also have used, is well known to
inhibit pigment granule aggregation, either by microinjection [Beckerle and Porter, 1982] or by permeabilization of
the cell membrane [Clark and Rosenbaum, 1982; Grundstro?m et al., 1985; Negishi et al., 1985; Ogawa et al.,
1987; Oshima et al., 1990], but its specificity is under
debate [Penningroth, 1986; Collins, 1994]. Here we have
shown that similar inhibitory effects were achieved with
microinjection of the 70.1 antibody and with the addition
of vanadate to permeabilized skin melanophores in the
studies on aggregation and maintenance of the spherical
CPM, while only the use of the dynein antibody could
show that dynein has a role in keeping dispersed pigment
evenly distributed. Our results on inhibition of aggregation is also similar to the older data with vanadate,
making it possible to discuss dynein as a general mediator
for pigment aggregation.
The mechanism behind maintenance of the spherical CPM has not been examined before, but a lack of
microtubule dependency in this process has been discussed (Schliwa and Bereiter-Hahn, 1975; Chen and
Wang, 1993). Our results show that disruption of microtubules with vinblastine, inhibition of dynein with vanadate, or microinjection of 70.1 antibody into cod melanophores with aggregated pigment, resulted in loosening of
the CPM. This shows clearly that microtubules and
dynein are responsible for maintenance of the spherical
CPM. Loosening of the CPM did not induce pigment
dispersion, implying that an opposite-directed motor
must be responsible for the dispersion process. This is in
agreement with Rodionov et al. [1991], who showed that
a kinesin-related protein was involved in melanophore
pigment dispersion in black tetra.
The normal dispersion process from the cell center
can be explained by two different mechanisms. One
possibility is that dynein is inactivated shortly after
loosening of the CPM and kinesin is induced to translocate pigment granules towards the cell periphery until
dynein is activated again. Together with kinesin, they
could accomplish the shuttling and uniformly spreading
of the pigment granules. The similar rates of pigment
dispersion in skin melanophores treated with vanadate
and control melanophores support this model. Another
possibility is that dynein is continuously active during
aggregation and dispersion, and kinesin is the target of
activation during dispersion, as speculated in a review by
Haimo and Thaler [1994], leading to overriding of dynein
by kinesin dynein during dispersion. It has been suggested from in vitro studies that kinesin could determine
the direction of movement of other vesicles by overriding
the activity of dynein [Vale et al., 1992; Muresan et al.,
1996]. The mechanism of action is unclear, but differences in time that the motors spend attached to microtubules during the ATPase cycle, and in affinity for vesicle
membranes, were discussed. This model is in agreement
Roles of Dynein in Pigment Transport in Melanophores
407
Fig. 8. Effects of microinjection of 70.1 mAb on maintenance of the
spherical central pigment mass and pigment granule dispersion in
cultured melanophores. The cells were preincubated in 1 然 NA to
induce pigment aggregation before injection. Uninjected melanophore
(a?c), injected with ascites fluid (d?f), and injected with 70.1 mAb
(g?i). d, g: Cells before injection. e, h: At 30 min after injection. f, i: At
20 min after transfer to fresh medium without NA. k: Arrow, deformed
shape of the central pigment mass in mAb injected cell after 1-h rest in
NA. i: Arrow, pigment translocated to the periphery of the cell after
transfer of the cell into medium without NA to induce pigment
dispersion. Bars 5 50 痠.
with the irregular pigment movement observed during
dispersion [Green, 1968; Murphy and Tilney, 1974].
Dispersed pigment granules are usually homogeneously distributed in melanophores and shuttle short
distances to and fro [Green, 1968], suggesting that some
force antagonize the transport of pigment completely to
the peripheral parts of the cell. This is in clear contrast to
the aggregation process, where all pigment granules are
transported rapidly to the central part of the cell. The
reason for the evenly distributed pigment granules has
hitherto been unclear. If only kinesin is active during
dispersion, the pigment granules would move to the
periphery of the cells and change the color of the animal.
Color changes of animals involve aggregation or dispersion of pigment throughout the cells and do not involve
pigment dispersion to the cell periphery [for review, see
Haimo and Thaler, 1994]. Our data that microinjection of
the dynein antibody into dispersed melanophores induced
a transport of melanosomes to the cell periphery suggest
that the minus end motor dynein not only is active during
aggregation, but also in the process of keeping pigment
granules evenly distributed in the cell. Vanadate also
induced a dispersion of pigment to the periphery (unpublished observations) but prolonged saponin permeabilization of the melanophores affected the melanophores
similarly, making it difficult to draw clear conclusions.
408
Nilsson and Wallin
distribution of dispersed melanosomes in cod melanophores. In light of our data, the possibility exists that
dynein is continuously active and kinesin the target for
regulation.
ACKNOWLEDGMENTS
Fig. 9. Hypothetical depiction of how pigment granule distribution is
regulated in melanophores. In the dispersed state of pigment distribution (dark gray melanophore), both dynein and kinesin are active and
try to transport pigment granules in opposite directions, leading to
uniformly spread pigment granules throughout the melanophore.
During aggregation (indicated by the white melanophore), and the
subsequent maintenance of the spherical central pigment mass, kinesin
is inactivated by a noradrenaline-induced mechanism, while dynein
remains active. In the dispersion process, kinesin either becomes active
again and dynein could be inactivated until a uniform distribution of
dispersed pigment is complete, or kinesin overrides a continuously
active dynein.
Pigment dispersion requires cAMP, while dephosphorylation seems to be a crucial component regulating pigment
aggregation [Rozdzial and Haimo, 1986; Thaler and
Haimo, 1990]. Vanadate is known to inhibit phosphatases
[Cyboron et al., 1982; VanEtten et al., 1974]. The effect of
vanadate might therefore either be a direct effect on
dynein, or an inhibition of phosphatase activity, with the
result that the melanophores remain dispersed, or hyperdispersed. The 70.1 antibody is specific to dynein,
making it a more useful tool in crude systems. Based on
the present results, our current hypothesis is that dynein
has an active role in aggregation, in formation and
keeping of a spherical CPM, and in the homogeneous
distribution of pigment throughout the cell, while kinesin
only is active in the dispersion process (Fig. 9).
To test the hypothesis with continuously active
dynein, one would have to block kinesin completely.
Rodionov et al. [1991] found that microinjection of
kinesin antibodies into melanophores suppressed dispersion, but the inhibition of kinesin did not lead to pigment
aggregation in those cells. However, the inhibition was
not complete, and one cannot exclude the possibility that
kinesin still might be able to override dynein. Further
studies focusing on pigment dispersion are therefore
needed to clarify the regulatory steps in this process.
In conclusion, our results show that dynein is
responsible for pigment aggregation and maintenance of
the spherical CPM and is involved in the homogeneous
We thank Elisabeth Norstro?m for introducing the
microinjection technique in our laboratory, microinjection experiments, and other excellent technical assistance; Malin Ba?ckstro?m for assisting vanadate experiments; and Dr. Mattias Sko?ld for statistical help. This
work was supported by grants from the Swedish Natural
Science Research Council, Helge Ax:son Johnsons Stiftelse, Nordenskjo?ldska Swedenborgsfonden, Kungl. och
Hvitfeldska stipendieinra?ttningen, Magnus Bergvalls stiftelse, Lars Hiertas Minne, and Hierta Retzius Stipendiefond.
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