Better explicating the strengths and shortcomings of these models will help refine projections and improve transparency in the years ahead.
Image credit: Witsawat.S.
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Communicated by Ruth Lehmann, New York University Medical Center, New York, NY, June 7, 2006 (received for review February 24, 2006)
Abstract
In the Drosophila visual system, the color-sensing photoreceptors R7 and R8 project their axons to two distinct layers in the medulla. Loss of the receptor tyrosine phosphatase LAR from R7 photoreceptors causes their axons to terminate prematurely in the R8 layer. Here we identify a null mutation in the Liprin-α gene based on a similar R7 projection defect. Liprin-α physically interacts with the inactive D2 phosphatase domain of LAR, and this domain is also essential for R7 targeting. However, another LAR-dependent function, egg elongation, requires neither Liprin-α nor the LAR D2 domain. Although human and Caenorhabditis elegans Liprin-α proteins have been reported to control the localization of LAR, we find that LAR localizes to focal adhesions in Drosophila S2R+ cells and to photoreceptor growth cones in vivo independently of Liprin-α. In addition, Liprin-α overexpression or loss of function can affect R7 targeting in the complete absence of LAR. We conclude that Liprin-α does not simply act by regulating LAR localization but also has LAR-independent functions.
The color-sensitive photoreceptors of the Drosophila visual system, R7 and R8, provide a simple system in which to study layer-specific axon targeting. Whereas the outer photoreceptors R1–R6 project their axons to the lamina, R7 and R8 project to the medulla, where R8 terminates in the more superficial M3 layer and R7 in the deeper M6 layer. This targeting occurs in two stages, with both R7 and R8 growth cones pausing in separate temporary layers before proceeding to their final positions (1). Several genes are known to contribute to the establishment of the R7 and R8 projection pattern. The transcription factor Runt is expressed in R7 and R8, and its misexpression is sufficient to target R2 and R5 to the medulla, suggesting that it controls the choice of optic neuropil (2). Endogenous expression of the homophilic cell adhesion molecule Capricious (Caps) in R8 or its ectopic expression in R7 directs these photoreceptors to terminate in the Caps-positive M3 layer (3). The transmembrane cadherin Flamingo (Fmi) is required for R8 targeting (4, 5), whereas loss of either N-cadherin (Ncad) (6) or one of the receptor protein tyrosine phosphatases (RPTPs), PTP69D (7) or LAR (8, 9), causes R7 to terminate inappropriately in the R8 layer.
Other functions of LAR include axonal patterning of photoreceptors R1–R6 (9) and embryonic motor neurons (10, 11), synapse morphogenesis at the larval neuromuscular junction (NMJ) (12), and polarization of actin filaments in the follicle cells surrounding the oocyte, which promotes egg elongation along the anterior-posterior axis (13, 14). It is unclear how LAR and other RPTPs signal within the cell to induce the cytoskeletal rearrangements that mediate these functions. Trio, a guanine nucleotide exchange factor for Rac (15), and Enabled (Ena), which regulates actin polymerization (16), show genetic interactions with LAR in both R7 targeting (8) and motor axon guidance (17). LAR can dephosphorylate both Ena and its antagonist, the cytoplasmic kinase Abelson (Abl) (17). Yeast two-hybrid screens for proteins that bind to the LAR intracellular domain identified both the human and Drosophila homologues of Liprin-α, a protein with an N-terminal coiled-coil domain and a C-terminal LAR-binding liprin homology domain (LHD) consisting of three sterile alpha motif domains (12, 18). Drosophila Liprin-α mutations have the same effects as LAR mutations on NMJ synapse morphology (12), suggesting that the two proteins act together.
Several studies suggest a role for Liprin-α in protein localization. Synaptic vesicle proteins such as synaptotagmin and synaptobrevin are mislocalized in neurons mutant for either Drosophila Liprin-α or the Caenorhabditis elegans Liprin-α homologue syd-2 (19, 20). In Drosophila, this phenotype reflects a requirement for Liprin-α in axonal transport of synaptic vesicles through its binding to the Kinesin-1 (Khc) motor protein (19). In cultured hippocampal neurons, Liprin-α promotes glutamate receptor targeting to synapses (21, 22). The observations that human Liprin-α2 promotes clustering of LAR within the plasma membrane in COS cells, and that SYD-2 and the C. elegans LAR homologue PTP-3 regulate each other’s localization along the nerve cord, suggest that localization also may be the mechanism by which Liprin-α influences LAR function (23, 24).
