Construction and characterization of a fryrescue transposon
The fry mRNA is predicted to be 10873 bp and much of it was not recovered in the Drosophila genome project cDNA collection. We assembled a fry minigene using a 5' fragment synthesized de novo, two middle fragments of cDNA derived from mRNA by RT-PCR and a 3' fragment of genomic DNA generated by PCR that contained 5 exons and 4 introns (see Methods). This minigene was subcloned into the pTWG vector and used to generate transgenic flies. The final transgene was tagged at its 5' end by six copies of the Myc epitope tag and at its 3' end by GFP.
To determine if the transgene encoded a functional protein we crossed UAS-myc-fry-GFP/CyO; fry1/TM6 and actin-gal4; fry2/TM6 flies. The resulting UAS-myc-fry-GFP/actinGAL4; fry1/fry2flies eclosed at the expected frequency (98.4%, 62/63 observed/expected). Thus, the transgene provided complete rescue of the lethality seen in fry1/fry2 flies (both of these alleles are likely null alleles). We examined adult wings from these flies and found that more than 99% of wing cells produced a single hair compared to the more than 4 hairs per cell seen with fry mutants (Figure 1A, B). The bristle phenotype of fry was completely rescued. Thus, the Myc and GFP tags did not interfere with the protein's function. In contrast, the UAS-myc-fry-GFP/actinGAL4; fry1/fry2 flies were both male and female sterile. This phenotype had 100 percent expressivity and penetrance. The paired ovaries of rescued females were often of different sizes, and the eggs produced were rounder than normal and were rarely laid (Fig. 1C). We suggest that the transgene was not expressed well in the ovary and that fry has an important function in egg morphogenesis.
The act-Gal4 driver used in the rescue experiments does not drive a high level of expression in most cell types. When we drove expression with stronger drivers such as ptc-Gal4 or ap-Gal4 the resulting animals appeared relatively normal, with the exception that driving expression using ap-Gal4 led to an occasional loss of thoracic macrochaetae. This is also seen in flies that carry ap-Gal4 without UAS-myc-fry-GFP although at a lower frequency. We observed an average of 4 dorsocentral bristles in wild type flies (n = 26) and UAS-myc-fry-GFP flies (n = 24), 3.8 (SEM = 0.038) in ap-Gal4 flies (n = 122) and 3.01 (SEM = 0.1) in flies that contained both ap-Gal4 and UAS-myc-fry-GFP (n = 86). The difference between the ap-Gal4 and ap-Gal4/+; UAS-myc-fry-GFP flies was highly significant (p < 0.001, Mann-Whitney Rank Sum test). On the whole however, our observations argue that increased Fry levels did not seriously affect most fly cells.
The Fry protein is intact in vivo
The Myc-Fry-GFP protein encoded by the transgene was characterized by Western blot analysis. When detected using anti-GFP antibody (Figure 2A, B), the protein ran close to the predicted size although the very large size of the protein (predicted to be 418 kDa) makes precise molecular weight estimation difficult. We confirmed that this protein contains the C-terminal Fry sequence by probing a separate Western blot using an antibody directed against the C terminal end of the endogenous Fry protein (Figure 2A) [10]. To confirm that the Myc-Fry-GFP protein is present as a single protein in vivo, we also probed paired Western blots with anti-Myc or anti-GFP antibodies. Both antibodies recognized a common band of approximately 400 kDa (Figure 2A) indicating that the protein was intact in vivo. We also found that we could pull down the full length Myc-Fry-GFP protein using a polyclonal rabbit-anti-GFP antibody and detect it using mouse-anti-GFP antibody (Figure 2B). This result opens up the possibility for additional experiments to study the biochemistry of Fry.
Fry Protein localization
We previously found the endogenous Fry protein had a punctate distribution in developing wing hairs [10]. When we examined Fry in growing bristles by immunostaining, we also found it to be punctate (Figure 3A3). In addition, we found Fry enriched at the distal tips of growing bristles (Figure 3A1, A2). We observed this for bristles on the head, thorax and wing and in both macrochaetae and microchaetae. We had previously noted that there was only modest co-localization of Trc and Fry in hairs [10] and we found this to also be the case in bristles (Figure 3H). Only a minority of Trc staining puncta also stained positively for Fry and similarly only a minority of Fry staining puncta also stained positively for Trc. There was no evidence of co-localization of Fry with either the actin or microtubule cytoskeletons (Figure 3A3, C1-4). Growing bristles stained intensely with an anti-acylated tubulin antibody suggesting that many bristle microtubules are stable.
