Department of Cell and Molecular Biology, Robert H. Lurie Comprehensive Cancer Center, and Center for Genetic Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA
Author for correspondence (e-mail: r-chisholm{at}northwestern.edu )
Accepted 4 January 2002
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Summary |
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Key words: Cytoplasmic dynein, Organelle transport, Molecular motor regulation, Green fluorescent protein, Live cell imaging
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Introduction |
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Previous evidence distinguishing between these models was based on
immunohistochemistry studies of fixed cells and tissues. Several studies have
indicated that for both dynein and kinesin-like motors, motor-cargo
interaction could be regulated by phosphorylation
(Dillman and Pfister, 1994;
Lee and Hollenbeck, 1995
;
Lin et al., 1994
;
Marlowe et al., 1998
;
Niclas et al., 1996
;
Sato-Yoshitake et al., 1992
),
although it is not clear whether the cell actually uses this mechanism to
regulate directional transport. Evidence for the regulation model came from
comparing the distribution of motors and specific groups of membranous
organelles. In neuronal axons, cytoplasmic dynein heavy chain antibody stained
vesicles accumulated both proximal and distal to a ligation, suggesting that
dynein associates with vesicles moving in both retrograde and anterograde
direction (Hirokawa et al.,
1990
). In endoplasmic reticulum (ER)-Golgi membrane traffic,
kinesin was immunolocalized not only to the membrane compartment destined for
Golgi-to-ER transport but also to those in the reverse direction
(Lippincott-Schwartz et al.,
1995
). Similar results have been shown by immunofluorescence
localization of cytoplasmic dynein in the ER-Golgi system
(Roghi and Allan, 1999
). In
melanocytes, both kinesin-like motors and dynein were detected by
immunoblotting on aggregated and dispersed populations of melanophores
(Nilsson et al., 1996
;
Reese and Haimo, 2000
). These
studies indicate that motors could localize to cargoes that potentially move
in a direction opposite to their polarity, suggesting that motors do not
dissociate from cargo moving in the wrong direction. However, actual motor
movement in both directions has not been directly visualized. Localization of
both types of motors on a single vesicle could occur on static cargo instead
of moving ones. Moreover, it is not clear how directional reversal occurs,
what time scale it involves nor what the dynamics of the associated motors
are. These questions cannot be easily addressed by static studies in fixed
samples but rather require dynamics analysis in living cells.
Several recent studies have focused on the dynamic behavior of specific
types of cargoes using GFP-fusions (Gross
et al., 2000; Presley et al.,
1997
; Suomalainen et al.,
1999
; Wubbolts et al.,
1999
; Ye et al.,
2000
). These studies have revealed that many cargoes displayed
bidirectional movement and could reverse direction of movement rapidly.
However, the behavior of the motor has not been studied. To characterize
dynein dynamics, we have analyzed the dynamics of a dynein intermediate chain
(IC)-green fluorescent protein (GFP) fusion. We have generated stable cell
lines that express the IC-GFP fusion in an IC-null background in
Dictyostelium discoideum and have shown that the IC-GFP fusion
functions normally. By time-lapse fluorescence microscopy, we observed that
dynein travels bidirectionally along the microtubules and that dynein remains
stably associated with the cargo during the rapid reversals of movement
direction. Our results provide support for a model in which both minus- and
plus-end-directed motors coexist on a given cargo and regulation of motor
activities controls the directionality of transport.
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Materials and Methods |
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Immunoblots and immunoprecipitations
The antibodies used in this study include: anti-Dictyostelium
dynein IC, IC144; anti-Dictyostelium dynein heavy chain, NW127; and
anti-Dictyostelium capping protein ß. These antibodies have been
previously described (Ma et al.,
1999). To screen for protein expression in the transformants,
protein samples were prepared from cells collected directly from the 96-well
plates. For immunoblots, protein samples were separated on 7.5%
SDS-polyacrylamide gels and transferred to PVDF membranes. Blots were blocked
in 5% nonfat milk and incubated with the IC144 antibody against dynein IC
(Ma et al., 1999
) followed by
an HRP-conjugated goat-anti-rat antibody. Blots were developed in
Renaissance enhanced chemiluminescence reagent (NEN Life Science
Products, Boston, MA) and exposed to X-ray film. For immunoprecipitations
(IPs), 4x107 cells were collected and washed twice with 15 mM
Na-KPO4 buffer, pH 6.5. Following resuspension in 1 ml IP buffer
(50 mM PIPES, pH 6.8, 5 mM EDTA, 100 mM NaF, 25 mM Na pyrophosphate, 2.5 mM
DTT, 1 mM PMSF, 50 µg/ml leupeptin, 50 µg/ml pepstatin, 1 mM ATP) cells
were lysed by sonication. The cell lysate was cleared by centrifugation at
38,000 g for 30 minutes at 2°C. Protein-A sepharose preincubated
with the IP antibody was added to the cell lysate and the mixture incubated
while rocking for at least 2 hours at 4°C. Dynein heavy chain antibody
NW127 was used to IP the dynein complex, whereas affinity-purified capping
protein ß antibody R18 was used to IP the dynactin complex.
