Department of Biology, Washington University, St. Louis, Missouri 63130
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Abstract |
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Abstract. Coordination of cellular organization requires the interaction of the cytoskeletal filament systems. Recently, several lines of investigation have suggested that transport of cellular components along both microtubules and actin filaments is important for cellular organization and function. We report here on molecules that may mediate coordination between the actin and microtubule cytoskeletons. We have identified a 195-kD protein that coimmunoprecipitates with a class VI myosin, Drosophila 95F unconventional myosin. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homologue identified of cytoplasmic linker protein (CLIP)-170, a protein that links endocytic vesicles to microtubules. We have named this protein D-CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment with microtubules. The similarity between D-CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they have a number of predicted sequence and structural features in common. 95F myosin and D-CLIP-190 are coexpressed in a number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same particulate structures, which resemble vesicles. They are also colocalized at the posterior pole of the early embryo, and this localization is dependent on the actin cytoskeleton. The association of a myosin and a homologue of a microtubule-binding protein in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important, leads us to speculate that these two proteins may functionally link the actin and microtubule cytoskeletons.
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Introduction |
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GLOBAL organization of the cell and the coordination of its physiology requires interaction between different cytoskeletal systems. During interphase, a typical eukaryotic cell has microtubules emanating from the centrosome located near the nucleus, which extend to the periphery of the cell, presumably interacting with the cortical actin filament meshwork. Microtubules during interphase are thought to be mainly required for the organization of the membrane systems (e.g., vesicular traffic and organelle movement). The actin-rich cortex is important for maintaining cell shape and for cellular movement.
There is increasing evidence of coordination between
the actin and the microtubule cytoskeletons (Langford,
1995; Koonce, 1996
). Data from a number of systems suggests that many cell types use a combination of microtubule and actin filament-based transport in vesicle and organelle trafficking. It is well established that microtubules
are required for long distance transport of cellular components. In contrast, the actin cytoskeleton is thought to be
required for more local traffic. The best evidence for
transport along both cytoskeletal systems is in neurons.
Vesicles appear to be transported along actin filaments in
mammalian growth cones (Evans and Bridgman, 1995
).
Furthermore, gelsolin, which promotes depolymerization
of actin filaments, has been shown to inhibit fast axonal
transport in this system (Brady et al., 1984
). In extruded
squid axoplasm, Kuznetsov et al. (1992)
observed what appeared to be the same vesicle moving along microtubules
and then, subsequently, along microfilaments. Inhibitor
studies provide evidence that mitochondria can move
along both actin filaments and microtubules in neurons in
vivo (Morris and Hollenbeck, 1995
). These data support
the idea that actin filament and microtubule-based transport cooperate to achieve proper organization of cellular components.
The same phenomenon may be occurring in other cell
types. In yeast, the mutant phenotype of the MYO2 gene,
which encodes an unconventional myosin, is suppressed by
overexpression of a kinesin-related protein. These two
proteins are colocalized in regions of active growth where
a polarized arrangement of actin plays an important role
(Lillie and Brown, 1992, 1994
). Microtubules are not normally required for this growth. Thus, the basis for suppression is not completely understood. However, the phenotypic suppression suggests that perhaps microtubule-based
transport can substitute for actin filament-based transport, under some conditions. In polarized epithelial cells,
Fath et al. (1994)
have isolated a population of vesicles
containing both myosin and microtubule motors. They speculate that proper transport of vesicles relies on both
microtubule and actin filament-based transport.
Previously, it has been shown that a class VI unconventional myosin, the Drosophila 95F unconventional myosin,
transports particles along actin filaments during the syncytial blastoderm stage of Drosophila embryonic development (Mermall et al., 1994). 95F myosin activity is required
for normal embryonic development (Mermall and Miller,
1995
). 95F myosin is also associated with particulate structures in other cells of the embryo later in development where it may also be involved in actin-based transport. To
investigate further the transport catalyzed by 95F myosin,
we have begun studies to identify proteins associated with
95F myosin that might be cargoes or regulators. In this
work, we have identified a protein that coimmunoprecipitates with 95F myosin. Sequence analysis reveals that this
protein is the Drosophila homologue of cytoplasmic linker protein (CLIP)1-170. CLIP-170 is believed to function as a
linker between endocytic vesicles and microtubules (Pierre
et al., 1992
). We have named this associated protein D-CLIP-190. Colocalization of 95F myosin and D-CLIP-190 at the
subcellular level at several times in development and in
cultured embryonic cells provides support for the in vivo association of these two proteins. The association of a myosin and a homologue of a microtubule-binding protein
suggests that these two proteins may act to coordinate the
interaction between actin filaments and microtubules.
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Materials and Methods |
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Immunoprecipitations
Immunoprecipitations (IPs) were performed according to Govind et al.
(1992) with minor modifications. The protocol is summarized below. Antibodies (900 µl of monoclonal supernatant or 50 µg of affinity-purified antibody) were incubated overnight at 4°C with 50 µl of protein A beads
(Bio-Rad Laboratories, Hercules, CA). The beads were washed twice in
500 µl of IP buffer. Whole embryo extracts were made from 0-3-h embryos (25°C) in IP buffer (150 mM NaCl, 20 mM Hepes, pH 7.5, 250 mM
sucrose, 1 mM Na molybdate, 1% NP-40) using 1 ml of embryos (~1 g) in
4 ml of buffer. We routinely used IP buffer containing 1% NP-40 because
more protein is immunoprecipitated than when 0.05% NP-40 is used; the
identical profile of proteins is immunoprecipitated under either condition.
The embryo homogenate was filtered through glass wool and spun at
5,000 g. The supernatant was diluted 1:3 in IP buffer and incubated overnight at 4°C with antibody protein A-beads. After this incubation the
beads were thoroughly washed with IP buffer by pelleting at 100 g and resuspending in 500 µl of IP buffer five times. The beads were suspended in SDS-loading buffer and boiled. Protein samples were analyzed by SDS-PAGE.
Immunoprecipitations from later embryos in which the nervous system is developing were also performed. Embryos were collected for 4 h at 25°C and then aged at 18°C for 16 h. Immunoprecipitations were then performed as described above. The same profile of proteins was immunoprecipitated from later embryos as early embryos. Immunoprecipitations from early embryos were generally used because more of the 195-kD protein is immunoprecipitated. Immunoprecipitations were also performed with different salt conditions with qualitatively similar results. When higher than 150 mM NaCl was used in IPs, the amount of the 195-, 190-, and 170-kD proteins was reduced relative to the level of 95F myosin, whereas decreased concentrations of salt increased the amount of the 195-kD protein present (data not shown).
Anti-95F IPs were performed using monoclonal 3C7 culture supernatant (Fig. 1) or affinity-purified rabbit polyclonal antibody (data not
shown). Immunoprecipitations with affinity-purified rabbit 195-kD
(
6D1a) antibody (see Results; Fig. 1), mouse antisera raised against gel-isolated immunoprecipitated 195-kD protein (data not shown), or mouse
antisera raised against 6D1a-glutathione-S-transferase (GST) fusion protein (data not shown) all immunoprecipitate the same profile of proteins.
Control immunoprecipitations were performed in parallel, in which either
protein A beads, or antibody or embryo extract were not included (data
not shown). A dorsal monoclonal antibody generously supplied by R. Steward and affinity-purified rabbit HRP antibody (Sigma Chemical Co.,
St. Louis, MO) were also used as controls in immunoprecipitations (Fig. 1).
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Western Analysis
Proteins were separated on 6 or 8% polyacrylamide gels. Afterwards,
SDS-PAGE proteins were transferred to nitrocellulose in glycine buffer
(150 mM glycine, 0.02% SDS, 25% methanol) overnight at 50 mA and 2 h
at 320 mA. To determine whether similar amounts of protein were
present in each lane, the blots were stained with Ponceau S red. After
blocking with 1% BSA in TBST (TBS [10 mM Tris, pH 8, 150 mM NaCl]/
0.05% Tween-20; Sigma Chemical Co.), blots were incubated for 1 h-overnight with primary antibody. Blots were rinsed three times and washed
four times for 15 min in TBST. Goat mouse secondary antibody conjugated to alkaline phosphatase (Boehringer Mannheim Biochemicals, Indianapolis, IN) was used at 1:5,000 to detect proteins. Alternatively, sheep
mouse secondary antibody conjugated to HRP (Amersham Corp., Arlington Heights, IL) was used at 1:2,000, and detection was performed using enhanced chemiluminescence (SuperSignal Substrate; Pierce, Rockford, IL). High molecular mass standards were from Bio-Rad (200, 116.2, 97.4, and 66.2 kD). The mouse
6D1a antisera was used at 1:2,500. The
95F myosin monoclonal antibody (3C7) culture supernatant was diluted
1:5.