Here we identify a likely null mutation in Drosophila Liprin-α that has the same effect on R7 photoreceptor targeting as LAR, yet does not affect two other LAR-dependent processes, egg elongation and motor axon guidance. Liprin-α physically interacts with LAR in embryos and cultured cells; however, our results do not support a primary role for Liprin-α in LAR localization. LAR localization to focal adhesions in cultured Drosophila cells and to photoreceptor growth cones in vivo is independent of Liprin-α. In addition, Liprin-α overexpression can partially restore R7 targeting, and removal of Liprin-α can further reduce R7 targeting in the complete absence of LAR, indicating that some functions of Liprin-α are independent of LAR.
Results
Liprin-α Is Required for Retinal Axon Targeting.
We reported an eyFLP-based mosaic screen in adult head sections designed to identify genes required in photoreceptors for their normal axonal targeting (8). In addition to a new allele of LAR (8), we also isolated a single allele of a second gene with a very similar phenotype (Fig. 1 B and C), which we named out of step (oos). We used P element-induced male recombination (25) to map the oos mutation to a small region that included the Liprin-α gene. oos mutants had a stop codon at amino acid 307 of Liprin-α, within the N-terminal coiled-coil domain (Fig. 1 D), making oos likely to be a null allele of Liprin-α. We confirmed the identity of oos by demonstrating that pan-neuronal expression of Liprin-α cDNA could rescue the oos targeting defect (Fig. 1 J). We therefore will refer to this allele as Liprin-α oos .
Fig. 1.
Liprin-α is required for R7 photoreceptor targeting. (A–C) Horizontal sections of adult heads carrying glass-lacZ to label all photoreceptors, stained with anti-β-gal. (A) WT. Photoreceptors project to the lamina (la) and medulla (me). R7 and R8 terminate in two distinct layers in the medulla (arrowheads). (B and C) In Liprin-α oos homozygous mutants (B) and in LAR null mutants (C), only one layer of termini is present in the medulla (arrowhead). (D) oos flies have a nonsense mutation predicted to truncate the Drosophila Liprin-α gene within the N-terminal coiled-coil domain. The C-terminal liprin homology domain (LHD) of Liprin-α binds to the PTP-D2 domain of LAR. The extracellular domain of LAR, containing three Ig domains and nine fibronectin type III domains, is not shown. Three tyrosine residues (Y) within the LHD of Liprin-α are highly conserved throughout evolution. (E) Tangential section of a retina containing a small Liprin-α oos mutant clone. The central R7 rhabdomere is present in both pigmented WT regions (yellow arrows) and unpigmented, mutant regions (red arrows). (F) Horizontal section of an adult head with Liprin-α oos mutant clones carrying the PanR7-lacZ reporter to label all R7 photoreceptors, stained with X-Gal. Liprin-α oos mutant R7 axons (brackets) terminate more superficially than surrounding WT R7 photoreceptors. (G and H) R1–R6 project from the retina (re) to the lamina (la) in both WT (G) and Liprin-α oos (H) horizontal sections of adult heads carrying the Rh1-lacZ reporter and stained with anti-β-gal. (I) shows a Liprin-α oos mutant whole-mount optic lobe at 24 h after puparium formation stained with anti-β-gal to reveal glass-lacZ expression. At this stage, most R7 termini are clearly separated from the R8 layer. A maximum intensity projection of six optical sections spanning ≈3 μm along the z axis is shown. (J) Horizontal head sections of the indicated genotypes carrying glass-lacZ were stained with anti-β-gal and scored for R7-targeting defects. In Liprin-α oos homozygous mutants, only 37% of R7 axons project beyond the R8 layer. Expression of HA-tagged Liprin-α from two independent transgene insertions (#21 and #43) in all neurons with elav-GAL4 or in R7 with sev-GAL4 rescued R7 targeting to almost the WT level. HA-Liprin-α expression driven in R8 by 109.68-GAL4 did not improve R7 targeting. Number of cartridges counted are given in parentheses in this and subsequent figures.
Liprin-α Is Autonomously Required for R7 Axons to Terminate in the Correct Target Layer.
