CHAPTER 2: MATERIALS AND METHODS
2.5 Cloning and expression of PTP52F, TER94, Src42a and DIAP1
cDNA sequence corresponding to full length PTP52F was obtained by reverse transcription of total mRNA from pupa formation stage of the fly. The full length WT and C1290S (CS) mutants were constructed with a hemagglutinin (HA) tag at the C
terminus and cloned into pAc5.1A (for protein expression in S2 cells) vector. Full length TER94-Flag and Src42A-Myc plasmids were constructed using the cDNA sequences obtained from reverse transcription of total RNA from PF stage of fly. All cDNAs were examined by sequencing. Full length DIAP1 construct was kindly obtained from Prof.
Kai Zinn, CalTech.
2.6 Cell Culture, Transfection, and Immunoprecipitation and immunoblotting
Drosophila S2 cells were routinely maintained in Schneider’s medium supplemented
with 10% fetal bovine serum. For transient transfection with the PTP52F expression vector, S2 cells (5x105 cells/6 cm plate) were incubated with a mixture of plasmid DNA (5 μg/6cm plate) and Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. For immunoprecipitation, the cells were lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 1% Nonidet P-40, 150 mM NaCl, 2 mM Na3VO4, and protease inhibitors. An aliquot of total lysate (1 mg) was precleaned with protein G-Sepharose (GE Healthcare) for 30 min at room temperature followed by immunoprecipitation with anti-HA antibody (clone HA-7, Sigma) at 4 °C for 3h followed by the elution of the beads with 2X sample buffer. For immunoblotting, 35 g of total protein was loaded with 2X sample buffer for each sample followed by gel running and blocking with primary antibody and secondary antibody. Rabbit anti-PTP52F antiserum and mouse anti-HA antibody (Sigma) was used in this study. DIAP1 antibody was kindly provided by Prof.
Bruce Hay from CalTech, TER94 antibody from Prof. Dennis McKearin, HHMI. We used the ubiquitin antibody from Cell signaling for immunobloting. 8% SDS gel was
used for all immunobloting experiments except for ubiquitin for which 6% SDS gel was used.
2.7 Phosphatase activity assay
Assays were performed as previously described [63] with a few modifications. Briefly, S2 cells overexpressing HA-tagged PTP52F-WT or PTP52F-CS were harvested in lysis buffer HA-PTP52F was immunoprecipitated from an aliquot of total lysate (1.5 mg) by HA antibody. The immunocomplex was incubated in phosphatase activity assay buffer (50 mM Hepes, pH 7.5, 1% NP-40, 10 mM DTT, and 20 mM p-nitrophenyl phosphate).
The reaction was carried out at 37 °C for 1h. After the reaction was terminated by 2 N NaOH, the phosphatase activity in the immunocomplex was measured by spectrometric analysis at 405 nm.
2.8 Extraction of RNA, cDNA Synthesis and RT-PCR
Total RNA was isolated from Drosophila tissue during different developmental stage using a Trizol reagent kit (Invitrogen, CA, USA) according to the manufacturer's instructions. The cDNA was synthesized from total RNA (~3μg) with Transcriptor reverse transcriptase (Roche Applied Science) using oligo (dT) primers according to the manufacturer's instructions. Initial screening of PTPs was carried out using reverse transcription PCR and the primers were designed spanning an intron to distinguish the product from genomic DNA and cDNA. The primer sequences are listed in Table-1.
2.9 Quantitative Real-Time PCR
The detailed screening of PTP52F mRNA expression levels during different stages of development were also quantified by real time PCR using a LightCycler instrument (Roche Applied Science) with the SYBR Green PCR Master Mix (Roche) in a one-step reaction according to the manufacturer's instructions. Primers were designed using the LightCycler Probe Design software 1.0 (Idaho Technology).The primer sequences used are listed below in Table-1. The melting curves and gel electrophoresis of the end products were obtained to confirm the PCR specificity and the correct size of the PCR band. The mRNA levels of target genes were normalized to the relative amounts of the housekeeping gene rp49 using the second derivative maximum method provided by the Relquant software (Roche).