-tubulin antibody (DM1A; gift of D. Kellogg and B. Alberts, University of California at San Francisco, San Francisco, CA) was diluted 1:10.
Cloning the Gene Encoding 195-kD Protein
Antisera to the 195-kD protein was obtained by injecting mice with protein isolated by immunoprecipitation. These IPs were subjected to SDS-PAGE, and the appropriate bands were isolated after Coomassie staining.
The gel slices were processed according to Amero et al. (1987). Approximately 5-10 µg of each of the proteins was injected at monthly intervals
into individual mice using standard protocols.
To obtain cDNA clones encoding the 195-kD protein, a Drosophila
ovarian cDNA gt11 expression library (Steinhaur et al., 1989
) was
screened according to standard methods (Synder et al., 1987
), using anti-
195-kD antisera from mice. Nine clones were isolated and plaque purified.
Southern analysis revealed that clones 8A2a and 6D1a cross-hybridize.
Drosophila 0-4- and 8-12-h embryo cDNA libraries (Brown and Kafatos,
1988
) were screened with the 6D1a clone. Cloning techniques were used
according to Sambrook et al. (1989)
. Standard Southern and Northern
analysis was used (Southern, 1975
). Random primed probes were used for
Southern and Northern experiments (Feinberg and Vogelstein, 1983
).
Nitrocellulose Antibody Affinity Purification
The 6D1a-GST fusion protein or 95F myosin immunoprecipitation reactions were subjected to SDS-PAGE and transferred to nitrocellulose. After staining the nitrocellulose with Ponceau S to visualize the protein, the
appropriate band was cut out and incubated with diluted antisera overnight at 4°C. The nitrocellulose strip was washed four times with TBST
and rinsed one time with 150 mM NaCl, and the antibody was eluted sequentially with 0.1 M glycine, pH 2.9, 2.4, and 2.2. The elutions were neutralized with 2 M Tris, pH 8.4.
Antiserum raised against gel-purified 195-kD protein was affinity purified by absorption to protein (either the 195- or 170-kD protein isolated
by immunoprecipitation) immobilized on nitrocellulose strips. Both the
195-kD antibodies and the
170-kD affinity-purified antibodies react
with the 195-, 190-, and 170-kD proteins on immunoblots of 0-3 h embryo
extract or
95F myosin IPs, suggesting that these proteins are related
(data not shown).
Isolation of 6D1a-GST Fusion Protein
The 6D1a cDNA subcloned into pGEX-1T (Pharmacia Biotech., Piscataway, NJ; Smith and Johnson, 1988
) was freshly transformed into
DH5
F
; colonies were picked and restreaked before fusion protein isolation. An overnight culture was diluted 1:10 in LB-Amp (50 µg/ml) and
grown at 37°C for 1 h. IPTG was added to a final concentration of 1.25 mM,
and the culture was further incubated at 37°C for 2.5 h. After harvesting,
the cells were resuspended in 1/6 vol of TEGK buffer (50 mM Tris, pH
8.0, 2 mM EDTA, 10% glycerol, 50 mM KCl, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin A). Lysozyme was added to a final concentration of 0.1 mg/ml, and the cell suspension was incubated at 30°C for 15 min. The lysozyme treatment was repeated once. Cells were further lysed by sonication. DTT was added to 15 mM. Cell debris was pelleted at 9,000 g
for 30 min. Glutathione beads were rehydrated and washed twice with
TEGK buffer. 50 ml of cell supernatant was incubated with 1 ml of beads
overnight at 4°C. The beads were washed three times with 10 ml of TEGK
buffer containing 1 mM DTT. The GST fusion protein was eluted by incubating the beads on ice for 10 min with 5 mM glutathione in TEGK buffer.
This procedure was repeated three to four times. Elutions were concentrated using a centricon-30 (Amicon Corp., Danvers, MA).
Antibodies
Antisera raised against D-CLIP-190 was obtained by injecting mice with
5-10 µg of the 6D1a-GST fusion protein at approximately monthly intervals using standard protocols. Rabbit polyclonal antisera was prepared by
Cocalico Biologicals, Inc. (Reamstown, PA); rabbits were immunized with
100 µg of the 6D1a-GST fusion protein and boosted with 50 µg of the fusion protein. Rabbit antibodies were affinity purified against a soluble
6D1a-maltose-binding protein fusion (MBP). To make this fusion protein, the 6D1a cDNA was subcloned into the EcoRI site of pMal-c2 vector
(New England Biolabs Inc., Beverly, MA). Bacterial cells transformed
with this construct and expressing the 6D1a-MBP fusion protein were lysed, and the resulting supernatant was incubated with amylose resin according to the manufacturer's protocol (New England Biolabs). The
6D1a-MBP column was preeluted with 0.1 M glycine, pH 2.4, neutralized
with 2 M Tris, pH 8.8, and washed with 1× PBS. The rabbit 6D1a-GST
sera diluted in PBS was incubated overnight with the resin. The resin was
washed with 1× PBS and the
6D1a antibody was eluted with 0.1 M glycine, pH 2.4. The antibody solution was neutralized with 2 M Tris, pH 8.8, dialyzed against 1× PBS, and concentrated using a centricon-30 (Amicon
Corp.).
Sequencing
Both strands of the 10A cDNA clone were completely sequenced by making an M13 library and randomly sequencing to at least twofold coverage
(Wilson et al., 1994). Single-strand templates were prepared and sequenced using a Prism fluorescent sequencing system (Perkin-Elmer
Corp., Norwalk, CT). Sequence data was recorded and analyzed using an
ABI 373-automated fluorescent DNA sequencer (Smith et al., 1986
). The
10A cDNA encodes the last 4 kb of the transcript. The remainder of the
cDNA sequence was obtained from clones 10C, 11A, and 11C by primer
walking. Double-strand templates of 10C, 11A, and 11C cDNA clones
were prepared and sequenced by the method of Sanger et al. (1977)
using
the Sequenase system (United States Biochemical Corp., Cleveland, OH)
and custom primers (GIBCO BRL, Gaithersburg, MD) spaced 200-300
nucleotides apart. The sequence data from all three clones was compiled
using the DNAStar program, SeqMan, to give at least twofold coverage
on each strand. The last 1 kb of sequence of both the 10C and 11A clones
was not determined as it overlaps that of the 10A clone.
Sequence databases were searched using BLASTX and BLASTP
(Altschul et al., 1990; Gish and States, 1993
). DNAStar programs EditSeq,
MegAlign, and Align were used for sequence analysis. Genetics Computer Group (Madison, WI) program Bestfit was used to compare CLIP-170 to D-CLIP-190 (Devereux et al., 1984
). Analysis of the
-helical
coiled-coil regions was done using an algorithm developed by Lupas et al.
(1991)
.
Microtubule Cosedimentation
Determination of the ability of D-CLIP-190 to cosediment with microtubules was performed according to Saxton (1994) with some modifications.
The protocol is summarized below. Whole embryo extracts were made
from 0-2-h embryos (25°C) in extraction buffer (0.1 M Pipes, pH 6.9, 0.9 M
glycerol, 5 mM EGTA, 2.5 mM MgSO4). The extract was clarified by centrifugation at 15,000 g for 40 min at 4°C. The resulting supernatant was
centrifuged at 50,000 g for 30 min at 4°C. To induce microtubule polymerization, 0.3 mM GTP and 20 µM taxol were added to this high speed supernatant. After agitation at room temperature for 20 min to allow microtubule polymerization and binding of microtubule-associated proteins, the
microtubules were sedimented through a sucrose cushion (20% sucrose
and 10 µM taxol in extraction buffer) by centrifugation at 23,000 g for 30 min at 4°C. The resulting supernatant and pellet were analyzed by SDS-PAGE and Western analysis. Embryo extract to which GTP and taxol had
not been added was processed in parallel as a control.