In wild-type optic lobes, the R7 and R8 photoreceptors terminate in two distinct layers, with R7 projecting deeper into the medulla than R8 (Fig. 1 A). In Liprin-α oos mutants, as in LAR mutants, the R7 layer was largely absent (Fig. 1 B); however, the Liprin-α oos phenotype was weaker than LAR, with 37% of R7 axons projecting beyond the R8 layer (Fig. 1 J) compared with 15% for LAR (8) (see Fig. 2 L). Despite the lack of terminals in the R7 layer, the R7 cell body was present at the correct location in tangential sections of eyes with Liprin-α oos clones (Fig. 1E). Liprin-α oos mutant R7 cells also expressed the appropriate rhodopsin genes; in mutant clones, axons expressing a lacZ reporter specific for the Rh3 and Rh4 genes (PanR7-lacZ) projected to the medulla but terminated prematurely in the R8 layer (Fig. 1F). Using the Rh1-lacZ reporter, we found that R1–R6 terminated in the appropriate target neuropil, the lamina, in the absence of Liprin-α (Fig. 1 G and H).
Fig. 2.
Interactions between LAR and Liprin-α. (A) Myc-tagged Liprin-α and HA-tagged LAR expressed ubiquitously under da-GAL4 control can be coimmunoprecipitated from embryonic extracts with anti-Myc. Input lanes contain 1% of the total protein used in the immunoprecipitation. (B–D) Liprin-α can be coimmunoprecipitated with full-length LAR or the LAR PTP-D2 domain from S2R+ cells. S2R+ cells were transfected with the indicated constructs. Immunoprecipitations were carried out with anti-Myc (B) or anti-HA (C and D). Input lanes contain 0.5% of the total protein used in the immunoprecipiation. (E) Liprin-α is tyrosine phosphorylated. S2R+ cells were transfected with HA- or Myc-tagged Liprin-α. Liprin-α was immunoprecipitated from lysates with anti-HA or anti-Myc. Western blots with anti-phosphotyrosine, anti-HA and anti-Myc are shown. (F–K) LAR and Liprin-α localize to the growth cones of R1–R6 (arrowheads) in third instar larval optic lobes. (F–H) WT. (I–K) GMR-GAL4; UAS-HA-Liprin-α. Photoreceptors are stained with anti-β-gal to reveal glass-lacZ expression (red in F, H, I, and K); anti-LAR staining (cyan in G and H) is detected on R1–R6 growth cones and in the medulla neuropil (asterisk); Liprin-α was localized to R1–R6 growth cones as monitored by anti-HA staining (cyan in J and K). (L) Horizontal head sections of the indicated genotypes carrying glass-lacZ were stained with anti-β-gal and scored for R7 targeting defects. LAR2127 /LARC12 null mutants were rescued by expression of full-length LAR but not by LAR lacking the D2 domain.
Mutations in both Ncad and LAR show a similar R7 targeting defect in adult optic lobes. However, the R7 targeting defect in Ncad mutants is already apparent at 17 h after puparium formation, whereas LAR mutant R7 cells project normally at this stage but retract to the R8 layer later in pupal development (1, 8, 9). We found that Liprin-α mutants had a normal R7 projection pattern at 24 h, a stage at which N-cad mutants show significant abnormalities but LAR mutants do not (Fig. 1I).
We next showed that Liprin-α acts autonomously in the R7 cell to direct its axon to the appropriate target layer. We found that expression of Liprin-α in R7 (and R3 and R4, which terminate in the lamina) with the sevenless (sev)-GAL4 driver rescued the R7 targeting defect in Liprin-α oos mutants as effectively as pan-neuronal expression with elav-GAL4 (Fig. 1 J). Conversely, expression only in R8 cells by using the 109.68-GAL4 driver (26) failed to rescue R7 targeting in Liprin-α oos mutants (Fig. 1 J).
A Direct Interaction Between LAR and Liprin-α Is Required for R7 Targeting.
Both human and Drosophila Liprin-α have been shown to bind to the LAR intracellular domain (12, 18). Several lines of evidence argue that this interaction is important for R7 targeting. First, both endogenous LAR and a tagged form of Liprin-α expressed in photoreceptors with GMR-GAL4 were transported to the growth cones of photoreceptors R1–R6 (Fig. 2 F–K). Second, we confirmed the presence of LAR and Liprin-α in the same protein complex by coimmunoprecipitation of epitope-tagged proteins from Drosophila embryos (Fig. 2 A) and S2R+ cells (Fig. 2 B and C). Consistent with previous findings for human LAR (18), the distal D2 phosphatase domain of LAR was sufficient to coimmunoprecipitate Liprin-α (Fig. 2 D). Third, the D2 domain is essential for LAR function in R7 targeting, because its absence in either the LARbypass truncation allele (27, 28) or a deleted LAR rescue construct (28) resulted in a strong R7 projection defect (Fig. 2 L). LARbypass generates a partially functional gene product, because its motor axon guidance phenotype is less penetrant than LAR null alleles (27) and it supports normal egg elongation (Fig. 6E, which is published as supporting information on the PNAS web site).