We expressed the UAS-myc-fry-GFP transgene in bristle cells using the neur-GAL4 driver and found that we could also observe the distal tip accumulation of Myc-Fry-GFP in thoracic microchaetae in vivo (Figure 3B1, B2). Surprisingly the distal tip enrichment of transgene-encoded protein was not obvious in macrochaetae in either in vivo imaging or immunostaining experiments. In previous experiments we had observed that the neur-Gal4 drives a higher level of target gene expression in macrochaetae than microchaetae, thus we suspected that the failure to observe distal enrichment could be due to high level of expression saturating tip binding sites. To test this hypothesis we generated UAS-myc-fry-GFP/Tub-Gal80ts; neur-Gal4/+ animals and grew these at 21°C. At this temperature the Gal80ts protein is active and blocks the ability of Gal4 to drive expression from the transgene. We collected white prepupae (WPP), aged these at 21°C for 48 hrs at 25°C and then moved these to 29°C for 1.5-2 hrs to briefly induce the expression of the transgene. This resulted in a much lower level of Myc-Fry-GFP accumulation (as judged by immunostaining) than in previous transgene experiments and in these preparations we could detect enrichment of Myc-Fry-GFP at the distal tips of some bristles (data not shown). We concluded that the tags did not alter the subcellular distribution of the Fry protein.
In the experiments where Myc-Fry-GFP was over expressed the protein appeared to be present in stripes along the long axis of the bristle (Figure 4A2, B2, E). This was also seen in living pupae by direct visualization of GFP (Figure 4E), thus the stripes were not a staining artifact. Large bundles of tightly cross-linked F-actin are also found in stripes oriented along the long axis [25]. To determine the relationship between the Fry stripes and the F-actin bundles we examined bristles stained with both anti-GFP antibody and phalloidin. It was clear that the stripes of Fry staining were in between the F-actin bundles (Figure 4A-D). It is possible that the stripes were simply a consequence of Fry being excluded from the highly crossed linked bundles of F-actin.
To localize the Fry protein in da neurons we expressed UAS-myc-fry-GFP using ppk-Gal4. We observed the apparent preferential accumulation of Fry-GFP at nodes of dendrites of da neurons in white prepupae (wpp) (Figure 5A). However, it seemed possible that the higher GFP levels could simply be due to the nodes being thicker. To test that we co-expressed a membrane tagged red fluorescent protein (mCD8-RFP) and indeed found that it also showed higher fluorescence at the nodes (Figure 5A), however the relative intensity of RFP and GFP varied across the dendrite and some nodes were enriched for Fry compared to RFP. A more extensive quantitative analysis will be needed to determine the significance of this observation. Previously it was found that the over expression of wild type Trc led to a decrease in the da neuron dendrite arbor compared to wild type [4]. That also appeared to be the case in when Fry was over expressed (Figure 5B), but we did not quantify the phenotype.
In our initial experiments it appeared that Fry was excluded from the nucleus (Figure 6A). To confirm that the area lacking Myc-Fry-GFP was in fact the nucleus we co-expressed Myc-Fry-GFP with the nuclear marker RedStinger (Figure 6B1). In the da neurons of these animals the Myc-Fry-GFP was almost completely found in the cytoplasm even though it was being over expressed (Figure 6B2). We also examined the distribution of Trc in da neurons using a UAS-trc-GFP transgene. In contrast to Myc-Fry-GFP, Trc-GFP preferentially accumulated in the nuclei of da neurons (Figure 6C, D), although it was also present in the cytoplasm. In contrast, in pupal wing cells we previously found Trc to be completely cytoplasmic [10]. This demonstrated that as is the case for NDR1 in mammalian cells the cytoplasmic versus nuclear localization of Trc (NDR1 in mammals) is cell type specific. The situation in da neurons is formally the same as in yeast daughter cells where Tao3 (fry homolog) is cytoplasmic and Cbk1 (trc homolog) is nuclear [3, 5].
We also generated a transgene that expressed the amino terminal 1637 amino acids of Fry tagged with an amino terminal 6XMyc (we refer to this protein as Myc-N-Fry). This fragment includes the most conserved region of Fry and was previously found to be sufficient for binding and co-immunoprecipitation with Trc [10]. The Myc-N-Fry protein showed a punctate localization in wing hairs and bristles (Figure 3D, E), but it did not extensively colocalize with endogenous Fry nor did it accumulate at the tips of bristles as endogenous Fry did (Figure 3D, E). However, Myc-N-Fry showed a high degree of colocalization with endogenous Trc. This was seen both in sensory bristles and wing hairs (Figure 3F, G). As expected Myc-N-Fry did not have any rescue activity nor did it act as a dominant negative (e.g. being expressed by a strong Gal4 driver did not result in a mutant phenotype). In addition it did not enhance or suppress the trcS292AT453A double mutant dominant negative phenotype when both were expressed using ptc-Gal4 (data not shown).
