(Received for publication, March 3, 1995; and in revised form, September 5, 1995)
From the
p150 is the largest polypeptide in the
dynactin complex, a protein heteromultimer that binds to and may
mediate the microtubule-based motor cytoplasmic dynein. Cloning of a
cDNA encoding p150
from rat revealed 31% amino
acid sequence identity with the product of the Drosophila gene, Glued. A dominant Glued mutation results
in neuronal disruption; null mutations are lethal. However, the Glued gene product has not been characterized. To determine
whether the Glued polypeptide is functionally similar to vertebrate
p150
, we characterized the Glued protein in
the Drosophila S-2 cell line. Antibodies raised against Glued
were used to demonstrate that this protein sediments exclusively at 20
S, and associates with microtubules in a salt- and ATP-dependent
manner. Immunoprecipitations from S-2 cytosol with the anti-Glued
antibody resulted in the co-precipitation of subunits of both
cytoplasmic dynein and the dynactin complex. An affinity column with
covalently bound Glued protein retained cytoplasmic dynein from S-2
cytosol. Based on these observations, we conclude that Glued is a
component of a dynactin complex in Drosophila and binds to
cytoplasmic dynein, and therefore the mutant Glued phenotypes
can be interpreted as resulting from a disruption in the function of
the dynactin complex.
The vertebrate dynactin complex is a 20 S protein heteromultimer
that is thought to be involved in mediating intracellular transport by
the microtubule-based motor protein, cytoplasmic
dynein(1, 2, 3, 4, 6) . The
dynactin complex consists of 10 polypeptides, of molecular mass
= 150, 135, 62, 50, 45, 42, 37, 32, 27, and 24
kDa(3, 5) . While these polypeptide were originally
identified as substoichiometric components of cytoplasmic dynein
preparations(7) , it was subsequently found that dynein and the
dynactin complex form distinct 20 S complexes in cytosol that were
separable by ion exchange chromatography (2) or
immunoprecipitation(3) . The demonstration of two distinct
complexes made unclear the relationship between dynein and the dynactin
complex in processes of intracellular motility. In an in vitro motility assay, although cytoplasmic dynein alone could move
microtubules along glass, both complexes were required to move vesicles
along microtubules(2) . Also, evidence from genetic studies
suggested that the two complexes were involved in similar processes in
vivo(8, 9, 10, 11, 12, 13) .
Furthermore, in vertebrate cells, components of both the dynactin
complex and cytoplasmic dynein were found to localize to the same
subcellular structures, including the centrosome during interphase, and
kinetochores and spindle microtubules during mitosis (2, 3, 14, 15, 16, 17) .
The recent demonstration that affinity matrices containing either
covalently bound p150, the largest polypeptide
in the dynactin complex(2, 3) , or covalently bound
dynein intermediate chain are capable of retaining intact cytoplasmic
dynein or dynactin complex, respectively(18) , provides a clear
demonstration that the two complexes are capable of a direct
biochemical interaction.
Several of the component polypeptides of
the vertebrate dynactin complex have been characterized. The 45-kDa
protein, centractin, shares 50% amino acid sequence identity with
skeletal muscle actin, and has been classified as a member of the Arp-1
family of actin-related
proteins(3, 15, 19, 20) . There is
also 1 mol of conventional actin present per dynactin
complex(5, 19) . The 37- and 32-kDa polypeptides of
the dynactin complex have been identified by antibody cross-reactivity
as the
and
subunits of the F-actin binding protein, capping
protein or CapZ(5) . Characterization of rat cDNA clones
encoding the 150-kDa polypeptide indicated that this protein shared 31%
amino acid sequence identity with the product of the Glued gene of Drosophila
melanogaster(21, 22) , and the 150-kDa rat
polypeptide was subsequently named
p150
(3) .