Liprin-α has 19 tyrosine residues, three of which are found at conserved positions in the LHD of mammalian and C. elegans Liprin-α homologues (Fig. 1 D). In S2R+ cells, tagged Liprin-α immunoprecipitated by using two different tags was recognized by anti-phosphotyrosine antibody (Fig. 2 E). Liprin-α is thus a potential substrate for LAR.
Liprin-α Is Not Required for LAR Localization in Photoreceptors.
Previous studies have concluded that Liprin-α binds to LAR to control its subcellular localization. In C. elegans syd-2 mutants, the LAR homologue PTP-3 fails to cluster at synapses (24), and in cultured mammalian cells, Liprin-α promotes the localization of LAR to focal adhesions (18, 23). We therefore analyzed the intracellular distribution of LAR in Liprin-α oos mutant photoreceptors. Despite its reported role in axonal transport of synaptic vesicle components (19), Liprin-α was not required for the transport of endogenous LAR protein to the growth cones of larval R1–R6 photoreceptors (Fig. 3 A–C). The strong expression of LAR on medulla neurons (8) prevented us from examining endogenous LAR protein within the R7 growth cone. Instead, we monitored the distribution of epitope-tagged LAR expressed in photoreceptors. We could not detect any difference between WT and Liprin-αoos mutants in hemagglutinin (HA)-LAR localization within the R7 or R8 termini in adult head sections (Fig. 3 D–I). Although the morphology of R7 growth cones that projected beyond the R8 layer was abnormal in Liprin-α oos mutants (Fig. 3 F and I Insets), HA-LAR was distributed into all regions of these growth cones that contained cytoplasmic β-gal (Fig. 3 D–F).
Fig. 3.
Liprin-α is not essential for LAR localization in vivo. (A–F) LAR can be detected on the growth cones of R1–R6 photoreceptors (arrowheads in A) in third instar larval optic lobes (A–C) and on R7 termini in the adult medulla (D–F) in Liprin-α oos mutants. All photoreceptors were labeled with anti-β-gal to reveal glass-lacZ expression (red in A, C, D, F, G, and I). LAR localization was monitored with anti-LAR antibody in the larval brain (cyan in B and C) where LAR is also strongly expressed in the medulla (asterisk). (D–I) Horizontal sections from adults carrying a GMR-HA-LAR insertion expressed in all photoreceptors were stained with anti-HA (cyan in E, F, H, and I). There is no obvious difference between the adult medulla of Liprin-α oos heterozygotes (G–I) and Liprin-α oos homozygous mutants (D–F) in HA-LAR distribution on R7 (open arrowheads) and R8 (filled arrowheads). (F and I Insets) Enlargements of R7 termini that have grown beyond the R8 layer to illustrate their aberrant shape in Liprin-α mutants.
Liprin-α Does Not Influence LAR Localization in S2R+ Cells.
To obtain higher resolution, we analyzed the intracellular distribution of LAR in adherent Drosophila S2R+ cells. In these cells, epitope-tagged Liprin-α colocalized with the focal adhesion marker Talin (29) (Fig. 4 A–C), consistent with the localization of human LAR and Liprin-α1 to focal adhesions in MCF7 cells (18). We found that even in the absence of cotransfected Liprin-α, HA-tagged LAR also localized to focal adhesions marked by Talin (Fig. 4 D–F). We were unable to detect endogenous expression of either Liprin-α or the related Liprin family member CG11206 in S2R+ cells by RT-PCR (Fig. 7A, which is published as supporting information on the PNAS web site). In case Liprin-α was expressed below the level of detection of this assay, we used RNA interference to knock down any potential endogenous Liprin-α mRNA. Expression of a double-stranded RNA hairpin construct complementary to Liprin-α (30) together with an HA-tagged form of Liprin-α strongly reduced HA immunoreactivity, demonstrating its effectiveness (data not shown). However, LAR still colocalized with Talin in the presence of this RNA interference construct (Fig. 4 G–I). Thus, LAR localization to focal adhesions in S2R+ cells requires neither endogenous nor ectopically expressed Liprin-α.
Fig. 4.
