(Received for publication, September 14, 1995; and in revised form, November 8, 1995)
From the
The 74-kDa intermediate chains (IC74) of the cytoplasmic dynein
complex are believed to be involved in the association of dynein with
membranous organelles. While each dynein molecule is thought to have
two or three IC74 subunits, at least six different IC74 protein
isoforms were found in dynein from brain. Therefore we investigated the
relationships of the brain cytoplasmic dynein IC74 isoforms and their
association in the dynein complex at the cellular level. We found that
cultured cortical neurons and glia express distinct IC74 isoforms. The
IC74 isoform pattern observed in dynein from cortical neurons was
generally similar to that found in dynein from adult brain, indicating
that there are different populations of cytoplasmic dynein in neurons.
Two IC74 isoforms were observed on two-dimensional gels of dynein from
glia, while a single glial IC74 mRNA was detected. Metabolic labeling
of glial dynein with P followed by treatment of the
isolated dynein with phosphatase in vitro demonstrated that
one of the glial IC74 isoforms is the product of the single glial IC74
mRNA and that the other is its phosphoisoform. A single mRNA product
and its phosphoisoform are therefore sufficient for constitutive dynein
function and regulation in glial cells.
Cytoplasmic dynein is a ubiquitous minus end-directed
microtubule-based motor protein(1, 2) . In neurons, it
is believed to be the motor for retrograde transport of membranous
organelles from the synapse to the cell body(3, 4) .
Retrograde transport is essential for the movement of neurotrophic
factors from the synapse to the cell body and for the recycling and
degradation of cellular components(5, 6) . The
cytoplasmic dynein complex is composed of two 530-kDa heavy chains,
which make up two globular heads, as well as intermediate chains of
74 kDa (IC74) (
)and 53-59 kDa(7) .
Molecular analyses indicate that portions of the cytoplasmic dynein
IC74 are related to the ICs from the outer arm dynein of flagella (8, 9, 10, 11) . In addition, the
antibody 74.1 directed against the IC74 subunit of cytoplasmic dynein
cross-reacts with a polypeptide of similar molecular mass in mammalian
cilia and flagella(8) . It is known that the two IC subunits
from the outer dynein arm of Chlamydomonas flagella are
located at the base of the molecule (12, 13) and that
one, IC78, participates in binding the outer arm to its cargo, the A
tubule of the axoneme outer doublet microtubules(12) . The
similarity of flagellar and cytoplasmic dynein ICs makes it likely that
the cytoplasmic dynein IC74 is also located at the base of the two
stalks and is involved in binding to cargo, such as membranous
organelles.
While quantitative analyses of the polypeptide
composition of the cytoplasmic dynein complex indicate that it contains
only two or three IC74 subunits per
molecule(4, 7, 14, 15) , we recently
identified at least six different IC74 isoforms in dynein
immunoprecipitated from adult rat brain(16) . Therefore, there
must be populations of cytoplasmic dynein in brain that differ in the
composition of their IC74 subunits. Since many of the brain IC74
isoforms were labeled in vivo with P, some of the
IC74 diversity is presumably generated by posttranslational
modification of a more limited number of polypeptides, and the role of
phosphorylation in the regulation of cytoplasmic dynein is under active
investigation (16, 17, 18) . Recently two
cytoplasmic dynein ic74 genes, and five alternative splice
mRNA variants were identified in brain (19) which suggested
that at least five distinct IC74 polypeptides can be synthesized.
Therefore, we sought to understand the circumstances which generate the
multiple IC74 isoforms and to determine if different populations of
cytoplasmic dynein could be identified.
