Correspondence to: Lawrence S.B. Goldstein, Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, Department of Pharmacology, 334 m/c 0683, University of California San Diego, La Jolla, CA 92093-0683., lgoldstein{at}ucsd.edu (E-mail), (619) 534-9702 (phone), (619) 534-9701 (fax)
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Abstract |
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Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97amino acid polypeptide that is 57% identical (70% similar) to the 105amino acid Chlamydomonas outer arm dyneinassociated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.
Key Words: axonal transport, mitosis, dynein, ATPase, nerve degeneration, flagella
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Introduction |
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INTRACELLULAR transport is facilitated by the movement of cytoplasmic dyneins and kinesins along ordered arrays of microtubules (
Dyneins, in particular, pose an important challenge because of the large numbers of associated proteins and diverse structural and functional roles (for review see
Cytoplasmic dynein is similarly complex and consists of a homodimer of heavy chains, a dimer of a WD repeat intermediate chains, four light intermediate chains (not present in the outer dynein arm), and several light chains. The 74-kD cytoplasmic dynein intermediate chain (IC74)1 has been shown to mediate the dyneindynactin interaction via direct association with p150glued (
Recent work suggests that dynein light chains (DLCs) may play crucial roles in dynein function and regulation. To date, two classes of cytoplasmic DLCs have been identified including Tctex1 (and the homologous rp3) and the highly conserved 10 kD/LC8 DLC (previously called the Mr 8,000 light chain). Both classes are also found associated with axonemal dynein. Tctex1 is a cytoplasmic and inner arm DLC thought to be involved in the meiotic drive of mouse t-haplotypes (B
(
To identify novel modulators of kinesin and dynein motor function, we took advantage of the observation that mutations in axonal transport motors of Drosophila share a common larval phenotype of posterior sluggishness and axonal cargo accumulation (
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Materials and Methods |
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Axoneme and Dynein Purification
Flagella were isolated from wild-type Chlamydomonas by standard methods (
Rat brain cytoplasmic dynein was isolated by ATP-dependent microtubule affinity (
Drosophila dynein was immunoprecipitated from 015 h embryo homogenates with the 74-1 antibody using a method similar to the one above. In brief, 0.6 g (wet weight) of dechorionated embryos were homogenized in 1 ml of lysis buffer (25 mM Tris-Cl, pH 8.0, 50 mM NaCl, 0.5% Triton X-100, 2 mM EDTA, 1 mM PMSF) containing 10 µg/ml aprotinin, 40 µg/ml bestatin, and 1 µg/ml leupeptin. The homogenate was split into two 400 µl aliquots to which 2.5 µg of 74-1 antibody was added to one sample (dynein immunoprecipitate), the other was mock-immunoprecipitated without antibody (bead control). Precipitation was performed with 10 µl of protein ASepharose 4B (Zymed Labs, Inc.) preblocked with 5% BSA in lysis buffer. The beads were washed five times with 20 vol of lysis buffer and the final immunoprecipitate was resuspended in 50 µl of SDS-PAGE loading buffer. 20 µl of each pellet were analyzed by Western blot as described below.
Chlamydomonas axoneme and rat brain dynein samples were electrophoresed in 515% acrylamide gradient gels. Drosophila samples were electrophoresed with tricine buffer in 10% acrylamide gels. The gels were either stained with Coomassie blue or blotted to nitrocellulose and probed with the 74-1 mAb to detect IC74 (
Analysis of Peptides from LC7
Purified outer arm dynein was concentrated in a Centricon 30 ultrafiltration unit (Amicon) that had been previously treated with 5% Tween 20 in TBS to reduce nonspecific protein binding. The sample was electrophoresed in a 515% acrylamide gradient gel and blotted to a polyvinylidene difluoride membrane (Immobilon Psq; Millipore Corp.). The LC7 band was excised and treated with trypsin in situ. Peptides eluting from the membrane were purified by reverse-phase chromatography using a C8 column and peptide masses determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Two peptides of sufficient purity were obtained and sequenced at the Protein Microsequencing Facility, University of Massachusetts Medical School.