Liprin-α is not essential for LAR localization in S2R+ cells. (A–F) Liprin-α and LAR colocalize with the focal adhesion marker Talin in S2R+ cells. S2R+ cells were transfected with HA-Liprin-α (A–C) or HA-LAR (D–F) and actin5c-GFP (blue in A and D) as a transfection marker. Cells were fixed and stained with anti-HA (B and E; red in A and D) and anti-Talin (C and F; green in A and D). (G–I) Liprin-α RNA interference did not alter LAR colocalization with Talin. S2R+ cells were transfected with HA-LAR and a double-stranded RNA hairpin construct directed against Liprin-α, and stained with anti-HA (H, red in G) and anti-Talin (I, green in G).
Liprin-α Is Not Required for All of the Functions of LAR.
The strikingly similar effects of LAR and Liprin-α mutations on R7 targeting, as well as on neuromuscular synapse morphogenesis (12), prompted us to investigate whether Liprin-α was required for other known functions of LAR. LAR has been shown to organize a network of actin filaments in the ovarian follicle cells that promotes egg elongation (13, 14); females lacking LAR thus lay short, rounded eggs (Fig. 6 B and E). In contrast, we found that females lacking Liprin-α laid eggs with the WT shape (Fig. 6 D and E). Egg elongation also does not require the D2 domain of LAR to which Liprin-α binds, because females carrying the LARbypass allele laid normally elongated eggs (Fig. 6E).
Zygotic LAR mutant embryos have a characteristic “bypass” phenotype in the embryonic motor axon projection (10), resulting from a failure of the ISNb branch of the projection to defasciculate from the ISN branch. Embryos lacking zygotic Liprin-α do not show this bypass phenotype (12). To test whether their normal development was due to perdurance of the maternal contribution of Liprin-α, we generated embryos lacking both maternal and zygotic Liprin-α; however, these embryos also did not display the bypass phenotype. Bypassing was not observed in 267 hemisegments examined in WT embryos or in 227 hemisegments examined in maternal/zygotic Liprin-α oos mutants. Liprin-α thus is not required for the function of LAR in this aspect of motor axon guidance.
Liprin-α Can Function Independently of LAR.
If Liprin-α acted by controlling LAR localization, it should have no effect in the absence of LAR. However, we found that overexpression of Liprin-α in photoreceptors in a LAR null mutant background could partially restore R7 targeting (Fig. 5 A). The converse was not true, because overexpression of LAR had no effect on R7 targeting in a Liprin-α mutant background (Fig. 5 C). In addition, clones lacking both Liprin-α and LAR had fewer correctly targeted R7 axons than clones homozygous for LAR (Fig. 5 B). These data show that both endogenous and overexpressed Liprin-α can promote some normal R7 targeting even in the complete absence of LAR, suggesting that Liprin-α may act both downstream of and in parallel to LAR.
Fig. 5.
Liprin-α acts partially in parallel to LAR in R7 targeting. (A–C) Horizontal head sections of the indicated genotypes carrying glass-lacZ were stained with anti-β-gal and scored for R7 targeting defects. (A) Liprin-α overexpression in all neurons by elav-GAL4 or in photoreceptors by GMR-GAL4 partially rescued the R7 targeting defect in LAR mutants. Asterisks indicate significant differences from LAR null with no rescue construct (P < 0.0001 by Student’s t test). (B) Whole-eye mosaics of the indicated mutations were generated with ey-FLP and FRT40, M (2)24F. In the absence of both Liprin-α and LAR, fewer R7 axons were correctly targeted than in the absence of either Liprin-α or LAR alone. Asterisks indicate a significant difference from LARc12 mosaics (P < 0.0001 by Student’s t test). (C) In a Liprin-α oos mutant background, we used elav-GAL4 to drive expression of the indicated constructs. ΔN-Liprin-α, which lacks the N-terminal coiled-coil domain, failed to rescue R7 targeting. Overexpression of LAR had no effect on R7 targeting.
These functions of Liprin-α might require interactions with other proteins involved in R7 targeting. The majority of protein-binding sites in human Liprin-α have been mapped to the N-terminal coiled-coil domain, which also mediates homodimerization. This domain likewise is required for homodimer formation in Drosophila, because a truncated form of Liprin-α lacking the N terminus failed to coimmunoprecipitate with full-length Liprin-α protein in S2 cells (Fig. 7B). Although the remaining C-terminal domain is sufficient to mediate binding to LAR (R. Farajian and J.E.T., unpublished data), expression of this truncation in vivo did not rescue the Liprin-α mutant phenotype (Fig. 5 C). These data suggest that R7 targeting requires Liprin-α dimerization or the interaction of other factors with the N terminus of Liprin-α.