Immunolocalization in Embryos
Immunolocalization was performed using a standard fluorescence microscope (Nikon, Melville, NY) or confocal imaging system (1024; Bio-Rad).
Embryos were fixed with 6% formaldehyde in PEM (0.1 M Pipes, pH 6.9, 1 mM EGTA, and 1 mM MgCl2) with an equal volume of heptane. The vitelline membrane was removed with methanol/50 mM EGTA. Embryos
were blocked with 1% BSA in PBST (1× PBS/0.05% tween 20) for 1-3 h.
Embryos were incubated in 95F myosin monoclonal culture supernatant
diluted 1:6 and
6D1a (
D-CLIP-190) rabbit affinity-purified antibody at
2 µg/ml. After overnight incubation at 4°C with primary antibody in 1%
BSA in PBST, the samples were incubated for 1 h with fluorescent-labeled
donkey
mouse and donkey
rabbit secondary antibodies (Chemicon International Inc., Waltham, MA). Embryos were stained with DAPI at 1 µg/
ml and mounted in 90% glycerol/PBS with 1 mg/ml p-phenylenediamine.
Cytochalasin D and Colchicine Treatment
Embryos were collected for 45 min, rinsed with Triton-salt buffer (0.4% NaCl, 0.1% Triton X-100), and dechorionated with 50% bleach. After thorough washing with distilled water and Triton-salt buffer, embryos were rinsed twice with 0.9% NaCl. Embryos were then treated with the desired concentration of drug in 2 ml 0.9% NaCl/2 ml octane. The NaCl solution was removed, and embryos were fixed in 12% formaldehyde/ PEM for 18-20 min. Embryos were then stored at 4°C or stained as described above.
When embryos were treated for 20-30 min with 10 µg/ml of cytochalasin D, the actin cytoskeleton appeared to be substantially depolymerized.
Treated embryos stained with phalloidin, which binds filamentous actin,
lacked normal actin structures found in untreated embryos such as the actin meshwork present in the cortex of early embryos and actin caps in later
embryos. The nuclei were disorganized in treated embryos as expected
because the axial expansion or spreading of nuclei is an actin-dependent
process (von Dassow and Schubiger, 1994). When embryos were treated
for 20-30 min with 10 or 20 µg/ml of colchicine, they lacked normal microtubule structures, such as the filamentous microtubule meshwork in the
cortex of early embryos and normal spindles during mitosis. Embryos
treated with cytochalasin D or colchicine under the same conditions were
stained with
D-CLIP-190 and/or
95F myosin antibodies; embryos were
also stained with DAPI to visualize the nuclei and assess whether the
drugs were effective.
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Results |
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Immunoprecipitation Identifies Several Proteins in Addition to 95F Myosin
To identify 95F myosin-associated proteins, we used 95F
myosin antibodies in IPs of extracts from 0-3-h embryos
or stage 14-17 embryos that are in the process of nervous
system development (data not shown). 95F myosin (band
5) is immunoprecipitated as expected; in addition, several
proteins of lesser abundance coimmunoprecipitate (Fig. 1,
95F myosin). The most prominent of these proteins are ~200 kD (band 1), 195 kD (band 2), 190 kD (band 3), and
170 kD (band 4). The coimmunoprecipitated proteins are
not detected in controls (Fig. 1, no antibody,
dorsal antibody, and
HRP antibody). The same set of proteins is
also immunoprecipitated from later embryos (data not
shown).
Antibodies to the 195-kD protein were raised against
SDS gel-isolated protein. This antiserum reacts with the
appropriately sized protein (band 2) on immunoblots of
0-3-h embryo extracts and immunoprecipitations performed
with 95F myosin antibody (
95F myosin IPs) (Fig. 2 A).
This antiserum also recognizes the 190- (band 3) and 170-kD
(band 4) proteins (also see Methods and Materials). The
amounts of the 190- and 170-kD proteins vary depending
on the conditions under which the extract is prepared and
in whole embryo extracts often only the 195-kD protein is
detected. Thus, it appears these proteins (band 3 and 4)
are breakdown products of the 195-kD protein (band 2).
The antisera raised against the 195-kD protein also recognizes the 200-kD protein (band 1). This reaction is probably due to contamination of the 195-kD antigen with the
200-kD protein when isolating the protein from the gel rather than immunological relatedness (see below).
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Cloning of the Gene Encoding the 195-kD Protein
To determine whether the 195-kD protein was associated
with 95F myosin in vivo, we had hoped to use an antibody
specific for the 195-kD protein to determine whether the
expression pattern and distribution of the 195-kD protein
overlapped that of 95F myosin. Unfortunately, the antibody raised against gel-isolated 195-kD protein was not
useful for immunolocalization. To provide a source of antigen and begin molecular characterization, we cloned the
gene encoding the 195-kD protein by screening an ovary
expression library with the 195-kD protein antisera (Synder et al., 1987
; Steinhaur et al., 1989
). Several clones were
isolated, including two (ov 6D1a and ov 8A2a) that overlapped based on Southern analysis. To determine which of
the several clones might encode the 195-kD protein, but
not other proteins recognized by the
195 kD serum, GST
fusion proteins made with cDNAs obtained from the
screen were used to affinity purify antibodies from the
mouse polyclonal serum raised against the 195-kD protein
and used to probe Western blots. These experiments suggested that the 6D1a clone encodes part of the 195-, 190-, and 170-kD proteins, but not the 200-kD protein.
We raised antibodies in mice against the 6D1a-GST fusion protein and used them to confirm that the 6D1a
cDNA clone does indeed encode the 195-kD myosin-associated protein. Duplicate immunoblots of 0-3-h embryo
extract (0-3 h EE), 95F myosin IPs (IP-
95F), and 6D1a-maltose-binding protein fusion (6D1a-MBP) were probed with several different antibodies/antisera. Anti-6D1a antiserum (
6D1a-GST; Fig. 2 B) recognizes the 6D1a-MBP
fusion protein indicating that the serum contains antibodies that react with the 6D1a protein as expected. In addition, the serum recognizes a protein of 195 kD (band 2) in
0-3-h embryo extract and the 195-kD protein (band 2) and
its breakdown products (band 3 and 4) in
95F myosin IPs. The pattern of bands (2-4) is identical to that seen when
the
195-kD gel-isolated protein antiserum (
195 kD; Fig.
2 A) is used to probe a duplicate blot, except that the
195-kD protein antiserum recognizes the 200-kD protein
(band 1) whereas
6D1a sera does not. The preimmune
sera from the mouse in which this antibody was raised was
used as a control (Fig. 2 C; PI(
6D1a-GST)). Thus, the
6D1a clone encodes at least part of the 195-kD protein. When
95F myosin monoclonal antibody is used to probe
immunoblots of the same samples, no cross-reactivity is
observed with the 200-kD protein, the 195-kD protein, or
the 6D1a-MBP fusion protein (
95F; Fig. 2 D). This result
suggests that the 195-kD protein coimmunoprecipitates
with the 95F myosin antibody because of its association
with 95F myosin and not because of cross-reactivity of the
95F myosin antibody.
Immunoprecipitation with Antibodies Raised against the 195-kD Protein Coimmunoprecipitate 95F Myosin
To determine if 95F myosin could be coimmunoprecipitated with the 195-kD protein antibody, affinity-purified
rabbit antibody raised against the 195-kD cloned fusion
protein (6D1a-GST; Fig. 1,
6D1a) or mouse polyclonal
antisera raised against the 195-kD protein isolated by IP
(data not shown) were used in IPs from early embryos.
The proteins present in these IPs are essentially identical
to those immunoprecipitated with
95F myosin antibodies. The 195-kD protein (band 2) and its breakdown products (band 3 and 4) are immunoprecipitated as expected.