Sepharose-beadbound immune complexes were collected by centrifugation and
washed four times with IP buffer. The final pellets were resuspended in 30
µl 2xSDS sample buffer (125 mM Tris, pH 6.8, 4% SDS, 10%
2-mercaptoethanol, 20% glycerol), boiled for 5 minutes, centrifuged and the
supernatant collected and analyzed by immunoblot.
Immunofluorescence staining
For tubulin staining, cells plated on coverslips were fixed in 1.85%
formaldehyde in 15 mM Na-KPO4, pH 6.5 for 5 minutes at room
temperature, then extracted in -15°C methanol for 5 minutes. The
coverslips were then stained with a rat anti--tubulin monoclonal
antibody (Serotech, Ltd.) followed by a lissamine-conjugated goatanti-rat
secondary antibody.
Image capturing and analysis
Cells expressing IC-GFP in an IC-null background were used for GFP imaging.
Living cells were flattened by agar overlay
(Yumura et al., 1984) and
imaged in a humid chamber. GFP signals were observed by fluorescence
microscopy with a 100x 1.4 N.A. PlanNeofluar oil objective. Images were
captured using Micromax 512 BFT cooled CCD camera (Princeton Instruments).
Time-lapse sequences were captured using the streaming mode, which allows 302
msecond intervals with 300 msecond exposures. Metamorph 4.0 (Universal
Imaging) was used for image capturing and data analysis.
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Results |
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|
|
IC-GFP is functionally comparable to wild-type IC
IC plays an important role in cytoplasmic dynein function. A major function
of dynein IC is to link dynein to its regulatory partner, the dynactin complex
(Karki and Holzbaur, 1995;
Ma et al., 1999
;
Vaughan and Vallee, 1995
). It
has previously been demonstrated that the N-terminal domain of IC is critical
for interacting with dynactin, and the C-terminal region is essential for
dynein association (Karki and Holzbaur,
1995
; Ma et al.,
1999
; Vaughan and Vallee,
1995
). To directly compare the ability of IC-GFP and native IC to
associate with dynein and dynactin, we performed immunoprecipitations using
cells that express both proteins. The ratio of fusion to native protein in
either dynein or dynactin immunoprecipitates was similar to their ratio in
cell lysates, suggesting that the IC-GFP fusion efficiently associates with
both complexes (Fig. 2b).
Moreover, cells expressing only IC-GFP were phenotypically indistinguishable
from wild-type cells with respect to growth rate, microtubule, spindle and
Golgi morphology and multicellular development, indicating that IC-GFP is
physiologically functional. Since it is known that over 90% of the IC in
Dictyostelium associates with the dynein complex
(Ma et al., 1999
), the IC-GFP
signal in these cells represents dynein's normal intracellular localization.
The IC-GFP distribution agrees with that previously reported for the dynein
heavy chain (Koonce and McIntosh,
1990
).
Dynein-containing structures move bidirectionally along microtubules
in living cells
IC-GFP expression allowed us to study dynein dynamics in living cells by
following GFP signals with time-lapse fluorescence microscopy. In still
images, dynein typically appeared as puncta distributed throughout the
cytoplasm, with a strong perinuclear accumulation that coincided with the
microtubule organizing center (MTOC) (Fig.
2c; Fig. 3). In
living cells, the highly dynamic dynein typically moved rapidly over long
distances along linear tracks extending radially from the MTOC, moving both
towards and away from the MTOC.
|
The linear tracks for dynein traffic most probably represent microtubules
for several reasons. First, in cells fixed and stained to display
microtubules, there is significant colocalization of dynein puncta and
microtubules (Fig. 3). Second,
dynein moves along microtubules in vitro. Third, living cells imaged with long
exposures (2 seconds or greater), or when a time series of images was
superimposed, showed dynein averaged into linear tracks closely resembling the
pattern of interphase microtubules (Fig.