As brain tissue is made up of many specialized cell types, the IC74 isoforms of two brain-specific cell types, cultured cortical neurons and glia, were investigated at the protein and mRNA levels. In addition, the role of phosphorylation in generating the IC74 isoforms in brain and the cultured cells was investigated. We report that neurons and glia express different IC74 isoforms. Two IC74 isoforms were found when cytoplasmic dynein from glia was analyzed by two-dimensional gel electrophoresis. One isoform is the product of the single dynein IC74 mRNA transcribed in glia, and the other is its phosphoisoform. Three additional ic74 gene products and their phosphoisoforms are found in neurons. These results demonstrate that there are cell specific differences in the expression of the cytoplasmic dynein IC74. Furthermore, given the number of ic74 gene products identified in cortical neurons, neurons must contain populations of cytoplasmic dyneins which have different IC74 polypeptide compositions and differences in polypeptide phosphorylation. However, all the cytoplasmic dynein molecules in glia have the same IC74 polypeptide and differ only in the extent of its posttranslational modification. These results have important implications for the mechanism of cytoplasmic dynein function.
The oligonucleotides were used in the
following four combinations: G1/S and G1/AS, G2/S1 and G2/AS1, G2/S1
and G2/AS2, and G2/S2 and G2/AS1. To distinguish the different PCR
products generated by these primers, the products of each PCR were
separated on 8% acrylamide gels, 1 TBE (89 mM Tris, 89
mM boric acid, 25 mM sodium EDTA) buffer. A HaeIII digest of pBR322 DNA (Marker V, Boehringer Mannheim)
was used to determine the sizes of the PCR products.
Figure 1: The cytoplasmic dynein IC74 isoforms of adult rat brain. An adult rat brain was homogenized in lysis buffer. The dynein was immunoprecipitated and analyzed by two-dimensional gel electrophoresis, and the gel was silver-stained, as described previously(16) . A, the IC74 region of the two-dimensional gel, showing the six resolved isoforms. B, diagram identifying the IC74 isoforms. The arrow points to the region known as the B1 spot, the arrowhead points to the A1 spot. The acidic and basic ends of the IEF pH gradient are indicated with a ``+'' and ``-'' on the top of Panel A.
Figure 2:
Phosphatase treatment of the cytoplasmic
dynein eliminates the IC74-A2 and -B2 spots. Adult brain cytoplasmic
dynein was labeled in vivo as described under ``Materials
and Methods.'' The radiolabeled dynein was immunoprecipitated and
divided into two aliquots. One aliquot was treated with the general
protein phosphatase, phosphatase, as described under
``Materials and Methods,'' and the other aliquot was treated
in the same manner, except that phosphatase was not added to the
reaction mixture. The IC74 isoforms were resolved by two-dimensional
gel electrophoresis, visualized by silver staining, and then analyzed
by autoradiography. Only the IC74 portion of the gel is shown. A, silver-stained gel of control dynein immunoprecipitated
from adult brain; B, autoradiograph of
P-labeled
control dynein; C, silver-stained gel of dynein treated with
phosphatase; D, autoradiograph of
P-labeled
dynein treated with
phosphatase. The arrows point to the
B1 spot and the arrowheads point to the A1 spot. After
phosphatase treatment, the A2 and B2 spots are not found, and no spots
are labeled with
P.
When
brain cytoplasmic dynein, labeled with P in vivo,
was analyzed on two-dimensional gels, label was found over all the
spots, except the B spot(16) . However, the extent to which
this posttranslational modification contributed to the diversity of the
IC74 isoforms seen in adult brain was unknown. To identify and
characterize the unphosphorylated IC74 isoforms, we treated
immunoprecipitated dynein with
phosphatase in vitro and
analyzed the resulting IC74 isoform pattern on two-dimensional gels (Fig. 2). To demonstrate the effectiveness of the phosphatase
treatment, brain cytoplasmic dynein was first labeled in vivo with
P, then immunoprecipitated and resolved on
two-dimensional gels. As previously reported(16) , label was
observed over all the spots except the B spot (Fig. 2, A and B).
Phosphatase treatment of the
P-labeled brain dynein removed all of the detectable
P (Fig. 2D). Coincident with the removal
of the
P, the A2 and B2 spots were no longer seen (Fig. 2C). Therefore, some of the adult rat brain IC74
isoform diversity is solely the result of posttranslational
modification of other isoforms. While four distinct polypeptides
corresponding to the A, A1, B, and B1 spots remain after
dephosphorylation, the A2 and B2 spots are generated by phosphorylation
of the other polypeptides.