Discussion
We have shown that Liprin-α is specifically required in the R7 photoreceptor for its axonal targeting to the correct layer in the medulla. R7 terminates inappropriately in the R8 target layer in the absence of either Liprin-α, LAR (8, 9), or the D2 domain of LAR to which Liprin-α binds. Liprin-α and LAR both localize to photoreceptor growth cones and are found in the same protein complex. These results imply that a protein–protein interaction between LAR and Liprin-α is important for R7 targeting. Our data suggest that this interaction primarily contributes to signaling downstream of LAR, rather than mediating localization of the LAR protein. However, Liprin-α also appears to act in a parallel, LAR-independent pathway.
The C. elegans Liprin-α homologue, syd-2, was identified through its effects on the localization of presynaptic proteins (20). Previous studies also have shown that in mammalian cells, LAR and Liprin-α colocalize at focal adhesion contacts (18), but in the absence of cotransfected Liprin-α, LAR is uniformly distributed throughout the plasma membrane (23). In contrast to these results, we could not detect an effect of Liprin-α on LAR localization. Despite the reported role for Liprin-α in axonal transport in motor neurons (19), endogenous LAR is correctly transported to the R1–R6 growth cones, and epitope-tagged LAR does not alter its distribution in R7 terminals in Liprin-α mutants. In S2R+ cells, LAR localizes to focal adhesions in the absence of cotransfected or detectable endogenous Liprin-α. It is unlikely that one of the other two Liprin family members encoded in the Drosophila genome can substitute for Liprin-α in LAR localization, because CG10743 is most homologous to human Liprin-β, which does not bind directly to LAR, and CG11206 is not expressed in S2R+ cells (Fig. 7A).
Because Liprin-α is not required for LAR localization but binds to LAR and has the same effect as LAR on R7 targeting, it may contribute to LAR signal transduction. Although the Liprin-α protein contains no predicted catalytic domains, human Liprin-α1 has been shown to undergo autophosphorylation (31), suggesting that it might act as a kinase. Alternatively, Liprin-α might promote the association of LAR with its substrates; mammalian Liprin-α family members have been shown to bind to a variety of synaptic molecules (21, 22, 32). Although the LAR substrate Ena binds directly to the intracellular domain of LAR (17), the Drosophila Trio homologue lacks the domains that mediate LAR binding by human Trio (15) and might require an adaptor protein such as Liprin-α. Liprin-α also might promote interaction of LAR with a cadherin/catenin complex, because β-catenin is a substrate for human LAR in COS cells (33). Finally, Liprin-α might directly regulate the phosphatase activity of LAR. The D2 domain of LAR and other RPTPs can inhibit their phosphatase activity, in some cases by promoting dimerization (34–37). Although the LAR intracellular domain crystallizes as a monomer (38), homotypic interactions between the LAR transmembrane and intracellular domains have been observed in biochemical assays (39). Binding of Liprin-α to LAR-D2 might reduce its ability to dimerize.
LAR does not require Liprin-α for all of its functions. The R7 mistargeting phenotype is stronger in LAR mutants than in Liprin-αoos mutants, although the Liprin-α oos allele is likely to be a null. More dramatically, egg elongation and ISNb motor axon guidance are unaffected in Liprin-α mutants but strongly affected in LAR mutants. Conversely, removal or overexpression of Liprin-α can alter the extent of correct R7 targeting even in the complete absence of LAR. These observations indicate that Liprin-α does not function exclusively as an adaptor for LAR or a regulator of LAR activity but can also act in a parallel pathway, perhaps by localizing other proteins necessary for R7 targeting. Upstream input for this parallel function might come from Ncad or the RPTP PTP69D, because mutations in either show a LAR-like R7 targeting defect in the adult (6, 7). However, Liprin-α could mediate only a subset of the functions of these molecules, because Ncad is required at an earlier stage (1) and Ptp69D mutants cause an R1–R6 mistargeting phenotype (40). LAR and PTP69D might have substrates in common that control R7 targeting, because a chimeric protein with the extracellular domain of LAR and the intracellular domain of PTP69D can rescue R7 targeting in LAR mutants (8). Taken together, these results show that the simple model that Liprin-α acts by localizing LAR to the appropriate region of the plasma membrane (23) is unlikely to be correct. Understanding the mechanism of Liprin-α function will lead to new insights into RPTP signaling and layer-specific axonal targeting.