In addition, 95F myosin (band 5) is coimmunoprecipitated. Neither 95F myosin nor the 195-kD protein are immunoprecipitated in controls (Fig. 1, no antibody,
dorsal
antibody, and
HRP antibody). The identities of the proteins in the Coomassie-stained gel (Fig. 1 A) were confirmed by immunoblot (Fig. 1 B). Thus, IPs with
95F myosin antibody and
195-kD protein antibody show that
these two proteins are associated in embryo extracts. The
200-kD protein (band 1) seen previously in
95F myosin
IPs is also present in
195-kD protein immunoprecipitations.
Sequence Analysis of cDNAs Encoding the 195-kD Protein
Based on Northern analysis with the 6D1a cDNA clone
(2.5 kb), the transcript that encodes the 195-kD protein is
expected to be ~6.1 kb (data not shown). To obtain
cDNA clones encoding the entire transcript, we screened
Drosophila 0-4- and 8-12-h embryonic cDNA libraries
(Brown and Kafatos, 1988) with the ov 6D1a cDNA clone
(Fig. 3 A). Because a full-length cDNA was not obtained, four overlapping cDNA clones, all from the 8-12-h cDNA
library, were sequenced using a combination of random
automated fluorescent DNA sequencing and primer walking using standard dideoxy sequencing methods (Fig. 3 A;
Materials and Methods).
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The composite cDNA is 5,999 bases in length with a single long open reading frame (ORF) predicted to encode a
protein of 1,690 amino acids. Because the largest transcript detected on Northern blots is ~6.1 kb, this composite cDNA is likely to be full length. The calculated molecular mass of this protein is 189 kD, which is in good
agreement with the size estimated from SDS polyacrylamide gels. The first ATG of this ORF is located at nucleotide 633 and is preceded by the sequence AAAA, which
matches the Drosophila translational initiation consensus
sequence (C/A, A, A, C/A; Cavener, 1987). After the large
ORF is a 294 nucleotide 3
untranslated region. At the end
of the 10A cDNA are 16 adenine residues; 23 nucleotides
5
of this stretch of adenines is the sequence AAUAAA, which is identical to the most commonly observed polyadenylation signal (Proudfoot and Brownlee, 1982
). Thus,
the composite cDNA extends to the poly(A) tail.
The 195-kD Myosin-associated Protein Is a CLIP-170 Homologue
When the sequence databases GenBank/EMBL/DDBJ,
PDB, Swiss Prot, and PIR were searched using BLASTP
(Altschul et al., 1990; Gish and States, 1993
), the proteins
that have the most significant similarity to the 195-kD protein are CLIP-170 (BLASTP score of 262 and a probability score of 6.1 × 10
106) and restin (BLASTP score of 262 and a probability score of 1.4 × 10
105). CLIP-170 was
identified in HeLa cells as a protein that links endocytic
vesicles to microtubules (Pierre et al., 1992
). Restin, which
was identified in human peripheral blood monocytes, has two isoforms, one identical to CLIP-170 and the other that
differs from CLIP-170 by an insert of 35 amino acids
(Bilbe et al., 1992
). A search of the yeast sequence database with either CLIP-170 or the 195-kD protein did not
reveal any proteins with such a high level of similarity at
the amino acid level.
An alignment of the sequence of the 195-kD protein and
CLIP-170 (Fig. 3) reveals that with the exception of 48 amino acids at the extreme amino terminus of the 195-kD
protein, the two proteins can be aligned along their entire
length. An amino-terminal domain of ~350 amino acids
and a carboxy-terminal domain of ~90 amino acids are
most similar (Fig. 4 A). These two domains are separated by a long region of predicted coiled coil (Lupas et al.,
1991). Overall, the 195-kD and CLIP-170 are 34.5% identical. If conservative substitutions are included, the similarity increases to 53%. Thus, the 195-kD protein appears
to be the Drosophila homologue of CLIP-170/restin. We
have named the 195-kD protein, based on its predicted
molecular weight, D-CLIP-190.
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As expected for homologous
proteins, the sequences of D-CLIP-190 and CLIP-170 predict a number of shared biochemical and structural features in addition to sequence motifs (see below and Figs. 3 B and 4). Both proteins can be divided into three regions:
amino- and carboxy-terminal domains, separated by a region predicted to form an extended -helical coiled coil
(Fig. 4 A; Pierre et al., 1992
). The amino-terminal region
has two copies of a 57-amino acid motif or repeat. These
motifs in CLIP-170 have been demonstrated to be capable
of binding to microtubules. Comparison of the motifs reveals even higher identity (rep1 - 48%; rep 2 - 50%) and
similarity (rep1 - 64%; rep 2 - 68%) than when the entire
amino-terminal regions are compared (Fig. 4 A). The microtubule-binding motif has been identified in a number of
other proteins that are known to interact with the microtubule cytoskeleton. Alignment of the D-CLIP-190 repeats
with those of CLIP-170 and some of these other proteins is
shown in Fig. 4 B.
The majority of the difference in the predicted size between the Drosophila and the human protein is in the second region, the coiled-coil domain. The coiled-coil region
of CLIP-170 extends from amino acid 350 to 1310, a region
of 960 amino acids. Regions of D-CLIP-190 that are predicted to form a coiled coil begin at amino acid 380 (Lupas
et al., 1991). However, four prolines, which are predicted
to break
helices, are closely spaced together at positions
469, 476, 477, and 480 (Fig. 3). Only one proline is present
in the coiled-coil domain of CLIP-170 (position 492). After these prolines, D-CLIP-190 is predicted to form an extended coiled coil until amino acid 1600. The coiled-coil
domain is 1,120 amino acids long in D-CLIP-190 compared to the 960-amino acid long region of CLIP-170; the
region from position 380-480 in D-CLIP-190 may form an
additional shorter domain of coiled coil. Not surprisingly, the level of similarity between CLIP-170 and D-CLIP-190
decreases in the coiled-coil domain (Fig. 4 A). However, a
short sequence near the start of the coiled-coil regions of
the two proteins is notably more similar than any other sequence in the coiled-coil domain (amino acids 370-393 in
D-CLIP-190 and amino acids 354-377 in CLIP-170) (Fig. 3
B). In addition, near the end of the predicted coiled-coil
regions is a stretch of sequence that is also more similar
(amino acids 1573-1594 in D-CLIP-190 and amino acids
1277-1298 in CLIP-170).
The third region, the carboxy terminus, is ~90 amino acids long and is the region most highly conserved between
CLIP-170 and D-CLIP-190 (Figs. 3 B and 4 A). This region
contains a putative metal-binding motif (CX2CX3GHX4C;
Copeland et al., 1984; Berg, 1986
; Pierre et al., 1992
) of unknown function, which is also shared with BIK1 (Fig. 4 C).
In addition to the metal-binding sequence, another stretch
of 23 amino acids is highly conserved between CLIP-170
and D-CLIP-190; 19/23 amino acids are identical (Fig. 4
D). This sequence contains several cysteines and a histidine and is reminiscent of the metal-binding domain already noted. However, the spacing between the second
cysteine and the histidine does not conform to the consensus for any metal-binding motifs that have been previously
identified (Klug and Schwabe, 1995
; Berg, 1986
). Correct
spacing of these critical amino acids is thought to be necessary for metal binding. Notably, one of the amino acids
that is not conserved is a cysteine in CLIP-170 and a
glutamic acid in D-CLIP-190. The similarity between the
two proteins in this motif is much greater than that in the
metal-binding region, indicating that this is likely to be a
functionally important motif.
D-CLIP-190 Cosediments with Microtubules
The similarity of D-CLIP-190 to CLIP-170 at the amino
acid level and, in particular, in the putative microtubule-
binding motifs predicts that D-CLIP-190 is capable of
binding to microtubules. To determine if D-CLIP-190 can
in fact associate with microtubules, the ability of D-CLIP-190 to cosediment with microtubules was assessed (Fig. 5).
Microtubules were polymerized using endogenous tubulin
by adding GTP and taxol to early Drosophila embryo extracts. A subset of proteins were cosedimented with microtubules in the presence of GTP and taxol (data not shown).