Figure 3:
Cultured glia and cortical neurons have
different IC74 isoforms. Cultured rat glia and cortical neurons were
metabolically labeled with 1 mCi of TransS-label for 1 h
as described under ``Materials and Methods.'' Cytoplasmic
dynein from the radiolabeled cultures was immunoprecipitated, resolved
by two-dimensional gel electrophoresis, and analyzed by
autoradiography. A, the IC74 region of three autoradiographs
showing cytoplasmic dynein from glia (left), neurons (center), and a mixture of equal counts/min of dynein from
glia and neurons (right). B,
S-labeled
dynein from glia was mixed with cold carrier dynein from rat brain, and
the combined sample was analyzed by two-dimensional gel
electrophoresis. The gel was stained with Coomassie Blue and then
analyzed by autoradiography. Left, Coomassie Blue-stained gel; right, autoradiograph of the gel showing the position of
S-labeled glial dynein IC isoforms. The arrows point to the B1 spot, and the arrowheads point to the
poorly resolved A1 spot. The glial spots co-migrate with the B and B1
spots. Cortical neurons, but not glia, have A
spots.
To directly compare the
neuronal and glial isoforms, approximately equal counts/min of
metabolically labeled glial and neuronal dynein were mixed, and the
combined sample was resolved by two-dimensional gel electrophoresis (Fig. 3A, MIXED). Two major spots of
approximately equal intensity, as well as a small amount of a slightly
more acidic and faster migrating spot, are identified on the
autoradiograph of the combined samples. This demonstrated that the
glial and neuronal IC74 isoform patterns overlap. From our analysis of S-labeled glial dynein, as well as the general pattern of
the spots in the combined sample, we conclude that the majority of the
B spot was contributed by the glial dynein and that the faint A spots
and majority of the B1 spot were derived from the cortical neuron
dynein.
In these initial studies, the glia and neurons were labeled for a relatively short time period. When glia were labeled for longer times (see Fig. 5C), the same IC74 isoform pattern was observed. Furthermore, the relative amounts of the glial IC74 isoforms seen in all the metabolic labeling experiments closely resembled the results observed when the gels were stained for protein (Fig. 4A). However, when cortical neurons were labeled for longer time periods the B2 spot was clearly identified, and the relative amount of the A isoforms appeared to increase (Fig. 5E). Nevertheless, dynein from cultured neurons still had a lesser amount of the A spots compared to dynein from adult brain. These results demonstrate that different cell types from brain have different cytoplasmic dynein IC74 isoforms.
Figure 5:
Phosphatase treatment of glial cytoplasmic
dynein eliminates the IC74 B1 spot. Cultured rat brain glia were
radiolabeled for 12 h with either 3 mCi of TransS-label or
3.0 mCi of [
P]orthophosphate, then the
cytoplasmic dynein was immunoprecipitated. Portions of each sample were
treated with
phosphatase, and all of the samples were analyzed by
two-dimensional gel electrophoresis and autoradiography, as above. The
relative amount of
P in the IC74 region of the glia gels
was calculated as described under ``Materials and Methods.''
Only the IC74 regions of the gels are shown. A, autoradiograph
of cytoplasmic dynein from glia labeled with
P showing the
control
P-labeling pattern. B, autoradiograph of
a portion of the same sample as in Panel A, treated with
phosphatase, showing the removal of
P from the
immunoprecipitated dynein. C, autoradiograph showing
S-labeled glial cytoplasmic dynein. D,
autoradiograph of a portion of the same sample as in Panel C,
treated with phosphatase, showing the loss of the B1 protein spot upon
removal of the phosphate from the dynein polypeptides. The A and B, and C and D pairs of images were
exposed to the same storage phosphor screen and were printed with the
same exposure settings. Rat cortical neuron cultures were labeled with
4 mCi of Trans
S-label for 18 h, then the dynein was
immunoprecipitated analyzed by two-dimensional gel electrophoresis and
autoradiography. E, autoradiograph of the IC74 region of
control cytoplasmic dynein from neurons. F, autoradiograph of
the IC74 region of phosphatase-treated cytoplasmic dynein from neurons.
