Fly genetics and generation of fob1 mutant
Ends-out homologous recombination was used for generating a fob
-null allele (Gong and Golic, 2003
). Stepwise, left (5,906 bp) and right (5,293 bp) flanking regions of the fob
gene were cloned into p[w25.2] vector yielding pw25-16Bko. Primers for amplification of left and right regions were: left, 5′-ATTTGCGGCCGCTGCGTGGTGAGTTTGCAC-3′ and 5′-ATTTGCGGCCGCGCTCAAGTTGTAAAATTGACTTTCT-3′, and right, 5′-TTGGCGCGCCGCACAGACATGGTCGCACTA-3′ and 5′-AACGTACGATCACGGCCAGTTACTCCAC-3′. A transgenic line carrying this donor on the second chromosome was selected for targeting (Gong and Golic, 2003
). Candidates for which the mini-white
gene of pw25-16Bko mapped to the third chromosome were analyzed by probing Southern blots, first with the entire fob
gene and then with dVps33B
to control for loading.
For infections and immunohistology, the following lines were used: Oregon-R (wt), fob1
/Df(3R)BSC547/TM6C, Sb1, eater
trans heterozygous [Df(3R)D605/Df(3R)Tl-I], da-Gal4/uas-Vps33B-RNAi, and da-Gal4/Vps16A-RNAi (Pulipparacharuvil et al., 2005
). Clones for car
-null cells were generated in the eye discs, which were stained as described previously (Akbar et al., 2009
). Gal4 drivers and deficiencies were obtained from the Bloomington Stock Center.
For rescue experiments, the fob coding unit was amplified using the primer set 5′-CCGCTCGAGATGGAGGAGCAGAAGCTGAT-3′ and 5′-GGGGTACCTTACAACTTGAGCTTGATGTTGTCC-3′. The unit was then cloned into the pUASt vector using XhoI and Acc65I, and the resulting uas-Myc-Fob transgene was expressed under control of Da-Gal4 or Srp-Gal4 in fob1
background. Alternatively, a genomic fragment containing sequences 1.8 kb upstream and 0.8 kb of downstream of the fob coding region was cloned into a derivative of pCaSp4 for the generation of a transgenic line. For Vps33B-RNAi experiments, a 381-bp inverted repeat (bp 1,180–1,561 of the Vps33B mRNA; available from GenBank/EMBL/DDBJ under accession no. NM_143138.1
) was clone into a modified pWIZ vector (Pulipparacharuvil et al., 2005
) and expressed in transgenic flies under uas/Gal4 control. Plasmids containing the fob, vps33B, and Car cDNAs templates had been generated by the Berkeley Drosophila Genome Project and were obtained from the Drosophila Genomics Resource Center.
(DH5α, amp resistance, GFP) and E. faecalis
cultures were grown overnight in Luria Bertani (LB) or brain heart infusion medium (BHI) medium at 37°C. Female virgin flies (5 d old) were injected (Schneider et al., 2007
) with 80 nl PBS containing a mean of 1,600 E. coli
= 0.1) or 200 E. faecalis
= 0.005). Sterile PBS was injected as a control. Injected flies (20 flies per vial) were reared at 25°C, 65% humidity, on yeast-agar-molasses food. Injections were performed with a pico-injector (model PLI-188; Nikon) fitted with glass capillary needles. Injections were performed in triplicate (total of 60 flies) for each group with either of the indicated microbes and PBS control on the same day. All injection experiments were repeated 8–10 times. For each survival curve, flies were counted every 24 h, and bars represent mean values with standard deviation. Data were analyzed using the SAS software (SAS Institute, Inc).
To determine bacterial load, flies were injected with E. coli
(DH5α, kanamycin resistant, five flies per data point) and homogenized after the indicated time (Schneider et al., 2007
). Serial dilutions were plated and colonies were counted for each time point. Data are plotted as boxes with whiskers. The mean is indicated with a diamond. The boxes indicate 25th and 75th percentiles; the bold line is the 50th percentile, whereas the whiskers show the complete range of the data.
pHrodo-E. coli bioparticles (Invitrogen) were suspended according to manufacturer’s instructions, and 80 nl were injected. After 2 h, flies were mounted and imaged on a microscope with 1.5× magnification (SZX12; Olympus). During imaging, exposure parameters were set such that for Oregon-R the brightest spots were not saturated. FITC-E. coli (catalogue No. F6694; Invitrogen) were used for phagocytosis and immunostaining experiments in hemocytes.