Table-1
Gene name Primer sequence
Ptp52F forward 5′-ATTGTTCAAGTTACCCAGTTTCGCG-3′
Ptp52F reverse 5′-TTTTTGGGAGAGGGAATGGCG-3′
Ptp99A forward 5′-AGATGGTCTGGGACCACAATGC-3′
Ptp99A reverse 5′-GCAAGAGGCTTGGATTAGTATCGGT-3′
Ptpmeg forward 5′-ATCCACCACATGCAGTACTTGGC-3′
Ptpmeg reverse 5′-TAGCGGAAGATTGTTGATTGCGT-3′
myopic forward 5′-TATTTCCAGAAAGCTGAAACGGC-3′
myopic reverse 5′-CATAGCGTTCTCTCAGCTCTCCAT-3′;
CG7180 forward 5′-GTGAATGGAAAATCGAGGGAACG-3′
CG7180 reverse 5′-AGCACCACCCGATTGTAGTCATA-3′
Ptp52F forward (real time) 5′-ATACGCCCAGTGATGAAGTTAAG-3′
Ptp52F reverse (real time) 5′-ATCGAGTAGGCTTCTGCTTCCA-3′
rp49 forward (real time) 5′-TACAGGCCCAAGATCGTGAA-3′
rp49 reverse (real time) 5′-ACGTTGTGCACCAGGAACTT-3′
2.10 Mass spectrometry based substrate trapping
The WT form and CS/DA mutant form of full length PTP52F with HA tag in the c-terminus were used for substrate trapping. The full length constructs were ectopically expressed in the S2 cells and the lysates were collected using the NP-40 lysis buffer. The over expressed PTP52F was immunoprecipitated using the HA-agarose beads (sigma) from ~2mg of total lysate. In the meantime Kc-167 cells stimulated with 100 μM pervanadate for 30 minutes was lysed at 4oC with buffer containing 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10% glycerol, 1%Triton X-100, 5 mM iodoacetamide (IAA) and protease inhibitors. Excess IAA in the total lysates was neutralized by DTT and further removed by a PD-10 column. IAA was used to drive the irreversible inhibition of PTP active site. We avoided using orthovanadate since the trace of unbound orthovanadate may inhibit the immobilized PTP52F in the subsequent step of substrate trapping. The immobilized PTP52F is allowed to react with the pervanadate treated total lysates (~4mg) for three hours at 4oC. To elute the potential substrates associated with PTP52F, beads were either boiled in SDS sample buffer or incubated with 10 μM sodium orthovanadate (Na3VO4) at room temperature for 10 min. Those eluted proteins were resolved in a SDS-PAGE gel and then subjected to immunoblotting with anti-pTyr antibody (4G10 from
Millipore) or subjected to silver staining for in-gel trypsin digestion followed by LC-MS/MS analysis. For in vivo substrate trapping, the full length PTP52F-WT and CS/DA were overexpressed, pulled down and checked for their specific association with endogenous TER-94.
2.11 In vivo substrate trapping from fly tissue
UAS-PTP52F –WT and UAS- PTP52F-DA transgenic lines were generated using full
length plasmids with HA tag in the c-terminal by microinjection and their over expression was confirmed in western blot by cross with Tubulin-Gal4 line. In order to do the in vivo substrate trapping we used the NP1-Gal4 to drive the midgut specific overexpression of PTP52F-WT and PTP52F-DA. White pupa were collected from the two crosses and the total lysates was obtained by grinding the fly tissues with lysis buffer containing 20 mM HEPES (pH 7.5), 1% Nonidet P-40, 150 mM NaCl, 10 mM NaF, 10 mM Na4P2O7, 10% glycerol and protease inhibitors. The PTP52F was pulled down using the HA-agarose beads for two hours at 4oC. To elute the potential substrates associated with PTP52F, beads were either boiled in SDS sample buffer, were resolved in a SDS-PAGE gel and then subjected to immunoblotting with TER94 antibody (kindly provided by Prof. Dennis McKearin, Howard Hughes Medical Institute).