The Drosophila Glued gene has been extensively characterized by traditional genetic approaches. The original dominant Glued mutation, Gl results in abnormal eye formation in heterozygotes, with a reduced number of facets and a smooth shiny surface(23) . These macroscopic aberrations result from gross disruptions of the optic lobe, as well as disorganization of axonal projections between the retinal cells and the medulla neuropil (24) . Homozygotes for Gl as well as null mutants at the Glued locus die by the first instar stage of development (24, 25) . The ability of embryos to survive to this stage is probably due to maternal contribution of Glued transcripts to the zygote(25, 26) . In situ hybridization studies showed that Glued is transcribed in virtually all tissues and at multiple stages of Drosophila development(26) . In addition, mosaic analyses of mutations induced at the Glued locus by somatic recombination showed a failure to recover homozygous cells in heterozygous flies(27) , implying that Glued is essential for the viability of individual cells.
Although Drosophila Glued and rat p150 share significant sequence
identity, the homology between them is discontinuous and
patchy(21) . Regions of p150
and the
Glued polypeptide that share particularly high sequence identity
include an N-terminal domain that has recently been shown to mediate
the binding of p150
to microtubules (16) and a highly charged C-terminal motif that is involved in
the binding of p150
to centractin (16) (Fig. 1). The conservation of these functional
domains supports the contention that the two polypeptides may be
involved in analogous cellular functions in vertebrates and Drosophila. However, related proteins have been identified
that also share extensive sequence identity with the microtubule
binding domain of p150
, including the human
endosome-microtubule linker protein, CLIP-170 (28) and the
yeast microtubule binding protein, BIK-1(29) . Glued,
p150
, and CLIP-170 also share remarkable
similarity in predicted secondary structure outside of this domain,
including extensive predicted
-helices with heptad repeats of
hydrophobic amino acids (Fig. 1). Therefore, based on sequence
comparisons alone it is unclear whether Glued and
p150
polypeptides are related members of a
large family of microtubule binding proteins, or if they share a
homologous function in the dynactin complexes of Drosophila and rat, respectively.
Figure 1:
Schematic comparison of
the structural features of rat p150, Drosophila Glued, and human CLIP-170. Hatched areas represent predicted
-helix, stippled areas represent
the conserved microtubule binding domain common to the three
polypeptides(16, 28) , and the black regions represent the putative centractin binding domain(16) . Thick bars above the maps represent the regions of the
proteins, expressed as bacterial fusions, that were used both as
immunogens for the production of polyclonal antisera as well as for the
construction of affinity matrices. The shaded region represents the overlap in amino acids between the constructs used
for formation of affinity matrices in the rat and Drosophila systems. This region may represent the minimal dynein intermediate
chain binding domain (see ``Discussion''). Thin arrows between proteins represent regions of their amino acid sequences
that were aligned and compared by the Lipman-Pearson method, with the
percent identity shared between proteins assigned to each region.
Predicted amino acid sequences were obtained from published cDNA
sequences(21, 22, 28) , and used to predict
secondary structure by the Garnier-Robson
method.
To date, there has been no biochemical
or cell biological characterization of the Glued gene product
in Drosophila. Therefore, we sought to determine whether Glued
and p150 are members of functionally
homologous dynactin complexes. To approach this question, antibodies
were raised to recombinant Glued protein and used to examine the
biochemical properties and cellular distribution of Glued in the
embryonically derived Drosophila Schneider 2 (S-2) cell
line(30) . We find that the Glued polypeptide is a member of an
oligomeric, microtubule-associated, dynactin complex which localizes to
the centrosome of cultured cells. In addition, like
p150
, we demonstrate that Glued is capable of
a direct interaction with Drosophila cytoplasmic dynein in S-2
cell cytosol. The similarity in biochemical behavior and subcellular
localization of these two proteins indicates that they are involved in
analogous cellular functions, and in turn, that dynactin function is
essential for cell viability in Drosophila.
