(The FASEB Journal. 1999;13:S221-S224.)
© 1999 FASEB
Lessons from the Melanophore
G. G. BORISY1 and
V. I. RODIONOV
Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, USA
1Correspondence: Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Dr., Madison, WI 53706, USA. E-mail: ggborisy{at}facstaff.wisc.edu
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INTRODUCTION
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THE ROLE OFthe centrosome in microtubule dynamics, as many other central problems
of modern cell biology, was inspired by the work of Keith Porter. One
particular system that he introduced to experimental analysis was the
fish melanophore. This cell, residing in the pigmented skin of fish or
amphibia, contains many thousands of granules of pigment and is a
device of nature by which prey attempt to evade their predators. When
stimulated by the hormone adrenalin, the pigment granules of the
melanophore aggregate to the center of the cell, reducing the cell’s
optical density and thus lightening the color of the skin tissue. The
melanophore can also disperse its pigment, restoring the darker
coloration. Excellent reviews of the biology of melanophores and their
contribution to our understanding of intracellular transport mechanisms
have been written by Schliwa (1
, 2
,) who began his studies
of this system independently in Munich, but for several years was an
investigator in Porter’s department in Boulder, Col.
The rapid and reversible aggregation-dispersion of pigment granules led
to the melanophore being used as an exemplar system for studying
microtubule-based transport. An early Porter student, Lew Tilney,
contributed importantly to our understanding that the melanophore
contained a radial array of ~1000–2000 microtubules
(3
4
5)
. Another Porter student, Dick McIntosh, later
established that the microtubules of the melanophore were of uniform
polarity—minus ends at the cell center and plus ends toward the cell
periphery (6)
. The microtubules of uniform polarity
provide tracks for molecular motors to drive motion of the granules.
The Porter lab was the first to obtain evidence that motors of the
dynein family drive the granules inward toward the minus end
(7)
while kinesin-related motors drive the granules
outward toward the plus end (8
9
10)
. Although most work on
melanophores has emphasized the role of microtubules in intracellular
transport, Schliwa, in an association with Porter, reported that
melanophores also contain an array of actin filaments
(11)
. Recently, this finding has surfaced in a new light.
Stimulation of plus-end motors is predicted to drive pigment granules
to the cell margin, yet melanophores achieve a uniform distribution.
This paradox suggested that the transport system was incompletely
understood. In attempting to draw a lesson from this paradox, we
demonstrated the existence of a microtubule-independent system in fish
melanophores. The system is based on actin filaments (12)
and myosin V (13)
and is used to achieve a uniform
distribution of pigment granules. Thus, the melanophore teaches us that
an actin-based motility system works in coordination with
microtubule-based transport, and this coordination may be of general
significance for organelle motility in cytoplasm.
Another contribution of Keith Porter was the early realization that the
origin and distribution of microtubules had to be under cellular
control (14)
. Porter postulated that "to influence the
shapes of cells and concomitantly the distribution of the cytoplasm,
the microtubules must 1) be distributed unevenly according
to some prescribed pattern, 2) be anchored at one end and
free to grow at the other, 3) be oriented, i.e., given a
direction in their growth from a point, 4) possess some
tendency to straightness, and 5) be limited in length." In
melanophores, because the microtubule array was radial, the organizing
principle was thought to be the centrosome—the centrioles plus the
surrounding pericentriolar material. The centrosome, by nucleating
microtubules and anchoring their minus ends, was thought to be
responsible for organizing them into a radial array.
Another student of Porter’s, Mark McNiven, revealed an unanticipated
dimension to understanding microtubule organization. The work of
McNiven and Porter developed a finding reported briefly 50 years
earlier by Matthews (15)
, but which had apparently been
forgotten. Matthews showed that it was possible to surgically cut a
melanophore so as to produce a piece of cytoplasm lacking the
centrosome. On stimulation of aggregation, the fragment aggregated its
pigment to its own center with approximately the same speed as the
nearby parental cell. Matthews showed that the centering activity of
the cytoplasmic fragment was completely independent of proximity to its
parental cell by physically destroying the parent with a needle, thus
removing it from the experimental theater. McNiven and Porter
demonstrated that the cytoplasmic fragments not only aggregated pigment
to their geometric centroid but also formed a radial array of
microtubules of correct polarity focussed at the centroid (16
, 17)
.