Our observations indicate that the interaction of Fry and Trc, defined by both genetic and biochemical experiments, is not reflected in a consistent and complete co-localization. Rather, the limited co-localization suggests the function of the proteins only involves a transient physical interaction
The Fry Protein is highly mobile
The myc-fry-GFP transgene allowed us to visualize Fry in living cells and we took advantage of this to assess the mobility of the protein in both bristles and dendrites of da neuron. We carried out FRAP (fluorescence recovery after photobleaching) experiments on both of these cell types. For bristles we used UAS-myc-fry-GFP/actinGAL4; fry1/fry2 pupae. In these animals the transgene encoded Fry rescued the recessive lethality of the fry mutation and the only functional Fry protein was GFP-tagged. For da neuron dendrites we used UAS-myc-fry-GFP/+; ppk-GAL4-UAS-mCD8-RFP/+ animals. When we examined live cells, Fry-GFP appeared to be in rapidly moving small particles (Additional Files 1 (movie S1), 2 (movie S2)). When we bleached segments of either of these cell types we observed a rapid recovery of fluorescence (less than one minute) (Figure 7A, B)(Additional Files 1 (movie S1), 2 (movieS2)). The movement was rapid enough and the particles small enough that we could not follow individual particles. In experiments where we bleached the internal segment it appeared to recover from both ends (Figure 7B, C). In experiments where we bleached the distal tip of bristles it recovered from the base to the tip (Additional File 3, Figure S1). Based on these experiments, we suggest that the movement of Myc-Fry-GFP was bi-directional.
Fry directly interacts with the C-terminal region of Trc
Previous data showed that the amino terminal fragment of Fry (including the Fry domain) and Trc could be co-immunoprecipitated from Drosophila S2 cells [10]. We subcloned this fragment of Fry into the pGADT7 vector and used the yeast-two-hybrid system to identify the region of Trc that mediated the interaction. We first tested a series of Trc deletion mutants. Surprisingly, we found that full length Trc interacted weakly if at all with Myc-N-Fry but that a truncated Trc (amino acids 1-404) that was missing the 60 most C-terminal residues interacted strongly (Figure 7A). This interaction was lost when a smaller Trc protein (amino acids 1-337) was used (Figure 8A). Thus, residues from the carboxy terminal region of Trc inhibited the two-hybrid interaction. One possibility was that in vivo the interaction of Fry and Trc is regulated by the phosphorylation of the C-terminal hydrophobic motif site (T453). To test this hypothesis we used mutant Trc proteins in the 2-hybrid system. We tested the interaction between Myc-N-Fry and trcT453A (dominant negative construct), trcT453D (constitutively active construct) or trcT453E (a second constitutively active construct) and found no difference compared to wild type Trc (Figure 8B). We therefore concluded that phosphorylation at T453 did not regulate the interaction between Trc and Fry. Another possibility to explain the lack of a strong 2 hybrid interaction between Trc and Myc-N-Fry is that a third Drosophila protein is part of the complex in the fly and in its absence the Trc carboxy terminal region inhibits the interaction. A Mob protein would be a candidate to be involved in such an interaction as Mob and Trc are known to interact. To further map the location of residues that inhibited the interaction with Myc-N-Fry we made a further set of Trc deletions and used them in the two-hybrid system (Figure 8C). We found that extending the deletion from amino acid 414 to 404 led to a strong interaction with Myc-N-Fry (Figure 8C) implicating residues from this region as being important. This 11 aa region is highly conserved with 7/11 residues identical between the fly, frog and human homologs and two of the differences are conservative (V-I, R-K) substitutions (Figure 8D2). Control western blot experiments established that the non-interacting mutant proteins were expressed (Additional File 4, Figure S2).
The data from our original set of Trc deletions indicated that residues from 338-404 were essential for the interaction. We generated and tested additional deletions and found that extending the deletion from aa 350 to 337 abolished the interaction (Figure 7C). These residues could contain the binding surface for the interaction or be required for the folding of the protein to allow other residues to bind to Myc-N-Fry. This region is within the catalytic domain (sub-domain X) [26], and based on the 3-D structure of the related AGC family kinase human Sgk, this region is exposed and available for interacting with other proteins (the 3D structure of Sgk is available at http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=75006). The region is highly conserved with 11/14 residues being identical between the fly, frog and human Trc/NDR proteins and one of the differences is conservative (R-K) Figure 7D1).
Previous experiments in the Drosophila system showed that Trc and Myc-N-Fry could be co-immunoprecipitated from S2 cells. We extended these experiments to see if either or both Myc-N-Fry or Myc-Fry-GFP would co-immunoprecipitate from wing discs or whole larvae. We used transgenic flies expressing both UAS-myc-fry-GFP and UAS-trc-FLAG, or both UAS- Myc-N-fry and UAS-trc-FLAG using the actin-GAL4 driver. We found that both Myc-N-Fry and Myc-Fry-GFP co-immunoprecipitated with Trc (Figure 9).