2.12 Immunofluorescence staining
S2 cells were plated and transfected as described above. After 48h of incubation, the cells were suspended and replated on Concavalin A (ConA) (0.5 mg/ml; C2010; Sigma) - coated glass coverslips and stained with anti-PTP52F antibody followed by Cy2-conjugated goat anti-rabbit IgG or (Jackson). For F-actin staining, cells were stained with
tetramethyl rhodamine isocyanate conjugated phallodin (Jackson). The samples were visualized using a Zeiss LSM 510 confocal microscope. Late third instar larval midguts were dissected in PBS and fixed im 4%paraformaldehyde for 30 minutes followed by, permeabilization with 0.3% Triton X-100 for 10 minutes. Blocking was done with 5%
goat serum in 0.3% Triton X-100 and stained with either PTP52F antibody or anti-DIAP1 antibody (kindly provided by Prof.Bruce Hay, Caltech) at 4oC overnight followed by Cy3-conjugated goat anti-mouse IgG -(Jackson) according to the protocol described in a previous study [64]. The samples were visualized using a Zeiss LSM 510 confocal microscope.
2.13 Light Microscopy
Midguts from APF4H pupae were dissected in PBS and fixed in 4% Para formaldehyde for 20 minutes and were mounted and examined using Olympus-BX51 microscope. For each set 30 flies (n=30) were scored for the phenotype analysis.
2.14 TUNEL staining
Midgut tissue was dissected from different stages of third instar larvae and fixed in 4%
paraformaldehyde in PBS for 30 min at RT. Samples were permeabilized in 100mM Sodium Citrate in PBTx (PBS + 0.1% Triton X100) at 37 oC for 30 min followed by the addition of TUNEL mix according to the manufacturer instructions (In Situ Cell Death Detection Kit, Roche) and incubated at 37oC for 1hr on a humid chamber. Tissues were stained with DAPI before mounting and were mounted in 90% glycerol/PBS with 0.5%
propyl gallate and TUNEL positive cells were detected by fluorescence microscopy (Olympus BX51).
CHAPTER 3: RESULTS AND DISCUSSION
3.1 Profiling of PTPs during Drosophila development by in-gel phosphatase activity assay
While individual PTPs have been implicated in the regulation of Drosophila development during embryogenesis, the expression and activity profile of PTPs at other developmental stages remain uncharacterized. Such data might provide more insight into the role of PTPs playing in the control of the developmental process. We used in-gel phosphatase activity assay to visualize the possible participation of PTPs at each developmental stage.
This assay displays a diverse array of active PTPs in total extracts of cells and tissues according to the molecular weights of these phosphatases resolved by SDS-PAGE. As shown in Figure 1, the overall PTP activity in the embryonic stage was significantly higher than in other developmental stages, suggesting that rapid protein tyrosine dephosphorylation plays a critical role in signal transduction during this stage. We found the activity of many PTPs to be diminished during the larva-pupa transition, and increased slightly in adult flies (Fig. 1). Although some PTPs were visible in adult flies, their activity was far weaker than the activity of those in embryos (Fig. 1). The biological implication of such interesting observation requires further investigation. Nonetheless, it should be addressed that all phosphatases detected by this assay format are likely to be non-receptor PTPs due to the inherited limitation of this technique [57, 58]. Indeed none of Drosophila receptor PTPs (RPTPs), which run greater than 100 kDa in SDS-gels according to the theoretical calculation (www.uniprot.org), was unraveled by the in-gel phosphatase activity assay (Fig. 1). This may be due to the refolding problem since during the process of experimental procedure we do denaturation followed by renaturation and receptor PTPs might not have refolded properly and hence cannot have
their activity. Obviously, other methods are needed to collect information for profiling RPTPs at different stages of Drosophila development.
3.2 Data mining from modENCODE to depict the mRNA expression profiles of RPTPs during Drosophila development
Since the protein expression profiles of RPTPs were not available, we switched our focus to other information such as the mRNA level of these phosphatases over various developmental stages. Flybase provides modENCODE temporal expression data for each gene [59, 60] (www.modencode.org). Since this database shows the expression pattern of every gene throughout the development of flies, is easily accessed, we decided to perform data mining to profile RPTPs using already existing information embedded in the Flybase.
We analyzed all RPTPs in the Drosophila genome, including dLAR, PTP4E, PTP10D, PTP52F, PTP99A, and PTP69D except dIA2, which is a naturally inactive phosphatase.
We examined the mRNA levels of these R-PTPs at various developmental stages (the embryo, early and late third instar larva, white prepupa, pupa and adult). As seen in Figure 2, all R-PTPs except PTP52F were highly expressed during embryogenesis.
