Proteomics analysis identifies orthologs of human chitinase-like proteins as inducers of tube-morphogenesis defects in Drosophila
Sandra G. Zimmerman, Gennifer E. Merrihew, Michael J. MacCoss, Celeste A. Berg. 2017 Proteomics analysis identifies orthologs of human chitinase-like proteins as inducers of tube-morphogenesis defects in Drosophila. Genetics Early online April 12, 2017
- Organism: Drosophila melanogaster
- Instrument: Thermo TSQ Vantage 2
Elevated levels of human chitinase-like proteins (CLPs) are associated with numerous chronic inflammatory diseases and several cancers, often correlating with poor prognosis. Nevertheless, there is scant knowledge of their function. The CLPs normally mediate immune responses and wound healing, and when upregulated, they can promote disease progression by remodeling tissue, activating signaling cascades, stimulating proliferation and migration, and by regulating adhesion. We identified Imaginal disc growth factors (Idgfs), orthologs of human CLPs CHI3L1, CHI3L2, and OVGP1, in a proteomics analysis designed to discover factors that regulate tube morphogenesis in a Drosophila melanogaster model of tube formation. We implemented a novel approach that uses magnetic beads to isolate a small population of specialized ovarian cells, cells that non-autonomously regulate morphogenesis of epithelial tubes that form and secrete eggshell structures called dorsal appendages. Differential mass-spectrometry analysis of these cells detected elevated levels of four of the six Idgf family members (Idgf1, Idgf2, Idgf4, and Idgf6) in flies mutant for bullwinkle, which encodes a transcription factor and is a known regulator of dorsal-appendage tube morphogenesis. We show that during oogenesis, dysregulation of Idgfs (either gain or loss of function) disrupts the formation of the dorsal-appendage tubes. Previous studies demonstrate roles for Drosophila Idgfs in innate immunity, wound healing, and cell proliferation and motility in cell culture. Here, we identify a novel role for Idgfs in both normal and aberrant tubulogenesis processes.
Three biological replicates each of bwk and wild-type samples were compared for differential abundance. One µg of peptides for each sample was measured using a Thermo TSQ Vantage 2 coupled with a Waters nanoACQUITY liquid chromatography system. A 75 µm fused silica column (Polymicro Technologies) was packed with 15 cm C12 Jupiter (Phenomenex) 4 µm reverse-phase beads and a 100 µm fused silica Kasil (PQ Corporation) frit trap loaded with 4 cm of C12 Jupiter. The gradient used for separation was a total of 90 minutes with buffer A containing 95% water, 5% acetonitrile and 0.1% formic acid and buffer B containing 95% acetonitrile, 5% water and 0.1% formic acid. Three analytical replicates were run for each sample with each set of replicates randomized. Quality control samples were run every sixth sample and in the beginning of the runs to assess column chromatography stability.
Flies of genotypes w; UAS-mCD8::GFP; c415-Gal4 +/TM3 Sb or w; UAS-mCD8::GFP; c415-Gal4 bwk151/TM3 Sb were reared at 25°C. Females of both genotypes, along with male siblings, were collected on the day of eclosion and fed with wet yeast paste for ~48 hours at 25°C prior to dissection. Ovaries were dissected in Ephrussi-Beadle Ringer’s (EBR) solution and kept on ice in a 2.0 mL Dolphin tube prior to cell separation. Ovaries were warmed (for about 10 minutes) to 25°C, rinsed three times with room temperature PBS, and dissociated with 1 mL of 5.9 units/mL elastase (Sigma E0258) per 200 ovaries for 1 minute and 30 seconds while gently triturating with a p1000 pipette tip. 1 mL of ice cold PBS was added and cells were centrifuged for 5 minutes at 250xg in a swinging bucket, refrigerated centrifuge and then resuspended in 1 ml of ice cold PBS. Cells were gently triturated for 5 minutes with a p1000 tip while keeping on ice. The single cell solution was filtered through a Filcon 50 µm syringe type filter (Filcon, BD Pharmingen) into a clean 2 ml dolphin tube, centrifuged at 250xg for 5 minutes and resuspended in 135 µl of PBS per 200 ovaries. 10 µL of magnetic beads [Miltenyi mouse CD8 (Ly2) coated beads] were added per 90 µL of cell solution, gently mixed, and incubated for 15 minutes at 4-8°C (not on ice, incubating on ice will slow down the binding of the beads to the cells). The cells were washed by adding 1-2 ml of PBS buffer, centrifuging at 250xg for 5 minutes and resuspending in 0.5 ml of PBS. A Miltenyi MACS separation column was placed in a magnet (Miltenyi magnet and stand) and equilibrated it by applying 500 µl of PBS buffer. A 30 µm pre-filter (Miltenyi) was rinsed two times with PBS, placed over the column, and the cell solution was applied through the pre-filter into the column. The flow-through (unlabeled cells) were collected as a control. The labeled cells in the column were washed by adding 500 µL PBS buffer three times. The column was removed from the magnet and placed over a clean 2.0 mL dolphin tube. To collect the labeled (CD8+) cells, 750 µL of PBS was added to the column and the cells were flushed out with the supplied plunger. A small sample of the cells was examined on the microscope removing 7.5 µl of cell solution and placing on a slide with 0.5 µL of 200µg/mL propidium iodide. A coverslip with a dab of vacuum grease on each corner was placed over cells and the total cells, dead cells, and GFP+ cells were counted. The purified fraction and the flow-through fraction (control) were centrifuged for 5 minutes at 250xg. To concentrate the sample, nearly all of the PBS was removed, so that there was just a very thin layer of PBS covering the cell pellet. The cells were frozen immediately at -80°C. After accumulating enough ovaries for three biological replicates for each genotype (~100 µg or 4000 ovaries per replicate), the cells were lysed by four cycles of thawing and freezing combined with sonicating in an ice-bath sonicator. The individual small samples were allotted into three replicates per genotype. The protein concentration was measured using a BCA assay. Cell lysates were resuspended in 0.1% RapiGest (Waters) in 50mM ammonium bicarbonate pH 7.8 and vortexed. Protein concentration was again measured with a BCA assay (Thermo). Sample was reduced with DTT (Sigma), alkylated with IAA (Sigma) and digested with trypsin (Promega).
Created on 4/20/17, 4:45 PM