Identification and Cloning of roadblock
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
(Hurd and Saxton 1996
; Gindhart et al. 1998
). Further characterization of roblz
revealed a complete absence of imaginal tissue, indicating a possible strong mitotic defect as well. To obtain robl
null alleles, deletions were generated from flanking P-elements that mapped near robl
. Homozygous null and roblz
hemizygote) animals die as late pupae; they also demonstrate a posterior larval sluggishness, a peculiar tail flipping phenotype, and accumulations of axonal cargo within their segmental nerves, as has been described for other axonal transport mutants in Drosophila
(Hurd and Saxton 1996
; Gindhart et al. 1998
). Additionally, the reduced size of imaginal tissue, rough pupal eyes, and missing bristle phenotypes seen in these animals are characteristic of mitotic mutants in Drosophila
. Thus, the robl
mutant phenotypes suggest roles for this gene in both axonal transport and mitosis.
Two overlapping deficiencies, robll(2)k10408 and roblc, identify the genomic interval encoding robl ( 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.
Figure 1 The robl genomic interval. (A) A diagrammatic map of the five genes identified in the genomic region around roadblock (accession number AF141921). The entire region has been sequenced and cDNAs have been obtained for robl and genes 1 and 4. Gene 3 is (more ...)
The genomic sequence of robl reveals a small three exon gene encoding a 97-residue polypeptide ( 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
outer dynein arm contains eight distinct light chain components (Piperno and Luck 1979
; Pfister et al. 1982
). Previously, we cloned and described all of these proteins except for LC7. To clone LC7, we purified and sequenced two tryptic LC7 peptides isolated from outer arm dynein ( A). Based upon this sequence, PCR primers were designed and an LC7 cDNA clone isolated. The largest cDNA clone was 864 bp in length ( B) and contained a single open reading frame of 105 residues with a predicted mass of 11,928 D and a calculated pI of 7.85. Both peptide sequences obtained from purified LC7 were found in this clone (26/26 residues correct) and were both preceded by the predicted basic residue. Three in frame stop codons were present upstream of the first Met residue and a 489-bp 3′ untranslated region, including a perfect copy of the Chlamydomonas
polyadenylation signal, followed the stop codon.
Figure 2 Molecular analysis of LC7 from the Chlamydomonas outer dynein arm. (A) Two tryptic peptides from outer arm dynein LC7 were completely sequenced, yielding a total of 26 residue assignments. The actual mass of each peptide is in agreement with the calculated (more ...)
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 ( 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 ( 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 (Harrison et al. 1998
). To confirm that the LC7 protein is a component of the outer arm, axonemes were prepared from mutants lacking specific dynein structures including the outer arm (oda9
), inner arm I1 (ida1
, and ida3
), and a subset of inner arms I2/3 (ida4
). Immunoblot analysis of these samples using a polyclonal LC7 antiserum revealed that the LC7 polypeptide was present in the mutants lacking inner arms, but was drastically reduced in the strain lacking outer arms ( A). Upon overexposure of the blot, a very small amount of LC7 could be detected in the outer armless axonemes. The origin of this minor fraction remains unclear as the LC7 protein could not be detected in sucrose gradient profiles of high salt extracts from outer armless strains (data not shown). Furthermore, sucrose gradient analysis of extracts from wild-type axonemes revealed that all the extracted LC7 comigrated with the outer arm at ~18 S ( B).
Figure 3 LC7 is a component of the outer dynein arm. (A) Flagellar axonemes were prepared from wild-type Chlamydomonas (WT) and from mutants lacking the outer arm (oda9), inner arm I1 (ida1, ida2, and ida3), and inner arms I2/3 (ida4). Samples were electrophoresed (more ...)
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
(Lipshitz et al. 1987
). The robl/LC7 similarity prompted us to look for additional robl-like genes in the NCBI GenBank. BLAST and comparative protein sequence analysis identified a large family of robl-like proteins conserved in Drosophila
, nematode, Chlamydomonas
, and three mammalian species ( and ). Four other robl-like genes, in addition to the bxd
late RNA, were identified in Drosophila
and are designated here by their cytological location: robl62A
, and robl60C
. In mammals, two classes of robl/LC7-like genes were identified by homology ( A). However C
apparently has only a single robl-like gene in its genome ( A). The differences between robl/LC7-like family members may suggest a possible functional distinction between the various members within an organism.