How does a radial array of microtubules become established in a
cytoplasmic fragment? Vladimir Rodionov and I recently investigated
this question and used modern digital fluorescence imaging techniques
to answer it (18)
. In intact melanophores, the
microtubules are always radially organized, both in the dispersed state
and in the aggregated state. But in a cytoplasmic fragment lacking the
centrosome with dispersed pigment, the microtubules are more or less
randomly arranged. Nevertheless, when adrenalin is presented to this
system, the pigment coalesces to the center and a radial array of
microtubules emerges (Fig. 1
). To explain this process, we have developed a model involving
microtubules, motors, and membranes (Fig. 2
). Pigment granules bear microtubule-dependent motors and are large
enough to bear a number of both plus and minus-end directed motors.
Thus, the granules may be considered to be multivalent in motors. They
interact with microtubules that may bind motors anywhere along their
length. Thus, microtubules may be considered as being multivalent in
motor binding sites. In the case of the pigment aggregation reaction,
granules are directed to the minus end of each microtubule they
interact with. If a granule cannot go to the minus end of all
microtubules simultaneously, then the microtubules move relative to the
granule, and a microaster results. Interactions between microasters
result in a pulling force between microasters resulting in their
coalescence into larger asters. If there is an interaction with the
surface, the larger asters will arrive at the same point, namely the
centroid, and a radial array of microtubules with pigment aggregated at
the center will result. Evidence for the centering process sensing the
membrane comes from changing the geometrical shape of the cell
fragment. When the fragment is shaped into a toroidal form, a medial
ridge develops between the inner and outer radii of the toroid (see
Fig. 3
of ref 18
). Thus, a remarkable lesson that the
melanophore teaches us is that the centrosome is not the total master
of organization in the cytoplasm. Rather, there are additional
principles, namely of self-organization and self-centering. In the case
of melanophore fragments, these properties are revealed as arising from
the interaction of dynamic microtubules with motors and the surface
membrane.
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Figure 1. Distribution of microtubules in the cytoplasmic fragments of
melanophores. Cytoplasmic fragments with dispersed (top) or aggregated
(bottom) pigment granules immunostained with tubulin antibody.
Microtubules were randomly arranged in the fragments with dispersed
pigment but formed radial array on pigment aggregation. Bar = 20
µm. From ref 18
.
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Figure 2. A model for self-organization of the radial microtubule array in
melanophore fragments. a) In dispersed state, pigment
granules (dots) are homogeneously distributed in the cytoplasm and
microtubules (arrows) are randomly arranged. b)
Adrenalin treatment triggers motion of pigment granules to minus ends
of microtubules. Motion of granules along microtubules and microtubules
relative to granules results in formation of microasters.
c) Interactions between microasters result in formation
of larger asters. Because of interaction with the surface, the larger
asters arrive at the same point, namely the centroid, and a radial
array of microtubules forms.
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Figure 3. Fluorescence imaging of microtubules in living CHO cytoplasts. In
centrosome-containing cytoplasts (upper row), microtubules showed
dynamic instability at their plus ends while free microtubules rapidly
shortened from their minus ends. In centrosome-free cytoplasts (middle
row), microtubules persistently grew at one end and shortened at the
other end. Injection of tubulin at low concentration resulted in
nonuniform incorporation of subunits along microtubules and produced
speckles along their length. Speckles remained stationary, indicating
that the mechanism of translocation was treadmilling (bottom row).
Black arrowheads point to shortening and white arrowheads to growing
ends. White arrows point to speckles. Numbers indicate time in seconds.
Bars = 1 µm. From ref 26
.
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Analysis of microtubule dynamics in cytoplasmic fragments of
melanophores taught another lesson. Direct observation by time-lapse
digital fluorescence microscopy showed that the radial array of
microtubules in the fragments was highly dynamic and that the aggregate
of pigment at the center appeared to function as a centrosome
equivalent (19)
. Microtubules were constitutively
nucleated by the pigment granule aggregate and they subsequently
elongated, growing toward the cell margin. More surprisingly,
microtubules stochastically became released from the pigment aggregate
and shortened from their trailing or proximal end, resulting in their
eventual depolymerization. This unexpected result prompted us to
examine more closely the dynamics pathway. If a microtubule’s leading
(plus) end arrived at the cell surface before it’s minus end released
from the cell aggregate, then the behavior observed was simply
shortening from the minus end. However, if the microtubule released
from that aggregate before its leading end arrived at the surface, the
microtubule appeared to translocate through the cytoplasm.