The arrows point to the position of the B1 spot, the arrowheads to the A1 spot.
Figure 4:
Analysis of the phosphorylation of
cortical neuron and glial cytoplasmic dynein IC74 isoforms. A,
cultured rat glia were labeled with 1.0 mCi of
[P]orthophosphate for 12 h, and the dynein was
immunoprecipitated and analyzed by two-dimensional gel electrophoresis.
The gel was silver-stained and analyzed by autoradiography. Left
panel, IC74 region of the stained gel; right panel,
autoradiograph of the gel, showing the labeling of the B1 spot. B, cultured cortical neurons were labeled with 2 mCi of
[
P]orthophosphate for 12 h, the radiolabeled
dynein was immunoprecipitated and mixed with cold carrier dynein from
brain, and the combined sample was analyzed by two-dimensional gel
electrophoresis. The gel was stained with Coomassie Blue and analyzed
by autoradiography. Left panel, IC74 region of the stained
gel; right panel, autoradiograph of the gel showing that the
neuronal dynein B1 spot is lightly labeled and the B2 spot is heavily
labeled. There is faint labeling over the A spots. The arrows point to the position of the B1 spot, the arrowheads to
the position of the A1 spot.
To determine if
the IC74 isoform resolved at the B1 spot was a phosphoisoform of the
polypeptide resolved at the B spot, glial cytoplasmic dynein was
treated with phosphatase in vitro. Cytoplasmic dynein was
immunoprecipitated from cultures metabolically labeled with either
[P]orthophosphate or
S-labeled
amino acids. One half of each of the labeled dynein samples was treated
with
phosphatase. Then the control and treated samples were
analyzed by two-dimensional gel electrophoresis. The results are shown
in Fig. 5. When the extent of
P-labeling of the
glial dynein IC74 subunit before and after
phosphatase treatment
was quantified, we found that the phosphatase removed
95% of the
P from the immunoprecipitated glial dynein (Fig. 5, A and B). Next, the
S-labeled IC74
polypeptides were analyzed. Following phosphatase treatment, only the B
spot was observed. No B1 spot was found (Fig. 5, C and D). This shows that, in glial cells, the cytoplasmic dynein
polypeptide migrating with the B1 spot is a phosphorylated form of the
polypeptide migrating with the B spot. Therefore, in contrast to brain,
glial cells have only one unmodified IC74 subunit. Quantitation of the
relative amounts of
S-label in the B and B1 spots of
dynein from cultured glia indicates that only
20% of the glial
IC74 polypeptide is phosphorylated at any one time.
When cytoplasmic
dynein from cortical neuron cultures was treated with phosphatase
the IC74 isoform pattern resembled that of similarly treated dynein
from brain (Fig. 5F). Four distinct spots were
observed, B, B1, A, A1. Interestingly the spots are more clearly
resolved than those observed for the untreated control (Fig. 5E). It is likely that this enhanced resolution
results when the protein spread out in the A2 and B2 spots migrates
with the other spots after the removal of the phosphate. Similar
changes in the resolution of the A spots are observed when embryonic
brain dynein, which also has relatively little of the A spots, is
treated with
phosphatase. (
)Taken together, the
phosphatase treatment of cytoplasmic dynein from brain, glia, and
neurons indicates that two polypeptides co-migrate with the B1 spot, a
phosphate-modified form of the polypeptide resolved at the B spot and
an additional polypeptide.
Figure 6: Identification of the IC74 isoform B spots as products of the ic74-2 gene. Cytoplasmic dynein was immunoprecipitated from adult brain, resolved by two-dimensional gel electrophoresis, transferred to poly(vinylidene difluoride) membrane, and probed first with anti-IC74-2 (A), and then without stripping the membrane, reprobed with antibody 74.1 (B). IC-2 reacts well with the B spots, including the B2 spot, but not with the A spots. The A1 spot is detected when the blot is reprobed with 74.1. To maximize the resolution of the spots when the blot was reprobed with antibody 74.1, the time the blot was exposed to film during ECL was kept to a minimum. Therefore, while the A1 spot is clearly visible, it appears smaller in size than on protein gels. The arrows point to the position of the B1 spot, the arrowheads to the A1 spot. The proteins resolved in the B spots are products of gene ic74-2.