Molecular Analysis of LC7
A portion of the LC7 coding region (~450 bp) was originally obtained from the first strand of cDNA made from RNA enriched for flagellar sequences using PCR. The forward primer 5'-GCGCGAATTCAAGAAGCACGAGATYATG-3' was designed from the peptide sequence (K)KHEIM using the Chlamydomonas codon bias and incorporated an EcoRI site and GC clamp at the 5' end. The oligo (dT) adaptor 5'-GCGCGTCGACTCGAGT20V-3' was employed as the reverse primer. The reaction was performed using Pfu DNA polymerase and standard buffer conditions with the following thermal profile: 96°C for 1 min, 50°C for 1 min, and 72°C for 1 min for 40 cycles followed by a final 10 min at 72°C. This PCR product was used to isolate a full-length clone from a ZapII Chlamydomonas cDNA library. Multiple clones were obtained and the longest sequenced on both strands using Sequanase v2.0 and a 7-deaza dGTP sequencing kit (U.S. Biochemical Co.). Southern and Northern blots were prepared and probed using standard methods.
LC7 Fusion Protein and Antibody Preparation
The LC7 coding region was subcloned into the pMAL-c2 vector by PCR-based cloning (New England Biolabs Inc.). This resulted in the COOH-terminal fusion of LC7 to the maltose-binding protein via a hydrophilic linker containing a Factor Xa cleavage site. Expressed protein was purified by amylose affinity chromatography and the entire fusion protein was used to raise antisera in rabbit R7178. Subsequently, electrophoretically isolated recombinant LC7 was used to blot purify the antisera using the minor adaptions to the method of
Identification of the roadblock Mutant
F3 lethal balanced ethyl methanesulfonate (EMS) mutant lines (cn bw l(2)EMS/Cyo) were obtained from the laboratory of Dr. Charles Zuker (University of California San Diego). The mutant larvae were examined 5 to 6 d after egg laying for sluggish crawling behavior. Roadblock was identified as a posterior larval sluggish mutant with a late third instar larval lethal phase (roblz allele). Preliminary studies identified an absence of imaginal tissue and extreme posterior paralysis in which larvae become completely paralyzed in the posterior, whereas the anterior remained noticeably mobile.
Cloning of the roadblock Gene
The robl gene was mapped approximately to cytological position 54 on the second chromosome of Drosophila by meiotic recombination. Screening of nearby lethal alleles obtained from the lab of Dr. Gerry Rubin (University of California Berkeley), identified l(2)k10408 as a robl allele (robll(2)k10408). Additionally a P-element mobilization screen with other nearby insertions generated another robl allele (roblc). Genomic sequence was rescued off the ends of robll(2)k10408 and roblc by inverse PCR (BDGP protocol; http://www.fruitfly.org/p_disrupt/) and used to identify Drosophila P1 genomic clones from the Berkeley Drosophila Genome Project (BDGP) using the P1 filter blot purchased from Genome Systems, Inc. The P1 clones in the robl genomic region (DS02323 and DS02859) were sequenced using an ABI 377 DNA sequencer. Analysis revealed large deletions in robll(2)k10408 and roblc that were partially overlapping, thus, identifying the robl genomic interval. Homozygous robll(2)k10408 and roblc genomic DNA were made from third instar larvae and used to confirm both deficiencies by PCR and Southern analysis. Sequencing and PCR analysis of roblz homozygous DNA revealed a 193-bp deletion identifying the robl gene. BLAST analysis using the BDGP database identified a full-length expressed sequence tag (EST) clone that encodes robl; this clone (LD34974; accession number AI061910) was ordered from Genome Systems, Inc. Sequence of the ~15-kb robl genomic interval and robl cDNA has been deposited at National Center for Biotechnology Information (NCBI) GenBank (accession numbers AF141921 and AF141920).