2d). It is unlikely that the movement of dynein was driven by
microtubule dynamics because the microtubule polymerization rate (0.05-0.35
µm/second; (Cassimeris et al.,
1988; Sammak and Borisy,
1988
)), depolymerization rate (0.07-0.6 µm/second;
(Cassimeris et al., 1988
;
Mitchison and Kirschner, 1984
;
Sammak and Borisy, 1988
)) and
treadmilling rate (50-60 µm/hour;
(Farrell et al., 1987
)) are too
slow to account for the observed speed of 1-2 µm/second. Also, microtubule
dynamics cannot explain several dynein puncta traveling along the same track.
Therefore, the linear movements probably represent dynein, which is likely to
be associated with the surface of cargo, moving along microtubules.
It has been well established that Dictyostelium interphase microtubules, like in most other eukaryotic cells, are organized in a radial array with their minus ends focusing on the microtubule organizing center (MTOC) in the center of the cell and their plus ends extended to the cell periphery. These established microtubule polarities were used to determine direction of dynein movement. Because the central mass of dynein-GFP colocalizes with the MTOC (Fig. 3) and because dynein moves along the radial array of microtubules, we refer to movements toward the central dynein mass as traveling towards microtubule minus ends, and movement away from the central mass as traveling towards microtubule plus ends. There are a small number of MTs that are curved back towards the center of the cell, resulting in the plus ends being away from the cell periphery. We have taken care to avoid using such MTs in our analysis.
As a minus-end-directed microtubule-based motor, dynein-engaged cargo would be expected to travel towards the MTOC. Indeed, we frequently observed dynein moving towards the central dynein mass along linear tracks (Fig. 4a). However, we also observed, almost equally, as many examples of dynein moving away from the central mass towards the cell periphery (Fig. 4b). The microtubule plus-end-directed movement of dynein is most probably a result of dynein being passively moved through its association with a cargo carried by a plus-end-directed motor. Dynein-associated structures moving along microtubules in the plus-end direction suggests that motors of opposing polarities co-exist on these cargoes.
|
The average velocity of dynein movement in either direction is similar,
with minus-end-directed movement occurring at 1.84±0.34 µm/second
(n=65) and plus-end-directed movement at a rate of 1.77±0.40
µm/second (n=55) (Fig.
5a). This result is consistent with previously measured
microtubule-dependent vesicle movements and in vitro motility of dynein and
kinesin-like motors in Dictyostelium (1.4-2.8 µm/second)
(McCaffrey and Vale, 1989;
Pollock et al., 1999
;
Pollock et al., 1998
;
Roos et al., 1987
). The normal
distribution of minus-end-directed velocities suggests that the movement is
driven predominantly by a single motor, most probably dynein. Dynein movements
were sometimes interrupted by short pauses, which occur more frequently in
plus-end-directed rather than minus-end-directed runs. Movement usually
resumed on the same track following the pause, suggesting that the cargo did
not fall off the track in the process. It should be noted that this analysis
is greatly facilitated by the relatively small numbers of microtubules in the
cytoplasm of Dictyostelium cells. This reduces the likelihood of
switching of cargo from one microtubule to another. In both directions,
dynein-associated structures can usually travel continuously along the same
track over a distance of several microns
(Fig. 5b), indicating that
movement in either direction is quite processive.
|
Dynein remains stably associated with cargo that reverses direction
of movement
Although most dynein travels unidirectionally over a long distance (up to 7
µm), some dynein-containing vesicles occasionally reverse the direction of
their movement along the same track (Fig.
6a). The properties of the plus-end-directed and
minus-end-directed elements of bidirectional movements are similar to those in
unidirectional movements described above. A direction reversal is usually
preceded by a very short pause (<1 sec) or no pause at all
(Fig. 6b). The intensity of the
corresponding dynein-GFP signal did not change during direction reversal. The
stable association of dynein with the cargo during the swift reversal of
direction argues against motor dissociation/association as a cause for change
in direction. Instead it suggests a rapid and coordinated switch between
opposing motor activities on the same cargo.
|
Dynein moves rapidly into and out of structures at both ends of the
microtubule
In addition to dynein traffic along the microtubule tracks, we also
observed interesting dynein dynamics at both the minus and plus ends of
microtubules. As mentioned above, cytoplasmic dynein was most concentrated
around the MTOC (Fig. 3), where
the minus ends of microtubules meet. This seems to be the result of dynein
accumulating at the ends of the microtubule tracks, as there is constant flow
of dynein into the MTOC. Probably because of the high microtubule density,
dynein dynamics were most active in this region. This central pool of dynein
was constantly exchanging with the rest of the cytoplasm, with punctate dynein
signals flowing into and out of the MTOC
(Fig. 7a). Some puncta
fluctuated near the MTOC, whereas others ran long distances.