Figure 7: Identification of brain IC74 mRNA by RT-PCR. A, diagram of ic74 gene 1 and gene 2 showing positions of splice regions and location of diagnostic PCR primers. B, diagram of a model gel of the relative position and molecular masses (in bp) of the PCR products predicted to result from each combination of primers. Each line on the gel indicates a PCR product. On the right side of the gel diagram are the predicted sizes of the individual PCR products in base pairs. On the left side of the gel diagram are the specific mRNAs which would generate the diagramed products. Lane 1, the IC74-1 primer set (S/AS) will produce a 140-bp product if mRNA for IC74-1A is present and a smaller 77-bp product if the alternatively spliced IC74-1B mRNA is present. The G2 lanes, for the analysis of gene ic74-2, three PCR combinations were used. Lane 2, with the IC74-2 primer set S1/AS1, each mRNA will produce a fragment of a different size. A fragment of 348 bp, diagnostic of the full-length mRNA IC74-2A; a fragment of 330 bp is diagnostic of the excision of region 5 (mRNA IC74-2B); a fragment of 270 bp is diagnostic of the excision of regions 5 and 7 (mRNA IC74-2C). Lane 3, the products of the S1/AS2 primer set are diagnostic for splicing of region 5. If the full-length IC74-2A mRNA is present, a product of 126 bp results, if there is splicing (mRNAs IC74-2B and IC74-2C), a product of 108 bp is produced. Lane 4, the products of primers S2/AS1 are diagnostic for splicing region 7. mRNAs IC74-2A and IC74-2B, which contain region 7, will generate products of 245 bp, while IC74-2C will generate a product of 185 bp. From the three IC74-2 reactions analyzed together, mRNA for IC74-2A produces products of 348 bp in lane 2, 126 bp in lane 3, and 245 bp in lane 4; IC74-2B mRNA gives products of 330 bp in lane 2, 108 bp in lane 3, and 245 bp in lane 4; IC74-2C gives products of 270 bp in lane 2, 108 bp in lane 3, and 185 bp in lane 4. Therefore, with this analysis, each IC74 mRNA is uniquely identified. C, ethidium-stained gel of the PCR products produced from adult rat brain mRNA isolated as described under ``Materials and Methods,'' using the primers described in A and B. Lane 1, using the probes for IC74-1, products of 140 and 77 bp are obtained, diagnostic of mRNA IC74-1A and IC74-1B, respectively. The band at >348 bp is unidentified and presumably an artifact. Lane 2, using probes for IC74-2, products of 348, 330, and 270 bp are obtained, diagnostic of the mRNAs, IC74-2A, -2B, and -2C. The products of lanes 3 and 4 confirm the interpretation of the lane 2 results. mRNA from adult rat brain has messages from each of the five known IC74 splice variants.
The RT-PCR procedure was first used to analyze mRNA isolated from adult rat brain. As seen in Fig. 7C, products of 140 and 77 bp are produced in the reaction designed to identify the gene ic74-1 mRNAs. This is the result predicted for the IC74-1A and IC74-1B mRNAs, respectively, and demonstrates that, as expected, they are present in RNA isolated from adult rat brain. Similarly, when the procedure was used to probe for the IC74-2 messages, products diagnostic of all three IC74-2 mRNAs are found in mRNA from adult brain. Therefore, when the procedure was used to screen adult brain mRNA, PCR products of the size predicted for each of the five known messages for the ic74 genes were obtained, verifying the utility of this procedure for identifying the five known mRNAs for IC74.