The apparent microtubule translocation suggested that it was driven by
molecular motors, but this possibility presented a paradox. Molecular
motors have been demonstrated to reside primarily on the pigment
granules (20)
, and all of the granules were aggregated at
the fragment center whereas the observed microtubule motion was in
granule-free cytoplasm. What was driving the apparent motion of these
cytoplasmic microtubules? One possibility was the existence of as yet
undiscovered motors in the cytoplasm driving microtubule transport
relative to a cytoplasmic matrix. Alternatively, the apparent transport
could be a reflection of a process that was of intense interest ~20
years ago, microtubule treadmilling, which means simultaneous growth
and shortening at the opposite ends of the polymer. The way to
discriminate between these two classes of mechanism is to place a
reference mark on the microtubule and determine whether the mark moved
or not. In the motor hypothesis, the microtubule lattice moves as a
whole and the reference mark would move at the same rate. In the
treadmilling hypothesis, the microtubule grows at its leading end and
shortens at its trailing end, and the mark would be stationary. We
created a reference mark on a fluorescent microtubule by photobleaching
(see Fig. 3
. of ref 19
). The results of the marking
experiments were clear. The mark remained stationary while the
microtubule apparently translocated, indicating that the apparent
motion was, in fact, treadmilling.
The phenomenon of treadmilling was first conceptualized for actin
filaments ~20 years ago (21)
and shortly afterward
discovered to also apply to microtubules (22)
. After an
initial period of excitement, the treadmilling seen in vitro
was thought not to be of any physiological relevance because it was too
slow (~1 µm/h) to account for the rapid changes in microtubule
organization observed in living cells. Also, a new concept, dynamic
instability (23)
was introduced, which conformed better
with experimental observations. In contrast, the treadmilling that we
observed in fish melanophore fragments was rapid—~4 µm/min, which
is a couple of orders of magnitude faster than that seen in
vitro. The rapidity of this phenomenon prompted us to reevaluate
microtubule dynamics What did the treadmilling signify? Was it a
curiosity of fish melanophore fragments, or was it of more general
significance?
In more recent experiments, we have extended exploration of microtubule
treadmilling by again drawing on heritage from the Porter lab. We
sought to determine whether microtubules in mammalian cells not
attached to the centrosome would treadmill. Porter and his associates
in the early 1970’s developed a procedure for enucleation of cells,
resulting in cytoplasmic fragments that they termed cytoplasts. The
procedure was to allow cells to adhere to a coverslip, then to treat
the cells with the actin-disrupting drug, cytochalasin, and to
subsequently centrifuge the cells in a direction normal to the plane of
the coverslip. Centrifugation resulted in the nucleus with some
adherent cytoplasm surrounded by a membrane, the karyoplast, going to
the pellet, leaving on the substrate a piece of membrane-bounded
cytoplasm—the cytoplast. These cytoplasts invariably contained the
centrosome (24)
. A modification of this procedure in which
the microtubule-disrupting drug, nocodazole, was combined with the
cytochalasin treatment resulted in cytoplasts that frequently lacked
the centrosome (25)
. We thought this was an ideal way to
test for the role of the centrosome in microtubule dynamics in
mammalian cells, as opposed to fragments of fish melanophores.
The results on mammalian cytoplasts (26)
reinforced the
conclusions drawn from the fish melanophore fragments. Microtubules in
Chinese hamster ovary (CHO) fibroblast cytoplasts containing
centrosomes displayed the characteristic dynamic instability showed by
the intact parental cells. In contrast, microtubules in fibroblast
cytoplasts lacking centrosomes displayed rapid treadmilling, at average
rates of ~11 µm/min (Fig. 3
). What these results signify is that treadmilling is a mechanism of
general significance and that the pattern of microtubule dynamics is
controlled by the centrosome. The treadmilling is indicative of an
underlying minus-end pathway contributing to microtubule turnover. It
remains to be established precisely how this minus-end pathway
contributes to cytoplasmic organization and function, but what we hope
has been made clear is that Keith Porter’s heritage is alive and well
through the experimental systems he introduced and through the ideas he
inspired.
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ACKNOWLEDGMENTS
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This work was supported by NIH grant GM-25062 to G.G.B. and NSF
grant MCB-9728252 to V. I .R.
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INTRODUCTION
REFERENCES