For immunoprecipitation, protein A-agarose beads (Life
Technologies, Inc.) were loaded with anti-Glued or
anti-p150 antibodies (16) in PBS at 4 °C
overnight. Cytosol was pre-cleared by incubation with unloaded protein
A beads for 30 min at 4 °C, prior to incubation with antibody-bound
beads for 3 h at 4 °C, with agitation. The beads were then rinsed
extensively with RIPA buffer without SDS and eluted with sample buffer
without
-mercaptoethanol. Protein eluates were analyzed by
SDS-PAGE and electroblotted onto Immobilon-P (Millipore). For some
immunoprecipitation experiments, Western blots were probed with
biotin-LC-hydrazide (Pierce) conjugated primary antibodies followed by
horseradish peroxidase-conjugated streptavidin (Pierce) and developed
using enhanced chemiluminescense (DuPont) or 3-amino-9-ethylcarbazole
and H
O
. For all other experiments, Western
blots were probed with rabbit primary antibodies followed by goat
anti-rabbit IgG and then rabbit peroxidase anti-peroxidase (Jackson
Immunologicals) and developed similarly. The anti-centractin antibody
has been described previously(18) , and the anti-dynein heavy
chain antibody DD1 (17) was the kind gift of Dr. E. Vaisberg.
For immunofluorescent localization of the Glued polypeptide,
coverslips of S-2 cells were fixed at -20 °C in methanol with
1 mM EGTA, blocked for at least 1 h in PBS with 10% normal
goat serum and 1% bovine serum albumin, and rinsed in PBS with 1%
bovine serum albumin. Coverslips were then incubated for 1 h at 37
°C in primary antibody, rinsed in PBS, incubated for 1 h in
fluorophore-conjugated secondary antibody (Jackson Immunologicals) that
had been preadsorbed against fixed S-2 cells, rinsed, and mounted in
Tris-buffered Mowiol 4-88 (Calbiochem) mounting media with 0.5% n-propyl gallate (Kodak) to retard photobleaching.
Microtubules were localized with the rat monoclonal anti-yeast tubulin
antibody YL1/2 (Serotec) and centrosomes were localized with rabbit
anti-Drosophila -tubulin antibody ((32) , the
kind gift of Dr. Y. Zheng). Immunofluorescence was performed on a Leica
TCS laser scanning confocal microscope equipped with a 100
/1.4
oil objective, a krypton-argon laser and fluorescein and Texas red
filters. Contrast enhancement and ``sharpening'' convolutions
were applied to images using the Leica software prior to recording on
T-MAX 100 film using a Focus Imagecorder Plus (Focus Graphics).
Figure 2:
Cross-reactivity of anti-Glued antibodies
with p150 from rat brain microtubule salt
extract. Proteins from whole S-2 cell cytosol (lane 1) or rat
brain microtubule 0.4 M NaCl extract (lane 2) were
resolved by 8% SDS-PAGE and the gel was stained with Coomassie Blue (A) or transferred to Immobilon-P and probed with
anti-Drosophila Glued antibodies (B). Molecular mass
in kDa is indicated on the left.
Mammalian
p150 may be isolated by extraction of rat brain
microtubules with either ATP or 0.4 M NaCl (21) . In
order to examine the microtubule binding properties of the Glued
protein, microtubules were assembled from endogenous tubulin in S-2
cytosol by addition of taxol. Because of the low level of tubulin in
S-2 cells as compared to neuronal tissue, excess exogenous
taxol-stabilized bovine brain microtubules were also added to the
assay. After incubation, the microtubules and associated proteins were
isolated by centrifugation, and following a buffer wash, the
microtubules were extracted with either 10 mM ATP or 0.4 M NaCl, and the resulting fractions were examined by immunoblot with
the anti-Glued antibody (Fig. 3, A and B). The
Glued polypeptide was observed to remain soluble in the cytosol until
microtubules were assembled, and then to co-sediment with microtubules (Fig. 3, A and B). Extraction of the pellet
with either 10 mM MgATP or 0.4 M NaCl released the
150-kDa Glued protein from microtubules into the supernatant. In
contrast, most of the 135-kDa Glued polypeptide did not co-sediment
with microtubules (Fig. 3B), and therefore very little
of the 135-kDa protein was extracted from microtubules by either ATP or
salt (Fig. 3B). The ATP- and salt-sensitive microtubule
binding of the Glued polypeptide was compared to the behavior of rat
brain p150
under similar conditions (Fig. 3, A and B). The p150
polypeptide
co-sedimented with microtubules, and was nearly fully released with
either ATP or elevated ionic strength, as had been previously noted for
the rat protein(7, 21) .
