Lysosomal Trafficking, Antigen Presentation, and Microbial Killing Are Controlled by the Arf-like GTPase Arl8b

Summary Antigen presentation and microbial killing are critical arms of host defense that depend upon cargo trafficking into lysosomes. Yet, the molecular regulators of traffic into lysosomes are only partly understood. Here, using a lysosome-dependent immunological screen of a trafficking shRNA library, we identified the Arf-like GTPase Arl8b as a critical regulator of cargo delivery to lysosomes. Homotypic fusion and vacuole protein sorting (HOPS) complex members were identified as effectors of Arl8b and were dependent on Arl8b for recruitment to lysosomes, suggesting that Arl8b-HOPS plays a general role in directing traffic to lysosomes. Moreover, the formation of CD1 antigen-presenting complexes in lysosomes, their delivery to the plasma membrane, and phagosome-lysosome fusion were all markedly impaired in Arl8b silenced cells resulting in corresponding defects in T cell activation and microbial killing. Together, these results define Arl8b as a key regulator of lysosomal cellular and immunological functions.

: Pulldown with GST-Arl8b identified members of the HOPS complex: Lysates from RAW macrophages were probed with GST-Arl8b or GST. Eluates were run on SDS-PAGE. Bands which appeared specifically in the GST-Arl8b lane were cut, partially digested, and analyzed by mass spectrometry. The number of unique peptides identified corresponding to each interaction partner is listed at right. We restricted our initial analysis to those genes where at least two unique peptides were identified in this manner. Previously, Arl8b has been shown to bind -tubulin. Encouragingly, we identified a number of tubulin subunits as potential binding partners of Arl8b. We also identified two members of the VPS-C core of the HOPS complex (see  Arl8b promoted the recruitment of VPS39 to lysosomes: HeLa cells were transfected with VPS39-HA alone or VPS39-HA with Arl8b, fixed, and stained with anti-HA (hVPS39) and anti-LAMP1. In control cells, hVPS39 distributed to the cytosol and did not colocalize with lysosomal marker LAMP1 (top row). In contrast, Arl8b overexpression resulted in a dramatic recruitment of hVPS39 to lysosomes (bottom row). B. Arl8b and VPS41 promoted the recruitment of VPS11 to lysosomes: HeLa cells were transfected with VPS11-GFP (top row), VPS11-GFP + Arl8b (middle row), or VPS11-GFP + Arl8b + Myc-VPS18 + VPS41-HA (bottom row). They were then fixed, permeabilized, and stained for the indicated markers. (top row) In control cells, hVPS11 exhibited a cytosolic staining pattern demonstrating little colocalization with lysosomal marker LAMP1. (middle row) Overexpression of Arl8b resulted in a partial recruitment of hVPS11 to Arl8b+ lysosomes. Note that a portion of VPS11 staining remained in Arl8b negative punctae. (bottom row) Overexpression of Arl8b, VPS41, and VPS18 resulted in near total recruitment of VPS11 to lysosomes as evidenced by the triple colocalization of VPS11, VPS41, and Arl8b staining. C. Arl8b and VPS41 promoted the recruitment of VPS16 to lysosomes: HeLa cells were transfected with VPS16-GFP (top row), VPS16-GFP + Arl8b (middle row), or VPS16-GFP + Arl8b + Myc-VPS18 + VPS41-HA (bottom row). They were then fixed, permeabilized, and stained for the indicated markers. (top row) Similar to VPS11 (part B above), VPS16 localized predominantly to the cytosol in control cells and did not display significant colocalization with lysosomal markers. (middle row) Overexpression of Arl8b resulted in partial recruitment of VPS16 to lysosomes. (bottom row) Overexpression of Arl8b, VPS41, and VPS18 resulted in near total recruitment of VPS16 to lysosomes as evidenced by triple staining for VPS16, VPS41, and Arl8b. Higher magnification insets are shown for clarity. All scale bars= 10 m. To address the possibility that LAMP1 on phagocytosed latex beads was adsorbed non-specifically during bead repurification we