Genomic and cDNA rescue construct lines were generated using standard techniques. The genomic construct (a 6.6-kb SpeI-KpnI fragment) was cloned into pP{CaSpeR 4} and the cDNA construct was cloned into pP{CaSpeR hs-ACT} (provided by Dr. Carl Thummel, University of Utah) by PCR cloning to introduce a 6xHis tag at the NH2 terminus (adding the amino acids: MGSSHHHHHHSSG). Multiple X chromosome insertion lines were obtained and used to test for rescue. The cDNA rescue construct (which is under control of an HSP70Bb promoter) was induced daily for 1 h at 37°C.
roadblock Fusion Protein and Antibody Preparation
The 6xHis-tagged robl fusion protein used in the cDNA rescue experiments was subcloned into the pET-14b vector by PCR-based cloning (Novagen, Inc.). Expressed protein was purified by Talon Superflow metal affinity resin according to the recommended protocol (CLONTECH Laboratories, Inc.), and then electrophoretically isolated and used to raise antisera in rabbit 6883. 10 mg of robll(2)k10408 homozygous and wild-type second/third instar larvae (wet weight) were extracted with SDS-PAGE loading buffer and analyzed by Western blot to confirm specificity of the anti-robl antibody.
robl/LC7 Sequence Analysis and Protein Comparison
All sequence assembly and protein comparison were performed using the GCG suite of software (Genetics Computer Group) and Sequencher 3.1 software (Gene Codes Corporation). Roadblock/LC7 family members were identified with the Drosophila robl sequence using BLASTP against the NCBI dbNR, tblastn against the NCBI dbEST, and tblastn against the BDGP DNA sequence database. All gene abbreviations here refer to those detailed in Figure 4. Drosophila robl22E (DS01020; accession number AC004276), robl37BC (DS00790; accession number AC005127), robl62A (DS02734; accession number AC004343), and robl60C (DS02336; accession number AC005718) were identified in BDGP genomic sequence and are apparently intronless genes, just like the related late RNA from the Bithoraxoid complex (accession number M27999). EST clones have been identified for late RNA-encoded bithoraxoid protein (bxd) (GH08635; accession number AI113381) and robl62A (GH15530; accession number AI292590) by BDGP from a Drosophila head cDNA library. The proteins T24H10.6 (accession number 995857) and bxd (accession number 290293) were identified from dbNR using blastp. Mouse, rat, and human ESTs identified, were compared by nucleotide sequence using tblastn against the species-specific NCBI GenBank dbEST to identify ESTs from identical genes; two different genes were identified in all three species (accession numbers from representative ESTs are given in Figure 4). The predicted translation of all mammalian ESTs was determined using DNA Strider (CEA); the small size of the genes meant that almost all ESTs translated into full-length protein. Protein comparison was done using the GCG pileup command to generate the dendrogram; the output MSF file was run through the BoxShade Server (http://www. isrec.isb-sib.ch/software/BOX_form.html) and the output EPS file was imported into Adobe Illustrator 6.0.
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Larval Segmental Nerve Immunostaining
Larval segmental nerve immunostaining was done as described by
Electron Microscopy of Larval Segmental Nerves
The method below is a hybrid of our standard protocol (
Larval Mitotic Brain Squash Analysis
Untreated third instar larval brain squash analysis was done as previously described (
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Results |
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Identification and Cloning of roadblock
roadblock (robl) was identified in a screen for novel axonal transport mutants in Drosophila melanogaster. The roblz EMS mutant allele is recessive lethal, dying at the third larval instar. The roblz homozygous larvae show a progressive posterior sluggish phenotype leading to complete posterior paralysis, a common phenotype of axonal transport mutants in Drosophila (
Two overlapping deficiencies, robll(2)k10408 and roblc, identify the genomic interval encoding robl (Figure 1 A). Sequencing of the entire genomic interval identified five putative gene candidates that may be affected by both deficiencies. To identify which gene encoded robl, we sequenced roblz and discovered a 193-bp deletion in the middle of a small transcription unit in the interval that we believe to be robl for several reasons. First, a 5-kb segment of this region that contains only robl, and one adjacent gene, was found to fully rescue all above-mentioned phenotypes in roblz hemizygotes. Second, this gene adjacent to robl was sequenced from roblz and found to be unaltered from the wild-type parental chromosome. In fact, this gene appears to be a robl pseudogene because it lacks any identifiable start codon. Third, robll(2)k10408 homozygotes are fully rescued by the genomic rescue construct that indicates that other genes in this interval are not essential and the observed phenotypes are robl-dependent. Finally, an NH2-terminal His-tagged robl cDNA construct under control of the hsp70Bb promoter fully rescues male roblz hemizygotes if given daily heat shock. Reducing the frequency of heat shocks results in a restoration of the described robl phenotype. This cDNA construct does not rescue an apparent female sterility seen in the rescued roblz hemizygotes, despite full rescue of all other observed robl phenotypes. Nevertheless, taken together, these data establish that the gene identified by the roblz deletion is roadblock.