|
Dynein also seems to accumulate at the plus ends of microtubules near the
cell cortex. We frequently observed elongated dynein-GFP structures associated
with microtubule plus ends in fixed cells
(Fig. 3). Here, the dynein
signal appeared as a comet-shaped structure, with an intensified end and a
tapered tail. Similar structures of dynein-GFP were observed in living cells
at the cell periphery. This localization resembles that of the
endosome-microtubule linker protein CLIP-170, which preferentially binds to
microtubule growing ends (Perez et al.,
1999). A similar localization has recently been observed in
Aspergillus nidulans, where dynein associates and moves with the
polymerizing ends of microtubules (Xiang
et al., 2000
), suggesting a potentially specialized structure at
the plus end of the microtubule to help associate cargo and dynein.
Interestingly, the microtubule tip decoration of dynein in
Dictyostelium seemed to be quite dynamic, with dynein flowing into
and out of the end structure along the microtubule
(Fig. 7b). The bidirectional
flow at both ends of the microtubule is continuous with dynein traffic along
the microtubules.
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Discussion |
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To ensure that the IC-GFP signal represent normal cytoplasmic dynein localization, we functionally replaced the endogenous dynein IC with the IC-GFP fusion using a two-step approach. We first selected cells expressing the IC-GFP fusion at a level similar to the endogenous IC and then disrupted the endogenous IC gene locus by gene targeting. Because Dictyostelium is a haploid organism and there is only a single dynein IC gene, endogenous IC expression was completely eliminated. As a result, IC-GFP replaces the endogenous IC at wild-type levels. We compared IC-GFP with wild-type IC for association with the dynein and the dynactin complex by immunoprecipitation. IC-GFP associated with dynein and dynactin with the same efficiency as wild-type IC. This ensures that IC-GFP represents the dynein complex. Cells expressing only IC-GFP showed normal growth rates, spindle morphology, microtubule and Golgi organization and multicellular development, supporting the idea that the IC-GFP provides normal dynein function.
Cytoplasmic dynein is well known as a microtubule-based motor, the evidence
for which mostly came from in vitro motility assays
(Lye et al., 1987;
Paschal et al., 1987
;
Schnapp and Reese, 1989
;
Schroer and Sheetz, 1991
).
Using the GFP-fusion, we have directly visualized dynein's movement along the
microtubules for the first time in living cells. Dynein travels along linear
microtubules tracks for several microns. Dynein traveled along microtubules
towards both microtubule plus ends and minus ends. Because cytoplasmic dynein
is the only minus-end-directed microtubule motor identified in
Dictyostelium so far and because the velocity of the
minus-end-directed movement of dynein signal is consistent with that of
dynein-dependent motility measured by in vitro motility assays, we infer that
the minus-end-directed movement of dynein is powered by dynein itself. The
plus-end-directed movement of the dynein signal, on the other hand, must be
driven by a plus-end-directed microtubule motor. The velocity of the
plus-end-directed dynein-associated cargo is consistent with that of the known
plus-end-directed, kinesin-like motors in Dictyostelium. Multiple
kinesin-like motors have been identified in Dictyostelium
(de Hostos et al., 1998
;
McCaffrey and Vale, 1989
;
Pollock et al., 1999
), and it
is hard to predict at this point which ones are responsible for the
plus-end-directed movement of dynein-associated structures.
The bidirectional movement of dynein in living cells strongly supports a
model for the regulation of vesicle directionality in which both plus-end and
minus-end-directed motors associate with a given vesicle. The fact that dynein
can reside on a structure being transported by a plus-end-directed motor
argues that motor dissociation is not necessary for the cargo to move in a
direction opposite to the motor's polarity. Further evidence for this comes
from dynein's stable association with structures that underwent several
directional reversals. Our observations are consistent with previous
immunohistochemistry of fixed samples showing that dynein or kinesin motors
colocalized with membranous compartments that were thought to move in a
direction opposite to the motor polarity
(Hirokawa et al., 1990;
Lippincott-Schwartz et al.,
1995
; Nilsson et al.,
1996
; Reese and Haimo,
2000
; Rogers et al.,
1997
; Roghi, 1999). Our study complements these early studies by
showing the dynamic association of dynein with cargo being transported by a
kinesin-like motor and during the rapid reversal of direction. Together, these
data strongly support a model in which directional movement is controlled by
regulating motor activity instead of motor-cargo association.