treated cells with the actin polymerization inhibitor cytochalasin D. Since phagocytosis depends on actin reorganization drug treatment abolishes phagocytosis. (left panel) Pre-treatment of RAW cells with 2 M cytochalasin D for 5 minutes resulted in a dramatic reduction of LAMP1 acquisition on latex beads following 60 minutes incubation. (right panel) The geometric MFI for LAMP1 on latex beads repurified 60 minutes after addition for both control and Arl8b silenced RAW cells is plotted with or without cytochalasin D pretreatment. Note that in both Arl8b silenced and control cells drug treatment drastically reduced the acquisition of LAMP1 on latex beads, indicating LAMP1 acquisition depended on actin polymerization. This strongly suggests LAMP1 acquisition on beads was due to phagocytosis followed by fusion with lysosomes. B. Phagocytosed beads isolated from Arl8b silenced cells did not show a delay in transferrin receptor acquisition. In contrast to LAMP1, acquisition of transferrin receptor on bead phagosomes isolated from Arl8b silenced cells was not delayed compared to control cells (compare B to Figure 7B). There was a slight increase in transferrin receptor levels detected on bead phagosomes isolated 30 minutes after infection in Arl8b silenced cells, suggesting a connection between phagosomal maturation and acquisition of lysosomal markers (LAMP1) and loss of early endocytic markers (transferrin receptor). We are happy to distribute vectors cloned in the lab. *=Each of these "control" shRNA hairpins was used in almost every experiment and found to give similar results. Additionally, results with transduction by these control shRNA were identical to nontransduced (uninfected) cells in all experiments except where both controls are shown (Figure 1b/1c). For clarity, all experiments in the manuscript utilize shMISSION transduced cells as the control unless otherwise noted. **=Figures targeting murine Arl8b utilize this hairpin unless otherwise noted ***=Figures targeting human Arl8b utilize this hairpin unless otherwise noted

Expression constructs:
Arl8b expression constructs were obtained from Origene and cloned into pcDNA3.1 using the BamH1 and EcoR1 restriction sites. Point mutants were designed and obtained using Stratagene site directed mutagenesis kits (Agilent). GFP-Rab7 and GFP-Rab9 expression constructs were obtained from Dr. Steve Caplan. GFP-RILP and GFP-ORLP1 were obtained from Dr. Jacques Neefjes. VPS expression constructs were obtained from Drs. J Wade Harper, Victor Faundez, and Robert Piper. All expression constructs were sequence verified using standard vector primers. GST-Arl8b, GST-Arl8b mutants, and GST-RILP were expressed from pGEX 4T-3 in BL-21 cells, purified using standard methods. His-Arl8b was cloned in pet15b vector using NdeI and BamH1 restriction enzymes and retransformed in BL-21 strain for protein production. Bacteria were induced with 0.5mM IPTG for 5 hours at 30 degrees C. After sonication, His-Arl8b was purified from the bacterial lysates using TALON cobalt resin (Clontech). Purified protein was eluted from the beads by using elution buffer (Clontech, HisTALON buffer set) Lentiviral transduction: For lentiviral transduction, U937 cells were plated in 96-well round bottom plates (10,000/well, Corning) in polybrene (8 g/mL, SIGMA), and mixed with 10 L of viral supernatant (Day 0). Puromycin was added after 24-48 hours at 2.5 g/mL for a minimum of three days to select transductants. Monocyte-derived DC were transduced with lentivirus by spinoculation at 2000 rpm, 30 minutes at 25 degrees C (Day 2), puromycin added (2.0 g/mL, Day 3) and analyzed for CD1 presentation (Day 6-7). HeLa and RAW cells were plated at 100,000/well in 6-well plates (Corning) in 5 g/mL Polybrene and transduced by addition of 100 uL viral supernatant. 24-48 hours later, puromycin was added at 2.5 g/mL (HeLa) and 5 g/mL (RAW) to select transductants and experiments performed on Days 5-21 following transduction.