In the absence of GTP and taxol neither microtubules nor
these proteins were pelleted. A substantial fraction of
D-CLIP-190 is present in the pellet in the presence of GTP
and taxol (Fig. 5). Without the addition of GTP and taxol,
D-CLIP-190 is not present in the pellet. Because the binding of CLIP-170 to microtubules is known to be regulated by phosphorylation (Rickard and Kreis, 1991), it is perhaps not surprising that not all of the D-CLIP-190 cosediments with microtubules. Thus, D-CLIP-190 can associate
with microtubules in vitro.
|
95F Myosin and D-CLIP-190 Are Coexpressed and Colocalized at the Subcellular Level in the Nervous System
If D-CLIP-190 is associated with 95F myosin in vivo, its distribution would be expected to overlap that of 95F myosin. To determine the expression pattern and subcellular localization of D-CLIP-190 at various stages of development, antisera raised against the cloned 195-kD protein (i.e., 6D1a-GST fusion) was used in immunolocalization studies. Overall, the distribution of D-CLIP-190 is very similar to that of 95F myosin at various stages of development, which is consistent with these two proteins being associated in vivo.
95F myosin is present in most if not all tissues throughout the lifetime of the fly. However, its level of expression in particular tissues is regulated, such that a subset of tissues express higher levels of protein at certain stages of embryonic development (K. Kellerman and K. Miller, unpublished observations). The overall immunostaining pattern of D-CLIP-190 and 95F myosin in stage 14-16 embryos is quite similar (Fig. 6). When viewed at low magnification, both proteins are enriched in the anterior structures of the head, the posterior spiracles, and the ventral nerve cord (Fig. 6, A-C).
|
The most striking example of their colocalization is the
enrichment of both proteins in the central nervous system
(CNS) located on the ventral side of the embryo. (Fig. 6,
D-F; the ventral side faces the viewer). The cell bodies of
the central nervous system form a broad, flat, compact region that surrounds the ladder-like connectives (longitudinal tracts) and commissures (transversal fibers that cross
the ventral midline between the connectives; Hartenstein,
1993). The connectives and commissures are formed by
axons from multiple neurons that are in large bundles as
they run anterior, posterior, and across the midline to innervate their targets. Axons that leave the CNS do so at
regular intervals, in a segmentally repeated manner. Both
95F myosin and D-CLIP-190 are enriched in the axonal
processes that make up the connectives and commissures
relative to overall staining levels in the embryo and in the
nerve cell bodies. This enrichment is also apparent in processes that exit the CNS. In contrast, the two are differentially enriched in what appear to be glial cells in different
positions (Fig. 6, D-F and G-I). 95F myosin, but not
D-CLIP-190, is enriched in the midline glial cells that serve
as guide cells for the formation of the commissures. Conversely, D-CLIP-190 is enriched in several cells that lie at the
extreme periphery of the CNS in a segmentally repeated
pattern that correlates with where the axons exit the CNS.
At this position are glial cells that guide the axons as they
exit the CNS. Thus, whereas in some cells or parts of cells the two proteins colocalize, in others they do not.
At higher magnification, the staining of the axons is
nonuniform. Strikingly, the two proteins are present in the
same particulate structures, possibly vesicles, in the neuronal processes. A ventral view of the axonal processes
that lie in the longitudinal and transverse fiber tracts is
presented in G-I. The anterior and posterior commissures
of two segmental repeats, as well as the longitudinal fibers
that connect them, are brightly stained with both antibodies. The individual axons that make up these tracts can occasionally be distinguished. Within these axons, punctate staining is observed with both antibodies. When the images are overlayed, it is clear that there is a one to one correspondence between the punctate spots that contain 95F
myosin and those that contain D-CLIP-190. In Fig. 6, J-L,
the anterior end and a stretch of about two segments
length of one of the connectives is seen in longitudinal section. Again, striking punctate staining is seen along the
length of each axon, with a clear colocalization of 95F myosin and D-CLIP-190 in the spots. In contrast, when embryos are double labeled with D-CLIP-190 and
HRP,
which stains neural cell membranes (Snow et al., 1987
), the
distribution of the two proteins appears rather different
(data not shown). Whereas D-CLIP-190 staining is punctate, HRP staining appears more homogeneous. Thus, the
punctate 95F myosin/D-CLIP-190 structures observed are
distinct from the distribution of a uniformly distributed
protein in axons of neurons of the CNS. Because the 95F
myosin/D-CLIP-190 punctate structures are present in the
axonal processes of neurons, it seems likely that they are
vesicular or organellar structures.
95F Myosin and D-CLIP-190 Are Colocalized at the Subcellular Level in Primary Embryonic Cultured Cells
Because subcellular localization is difficult to discern in
whole embryos, we also have colocalized 95F myosin and
D-CLIP-190 in primary cultures of embryonic cells. These
cultures contain several different cell types, including myoblasts, which eventually fuse to form myotubes with sarcomeric structures, neurons that elaborate extensive processes, and hemocytes, a macrophage-like cell that has a
flat, spread morphology (Cross and Sang, 1978). Cultures
made from gastrulating embryos were fixed and stained with antibodies specific for the two proteins (Fig. 7). We
see a number of interesting colocalizations in the different
cell types. Neurons show 95F myosin and D-CLIP-190 particles like those seen in intact embryos (not shown). Colocalization in hemocytes is quite striking (A-C; well spread
cell in D-F). There are a number of large inclusions in the
cytoplasm, possibly vesicles or other organelles, that stain
brightly for both proteins. In myoblasts, we also see these
cytoplasmic organelles (less well spread cells in D-F) in
which both proteins are present. Whereas 95F myosin
staining appears to be primarily or exclusively confined to
these organelles, D-CLIP-190 is clearly present in other
areas of the cell. Particularly prominent is the D-CLIP-190
staining in the region of the nucleus. 95F myosin is not enriched in this area. We conclude that 95F myosin and
D-CLIP-190 are colocalized in a subset of organelles in
several cell types.
|
95F Myosin and D-CLIP-190 Are Colocalized at the Posterior Pole of the Early Embryo
One of the most surprising features of the localization pattern of D-CLIP-190 is its distribution before migration of the nuclei to the cortex in precellularization embryos. In unfertilized eggs and during nuclear cycles 1-8 of embryogenesis, D-CLIP-190 protein is present at the posterior pole (Fig. 8 A). Consistent with the association of the two proteins, 95F myosin is also enriched at the posterior pole (Fig. 8 B). Because of the high level of protein present in the cortex of the embryo, the enrichment of 95F myosin at the posterior pole is not as striking as that of D-CLIP-190 protein; however, we consistently observe a higher level of protein at the posterior than in the adjacent cortex.
|
|
The Posterior Localization of D-CLIP-190 and 95F Myosin in the Early Embryo Is Dependent on the Actin but Not the Microtubule Cytoskeleton
The localization of D-CLIP-190 and 95F myosin at the
posterior of this large syncytial cell gave us the opportunity to test whether the localization of these two proteins
is dependent on the actin and microtubule cytoskeletons.
As associated proteins, one might expect them to behave
similarly in response to disruption of the cytoskeleton.
Therefore, early embryos were treated with cytochalasin
D to disrupt the actin cytoskeleton or colchicine to disrupt
microtubules (Limbourg and Zalokar, 1973).
When embryos were treated with cytochalasin D, the actin cytoskeleton appeared to be substantially depolymerized (data not shown; see Materials and Methods). In addition, the nuclei were not distributed normally in treated
embryos; this is expected if the cytochalasin D depolymerization was effective because the axial expansion or
spreading of nuclei is an actin-dependent process (von
Dassow and Schubiger, 1994). When such embryos were
stained with
D-CLIP-190 or
95F myosin antibodies, the
localization of these two proteins at the posterior pole was
substantially disrupted (Table I, Fig. 8, D-F). Most cytochalasin D-treated embryos had little or no D-CLIP-190
or 95F myosin present at the posterior pole. A low level of
D-CLIP-190 and 95F myosin may remain in some treated
embryos because cytochalasin D does not depolymerize
all actin filaments in some cases (Cooper, 1987
). From
these results we conclude that the posterior localization of
both D-CLIP-190 and 95F myosin is dependent on the actin cytoskeleton.