Figure 3:
Partitioning of the Drosophila Glued and rat p150 proteins during a
microtubule-associated protein preparation from S-2 cells (S-2) or rat
brain (Rat). Samples from the preparation were analyzed by 8% SDS-PAGE,
electroblotted to Immobilon P, the blot stained with Coomassie Blue (A), destained, and probed with anti-Glued (S-2, B)
or anti-p150
antibodies (Rat, B). S-2
cell cytosol was prepared by two successive rounds of centrifugation (45k, 45,000
g; 145k, 145,000
g; S, supernatant; P, pellet). ATP
was depleted in the cytosol, microtubules were assembled, and
exogenous, taxol-stabilized, bovine brain microtubules were added.
Microtubules were then sedimented and washed (MT; S, post-microtubule supernatant; P, buffer washed
microtubule pellet). The microtubule pellet was then extracted of
associated proteins with either 10 mM ATP (ATP; S,
ATP extract of microtubules; P, post-ATP extracted pellet) or
0.4 M NaCl (NaCl; S, NaCl extract of microtubules, P, post-NaCl extracted pellet). Rat brain (Rat) was processed
similarly, but only the 10 mM ATP (ATP, S and p) and 0.4 M NaCl (NaCl, S and P)
extracts of microtubules are shown for comparison. The ATP extract of
S-2 microtubules was then sedimented through a 5-25% sucrose
gradient and the fractions analyzed by 8% SDS-PAGE and immunoblotting
with anti-Glued antibodies (C). Arrowhead corresponds
to the position of a 150-kDa molecular mass
marker.
The ATP eluate of
microtubules from S-2 cell extracts was further fractionated on
5-25% linear sucrose density gradients. Immunoblots of the
gradient fractions indicated that Glued sedimented at 20 S (Fig. 3C). This result suggests that
microtubule-associated Glued polypeptide is part of a large protein
complex. To determine whether the Glued polypeptide exists in S-2
cytosol exclusively as a component of a macromolecular complex, whole
cytosol was fractionated on a 5-25% sucrose density gradient.
Analysis of gradient fractions by immunoblotting revealed that both the
150- and 135-kDa Glued polypeptides sedimented solely as a peak at
20 S (Fig. 4). The absence of detectable immunoreactivity
at a lower sedimentation coefficient suggests that there is not a
significant fraction of free Glued subunits in S-2 cytosol.
Figure 4: Sedimentation behavior of the Glued polypeptide in S-2 cell cytosol. Whole S-2 cell cytosol was subjected to centrifugation through a 5-25% sucrose density gradient, and the gradient fractions analyzed by 8% SDS-PAGE, electroblotted to Immobilon-P, and the blot Stained with Coomassie Blue (A). The blot was destained and probed with anti-Glued antibodies (B). Gradients were calibrated with catalase (11.3 S), thyroglobulin (19 S), and glutamate dehydrogenase (26.6 S) as standards, and molecular mass in kDa is indicated on the left.
To
further examine the native protein interactions of the Glued
polypeptide, immunoprecipitations from S-2 cytosol were performed under
nondenaturing conditions. Anti-Glued antibodies were observed to
precipitate proteins of 150, 143, 135, 72, and 45 kDa (Fig. 5).