For qRT-PCR experiments, RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. For anti-microbial peptide measurements, RNA was isolated from five flies after injection (6 h for E. coli and 12 h for E. faecalis). qRT-PCR was performed using a DNA-free, high-capacity cDNA reverse transcription kit (Fast SYBR Green master mix; Applied Biosystems) and a Fast Real-Time PCR System (7500; Applied Biosystems). Each data point was repeated three times beginning from injection. Values were normalized first with rp49 as an internal control and then expressed as fold change compared with flies injected with PBS as control. The following primer sets were used for amplification: fob left, 5′-TATTGGAACCGATCCTCTCG-3′; fob right, 5′-CACCAGTTTCAATGCCTCCT-3′; Ca left, 5′-CCATATCAGCCGCATTTCTT-3′; Ca right, 5′-AAGCTGGCATCGTTCTGACT-3′; CG7829 left, 5′-CAGGAACCTACTGGGCAAAA-3′; CG7829 right, 5′-AGTAGACTCCCGGCTTGTCC-3′; CG7802 left, 5′-GTCGCGACATCGACACTTC-3′; CG7802 right, 5′-CGTTGGCAGTGAATGTGGT-3′; Attacin A left, 5′-TGCAGAACACAAGCATCCTAA-3′; Attacin A right, 5′-TAAGGAACCTCCGAGCACCT-3′; Cecropin A1 left, 5′-TCTTCGTTTTCGTCGCTCTC-3′; Cecropin A1 right, 5′-ACATTGGCGGCTTGTTGAG-3′; Defensin left, 5′-GATGTGGATCCAATTCCAGA-3′; Defensin right, 5′-CTTTGAACCCCTTGGCAAT-3′; Diptericin left, 5′-ACCGCAGTACCCACTCAATC-3′; Diptericin right, 5′-CCATATGGTCCTCCCAAGT-3′; G Drosocin left, 5′-TTCACCATCGTTTTCCTGCT-3′; Drosocin right, 5′-GGCAGCTTGAGTCAGGTGAT-3′; Drosomycin left, 5′-GTACTTGTTCGCCCTCTTCG-3′; Drosomycin right, 5′-ACTGGAGCGTCCCTCCTC-3′; rp49 left, 5′-ATCGGTTACGGATCGAACAA-3′; and rp49 right, 5′-GACAATCTCCTTGCGCTTCT-3′.
Hemocyte isolation and phagocytosis experiments
Hemocytes were collected from 60–80 wandering third instar larvae in Schneider’s Drosophila medium (10% heat-inactivated FBS) containing glass-bottom culture dishes. Cells were allowed to settle down for 15 min and washed with Schneider’s media followed by incubation with the indicated bacteria or dextran.
After a 15-min incubation at 4°C, unbound bacteria were washed out and phagocytosed bacteria were chased for the various times. (a) 15 min to measure initial uptake, after which extracellular fluorescent bacteria were quenched with Trypan blue. (b) To visualize late stage phagosomes in life cells, we chased for 30 min, after which we collected images for 15 min. We call this a “30–45 min chase.” (c) To capture early or late stage phagosomes by immunofluorescence staining, we fixed after 15–25 min or 30–45 min of chase. (d) We chased for 45 min to analyze bacterial persistence in cells, as at that time most bacteria were digested in wild-type hemocytes. Subsequently, cells were fixed (4% PFA) and counterstained with Phalloidin–Alexa Fluor 546 (Invitrogen). Oregon-R was used as control in all assays. (e) For electron microscopy analysis of phagosome maturation, we chased for 30 min. Cells were fixed with a mixture of 1.5% glutaraldehyde and 2.5% PFA for 2 h, then stained with osmium tetroxide, dehydrated, and embedded in epon. Sections (60–70 nm) were stained with uranyl acetate, dried, and viewed under a transmission electron microscope (120kV; Technai G2 spirit; FEI) with an 11-megapixel Morada camera (Olympus). Data were collected from two independent sets of experiments with equivalent results. Quantification was performed in triplicate, with a representative example shown in .