GST pulldowns: GST, GST-Arl8b, and GST-Arl8b mutants were bound to GST beads. Lysates were prepared in 0.5% CHAPS buffer containing GTPγS and were incubated with purified bound GST protein overnight followed by washes with PBS or PBS containing GTPγS. Eluates were run on SDS-PAGE and subjected to electrospray ionization tandem mass spectrometry (LC-MS/MS) to discover Arl8b interacting proteins (Supplementary Figure 4) or were western blotted with anti-HA antibody (Figure 4). His-Arl8b was first incubated with 0.5mM GTPS in a nucleotide loading reaction. The GTP-loaded Arl8b was then incubated with Talon resin for 30 mins at 4 degrees followed by blocking of beads with 5% BSA for 1 hour. Incubation of GST proteins : GST, GST-Vps41 (Novus biologicals) or GST-RILP-Rab7 Binding domain was performed at 4 degrees C for 3 hours in 20mM Tris-HCl Ph 7.4 , 150Mm NaCl, 1mM MgCl2, 5% Glycerol , 0.5% NP-40. After the incubations, beads were pelleted by centrifugation (500g for 4 minutes at 4 degrees) followed by three washes in 25mM HEPES, 300mM NaCl, 90mM KCl, 1mM MgCl2, 0.2%Triton X-100 + 0.5mM GTPS and then analyzed by SDS-PAGE. Western blotting was performed using anti-GST-HRP antibody (Millipore) to detect the bound proteins. Coomassie brilliant blue and Silver stain were performed to detect His-Arl8b and GST-tagged proteins respectively.
Yeast two hybrid analysis: The Saccharomyces cerevisiae strain AH109 (Clontech) was maintained on Yeast extract, Peptone, Dextrose (YPD) agar plates. Transformation was carried out by the lithium acetate procedure as described in the instructions for the MATCHMAKER two-hybrid kit (Clontech). For colony growth assays, AH109 cotransformants were streaked on plates lacking leucine and tryptophan and allowed to grow at 30 degrees C for 5 days until colonies were large enough for further assays. An average of three to four colonies was then chosen and suspended in water, equilibrated to the same optical density of 600 nm and replated on plates lacking leucine and tryptophan (+HIS) as well as plates also lacking histidine (−HIS).

Arl8b antisera and western blotting
To generate Arl8b antisera, peptide was conjugated to KLH as a protein carrier, injected into rabbit, and boosted bi-weekly for 6 weeks. Rabbit IgG was then affinity purified against peptide. Lysates from 10 7 U937 or 10 7 RAW cell equivalents/lane were made in 0.5% Triton X-100 and analyzed on 15% SDS-PAGE, transferred to PVDF membranes (Bio-Rad) and blotted with Rabbit anti-Arl8b IgG (1 ug/mL) followed by donkey-anti rabbit HRP (Jackson Labs) at 1:30,000 dilution. Membranes were stripped and reprobed with anti-human -actin (Abcam) for human cell lysates or anti-murine GAPDH (Abcam) followed by appropriate secondary HRP conjugates (Jackson Labs). All blots were developed with ECL reagents and exposed to film Colocalization analysis using MetaMorph Imaging software: Images from control or Arl8b silenced cells were acquired using identical laser power and gain settings on a single day within an experiment. Each channel (R,G, B) was considered separately with each pixel assigned an intensity value 0-255 with great care taken to assure that no pixel was saturated in any image used for quantification (no values = 255). A lower threshold is set whereby areas of the image not containing cells or staining are excluded, and the same threshold was always applied to control and Arl8b silenced cells. This value was 10-30 depending on the stain and never excluded visibly positive regions. The software then calculates the area overlapping between two channels where both stains are positive over the lower threshold and reports it in percentage terms. This analysis was utilized to give the reported co-localization values in Figures 2, 5, 7, and Supplementary Figure 2.
Phagosomal Assays 3 m latex beads were purchased (Polysciences Inc) and coated by rotation with .4 mg/mL murine IgG (Sigma) for 1 hour at 37°C and added to RAW cells at a ratio of 10 beads/cell. Bead/cell mixtures were centrifuged at 300g to synchronize uptake. Following incubation for indicated times at 37°C, coverslips were washed 3x in complete DMEM to remove unbound beads, and fixed and stained as described. E. coli (EPEC E1348/69, gift from Lynn Bry, Harvard University) were added to RAW cells at multiplicity of infection (MOI) of 20 and centrifuged at 300g for 5 mins. The mix was then incubated for 60 mins at 37°C, 5% CO2 followed by treatment with 100 g/mL gentamycin and chase