Materials and Methods
Genetics.
The Liprin-α oos mutant was isolated in the mosaic screen described in ref. 8. The oos mutation was mapped between the insertion sites of P{EP}EP2141 at 27A1 and P{EP}CG17378EP2338 at 27B1 (FlyBase) by site-specific male recombination (25). The Liprin-α coding region was amplified by PCR from homozygous oos genomic DNA and sequenced. All flies were raised at 25°C. Fly stocks used include GMR-HA-LAR, UAS-LARΔC, FRT40, M (2)24F, arm-lacZ, LAR2127 , and LARc12 (8); LARbypass (27); UAS-LAR (10); UAS-LAR-ΔPTPD2 (28); Rh1-lacZ (41); PanR7-lacZ (a gift from Claude Desplan, New York University); glass-lacZ (42); elav-GAL4, sev-GAL4, T155-GAL4, daughterless (da)-GAL4, and Df(2L)Exel7027 (FlyBase); 109-68-GAL4 (26); ey-FLP (7); hs-FLP122; and P[w+ ; ovoD1 ] 2L P[hsneo; ry+ ; FRT] 2L /S Sp Ms (2)M bwD /CyO (43).
Cell Culture.
S2 or S2R+ cells were grown in Schneider’s Drosophila Medium (GIBCO Invitrogen) with 10% heat inactivated FBS/50 units/ml penicillin-50 μg/ml streptomycin (GIBCO Invitrogen) at room temperature. S2R+ cells were removed from the culture flask with Trypsin-EDTA (GIBCO Invitrogen). Transfections were performed with Effectene (Qiagen, Valencia, CA). Protein expression from UAS promoter constructs was driven by actin5c-GAL4VP16 (44). Efficiency of transfection was verified by cotransfection with actin5c-GFP plasmid.
Transgenes and Expression Constructs.
UAS-Liprin-α was made by cloning the full-length cDNA from clone LD27334 [Drosophila Genomics Resource Center (DGRC)] into pUAST. For UAS-HA-Liprin-α, an N-terminal HA tag was introduced by PCR. For UAS-MYC-Liprin-α, a fragment containing five copies of the Myc tag was amplified by PCR from Egalitarian-Myc (45) and added to the N terminus of Liprin-α. The N-terminal deletion in UAS-ΔN-Liprin-α uses the endogenous ATG coding for M897 as the start codon and was modified by PCR for cloning into pUAST-HA. A full-length cDNA encoding the Liprin CG11206 was excised from clone RE30521 (DGRC) and ligated into pUAST. For the Liprin-α and CG11206 RNA hairpin constructs (UAS-RNA interference), two identical fragments of each cDNA were PCR-amplified and cloned together in a sense-antisense orientation into pUAST. Template sequences correspond to amino acids 278–540 for Liprin-α and 143–568 for CG11026. UAS-HA-LAR was made by cloning the N-terminally HA-tagged LAR sequence from GMR-HA-LAR (8) into pUAST. UAS-HA-LAR-D2 was constructed by PCR to contain a N-terminal HA tag followed by LAR amino acids 1670–2029.
Coimmunoprecipitation and Phosphotyrosine Detection.
For coimmunoprecipitations from embryonic extracts, embryos carrying the da-Gal4 driver and either UAS-HA-LAR alone or both UAS-HA-LAR and UAS-Myc-Liprin-α were collected overnight. Dechorionated embryos were homogenized in ice-cold IP buffer (50 mM Tris, pH 7.5/150 mM NaCl/1 mM NaF with one “complete mini, EDTA-free protease inhibitor mixture” tablet (Roche) per 10 ml). For coimmunoprecipitations out of S2 or S2R+, cells were transiently transfected with the relevant constructs. After 48 h, cells were rinsed in ice-cold PBS and then lysed in ice-cold IP buffer (50 mM Tris, pH 7.5/150 mM NaCl/1 mM NaF/1 mM PMSF/1 mM NaVO 4 /200 μm each of aprotinin, leupeptin, and pepstatin/1% Nonidet P-40). Protein complexes were immunoprecipitated with 10 μg of mouse anti-Myc 9E10 (Santa Cruz Biotechnology) or 10 μg of rat anti-HA 3F10 (Roche) and protein-G agarose beads (Roche) according to the manufacturer’s instructions, separated by SDS/PAGE, and probed with mouse anti-Myc 9E10 (1:8,000) or rat anti-HA 3F10 (1:1,000) in Tris-buffered saline/0.2% Tween 20 with 5% dry milk, or anti-phosphotyrosine (1:1000, 4G10; Upstate Biotechnology) in TBS, 0.2% Tween 20 with 5% BSA. Secondary antibodies used were donkey anti-mouse-horseradish peroxidase (1:4,000) or donkey anti-rat-horseradish peroxidase (1:2,000) (Jackson ImmunoResearch). Signal was detected by enhanced chemiluminescence (Pierce).