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 (Hurd and Saxton 1996
; Gindhart et al. 1998
). The accumulation phenotype correlates with the other common axonal transport phenotypes in Drosophila
, tail flipping and posterior paralysis. It was proposed that these mutants disrupt the processive movement of their cargo within the axon, causing the axons to swell, filling with transported axonal material. Immunostaining of roblz
/null hemizygous and robl
null homozygous larvae reveals frequent accumulations of synaptotagmin (SYT) ( and ) and choline acetyltransferase (ChAT) ( and ) in the larval segmental nerves. In contrast, SYT ( A) and ChAT ( B) show only a low background level staining in wild-type segmental nerves. Additionally, axonal transport motors (of the kinesin I and kinesin II family), cysteine string protein, and a marker for endocytic traffic are also observed to accumulate in the axons of robl
mutants (data not shown). Thus, robl
mutants have a gross phenotype similar to that previously described for axonal transport mutants in Drosophila
; a progressive larval posterior paralysis, tail flipping, and segmental nerve axonal cargo accumulation.
Figure 5 Coimmunostaining of larval segmental nerves for (left column) SYT and (right column) ChAT revealed distal axonal accumulations of synaptic cargo in robl mutants. (A and B) In wild type, there is only a low background staining observed. (C and D) Segmental (more ...)
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 ( F) with staining observed in segmental nerves in the posterior of the larvae ( 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 ( E) with staining observed in segmental nerves at the posterior region of the larvae ( 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 SYT–ChAT co-immunostaining analysis. Since ChAT is expressed only in sensory neurons, SYT–ChAT 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 anterior–posterior 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 ~1–5% 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 ~200–400% 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 (Hurd and Saxton 1996
). The nerves of roblz
/null (hemizygote) larvae also contain swollen axons that have become filled with axonal cargo ( and ). These swollen axons are on average twice the diameter of the largest axon observed in wild type ( B). While the axonal swellings observed in khc
mutants vary in size, their content characteristics are uniform, containing all observed membrane bound axonal content (Hurd and Saxton 1996
). In addition to these multicomponent axonal accumulations ( D), robl
mutants also have a small subset of single component axonal accumulations ( C). These single component accumulations contain almost exclusively small clear vesicles and tend to be smaller on average than the multicomponent accumulations. These small clear vesicles may represent a class of cargo that is particularly sensitive to retrograde transport failure in robl
mutants. In support of this idea, when the synaptic area is examined by EM, there is an approximate twofold increase in the number of similar appearing small clear vesicles observed (data not shown).
Figure 6 Transmission EM cross-sections of robl mutant third instar larval segmental nerves revealed two classes of axonal cargo accumulations and severe axonal loss and nerve degeneration. (A) Nerves from roblz hemizygous larvae had axons that swelled with transported (more ...)
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
(Hurd and Saxton 1996
). All observed roblz
hemizygous larvae show at least mild axonal loss ( A). When the segmental nerves from the most severely sluggish roblz
hemizygous larvae are analyzed by EM, extensive axonal loss and nerve degeneration is observed ( E). Furthermore, the segmental nerves from roblz
homozygous larvae always show extensive axonal loss and nerve degeneration ( F). The basis for this axonal loss and nerve degeneration is unclear; however, we have observed a few large multilamellae structures (~1/10 nerve diameter) indicating a possible phagocytic component to the axonal loss and nerve degeneration (data not shown).
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 ( 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.
Figure 7 Severe mitotic defects were revealed in robl mutants examined by third instar larval brain squash analysis. Examples of typical wild-type mitotic figures are shown (designated by arrows): (A) a normal prometaphase figure, (B) a normal metaphase figure, (more ...)
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 ( and ). Additionally, many of the polyploid figures showed hypercondensation of their chromosomes ( F). Abnormal anaphase figures were also observed with hypercondensed chromosomes and disorganization of the chromosomes around the presumptive poles ( 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 ( 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 ( and ). In addition, we observe apparent telophase bridging in which DNA has become trapped between two dividing nuclei ( 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.
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
(Phillis et al. 1996
; McGrail and Hays 1997
). Attempts to rescue the robl
sterility phenotype by giving the cDNA-rescued females mild heat shock (to induce expression of robl
) failed. Since the genomic construct fully rescues the female fertility defect, female sterility is likely a real robl
mutant phenotype; robl
cDNA under heat shock control is likely failing to provide appropriate levels of robl protein in the needed cells because of the inadequacy of non-native promoter control.
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
), 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 ( 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 (King et al. 1996a
, King et al. 1998
). To further address the association of the robl/LC7-like protein with cytoplasmic dynein, the ATP eluate was sedimented through a 5–20% sucrose gradient. Immunological analysis of the resulting fractions revealed that the robl/LC7-like protein sedimented at ~18 S and precisely copurified with the IC74 component of cytoplasmic dynein ( B).
Figure 8 A robl/LC7-like protein is present in cytoplasmic dynein. (A) Western blot analysis was performed on samples from the fractionation of a rat brain homogenate. Blots were probed with mAb 74-1 and the R7178 rabbit polyclonal to detect IC74 of cytoplasmic (more ...)
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 ( and ). 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 ( E). These results demonstrate that a robl/LC7-like protein is indeed a component of cytoplasmic dynein from Drosophila and mammalian brain.