This was compared to immunoprecipitations from rat brain cytosol
performed in parallel with anti-rat p150 antibodies,
which precipitated detectable polypeptides of 150, 135, and 45 kDa as
assayed by Coomassie staining. Western analysis of the S-2 cell
immunoprecipitate demonstrated that the 150/143/135-kDa triplet was
reactive with anti-Glued antibodies, while in the immunoprecipitate
from rat brain, the 150/135-kDa polypeptides reacted with
anti-p150
antibodies. Antibodies to Dictyostelium cytoplasmic dynein (17) and human centractin (18) reacted weakly with bands of >300-kDa and 45-kDa
polypeptides in the Drosophila immunoprecipitate, respectively
(not shown), while the 45-kDa rat protein also reacted with
anti-centractin antibodies (not shown).
Figure 5:
Comparison of native immunoprecipitations
with either anti-Glued or anti-p150 antibodies. Protein A beads were loaded with either
anti-Glued (lanes 1) or anti-p150
(lanes 2) antibodies and used to immunoprecipitate
proteins from S-2 or rat cytosol, respectively. The immunoprecipitate
was analyzed by 8% SDS-PAGE and the gel stained with Coomassie Blue (C.B.), or electroblotted to Immobilon-P and probed with
biotinylated anti-Drosophila Glued (anti-Glued) or
anti-p150
antibodies. The 66-kDa band in the
rat immunoprecipitates is bovine serum albumin, which was added to the
antibodies to stabilize them during affinity purification, and the
prominent
50-kDa band in both immunoprecipitates is the IgG heavy
chain. Molecular mass in kDa is indicated on the left.
Figure 6: Chromatography using a Glued protein affinity matrix. A bacterially expressed fragment from the C terminus of Glued was covalently linked to Sepharose beads to construct the affinity matrix. S-2 cytosol was prepared (Cytosol, lanes 1 in A and B)) and loaded onto a 0.5-ml bed volume Glued affinity column. The flow-through was collected (Flow, lanes 2 in A and B), and the column washed extensively with PHEM buffer plus 50 mM NaCl, and the final 1.5 ml of the wash collected (Wash, lanes 3 in A and B). The column was then eluted with 0.5 M NaCl (0.5M, lanes 4 in A and B) and then 1.0 M NaCl (1.0M, lanes 5 in A and B). The eluates were concentrated by methanol precipitation and analyzed by 8% SDS-PAGE, transferred to Immobilon-P, stained with Coomassie Blue (A) and then probed with antibodies to dynein heavy chain (B, anti-DHC). The anti-dynein heavy chain antibody had only a low level of reactivity with Drosophila cytoplasmic dynein, and thus dynein in whole cytosol (lanes 1) was not detectable. Molecular mass in kDa is indicated on the left.
Figure 7:
Indirect immunofluorescence localization
of Glued in S-2 cells. The distribution of Glued protein in interphase
S-2 cells is shown in A, while anti--tubulin (32) is localized in a similar population of cells in B. In C and D, a metaphase S-2 cell was
double-labeled with antibodies to tubulin to localize microtubules (C) and anti-Glued antibodies (D).
We have examined the immunocytochemical and biochemical
behaviors of the Glued protein in the embryonically derived S-2 cell
line from Drosophila. The results of these studies indicate
that Drosophila Glued and mammalian p150 are
functionally homologous members of a dynactin complex and that each
interacts directly with the microtubule-based motor protein,
cytoplasmic dynein. Immunocytochemical evidence for the similarity
between the two polypeptides was demonstrated by inter-species antibody
cross-reactivity, as well as similar subcellular localization patterns.
Polyclonal antibodies raised to the C-terminal region of the Glued
protein react with rat p150
. This indicates that the
two share epitopes in regions that are only 24% identical at the
primary sequence level (see Fig. 1) and that higher order
structure may be conserved between Glued and p150
.