Immunohistochemistry.
Primary antibodies used were rabbit-anti-β-gal (1:2,500; Cappel); mouse-anti-LAR (8C4, 1:5; ref. 46); rat-anti-HA (3F10, 1:50; Roche); and rabbit-anti-Talin (1:500; ref. 29). All primary antibody incubations were performed overnight at 4°C with 5% normal donkey serum. Fluorescent-conjugated secondary antibodies (Jackson ImmunoResearch) were used at 1:250 for 2 h at room temperature. Fluorescent images were collected on a Leica TCS NT confocal microscope (or a Zeiss LSM510; Fig. 1 I and 5 B) and processed by using photoshop (Adobe Systems, San Jose, CA). S2R+ cells were stained 48 h after transfection. After reseeding in eight-well Lab-Tek II Chamber slides (Nunc), cells were allowed to adhere for 7 h, fixed with 4% formaldehyde in PBS (pH 7.2), and permeabilized in PBS/0.2% Triton before antibody incubation. Third instar larval brains and pupal eye-brain whole mounts were dissected in ice-cold PBS, fixed in 2% formaldehyde for 30 min (larvae) or 1 h (pupae) at room temperature, and washed several times in PBS/1% Triton before antibody incubation. Adult head sections were stained as described in ref. 7. Tangential sections of adult retina were performed as described in ref. 47.
Quantification of R7 Targeting Defect.
Fluorescent image stacks of 12-μm adult head sections labeled for glass-lacZ were gathered in 1-μm steps. Maximum intensity projections were obtained and termini projecting beyond the R8 layer were scored as “R7 correctly targeted.” Termini in the R8 layer were counted as total cartridge number per section. Groups of two to six genotypes were counted blind. Cartridges from at least five heads were scored except for Fig. 1 J: R7-HA-Liprin-α#43 (two heads).
Quantification of ISNb Guidance.
Females with Liprin-α oos homozygous germ-line clones were generated as described in ref. 43 and mated with males carrying the Liprin-α oos mutation over a CyO-GFP or CyO-lacZ balancer chromosome. Embryos lacking both maternal and zygotic Liprin-α were identified by the absence of GFP fluorescence or β-gal immunoreactivity (rabbit-anti-β-gal, 1:25; Cappel). Embryos were stained with anti Fasciclin-II antibody (1D4; Developmental Studies Hybridoma Bank) with horseradish peroxidase-coupled secondary antibodies used at 1:100 (Jackson ImmunoResearch). Stage 16 or 17 embryos were scored for ISNb targeting as described in ref. 48.
Acknowledgments
We thank Erika Bach, Nicholas Brown, Barry Dickson, Claude Desplan, Richard Hynes, Caryn Navarro, David Van Vactor, Kai Zinn, the Bloomington Drosophila stock center, and the Developmental Studies Hybridoma Bank for fly stocks and reagents and Kwang-Min Choe and Thomas Clandinin for communicating results before publication. The manuscript was improved by the critical comments of Inés Carrera, Reza Farajian, Nina Leeds, and Jean-Yves Roignant. This work was supported by National Science Foundation Grant IBN-9982093 and March of Dimes Birth Defects Foundation Grant 1-FY04-101 (to J.E.T.).
Footnotes
- §To whom correspondence should be addressed. E-mail: treisman{at}saturn.med.nyu.edu
-
↵ †Present address: Institut de Biologie du Developpement de Marseille, Campus de Luminy Case 907, 13288 Marseille Cedex 9, France.
-
Author contributions: K.H., C.M.-Z., H.S., and J.E.T. designed research; K.H. and C.M.-Z. performed research; K.H., C.M.-Z., H.S., and J.E.T. analyzed data; and K.H. and J.E.T. wrote the paper.
-
Conflict of interest statement: No conflicts declared.
- Abbreviation:
- RPTP,
- receptor protein tyrosine phosphatase.
- © 2006 by The National Academy of Sciences of the USA
References
Liprin-α has LAR-independent functions in R7 photoreceptor axon targeting
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