Embryos were also treated with colchicine to assess the effect of depolymerizing the microtubule cytoskeleton on localization at the posterior pole of D-CLIP-190 and 95F myosin. Microtubules appeared to be substantially depolymerized by colchicine treatment (data not shown; see Materials and Methods). The distribution of 95F myosin appeared to be unaffected by disrupting the microtubule cytoskeleton (Table I). D-CLIP-190 also appears unaffected, although at higher concentrations of colchicine (20 µg/ml), the protein does appear somewhat more diffuse. Thus, the posterior localization of D-CLIP-190 and 95F myosin does not appear to be dependent on the microtubule cytoskeleton. Embryos were also treated with both cytochalasin D and colchicine. Because this treatment did not completely abolish localization in the majority of embryos, we conclude that D-CLIP-190 is not substantially more affected when compared to treatment with cytochalasin D alone (Table I).
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Discussion |
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We have identified a 195-kD protein, D-CLIP-190, that
coimmunoprecipitates with a class VI myosin, Drosophila
95F unconventional myosin. The same profile of proteins
are immunoprecipitated from embryo extracts with 95F
myosin and
D-CLIP-190 antibodies. The fact that these
two proteins are coimmunoprecipitated with antibodies against either protein argues that their association in vitro is specific. Cloning and sequencing of the gene encoding
the 195-kD protein revealed that it is a homologue of the
human protein, CLIP-170, which is involved in microtubule-vesicle interactions (Pierre et al., 1992
). In addition
to coimmunoprecipitation data, immunolocalization studies support the contention that these two proteins are associated. D-CLIP-190 is coexpressed and colocalized with
95F myosin at several times in development and in a number of tissues. In the central nervous system, 95F myosin
and D-CLIP-190 are coexpressed in a subset of cells and
associated with the same particulate structures/vesicles in
axonal processes of neurons. They are also colocalized in
the same subcellular structures in primary embryonic cultured cells. Interestingly, the proteins are colocalized at
the posterior of the early embryo. The association of this
CLIP-170 homologue and an unconventional myosin suggests that these proteins may be important in coordination
of certain microtubule and actin filament-based processes
in cells.
D-CLIP-190 Is a Homologue of CLIP-170
Comparison of the amino acid sequence of D-CLIP-190
and CLIP-170 reveals that the two proteins are predicted
to have the same overall structure and to share sequence
motifs throughout their length. A predicted long -helical
coiled coil separates the relatively highly conserved amino-
and carboxy-terminal domains of the proteins. CLIP-170
was originally isolated as a protein that mediates binding of endocytic vesicles to microtubules in vitro (Rickard and
Kreis, 1990
; Scheel and Kreis, 1991
). When the CLIP-170
cDNA is transformed into HeLa cells, CLIP-170 binds
along the length of microtubules; however, normally it localizes to structures at the plus ends of peripheral microtubules. The amino-terminal domain of CLIP-170 possesses
two copies of a motif that has been demonstrated to mediate binding to microtubules both in vitro and in vivo
(Pierre et al., 1992
, 1994
). Binding to microtubules in vitro
has been shown by assaying the binding to purified microtubules of recombinant CLIP-170, including versions in
which the microtubule-binding domains have been mutated. Because of the high degree of similarity between the
two proteins in these motifs, it is likely that D-CLIP-190 is
also capable of binding microtubules. Consistent with this idea, D-CLIP-190 cosediments with microtubules. Interestingly, the carboxy-terminal region of CLIP-170 is
thought to be required for its association with cytoplasmic
structures that accumulate at the plus ends of peripheral
microtubules (Pierre et al., 1994
). It is possible that the two
conserved sequences in this region of CLIP-170/D-CLIP-190
are involved in mediating binding to other proteins on vesicles or other cellular structures.
The Posterior Localization of D-CLIP-190 and 95F Myosin Are Similarly Affected upon Disruption of the Cytoskeleton
The localization of D-CLIP-190 and 95F myosin at the
posterior pole of the precellularization embryo is dependent on the actin cytoskeleton but not the microtubule cytoskeleton. The similarity in the behavior of D-CLIP-190
and 95F myosin when the cytoskeleton is disrupted provides additional support that these two proteins are associated in vivo. However, assuming that 95F myosin binds actin filaments and D-CLIP-190 binds microtubules at the
posterior pole of the embryo, it is rather curious that disruption of microtubules does not substantially disrupt the
localization of at least D-CLIP-190. This result could be
due to incomplete depolymerization of microtubules, such
that the two proteins remain bound at the posterior via
colchicine-resistant microtubules. However, the observed
behavior could also be explained by the fact that human
CLIP-170 appears to associate with microtubules only
transiently (Rickard and Kreis, 1996). When microtubules are disrupted, D-CLIP-190 may still be part of a complex
with 95F myosin and bound to the actin cytoskeleton and,
thus, still present at the posterior pole. When the actin cytoskeleton is disrupted, however, the complex may not remain at the posterior pole because D-CLIP-190 is not stably bound to microtubules; therefore, the complex may
then diffuse from the posterior pole.
Both the microtubule and actin cytoskeleton are thought
to be involved in localization of determinants (Yisraeli et
al., 1990; Erdélyi et al., 1995
; Pokrywka, 1995; Guo and
Kemphues, 1996
; Tetzlaff et al., 1996
). In Drosophila the
cytoskeleton plays important roles during oogenesis and
early embryogenesis when axial polarity is being established. A group of genes has been characterized that are
required for posterior polarity and germ cell formation, both of which require the localization of determinants at
the posterior pole (St. Johnston, 1993
). Many of the gene
products encoded by these posterior group genes such as
oskar are present at the posterior of the oocyte and early
embryo. During oogenesis, microtubules are required for
localization of several mRNAs including oskar (Theurkauf et al., 1993
; Pokrywka, 1995). The actin cytoskeleton may also be involved in localization because oskar mRNA
is not localized in normal amounts to the posterior pole in
oocytes and early embryos from females mutant for cytoplasmic tropomyosin (Erdélyi et al., 1995
; Tetzlaff et al.,
1996
). In addition, when the actin cytoskeleton is disrupted with cytochalasin D, pole plasm components are
not stably maintained at the posterior of the early embryo
(Lantz, V.A., S. Clemens, and K. Miller, manuscript submitted for publication). These results suggest that RNA
localization at the posterior of the Drosophila embryo may
require coordination of the actin and MT cytoskeletons.
This potential coordination of actin- and microtubule-based processes is not unique to Drosophila. In Xenopus,
microtubules are responsible for Vg1 mRNA transport
whereas actin filaments are required for its maintenance at
the vegetal pole (Yisraeli et al., 1990). The localization of
95F myosin and D-CLIP-190 at the posterior pole raises
the possibility that they could be involved in stable association of pole plasm components at the posterior pole
through their association with actin filaments. Further
studies will be required to provide a functional link between these two proteins and posterior patterning.
A Family of Linker Proteins May Mediate Interactions between Different Cellular Components
CLIP-170 and its Drosophila counterpart appear to be a
member of, at present, a small family of proteins that includes DP-150Glued and BIK1 (Rickard and Kreis, 1996).
The other members of the family share only limited regions
of sequence similarity and a subset of structural features
with D-CLIP-190/CLIP-170. DP-150Glued is known to be part
of a larger complex, the dynactin complex, which stimulates dynein-mediated motility of vesicles in vitro (for review
see Schroer, 1996
). Biochemical evidence suggests that
DP-150Glued binds microtubules via the single microtubule-binding motif in the amino-terminal region and is a key
protein in linking components of the complex together.
D-CLIP-190/CLIP-170 may act in a manner analogous
to DP-150Glued, as a linker protein between microtubules
and in this case the actin cytoskeleton via 95F myosin. Because CLIP-170 is located at the peripheral end of microtubules near the actin cortex, it is possible that this protein
and a class VI myosin like 95F myosin may provide a direct link between microtubules and the actin cortex. This
complex could function to anchor microtubules in the actin cortex (Koonce, 1996). Such binding may be important
for microtubule cytoskeleton organization and microtubule stability. Alternatively, it is possible that the 95F myosin/D-CLIP-190 complex functions at a transition point
for the transfer of vesicles or other cytoplasmic structures
transported along microtubules to actin filaments in the
cortex or vice versa. At the posterior pole of the oocyte, such a link could be required to allow transfer of posterior
proteins/mRNAs from microtubules along which they
were transported to actin filaments where they can be anchored at the posterior. Maintaining this link, which initially forms during oogenesis through early embryogenesis, could be important for the stable localization of
posterior pole plasm components.