In biochemical assays, we have observed that like rat
p150, Drosophila Glued is a microtubule
binding protein that can be extracted from microtubules with either ATP
or elevated ionic strength, and that both proteins exist exclusively as
members of multimeric 20 S complexes in their respective species. This
distinguishes Glued from the related microtubule binding protein,
CLIP-170, which exists in cytosol as a dimer, with a sedimentation
coefficient of 5.7 S(28) . The data presented here also
demonstrates that there are functionally distinct isoforms of this
polypeptide in Drosophila. We noted that it was primarily the
150-kDa form of the Glued protein that associated with microtubules,
while the 135-kDa form did not. Based on observations made on the p150
and p135 isoforms expressed in human neurons, we speculate that p135 in Drosophila is an alternatively spliced isoform lacking the
microtubule-binding motif (
)that we previously characterized
in the rat p150
polypeptide(16) .
In addition to its ability to associate with microtubules, the Glued polypeptide was also found to be capable of binding to intact cytoplasmic dynein complex from S-2 cytosol. This was demonstrated by the specific retention of cytoplasmic dynein heavy chain, as well as several polypeptides similar in molecular mass to vertebrate cytoplasmic dynein subunits, by an affinity matrix constructed of a bacterially expressed fragment of the Glued polypeptide. This result provides further insight into the recent observation of a genetic interaction between Dhc64C, the gene encoding cytoplasmic dynein heavy chain, and Glued(44) in Drosophila, in that it demonstrates a biochemical interaction between the two gene products.
We have recently used a similar
method to demonstrate in rat that the interaction between the dynactin
complex and cytoplasmic dynein is mediated by direct binding of
p150 to the 74-kDa dynein intermediate
chain(18) . In that study, we showed that the 766 amino acids
spanning the predicted extended
-helix (amino acids 133-899)
of p150
(see Fig. 1) were sufficient for
mediating the interaction with cytoplasmic dynein(18) . In the
present study, the Glued affinity matrix was made with a 720-amino acid
C-terminal fragment (amino acids 599-1319, see Fig. 1) of the
Glued protein. This suggests that the minimal dynein intermediate chain
binding domain may be represented by the overlap, amino acids
599-899, in the constructs used in the respective binding
experiments in the rat and Drosophila systems.
Immunoprecipitations performed under nondenaturing conditions with
the anti-Glued antibody were used to further characterize the
polypeptides that interact with Glued in the Drosophila 20 S
complex. In this experiment we observed the co-immunoprecipitation of
polypeptides of 150, 143, and 135 kDa, which reacted with our
anti-Glued antibody. The 143-kDa form of Glued may represent a
differentially phosphorylated form of the protein or a proteolytic
fragment of the 150-kDa protein. That this form was found only in the
immunoprecipitation experiments may be due the use of a different
buffer system than was utilized for the rest of the experiments in this
study. The 45-kDa protein that was observed in the anti-Glued
immunoprecipitate is likely to be the Drosophila centractin
homolog, 87c, as described by Fyrberg et al.(33) . The
identity of the 45-kDa protein is substantiated by its weak
cross-reactivity with an anti-human centractin antibody. We also noted
that dynein heavy chain was present in the anti-Glued
immunoprecipitate, and this was most likely mediated by the
Glued-cytoplasmic dynein interaction demonstrated more directly by
affinity chromatography. While co-precipitation of dynein and the
dynactin complex was not observed in the parallel immunoprecipitation
experiment in rat brain, the anti-rat p150 antibody
used in this experiment was raised to the middle third (amino acids
133-899) of the p150
protein(16) . We
have preliminary results indicating that this antibody specifically
blocks the dynein-dynactin complex interaction, (
)thus it is
probable that the antibody would also block the co-immunoprecipitation
of the two complexes.
The demonstration that the product of the Drosophila gene Glued is functionally homologous to
the vertebrate p150 polypeptide is significant, because
the well characterized mutations at the Glued locus may now be
considered as resulting from a disruption in dynactin complex function.