The genomic sequence of robl reveals a small three exon gene encoding a 97-residue polypeptide (Figure 1 B). The 193-bp deletion found in roblz removes portions of intron 2 and exon 3. Interestingly, this deletion results in a robl allele that is more severe than null alleles. The increased severity of roblz homozygous animals compared with roblz hemizygotes or homozygous null animals suggests that this internal deletion is a recessive neomorphic allele that poisons intracellular transport. In fact, roblz homozygotes cannot be fully rescued by the genomic or cDNA rescue constructs. Thus, two copies of the roblz mutation act in a dominant fashion to inhibit the action of wild-type robl. An alternative explanation for the inability to rescue roblz homozygotes would be a secondary lethal lesion on the roblz chromosome. However, we have confirmed the absence of any other lethal complementation groups on the roblz chromosome by recombination mapping (data not shown).
Chlamydomonas LC7 Is an Outer Arm Dynein-associated Protein
The Chlamydomonas outer dynein arm contains eight distinct light chain components (
Genomic Southern blot analysis revealed a single band in both BamHI- and SmaI-digested DNA, suggesting that there is a single LC7 gene in Chlamydomonas (Figure 2 C). As is characteristic of flagellar proteins, Northern analysis revealed one message of ~0.95 kb that was greatly upregulated in cells that were actively regenerating their flagella (Figure 2 D).
The outer arm dynein samples used to obtain LC7 peptide sequences also contained inner dynein arm I1. This dynein partially cofractionates with the outer arm and is now known to contain light chain components (
A Family of robl/LC7 Proteins Is Conserved from Nematode to Man
The cloning of roadblock and LC7 revealed these proteins to be 57% identical and 70% similar. Additionally, both proteins are related to the predicted protein sequence from the late RNA of the Drosophila bithoraxoid complex (bxd); robl is 30% identical and 42% similar to bxd; LC7 is 26% identical and 39% similar to bxd. However, no known function has been attributed to this coding transcript from bxd (
robl Mutants Have a Distal Biased Axonal Transport Defect
Mutations in robl cause phenotypes similar to other axonal transport mutants in Drosophila. Previous analysis of kinesin heavy chain (khc) and kinesin light chain mutants demonstrated massive accumulations of axonal cargo and motors distributed randomly along the entire length of the larval segmental nerves. These accumulations were shown to be massive local axonal swellings that fill with organelles and vesicles (
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In roblz mutants, unlike previously described axonal transport mutants, there is a strong tendency for the synaptic cargo to accumulate at the distal regions of axons with only infrequent proximal accumulations. This distal bias can be inferred from the organization of the Drosophila larval nervous system. The larval segmental nerves are anti-parallel bundles of mostly cholinergic sensory neuron axons and noncholinergic motor neuron axons. The (ChAT and SYT expressing) sensory neurons project axons from peripheral cell bodies towards the anterior into the ventral ganglion (VG), whereas the (SYT expressing but ChAT lacking) motor neurons project axons in the opposite direction from cell bodies in the VG towards the posterior and peripherally where they form neuromuscular junctions.