When RNA from glia was analyzed by the RT-PCR procedure (Fig. 8A) no message for either IC74-1 mRNA was readily detected. However, a product of the size predicted for mRNA IC74-2C was observed in the analysis with the general IC74-2 primers. Further analysis with the primer reactions specific for each of the alternatively spliced regions confirmed the identification of IC74-2C mRNA in glia. mRNA from cortical neurons contained messages for IC74-1A, IC74-1B, IC74-2B, and IC74-2C (Fig. 8B). The amount of product diagnostic for mRNA IC74-2C was very weak and did not reproduce well photographically. However, it was clearly visible upon further cycles of amplification (not shown). No specific products diagnostic for mRNA IC74-2A were detected using mRNA isolated from neurons.
Figure 8: Analysis of glial and neuronal 74-kDa IC mRNA by RT-PCR. Glia has mRNA only for IC74-2C. As described under ``Materials and Methods,'' mRNA was isolated from cultured cells and RT-PCR with oligo(dT) was used to produce cDNA from the RNA. PCR using the oligonucleotides described in Fig. 7was used to generate different sized products, diagnostic for each of the mRNAs. A, RNA from cultured glia. Lane 1, analysis of IC74-1 products, none are discernable. Lane 2, analysis of all IC74-2 products, a product of 270 bp diagnostic of IC74-2C is identified. Lane 3, analysis of mRNA based on the excision of region 5. A product of 108 bp diagnostic of either IC74-2B or IC74-2C is observed. Lane 4, analysis of mRNA based on excision of both regions. A product of 185 bp is found diagnostic of IC74-2C. Therefore glia has a product of the sizes diagnostic only for IC74-2C. B, mRNA from cultured cortical neurons. Lane 1, analysis of IC74-1, products of 140 and 77 bp are observed diagnostic of mRNA IC74-1A and -1B. As in Fig. 7C, the >348-bp fragment is unidentified. Lane 2, analysis of IC74-2, a product of 330 bp diagnostic of IC74-2B is identified. A very faint product of 270 bp, diagnostic of IC74-2C which did not reproduce well was also observed. Lane 3, analysis of mRNA based on the excision of region 5. A product of 108 bp diagnostic of either IC74-2B or IC74-2C is observed. Lane 4, analysis of mRNA based on excision of both regions. A product of 245 bp is found diagnostic of IC74-2A or IC74-2B. A product of 185 bp, diagnostic of IC74-2C, which did not reproduce well, was also observed. Therefore cortical neurons have products of the sizes diagnostic for IC74-1A, -1B, -2B, and -2C. No IC74-2A mRNA was detected.
The cytoplasmic dynein protein complex is believed to contain two or three IC74 polypeptides per molecule(4, 7, 14, 15) . However, we previously reported that at least six IC74 isoforms are resolved on two-dimensional gels of dynein isolated from adult rat brain(16) . Furthermore, five alternatively spliced IC74 mRNAs have been identified(19) . We therefore initiated this study to investigate the relationships of the various IC74 isoforms and their association in the dynein complex. Our analysis has identified several factors which account for the two-dimensional gel IC74 spot pattern. First, individual cell types express different IC74 mRNAs and their polypeptide products. One mRNA, IC74-2C, is expressed in glia, while four mRNAs are found in neurons. Second, a fraction of the molecules in each IC74 polypeptide pool is phosphorylated. In brain and neurons, the A2 and B2 spots originate by the phosphorylation of the other isoforms. Finally, in at least one instance, different IC74 isoforms co-migrate at the same spot on two-dimensional gels. This is the first report of differences at the cellular level in the subunits of cytoplasmic dynein from the same species and tissue and demonstrates the existence of different pools of cytoplasmic dynein in neurons.
The cytoplasmic
dynein IC74 isoforms from cultured glia and cortical neurons are
remarkably different. One IC74 mRNA is transcribed in glia, the IC74-2C
message. The polypeptide product of this mRNA migrates at the B spot.
We will hereafter refer to this polypeptide as the IC74-2C isoform.