As the null mutation is lethal(25) , this indicates that
expression of the dynactin complex is required, and that the complex
plays an essential role in higher eukaryotes. This is in marked
contrast to the recent observations in Saccharomyces cerevisiae and Neurospora crassa, in which disruption of dynactin
complex function resulted in nonlethal
phenotypes(10, 11, 12) . In yeast, mutations
in either dynein heavy chain or Arp1 (centractin) resulted in the
mis-segregation of duplicated nuclei between mother and
bud(8, 9, 10, 11) , while in
filamentous fungi, disruption of either dynein or dynactin complex
results in the block of nuclear migration along the
hyphae(12, 13) .
In higher eukaryotes, however,
disruption of dynactin complex function has more severe consequences.
The principal phenotype of adult heterozygote mutants of the dominant Gl allele are disruptions in neuronal systems such
as the optic lobe and eye(24) . There is a body of evidence
from biochemical and cell biological studies that both
p150
and cytoplasmic dynein play a role in basic
neuronal function. Both of these polypeptide complexes are highly
enriched in brain tissue from a variety of sources. In rat, in situ hybridization studies have shown that the p150
component of the dynactin complex is expressed at high levels in
developing brain and eye tissues (34) , while in Drosophila embryos, cytoplasmic dynein is concentrated in the developing
nervous system(35) .
In the neuron, cytoplasmic dynein is a
microtubule-based motor molecule that is believed to participate in the
retrograde transport of organelles in the
axon(36, 37, 38) , and the dynactin complex
may mediate this process. The involvement of the dynactin complex in
axonal transport is supported by our recent observations that
p150 is localized along the length of axonal processes
in cultured human neuronal cells.
Thus, it may be that the
defects in optic neuronal architecture and the aberrant cell patterning
observed in Glued mutants (24) are the result of a
defect in retrograde transport along the axon. Growth factors required
for the survival of neurons (39) are transported from the
growth cone to the cell body by fast retrograde transport(40) .
Disruption of growth factor transport may be the direct cause of
aberrant neuronal outgrowth observed in the Gl
mutant.
In addition to the strong evidence for the involvement
of Glued in neuronal function, the cell lethal phenotype of Gl homozygotes observed in mosaic analyses (27) , as well as the lethality of the null mutation at this
locus (24, 25) suggest that the Glued gene
product is also required for an essential function in non-neuronal
cells. There is mounting evidence from a variety of species that
cytoplasmic dynein participates in some aspect of mitosis, such as
spindle assembly, nuclear migration, or chromosome-to-pole
motility(8, 9, 10, 11, 12, 13, 14, 17, 41) .
It is therefore likely that the dynactin complex is also involved in
cell division. This is supported by the observations in Drosophila that cytoplasmic dynein in somatic cells (35) and
oocytes(42) , as well as Glued in S-2 cells (this study), all
localize to spindle poles and kinetochore microtubules during mitosis.
The demonstration of a Drosophila dynactin complex also
sheds light on the molecular mechanisms of the Glued mutations. Genetic analyses of Glued strongly suggests
that certain mutations affect the ability of the gene product to
participate in protein-protein interactions as a member of an
oligomeric complex (27) . The original dominant negative Gl mutation arises from the insertion of a B104
transposon into the coding region of the gene, resulting in a truncated
protein in which native sequence is substituted by transposon-coded
sequence at the C terminus of Glued(43) . This results in the
truncation of a highly conserved motif in Glued which has been
implicated in centractin binding(16) . Thus, the protein
product of Gl
may be unable to form dynactin
complexes due to an inability to bind to centractin. However, cellular
transfections with similar truncations of p150
did not
produce a dominant negative phenotype (16) , and so the
substitution of native sequence by transposon-coded sequence in Gl
may be responsible for disrupting dynactin
complexes, or may produce poisoned complexes that are unable to
interact with either cytoplasmic dynein or microtubules. Our
demonstration of a dynactin complex in Drosophila will now
allow the targeted analysis of the function of the dynactin complex at
the level of the organism as well as during development.