When motors of opposing polarity are present on the same cargo, the
direction of movement would depend on which of the motors is functional or
exerts dominant function. This regulation could occur by regulating the
force-generating function of the motor or by regulating the ability of the
motor to interact with microtubules. The fast direction reversals suggest that
such regulation must be able to rapidly coordinate activity of different
motors on the same cargo, that is, upregulating the activity of one group of
motors while downregulating the opposite group of motors at the same time.
Post-translational modification would be a convenient way to perform such
regulation. Phosphorylation/dephosphorylation of motors or associated
proteins, for example, could differentially regulate motor activity under
various circumstances (Dillman and Pfister,
1994; McIlvain et al.,
1994
). Recently, Reese and Haimo suggested that the ability of
dynein and kinesin II to bind to microtubules varies on aggregated or
dispersed pigment granules in melanocytes. The microtubule-binding activity of
these motors could be regulated in vitro by kinase and phosphatase
(Reese and Haimo, 2000
),
suggesting that phosphorylation could regulate motor activity and thus control
directional transport of pigments.
In interphase, dynein is associated mostly with membranous organelles
including endosomes, lysosomes and intermediate compartments in the ER and
Golgi compex (Hirokawa, 1998).
Thus the majority of the dynein traveling along microtubules seems likely to
be membranous vesicles. However, dynein may also associate with other types of
cargo, such as nonmembranous protein particles. For example, neurofilaments
display bidirectional movement along microtubules in neuronal axons
(Prahlad et al., 2000
;
Roy et al., 2000
;
Wang et al., 2000
). These
movements may in part be mediated by cytoplasmic dynein
(Dillman et al., 1996a
;
Dillman et al., 1996b
). Thus,
we cannot exclude the possibility that dynein may be associated with
non-membranous particles. However, since Dictyostelium appears not to
contain intermediate filaments, it is unclear which proteins might reside in
such particles.
The dynamics we observe for dynein also raises questions regarding the
processivity of dynein-driven transport. In vitro, dynein's processivity is
limited (King and Schroer,
2000). Yet in living cells, as shown in
Fig. 4b, dynein-containing
structures frequently travel several microns in a single run without falling
off the track. This indicates that, in vivo, dynein is capable of driving
processive cargo movement. Several factors might contribute to dynein's
increased processivity in vivo. Dynactin has been shown to increase the
processivity of dynein by two-fold (King
and Schroer, 2000
). In addition, multiple dynein molecules might
be engaged on the same cargo to promote processive movement.
In addition to the bidirectional traffic of dynein along the microtubules,
the two ends of microtubules are also points of active bidirectional traffic:
dynein accumulated at, and appeared to move into and out of, the MTOC and the
microtubule plus ends. Microtubule minus ends meet at the MTOC, therefore
dynein accumulation there is likely to be the result of motor accumulation at
the end of the track. The microtubule plus end accumulation of dynein was
first reported in Aspergillus by Xiang et al.
(Xiang et al., 2000), and we
observed a similar accumulation in Dictyostelium. This pattern of
dynein localization resembles that of the endosome-microtubule linker protein,
CLIP-170, which preferentially binds to microtubule growing ends
(Perez et al., 1999
). It has
been suggested that a specialized structure containing CLIP-170, dynein and
dynactin might reside at the microtubule plus ends to help load endosomes to
dynein motors (Valetti et al.,
1999
).
Dynein dynamics in living cells suggest a simplified view of motor traffic and recycling (Fig. 8). Dynein, as a minus-end-directed motor, transports cargo along the microtubule tracks towards the cell center and accumulates at the end of the tracks (MTOC). This central pool of dynein must somehow be recycled to the cell periphery. One efficient mechanism of recycling would be for dynein to ride along the same track back to the cell periphery by associating with cargo being transported by a plus-end-directed motor. This mechanism is supported by the observation that dynein travels toward the cell center with almost the same frequency as it travels toward the cell surface. This recycling mechanism would maintain a constant supply of the motor at places in need. Plus-end-directed motors might take a similar approach (i.e., utilizing dynein) to be recycled. It will be interesting to see if disruption of one motor would affect the recycling and thus the function of the opposite motor. Being an integral part of the motor traffic, the MTOC and the plus ends of microtubules seem to form a big central pool and many smaller peripheral pools of dynein in the cytoplasm. As much of the dynein movement either initiated or ended at the MTOC or microtubule plus ends, the ends might serve as switch stations to load cargo onto motors for a new round of transport and at the same time recycle the opposite motor back to its initiation station. The mechanisms responsible for loading and unloading of motors at the microtubule ends will be an important target for future study.
|
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Acknowledgments |
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Footnotes |
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