Previously we reported that the isoelectric point of the B spot was pH
4.9(16) . This is in reasonable agreement with the value of pH
5.16 calculated by a computer program for the product of the IC74-2C
mRNA (Expasy Server, University of Geneva). Approximately 20% of the
IC74-2C isoform is phosphorylated in vivo. This produces a
phospho-IC74-2C isoform that co-migrates with the B1 spot. Cultured
glia show no evidence of the phosphoisoform that migrates with the B2
spot. The two-dimensional gel IC74 spot pattern of dynein from cultured
cortical neurons is generally similar to that of dynein from adult
brain. Treatment of cytoplasmic dynein from cortical neurons with
phosphatase in vitro yielded the same four discrete IC74 spots
observed in phosphatase treated brain dynein. However, a lesser amount
of the proteins migrating in the A spots was found when cytoplasmic
dynein is immunoprecipitated from cultured cortical neurons than was
found in adult brain. This is not an artifact of labeling cultured
cells with S-labeled amino acids. Rather, the expression
levels of several of the neuronal IC74 polypeptides which resolve at
the A, A1, B, and B1 spots change during brain development.
The relative amounts of the IC74 isoforms of cytoplasmic dynein
isolated from cultured cortical neurons, prepared from cerebral
cortexes obtained on the 18th day of gestation, are very similar to
those of dynein immunoprecipitated from 18th day of gestation brains or
cortexes. The RT-PCR assay demonstrated that four of the five known
brain IC74 mRNAs are expressed in the cultured cortical neurons. The
IC74-2A mRNA was not found in mRNA prepared from cultured cortical
neurons. Interestingly, it was also not found in mRNA prepared from
embryonic or newborn brain through P5,
indicating that the
expression of this IC74 isoform is also developmentally regulated,
although with a different time period than that of the other neuronal
IC74 isoforms.
Analysis of these results allowed us to deduce a
correlation between the IC74 mRNA products and the two-dimensional gel
spot pattern (summarized in Table 1). As shown above, the product
of IC74-2C migrates at the B spot, and its phosphoisoform, which is
eliminated by phosphatase treatment, migrates at the B1 spot. In
contrast, the B1 spot of cytoplasmic dynein isolated from neurons and
adult brain is still observed after phosphatase treatment. This
indicates that some IC74 isoforms co-migrate on two-dimensional gels.
The B1 spot observed when dynein is isolated from adult rat brain or
cultured neurons must contain an unphosphorylated polypeptide as well
as the phospho-IC74-2C isoform. Since the antibody specific for ic74-2 gene products recognizes all the B spots, this neuronal
IC74 polypeptide co-migrating with the B1 spot is most likely the
product of an IC74-2 mRNA. Two IC74 mRNAs are expressed in cortical
neurons, IC74-2C and IC74-2B. Therefore, it is likely that the
unphosphorylated polypeptide co-migrating with the B1 spot is the
product of the IC74-2B mRNA. Evidence supporting this conclusion comes
from the demonstration that the IC74-2B message is found in all tissues
that have a B1 spot after phosphatase treatment, including those with
no IC74-1 messages.
We were unable to identify the B2
spot in analyses of either S-labeled or
P-labeled cytoplasmic dynein from glia. This suggests that
phosphorylation of the IC74-2C isoform does not generate the B2 spot.
The B2 spot was observed in cytoplasmic dynein from neurons. It
therefore appears likely that the B2 spot is the phospho-IC74-2B
isoform. However, the possibility that a cortical neuron kinase, not
present in glia, generates the B2 spot by hyperphosphorylating the
IC74-2C isoform cannot be ruled out. The observation that polypeptides
migrating in the A arc of spots are not detected by the IC74-2-specific
antibody suggests that the polypeptides resolved in the A arc of spots
are the products of the two IC74-1 mRNAs. Given that cortical neurons
have both the IC74-1A and IC74-1B messages, it appears likely that one
corresponds to the protein of the A spot and the other to the A1 spot.
This was confirmed by further study.
The polypeptide
resolved at the A spot is the IC74-1B isoform and the A1 spot is the
IC74-1A isoform.