PTP4E, PTP10D and PTP69D levels were particularly pronounced in embryos (Fig. 2A and 2B), suggesting they may play essential roles in the control of developmental events at this time. These findings are in agreement with those of two recent studies reporting that PTP4E, PTP10D and PTP69D act in coordination of axon guidance during embryogenesis and that they have redundant and compensating functions [42]. Perhaps the most interesting observation in the profiling was the gradually increased expression of PTP52F from the embryonic stage to the larva-pupa transition (Fig. 2A and 2B ), at
which time metamorphosis begins and most larval tissues are readily remodeled for the development to adult flies [52]. The discovery that PTP52F is specifically expressed at this particular stage of development suggested that receptor PTPs may be involved in the control of metamorphosis, despite a very low activity of non-receptor PTPs being observed at this time of pupal formation (Fig. 1).
3.3 Tissue distribution of RPTPs during third instar larval stage
We wanted to examine the expression of RPTPs in various tissues of Drosophila, particularly focusing on the tissue distribution of PTP52F, which is highly expressed during larva-pupa transition (Fig. 2). We used data mining to profile the expression of R-PTPs in the third instar larval tissues including salivary gland, CNS, trachea, tubule, hindgut and midgut using the Fly Atlas database, which provides the most comprehensive collection of the mRNA level data on each gene in each tissue during III instar larva and adult stages of development [61] (www.flyatlas.org). Data were mined using three criteria: (1) mRNA SIGNAL, the abundance of mRNA; (2) mRNA ENRICHMENT, mRNA of the specific gene compared to total mRNA of the whole flies; and (3) the Affymetrix PRESENT CALL, the number of time a specific gene was detectably expressed out of four arrays being analyzed. To summarize our findings, we presented a simplified anatomy of the third instar larvae showing major tissues with relative expression levels of R-PTPs (Fig. 3). Four out of six RPTPs (dLAR, PTP4E, PTP99A and PTP69D) were detected in the CNS, suggesting they play important roles in the neuronal formation not only during embryogenesis but also at the beginning of metamorphosis. To our surprise, we found PTP52F to be exclusively expressed in gut tissues, and to be particularly enriched in the midgut (Fig. 3). Since this kind of tissue
distribution of RPTPs had never been recorded in flies, we hypothesized that PTP52F may have a specific roles in the regulation of gut tissue during larva-pupa transition. Thus, the remainder of the study was devoted to the characterization of PTP52F and the study of its potential involvement in Drosophila development.
3.4 PTP52F is highly enriched in midgut during larva-pupa transition
Based on our analysis from microarray and NGS data mining for Drosophila PTPs, we identified, Ptp52F, to have changed its expression pattern during larva-pupa transition. In order to confirm the microarray analysis, we did the RT- PCR during the developmental stages of the fly. We used the total RNA extracts from different stages of fly and probed the Ptp52F gene by PCR. The primers were designed to cover an intron so that the product from genomic DNA and cDNA can be distinguished based on the size. The RT-PCR results showed that Ptp52F is increased during larva-pupa transition (Fig. 4A). We further confirmed its expression pattern by real-time PCR (Fig. 4B). We found a sharp increase in Ptp52F RNA during the white pupa formation (PF) stage (Fig. 4B). We used the antibody that we generated to examine the expression of endogenous PTP52F in total protein extracts isolated from both WT and PTP52F knockdown (RNAi) flies at the third instar larval stage, when mRNA of PTP52F was robustly enriched (Figs. 2 and 3). As shown in Figure 4C, the specific band at ~200 kDa appeared only in the WT flies but not in the RNAi line, suggesting that endogenous PTP52F protein was indeed expressed during the larva-pupa transition. The excellent performance of this antibody indicated that we could use it to further characterize PTP52F in developing flies with various genetic backgrounds. The results of our data mining of the Fly Atlas (Fig. 3) suggested there would be a robust level of PTP52F protein in the prepupal midgut tissue. To findout, we performed immunofluorescence staining with anti-PTP52F antibody. As shown in Figure 4D, there was a strong signal of PTP52F in the midgut of WT flies, suggesting a high level of mRNA at this developmental stage (Figs. 2 and 3) might lead to the enhanced expression of its protein in larval epidermal cells or adult epidermal progenitor cells, both which are major types of cells in the larval midgut [65]. Interestingly, the staining of PTP52F protein was highly enriched in the midgut but much weaker in the hindgut region (Fig. 4D), consistent with the information of its mRNA distribution provided by the Fly Atlas (Fig. 3).