In roblz hemizygous larvae, ChAT accumulations were found predominantly in the distal portions of the sensory axons (the anterior region of the larval segmental nerves) as seen by comparing staining at the anterior VG (Figure 5 F) with staining observed in segmental nerves in the posterior of the larvae (Figure 5 H). SYT shows a gradual increase in the frequency of accumulations toward the distal portions of the motor axons (the posterior region of the larval segmental nerves) as seen by comparing the staining at the anterior VG (Figure 5 E) with staining observed in segmental nerves at the posterior region of the larvae (Figure 5 G). Thus, the frequency of ChAT accumulations is inversely correlated with the distance from the VG, whereas SYT accumulations show the opposite correlation.
We further analyzed this distal enrichment of axonal accumulations by SYTChAT co-immunostaining analysis. Since ChAT is expressed only in sensory neurons, SYTChAT co-accumulations can only occur in sensory neuron axons. In addition, most (~95%) of ChAT accumulations along the length of the nerves co-immunostain with SYT, supporting a view that most ChAT negative SYT accumulations occur in motor axons. Co-immunostaining demonstrated that 71% of anterior SYT cargo accumulations are ChAT positive. Thus, most anterior SYT accumulations are occurring in the distal regions of sensory axons and not the proximal region of motor axons. In contrast, only 16% of the posterior SYT accumulations are ChAT positive. Thus, most of the posterior SYT accumulations are likely occurring in the distal regions of motor axons and not the proximal regions of sensory axons. Therefore, the combined observations of an anteriorposterior accumulation frequency gradient, the majority of anterior SYT accumulations occurring in sensory axons, whereas the majority of posterior SYT accumulations occurring in motor axons, demonstrates that there is a strong propensity for synaptic axonal cargo accumulation to occur in the distal regions of axons in roadblock mutants.
Comparative analysis of robl null, roblz hemizygous, and roblz homozygous nerves revealed that as the number of roblz alleles is increased, the number of observed SYT and ChAT accumulations decreased. The roblz homozygous larvae have fewer axonal accumulations, ranging from ~15% than that observed for hemizygotes (data not shown). A similar distal enrichment in accumulations is observed for roblz homozygotes, as has been described above for roblz hemizygotes. Homozygous robl null larvae show a significant increase in axonal accumulations, ranging from ~200400% than that observed for hemizygotes (data not shown). However, the ChAT accumulations in robl null homozygotes appear more uniformly distributed, despite obvious distal-enriched SYT accumulations. Perhaps, the large number of axonal accumulations observed in the robl nulls obscures the distal bias; alternatively, sensory neuron axons (ChAT positive axons) may be affected differently in robl nulls.
robl Mutants Have Massive Axonal Loss and Nerve Degeneration
We used EM to examine the morphology of the axonal swellings in segmental nerves from robl mutants. Previously, transmission EM of larval segmental nerves from khc mutants revealed that these massive axonal swellings are filled with all types of identifiable axonal cargo (
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The robl mutants also have severe axonal loss and nerve degeneration that is not observed in khc mutants, despite the fact that khc mutant axonal swellings are more numerous and on average twice the size of those observed in robl (
roadblock Is a Severe Mitotic Mutant and Female Sterile Mutation
The first indication of a mitotic defect in robl mutants was the observation of a complete absence of the mitotically active tissues (imaginal tissues) in roblz homozygous larvae. Additionally, roblz hemizygous and robl null animals that survive into late pupal stages, demonstrate rough pupal eyes (Figure 7 L), missing bristles (data not shown), and reduced size of imaginal tissue (data not shown). These observations are consistent with a mitotic defect in Drosophila.