For phagocytosis experiments, n refers to the number of independent experiments that were quantified.
To measure acidity (pH), heat-killed E. coli
(DH5α) were colabeled with the pH-sensitive fluorophore Oregon green 488 and the pH-insensitive carboxy-tetramethylrhodamine (Vergne et al., 1998
). To calibrate their pH-dependent fluorescence, bacteria were suspended in phosphate-citrate buffer, pH 2.5–7.0, and imaged using a 63×, NA 1.4 lens on a confocal microscope (LSM510; Carl Zeiss, Inc.). Fluorescence ratios were determined for individual bacteria using ImageJ. To measure changes in phagosome pH, fob1
or wild-type hemocytes were incubated with double-labeled bacteria (2–4 × 107
) at 4°C for 15 min. After a 10-min or 30-min chase, fluorescence ratios were measured from intact bacteria that did not appear degraded as indicated by diffuse fluorescence.
Immunofluorescence and dextran internalization
Hemocytes were incubated with dextran–Alexa Fluor 488 or dextran–Alexa Fluor 594 (10 kD, 1 mg/ml) for 5 min in PBS, pH 7.4, for monitoring fluid phase endocytosis. Free dextran was removed by washing extensively, and cells were chased for 90 min in Schneider’s Drosophila medium with 10% heat-inactivated FBS. After that chase, cells were either incubated with LysoTracker (GFP-certified Lyso-ID red lysosomal detection kit; Enzo Biochem, Inc.) or allowed to phagocytose GFP-tagged E.
coli or latex beads to measure fusion of lysosome to phagosome.
For immunofluorescence staining after phagocytosis, hemocytes were fixed with 4% PFA for 45 min, then washed with PBS with 0.3% saponin. Samples were stained with the indicated primary antibodies rabbit anti-Hook (1:250; Sunio et al., 1999
), rabbit anti-Avl (1:1,000; Pulipparacharuvil et al., 2005
), rabbit anti–Rbsn-5 (1:5,000; a gift from A. Nakamura; Institute of Physical and Chemical Research Center for Developmental Biology, Kobe, Japan; Tanaka and Nakamura, 2008
), rabbit anti-Rab7 (1:3,000; a gift of P. Dolph; Dartmouth College, Hanover, NH; Chinchore et al., 2009
); and secondary antibodies labelled with Alexa Fluor 568 and Alexa Fluor 647 and mounted in Vectashield (Vector Laboratories).
Fluorescence images were captured with a 63×, NA 1.4 lens on an inverted confocal microscope (LSM510 Meta; Carl Zeiss, Inc.) at room temperature (21°C). All digital images were imported into Photoshop (Adobe) and adjusted for gain, contrast, and gamma settings.
The LIFETEST procedure of SAS 9.2 (SAS Institute Inc.) was used to analyze survival curves by the Kaplan-Meier method. Logrank comparisons were used to assess significance of differences between curves. Student’s t tests (two-tailed) were used to determine the statistical significance of differences between colony-forming unit counts and changes of Avl/Rbsn-5, Rab7, and Hook localization to phagosomes was identified by bacterial content. At least 50 cells with phagosome structures containing bacteria were used for each of the quantifications. Phagosomes in >100 cells were used for the quantification of dextran/LysoTracker or dextran/bacteria.
Online supplemental material
Fig. S1 shows an in vitro calibration curve for the ratiometric signal from Oregon green/Rhodamine-labeled bacteria with different pH values. Fig. S2 shows the lack of changes in endosomal trafficking or autophagosome maturation in fob
mutant cells and compares it to the reduced lysosomal delivery after loss of Vps16A or Carnation function. Fig. S3 shows in vivo binding of Fob and Vps33B and the reduced survival of E. coli
–injected flies after Vps33b knockdown. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201008119/DC1