Because these two proteins colocalize in structures that most likely include other proteins and perhaps lipids, it is not clear whether their association is direct. The ability to coimmunoprecipitate 95F myosin and D-CLIP-190 in 1% NP-40 would tend to suggest that these two proteins are directly associated. Further biochemical studies suggesting direct binding would support either of the above models.
Coordination of Microtubule and Actin-based Transport
Because 95F myosin has been implicated in transport and
the vertebrate homologue of D-CLIP-190, CLIP-170, is
suspected of being involved in transport (Rickard and
Kreis, 1996), the colocalization in axons, where vesicle/organelle transport along both actin and microtubules has
been observed, is particularly intriguing. The particulate
distribution of both 95F myosin and D-CLIP-190 in nerve
processes in the embryo and also in cultured cells from Drosophila embryos is consistent with these structures being vesicles. CLIP-170 has been suggested to participate in
loading endocytic vesicles on to the plus ends of microtubules (Rickard and Kreis, 1996
). No previous data exist
that suggest an interaction of CLIP-170 with the actin cytoskeleton. However, the plus ends of peripheral microtubules to which the CLIP-170-associated vesicles appear to
bind are adjacent to the actin filament-rich cortex. One
function for D-CLIP-190 and 95F myosin in the same
structures may be to coordinate the transport of specific
cargoes along both microtubules and actin filaments to facilitate their proper localization in the cell.
There is some evidence of a role for unconventional myosins in vesicle traffic (Fath and Burgess, 1994; Langford,
1995
). In the nervous system, in particular, in a variety of
organisms, there is increasing evidence for vesicular and
organellar transport along both microtubules and microfilaments (Brady et al., 1984
; Lillie and Brown, 1992
; Kuznetsov et al., 1992
; Fath et al., 1994
; Morris and Hollenbeck, 1995
; Evans and Bridgman, 1995
). More recently, in
yeast, class I myosins have been implicated in endocytosis (Geli and Riezman, 1996
). MYO2 (Myosin V) mutants
have defects that have been interpreted as recycling defects (Govindan et al., 1995
). Furthermore, expression of
activated RhoD, a small GTPase, causes reorganization of
the actin cytoskeleton and affects endosome motility and
distribution (Murphy et al., 1996
). The D-CLIP-190/95F myosin complex could serve to permit movement of vesicles from one cytoskeletal filament type to the other during secretion, transport, and/or endocytosis.
One model for how transport may occur in neurons is that long range movement occurs along microtubules whereas actin-based transport is required for traversing the actin-rich cortex along the axon periphery and at the nerve terminal. Thus, a myosin, like 95F myosin, and a microtubule motor, like a kinesin-related protein or dynein, would be present on the same vesicle. When a vesicle is being transported along the axon, it would primarily use its microtubule motor(s), potentially being attached or regulated through association with D-CLIP-190. Actin-based transport would only come into play when the vesicle encounters the relatively microtubule-free, actin-rich peripheral cytoplasm. Once a vesicle reached the terminal, actin-based transport events, mediated by 95F myosin, would then be required to bring it to its final target. Actin-based transport might also be important in local recycling of vesicles at the nerve terminal.
We have shown that D-CLIP-190, a microtubule-binding protein and CLIP-170 homologue, and the 95F (class VI) unconventional myosin are present in the same complex. Our studies reveal a potentially important link between the actin and microtubule cytoskeletal systems. They support other studies that have provided evidence for coordination between actin and microtubules-based cellular processes.
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Footnotes |
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Received for publication Received for publication 29 July 1997 and in revised form 24 November 1997..
We are especially grateful for the help of J. Verbsky and C. Cheney (both from Washington University, St. Louis, MO) with automated fluorescent DNA sequencing. In addition, we thank the Department of Genetics at Washington University School of Medicine for use of the ABI Prism sequencing system. We thank J. Diani and the animal facility in the Biology Department at Washington University for injecting and bleeding the mice. We thank R. Steward for the dorsal antibody. We thank J. Cooper, J. McNally, C. Cheney, C. Wagner, R. Hopmann, J. Hicks, B. Clifford (all from Washington University), and T. Schroer (Johns Hopkins University, Baltimore, MD) for helpful suggestions on the manuscript. ![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410 |
2. | Amero, S.A., T.C. James, and S.C.R. Elgin. 1987. Raising antibodies to protein bands in gels. In Methods in Molecular Biology. Vol. 3. J.M. Walker, editor. Humana Press, Inc., Clifton, NJ. 355-362. |
3. | Berg, J.M.. 1986. Potential metal-binding domains in nucleic acid binding proteins. Science. 232: 485-487 |
4. | Berlin, V., C.A. Styles, and G.R. Fink. 1990. BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae colocalizes with tubulin. J. Cell Biol. 111: 2573-2586 [Abstract]. |
5. | Bilbe, G., J. Delabie, J. Bruggen, H. Richener, F.A.M. Asselbergs, N. Cerletti, C. Sorg, K. Odink, L. Tarcsay, W. Wiesendanger, C. DeWolf-Peeters, and R. Shipman. 1992. Restin: a novel intermediate filament-associated protein highly expressed in the Reed-Sternberg cells of Hodgkin's disease. EMBO (Eur. Mol. Biol. Organ.) J. 11: 2103-2113 [Abstract]. |
6. | Brady, S.T., R.J. Lasek, R.D. Allen, H.L. Yin, and T.P. Stossel. 1984. Gelsolin inhibition of fast axonal transport indicates a requirement for actin microfilaments. Nature. 310: 56-58 |
7. | Brown, N.H., and F.C. Kafatos. 1988. Functional cDNA libraries from Drosophila embryos. J. Mol. Biol. 203: 425-437 |
8. | Cavener, D.R.. 1987. Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15: 1353-1361 [Abstract]. |
9. | Cooper, J.A.. 1987. Effects of cytochalasin and phalloidin on actin. J. Cell Biol. 105: 1473-1478 |
10. | Copeland, T.D., M.A. Morgan, and S. Oroszlan. 1984. Complete amino acid sequence of the basic nucleic acid binding protein of Feline Leukemia Virus. Virol. 133: 137-145 . |
11. | Cross, D.P., and J.H. Sang. 1978. Cell culture of individual Drosophila embryos. J. Embryol. Exp. Morphol 45: 161-172 |
12. | Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387-395 [Abstract]. |
13. | Erdélyi, M., A.-M. Michon, A. Guichet, J.B. Glotzer, and A. Ephrussi. 1995. Requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization. Nature. 377: 524-527 |
14. | Evans, L.L., and P.C. Bridgman. 1995. Particles move along actin filament bundles in nerve growth cones. Proc. Natl. Acad. Sci. USA. 92: 10954-10958 [Abstract]. |
15. | Fath, K.R., and D.R. Burgess. 1994. Membrane motility mediated by unconventional myosin. Curr. Opin. Cell Biol. 6: 131-135 |
16. | Fath, K.R., G.M. Trimbur, and D.R. Burgess. 1994. Molecular motors are differentially distributed on Golgi membranes from polarized epithelial cells. J. Cell Biol. 126: 661-675 [Abstract]. |
17. | Feinberg, A.P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13 and 137:266-267. |
18. | Geli, M., and H. Riezman. 1996. Role of type I myosins in receptor-mediated endocytosis in yeast. Science. 272: 533-535 [Abstract]. |
19. | Gish, W., and D.J. States. 1993. Identification of protein coding regions by database similarity search. Nat. Genet. 3: 266-272 |
20. | Govind, S., A.M. Whalen, and R. Steward. 1992. In vivo self-association of the Drosophila rel-protein dorsal. Proc. Natl. Acad. Sci. USA. 89: 7861-7865 [Abstract]. |
21. | Govindan, B., R. Bowser, and P. Novick. 1995. The role of Myo2, a yeast class V myosin, in vesicular transport. J. Cell Biol. 128: 1055-1068 [Abstract]. |
22. | Guo, S., and K.J. Kemphues. 1996. A non-muscle myosin required for embryonic polarity in Caenorhabditis elegans. Nature. 382: 455-458 |
23. | Hartenstein, V. 1993. Atlas of Drosophila Development. Cold Spring Harbor Press, Cold Spring Harbor, NY. |
24. | Higgens, D.G., A.J. Bleasby, and R. Fuchs. 1992. CLUSTAL V: improved software for multiple sequence alignment. Comp. Appl. Biosci. 8: 189-191 [Abstract]. |
25. | Holzbaur, E.L.F., J.A. Hammarback, B.M. Paschal, N.G. Kravit, K.K. Pfister, and R.B. Vallee. 1991. Homology of a 150K cytoplasmic dynein-associated polypeptide with the Drosophila gene Glued. Nature. 351: 579-583 |
26. |
Klug, A., and
J.W.R. Schwabe.