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To examine the mitotic defect further, third instar untreated (no hypotonic or colchicine treatment) larval brain squashes were performed. This procedure permits observation of dividing neuroblasts within the larval central nervous system by staining with a fluorescent DNA dye and allows quantitation and characterization of the mitotic figures. The analysis revealed significant mitotic defects in roblz hemizygous larvae. Numerous polyploid mitotic figures were observed (Figure 7D and Figure E). Additionally, many of the polyploid figures showed hypercondensation of their chromosomes (Figure 7 F). Abnormal anaphase figures were also observed with hypercondensed chromosomes and disorganization of the chromosomes around the presumptive poles (Figure 7 G). As anticipated, since the mutant survives until late pupal stages, apparently normal mitotic figures were also observed (not shown). The mitotic index in this mutant is fivefold higher than wild type (Figure 7 K). This increased mitotic index is due to an increased number of figures from all mitotic phases counted (prometaphase, metaphase, and anaphase). An elevated mitotic index for all phases, coupled with the variety of defective structures suggests defects in multiple stages of mitosis.
Larval brain squash analysis on the roblz homozygotes also revealed a profound mitotic defect; in addition to the lack of imaginal tissue, there is a striking absence of prometaphase and metaphase mitotic figures. Only infrequent defective anaphase and telophase figures are seen. The few anaphase figures have severe bridging and lagging chromosomes (Figure 7H and Figure I). In addition, we observe apparent telophase bridging in which DNA has become trapped between two dividing nuclei (Figure 7 J). The failure to observe any prometaphase or metaphase figures prompted us to perform a larval brain squash on colchicine-treated brains. This procedure, which blocks cells in metaphase, resulted in an approximate doubling of the observed number of metaphase figures and a decrease in the observed frequency of postmetaphase figures in wild-type controls. However, in roblz homozygotes, we never observed a prometaphase or metaphase figure in treated third instar larval brains, yet the low frequency of observed defective anaphase and telophase figures remained unchanged from untreated brains. These data strongly suggest that third instar roblz homozygote larvae lack cells capable of division and the few figures observed represent cells arrested in mitosis.
Female roblz hemizygous flies rescued to adulthood by the 6xHis-tagged cDNA construct under heat shock promoter control show a female sterile phenotype. However, this same allelic combination is fully rescued by the robl genomic rescue construct, presumably under native robl promoter control. Female sterility is commonly observed in mutants of cytoplasmic dynein components in Drosophila (
roadblock and a Mammalian robl/LC7-like Protein Are Associated with Cytoplasmic Dynein
Previously, the highly conserved LC8 protein and Tctex1 were found in both cytoplasmic and flagellar dyneins. The robl mutant phenotypes and the identification of a homologous sequence in organisms lacking motile cilia/flagella (C. elegans), raised the obvious possibility that robl/LC7-like proteins may be present in cytoplasmic dynein. Accordingly, we examined samples from the stepwise ATP-dependent microtubule affinity purification of cytoplasmic dynein from rat brain homogenates for the presence of a robl/LC7-like protein (Figure 8 A). The R7178 antibody detected a single band of Mr ~12,000 in the initial microtubule pellet. Some robl/LC7, and a similar fraction of IC74, remained in the supernatant. Most of the robl/LC7 protein co-purified with microtubules through a buffer wash and GTP elution. Some of the protein was eluted from microtubules with ATP and nearly all of the remainder could be stripped by treatment with 1 M NaCl. In contrast, most IC74 was ATP-eluted. We previously observed that different DLCs do not show precisely the same pattern during elution from microtubules, perhaps because they mark specific subsets of cytoplasmic dynein with distinct microtubule binding characteristics (
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To confirm this association, cytoplasmic dynein, dynactin, and kinesin from rat brain homogenates and cytoplasmic dynein from Drosophila embryo homogenates were immunoprecipitated with specific mAbs. The robl/LC7-like protein was pelleted only in the cytoplasmic dynein samples, no association was seen with dynactin, kinesin, or the bead controls (Figure 8C and Figure D). The 6883 antiserum raised against the Drosophila robl protein detected a band of Mr ~12,000 from Drosophila embryonic and larval homogenates. This band was not present in homogenates from robl null larvae, indicating that the band seen by this antibody is the product of the robl gene (Figure 8 E). These results demonstrate that a robl/LC7-like protein is indeed a component of cytoplasmic dynein from Drosophila and mammalian brain.