3.5 PTP52F is an ecdysone response gene
Since the molting hormone ecdysone plays an important role during larva-pupa transition, we further checked the role of ecdysone in Ptp52F expression. We used an EcR heterozygous mutant to do the real-time PCR for Ptp52F and we found that inducible expression of Ptp52F during pupa formation is highly reduced in the EcR mutant compared to WT flies (Fig. 5A). We also found that the protein expression of PTP52F was also highly reduced in EcR mutant compared to the WT fly during the PF stage (Fig.
5B). Based on our previous studies we have identified some potential EcR response elements in the promoter region of Ptp52F [66]. Hence we used the Kc167 cells to do the ecdysone stimulation in-vitro. Kc167 cells were stimulated with ecdysone for different time points from 0-24 hours. Even though we see little increase in the early time points, the inducible expression of PTP52F was very clear at 24 hrs confirming the role of ecdysone in the inducible expression of PTP52F (Fig. 5C). Taken together the results from our studies and also from previous studies from Thummel’s lab [51] clearly show
that PTP52F is an ecdysone response gene and it is increased during the PF stage in the fly during which the ecdysone pulse is high.
3.6 PTP52F mutants show delay in midgut metamorphosis
Based on our earlier studies we already confirmed that the PTP52F is highly enriched in the midgut during the larva-pupa transition. Ecdysone triggers and directs metamorphosis during the pre-pupal and pupal transitions. Ecdysone binds to EcR and activates a set of early response genes triggering tissue specific responses during metamorphosis [65]. The midgut is one of the major tissues that undergo extensive metamorphosis under ecdysone stimulation during larva-pupa transition. Since Ptp52F is highly enriched in the midgut and is an ecdysone response gene, we next asked whether PTP52F have any functional role in midgut metamorphosis. First we did the immunofluorescence experiment to analyze the inducible expression of PTP52F in midgut during larva-pupa transition. Our data showed that the PTP52F was increased during PF and APF-2h compared to <5h BPF (Fig. 7A). We also noticed that PTP52F is highly enriched in the gastric ceaca of the midgut which will be destroyed by cell death during metamorphosis. As shown by earlier studies the midgut will undergo cell death during metamorphosis and its size is highly condensed during APF-4h compared to PF or <5H BPF and this process is shown in representative images in Fig. 6. We can also notice that the gastric ceaca is completely destructed at APF-4h. EcR, the key mediator of ecdysone action is known to regulate midgut metamorphosis. Mutation or loss of function of EcR results in the delay of midgut metamorphosis. The size of midgut still does not condense at APF-4h and has been shown by Thummel’s group [67]. Our results also shows that the over expression of dominant negative EcR lead to a delay in midgut metamorphosis and hence we can see
the gastric ceaca at APF-4h compared to the control (Fig. 7B upper panels). Correlating to its inducible expression the PTP52F-KD lines as well as the PTP52F-CS mutant lines (loss of phosphatase activity) showed a delay in the midgut metamorphosis (Fig. 7B middle panels). Also we have checked the midgut metamorphosis in transheterozygous mutants, and all the trans-heterozygous mutants show delayed gastric ceaca degradation compared to the heterozygous lines (Fig. 7B lower panels). Whole length of the gut in KD and mutant line is longer than the control. Gastric ceaca is also seen clearly compared to the control. We used gastric ceaca as an indicator to show the delayed metamorphosis of the gut. This phenotype clearly suggests that the ecdysone induced PTP52F is involved in regulation of midgut metamorphosis. The protein levels of EcR and PTP52F showed the corresponding increase and decrease according to the lines used representing the degree of knockdown and over expression in Fig. 7C.
3.7 Ectopically expressed PTP52F is an active, plasma membrane-localized phosphatase
We performed sequence analysis on the full-length cDNA of Ptp52F gene that we cloned from flies and found it to be identical to the one published previously [45]. As shown in
We performed sequence analysis on the full-length cDNA of Ptp52F gene that we cloned from flies and found it to be identical to the one published previously [45]. As shown in