1995.
Zinc fingers.
FASEB (Fed. Am. Soc. Exp.
Biol.) J.
9:
597-604
|
27. | Koonce, M.P.. 1996. Making a connection: the "other" microtubule end. Cell Motil. Cytoskeleton. 35: 85-93 |
28. | Kuznetsov, S.A., G.M. Langford, and D.G. Weiss. 1992. Actin-dependent organelle movement in squid axoplasm. Nature. 356: 722-725 |
29. | Langford, G.M.. 1995. Actin- and microtubule-dependent organelle motors: interrelationships between the two motility systems. Curr. Opin. Cell Biol. 7: 82-88 |
30. | Lillie, S.H., and S.S. Brown. 1992. Suppression of a myosin defect by a kinesin-related gene. Nature. 356: 358-361 |
31. | Lillie, S.H., and S.S. Brown. 1994. Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae. J. Cell Biol. 125: 825-842 [Abstract]. |
32. | Limbourg, B., and M. Zalokar. 1973. Permeabilization of Drosophila egg. Dev. Biol. 35: 382-387 |
33. | Lupas, A., M. Van Dyke, and J. Stock. 1991. Predicting coiled coils from protein sequences. Science. 252: 1162-1164 |
34. | Mermall, V., J.G. McNally, and K.G. Miller. 1994. Transport of cytoplasmic particles by an unconventional myosin in living Drosophila embryos. Nature. 369: 560-562 |
35. | Mermall, V., and K.G. Miller. 1995. The 95F unconventional myosin is required for proper organization of the Drosophila syncytial blastoderm. J. Cell Biol. 129: 1575-1588 [Abstract]. |
36. | Morris, R.L., and P.J. Hollenbeck. 1995. Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons. J. Cell Biol. 131: 1315-1326 [Abstract]. |
37. | Murphy, C., R. Saffrich, M. Grummt, H. Gournier, V. Rybin, M. Rubino, P. Auvinen, A. Lutcke, R.G. Parton, and M. Zerial. 1996. Endosome dynamics regulated by a Rho protein. Nature. 384: 427-432 |
38. | Pellman, D., M. Bagget, H. Tu, and G.R. Fink. 1995. Two microtubule-associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae. J. Cell Biol. 130: 1373-1385 [Abstract]. |
39. | Pierre, P., J. Scheel, J.E. Rickard, and T.E. Kreis. 1992. CLIP-170 links endocytic vesicles to microtubules. Cell. 70: 887-900 |
40. |
Pierre, P.,
R. Pepperkok, and
T.E. Kreis.
1994.
Molecular characterization of
two functional domains of CLIP-170 in vivo.
J. Cell Sci.
107:
1909-1920
|
41. | Pokrywka, N.J., and E. Stephenson. 1995. Microtubules are a general component of mRNA localization systems in Drosophila oocytes. Dev. Biol. 167: 363-370 |
42. |
Proudfoot, N.J., and
G.G. Brownlee.
1982.
3![]() |
43. | Rickard, J.E., and T.E. Kreis. 1990. Identification of a novel nucleotide-sensitive microtubule binding protein in HeLa cells. J. Cell Biol. 110: 1623-1633 [Abstract]. |
44. |
Rickard, J.E., and
T.E. Kreis.
1991.
Binding of pp170 to microtubules is regulated by phosphorylation.
J. Biol. Chem.
266:
17597-17605
|
45. | Rickard, J.E., and T.E. Kreis. 1996. CLIPs for organelle-microtubule interactions. Trends Cell Biol. 6: 178-183 . |
46. | Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. |
47. | Sanger, F., S. Nicklen, and A.R. Coulson. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74: 5463-5467 [Abstract]. |
48. | Saxton, W.M.. 1994. Isolation and analysis of microtubule motor proteins. Methods Cell Biol. 44: 279-288 |
49. |
Scheel, J., and
T.E. Kreis.
1991.
Motor independent binding of endocytic carrier
vesicles to microtubules in vitro.
J. Biol. Chem.
266:
18141-18148
|
50. | Schroer, T.A.. 1996. Structure and function of dynactin. Semin. Cell Dev. Biol. 7: 321-328 . |
51. | Smith, D.B., and K.S. Johnson. 1988. Single step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 67: 31-40 |
52. | Smith, L.M., J.C. Sanders, R.J. Kaiser, P. Hughes, C. Dodd, C.R. Connell, C. Heiner, S.B.H. Kent, and L.E. Hood. 1986. Fluorescence detection in automated DNA sequence analysis. Nature. 321: 674-679 |
53. | Snow, P.M., N.H. Patel, A.L. Harrelson, and C. Goodman. 1987. Neural-specific carbohydrate moiety shared by many surface glycoproteins in Drosophila and grasshopper. J. Neurosci. 712: 4137-4144 . |
54. | Southern, E.M.. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517 |
55. | St. Johnston, D. 1993. Pole plasm and the posterior group genes. In The Development of Drosophila melanogaster. M. Bate and A. Martinez Arias, editors. Cold Spring Harbor Press, Cold Spring Harbor, NY. 325-364. |
56. | Steinhaur, W.R., R.C. Walsh, and L.J. Kalfyan. 1989. Sequence and structure of the Drosophila melanogaster ovarian tumor gene and generation of an antibody specific for ovarian tumor protein. Mol. Cell Biol. 9: 5726-5732 |
57. | Swaroop, A., M. Swaroop, and A. Garen. 1987. Sequence analysis of the complete cDNA and encoded polypeptide for the Glued gene of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA. 84: 6501-6505 [Abstract]. |
58. | Synder, M., S. Elledge, D. Sweetser, R.A. Young, and R.W. Davis. 1987. Lambda gt11: gene isolation with antibody probes and other applications. Methods Enzymol. 154: 107-128 |
59. | Tetzlaff, M.T., H. Jäckle, and M.J. Pankratz. 1996. Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation. EMBO (Eur. Mol. Biol. Organ.) J. 15: 1247-1254 [Abstract]. |
60. |
Theurkauf, W.E.,
B.M. Alberts,
Y.N. Jan, and
T.A. Jongens.
1993.
A central
role for microtubules in the differentiation of Drosophila oocytes.
Development (Camb.).
118:
1169-1180
|
61. | Truehart, J., J.D. Boeke, and G. Fink. 1987. Two genes required for cell fusion during cell conjugation: evidence for a pheromone-induced surface protein. Mol. Cell Biol. 7: 2316-2328 |
62. | von Dassow, G., and G. Schubiger. 1994. How an actin network might cause fountain streaming and nuclear migration in the syncytial Drosophila embryo. J. Cell Biol. 127: 1637-1653 [Abstract]. |
63. | Wilson, R., R. Ainscough, K. Anderson, C. Baynes, M. Berks, J. Bonfield, J. Burton, M. Connell, T. Copsey, J. Cooper, et al . 1994. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 368: 32-38 |
64. | Yisraeli, J.K., S. Sokol, and D.A. Melton. 1990. A two step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA. Development (Camb.). 108: 289-298 [Abstract]. |