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Discussion |
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We have identified a new family of axonemal- and cytoplasmic dyneinassociated proteins. This family was identified by two independent means: the biochemical isolation and cloning of the Chlamydomonas dyneinassociated LC7 polypeptide and the identification and cloning of a Drosophila axonal transport mutant, roadblock. Our discovery of a new family of DLCs with roles in axonal transport, flagellar motility, and mitosis has intriguing implications.
The Structural Organization of Dyneins
With this report, all the known components of Chlamydomonas outer arm dynein have now been sequenced and a complete list of the properties of outer dynein armassociated DLCs can be made (Figure 9 A). The outer dynein arm consists of three heavy chains that form the globular heads and stems of the particle. Each heavy chain is tightly associated with one or more light chains. Located at the base of the structure are two intermediate chains (IC1 and IC2) and several additional light chains including a member of the Tctex1 protein family (LC2) together with multiple copies of the LC8 polypeptide and its homologue LC6. The LC7 protein is not tightly associated with any heavy chain and appears to form part of the intermediatelight chain complex located near the base of the particle (
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Examination of outer armless mutants revealed that a very small amount of the LC7 protein was still incorporated into the axoneme. The origin of this pool remains unclear at present. It did not appear to derive from inner arm I1 as it could not be detected in salt extracts of axonemes lacking outer arms. It may represent a small pool of LC7 that is mistransported to the axoneme in the absence of the remainder of the outer arm. Alternatively, it may be associated with some other axonemal enzyme such as the DHC1b-like dynein that is responsible for retrograde intraflagellar transport (
The cytoplasmic dynein particle is built around two ~520-kD heavy chains that form the stems and globular heads of the complex. Associated with the stems are a series of accessory proteins (Figure 9 B). These are now known to include two IC74s, two copies of the Tctex1 light chain (or of the related rp3 protein), one dimer of the highly conserved LC8 protein, and perhaps a 22-kD polypeptide (the position of which is speculative). The present study indicates that cytoplasmic dynein also contains a robl/LC7-like protein. Since axonemal and cytoplasmic dyneins utilize homologous intermediate chain genes, it is likely that robl/LC7 associates with IC74. By analogy with flagellar outer arm dynein, we propose a cytoplasmic dynein organizational model where robl/LC7 is located at the base of the dynein particle (Figure 9 B).
Cellular Functions that Require the robl/LC7 Family of Proteins
Previous work has provided strong evidence that cytoplasmic dynein plays an important role in retrograde axonal transport (for review see
Dynein is also thought to play a role in chromosome alignment and mitotic spindle assembly (
A role for dynein in the later stages of mitosis remains controversial. Cytoplasmic dynein heavy chain antibody injection experiments in mammalian cells failed to identify a role for dynein in anaphase chromosome movements (
ESTs belonging to two mammalian robl/LC7-like gene classes have been found from a wide assortment of embryonic, adult, and germline tissues (Figure 4 A). We identified >100 independent human ESTs in dbEST that encode a robl/LC7-like gene belonging to the first class (e.g., accession number hum424E02B). These ESTs are found from a wide array of tissues with unique and heavy intracellular transport needs such as: neural tissues (fetal and adult brain and retina), tissues with a heavy transcytosis burden (liver, spleen, kidney, placenta, and breast), a tissue involved in pigment dispersion (melanocyte), and mitotically active tissues (fetal and tumor tissues). Also, the rat robl/LC7-like gene from this first class was identified in the NCBI GenBank as being expressed in light-stimulated visual cortex (accession number 3288881). The robl/LC7-like gene identified by nine independent human ESTs of the second class (e.g., accession number AA446298) were found in a smaller subset of tissue types. This second class is found in human testes (6 of 9 clones) and tumor tissues (germ cell and kidney tumor tissues). Perhaps the testes expression may indicate a role for the second class with axonemal dynein, whereas the broad tissue expression may indicate a role for the first class with cytoplasmic dynein.