The best studied dynein ICs are the outer arm dynein ICs of Chlamydomonas flagella, which serve as a model for understanding the role of the cytoplasmic dynein ICs(8, 9, 12, 13, 22) . There are two ICs per outer arm dynein, IC68 and IC79(22, 23) . These are the products of distinct, although related genes(9, 10, 11) . The two ICs associate with one another are located at the cargo binding end of the dynein molecule and contribute to the assembly of the dynein complex(12, 13, 22) . The flagellar ic69 and ic78 genes are related to the ic74 genes of cytoplasmic dynein (8, 9) . The flagellar IC78 binds the dynein complex to its sole cargo, the A microtubule(12) . Interestingly, only one isoform of each IC is observed on two-dimensional gels(24, 25) . Since the cargo of cytoplasmic dynein includes a variety of membranous organelles, it seemed possible that the different IC74 isoforms were involved in binding cytoplasmic dynein to specific organelles. However, glia express only the IC74-2C mRNA. Therefore, the presence of only the IC74-2C polypeptide in the dynein complex is sufficient for the constitutive dynein-powered movement of membranous organelles including mitochondria, endosomes, and endoplasmic reticulum-Golgi traffic in cultured cells(26, 27, 28, 29, 30) . The results presented here further demonstrate that, unlike flagellar dynein, the products of both ic74 genes are not necessary for general cytoplasmic dynein function.
The observation that 20% of the single IC74 polypeptide found in glia, the IC74-2C isoform, is phosphorylated is consistent with our previous identification of differences in the phosphorylation of cytoplasmic dynein associated with anterograde organelles and that of the whole cell pool(16) . Since cytoplasmic dynein from glia has a single IC74 polypeptide, the study of glial dynein should simplify further investigations into the role of phosphorylation of the IC74 subunit on the functional properties of dynein. Recent studies suggest that phosphorylation of the kinesin heavy and light chains in vivo correlates with the association of kinesin with membranous organelles(31, 32) . Genetic studies on the outer arm flagellar dynein support the hypothesis that the IC subunits regulate dynein function (22) . Furthermore, it was recently realized that the conserved portions of the flagellar and cytoplasmic genes are a series of WD repeats in the COOH-terminal portion of all three molecules(9, 10) . WD repeats are believed to be important for subunit-subunit interactions in protein complexes and, interestingly, all the other proteins with WD repeats are regulatory proteins(33) .
Since dynein-based membrane traffic in a
glial cell functions with only the ic74-2c gene product, the
roles of the three additional neuronal IC74 polypeptides and their
phosphoisoforms in cytoplasmic dynein function remain to be determined.
Interestingly, multiple isoforms of both the heavy and light chains of
bovine brain kinesin have also been identified on one- and
two-dimensional gels(34) . In an analysis of kinesin from rat
brain, Cyr et al.(35) identified a single kinesin
light chain gene and three alternative splice variants and suggested
that the alternative isoforms may be involved in binding to different
organelles. Subsequent work on the light chains from other species has
yielded similar results(36, 37, 38) . Elluru et al.(39) have examined the anterograde axonal
transport of the two kinesin heavy chain isoforms. They find that one
kinesin heavy chain isoform is predominantly associated with
anterogradely moving synaptic vesicles, while the other is associated
with mitochondria. While two of the neuronal IC74 polypeptides are
found in at least one other tissue, one is specific to neurons. Cytoplasmic dynein has two to three IC74 subunits per molecule.
Therefore, there must be distinct populations of cytoplasmic dynein in
neurons. It is tempting to speculate that the additional IC74 isoforms
may confer a regulatory or functional specificity on the dynein complex
needed in neurons and some other cells, but not glia. Neurons are
specialized for long distance axonal transport, and the presence of
additional IC74 isoforms may be related to the specialized cargo moved
in retrograde axonal transport, or the regulation of retrograde axonal
transport. Interestingly, we find that all the neuronal dynein IC74
isoforms are present in axons(16) . However, understanding the
role of the various neuronal cytoplasmic dynein IC74 isoforms remains a
challenge. The demonstration of basic differences in the IC74 subunits
of cytoplasmic dynein from glia and neurons should also raise a
cautionary note concerning biochemical studies of dynein from mammalian
brain, as dynein from this source is a mixed population of the motor
protein.