Together, the genetic and expression analyses suggest that the robl/LC7 family is important for many aspects of intracellular transport. In Drosophila, the mutant phenotypes found thus far suggest that the robl gene is required for axonal transport and mitosis. In addition, the female sterility defect seen in some genetic combinations suggests a role for robl in oocyte development. This finding is consistent with previous evidence that dynein plays a role in oocyte differentiation in Drosophila (
Possible Roles of the robl/LC7 Family in Dynein Function
Our work on robl/LC7 adds to a growing body of evidence supporting modulatory roles for DLC proteins in dynein-mediated movements. Specifically, the observation that DLC phenotypes are not as severe as dynein heavy chain phenotypes, the structural placement of DLCs at key positions in dynein, and the nonequivalent phenotypes among DLC mutants, supports this view. For example, other than female sterility, robl mutants have no apparent phenotypic similarities to the 10-kD/LC8 DLC (ddlc1) mutants in Drosophila (
In view of the dynein intermediate chains' possible structural role in linking motor activity to cargo binding activity, they may be a key regulatory target of the dynein complex. For example, IC74 mediates the binding of dynein to dynactin via a direct interaction with the p150glued subunit (
In support of a modulatory role for robl, there is evidence that loss of robl does not eliminate dynein function. For example, previous clonal analysis of the null allele robll(2)k10408 (before cloning of the gene) showed reduced clone size but a normal frequency of clone generation (
There is evidence that robl actively modulates dynein activity. The roblz homozygotes have a significantly stronger phenotype than robl nulls, exhibiting a complete absence of imaginal tissue, and eventually complete posterior larval paralysis and larval lethality. Yet, roblz/null animals are phenotypically similar to robl null animals, exhibiting only a slight reduction in the size of imaginal tissue, distal larval sluggishness without eventual paralysis, and survival to the late pupal stages. The accumulations of axonal cargoes in microtubule-based motor mutants in Drosophila may be caused by decreased processivity of cargo whose transport is directly dependent on the motor affected by the mutation, resulting in a buildup of other axonal cargo around this stalled cargo. The observed correlation of fewer axonal accumulations occurring in larvae with more copies of the roblz allele may suggest that fewer cargoes are entering the axons in these alleles. Thus, fewer accumulations may occur because there is less robl-dependent cargo within the axons. The concentration dependence of the roblz phenotype suggests that this robl allele interferes actively and directly with dynein function. Furthermore, since roblz can result in phenotypes worse than robl nulls, this aberrant DLC apparently has the ability to alter the functions of the dynein holoenzyme.
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Footnotes |
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Reprint requests may be sent to either L.S.B. Goldstein or S.M. King.
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Acknowledgements |
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We thank Kevin Pfister (University of Virginia Health Science Center, Charlottesville, VA) for cytoplasmic dynein samples, the University of California San Diego (UCSD) Immunocytochemistry/EM Core and Marilyn Farquhar (UCSD) for use of EM facilities, and MaryAnn Martin and Bill Saxton (both from Indiana University, Bloomington, ID) for discussions and sharing of unpublished work. We express sincere appreciation to Charles Zuker, Edmund Koundakjian, David Cowan, and Robert Hardy (all from UCSD) for providing the balanced EMS mutant lines used to identify roblz.
A. Bowman is supported by the UCSD Pharmacology Training Grant and is a Markey Research Fellow. L. Goldstein is an investigator of the Howard Hughes Medical Institute. This study was supported by GM35252 (to L. Goldstein) and GM51293 (to S. King) from the National Institutes of Health.
Submitted: January 28, 1999; Revised: May 26, 1999; Accepted: June 4, 1999.
1.used in this paper: BDGP, Berkeley Drosophila Genome Project; bxd, late RNA encoded bithoraxoid protein; ChAT, choline acetyltransferase; DLC, dynein light chain; EMS, ethyl methanesulfonate; EST, expressed sequence tag; IC74, 74-kD cytoplasmic dynein intermediate chain; khc, kinesin heavy chain; NCBI, National Center for Biotechnology Information; robl, roadblock; SYT, synaptotagmin; VG, ventral ganglion
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