Lysosomal trafficking, antigen presentation, and microbial killing are controlled by the Arf-like GTPase Arl8b - PubMed (original) (raw)

Lysosomal trafficking, antigen presentation, and microbial killing are controlled by the Arf-like GTPase Arl8b

Salil Garg et al. Immunity. 2011.

Abstract

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.

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Figures

Graphical abstract

Figure 1

shRNA Silencing of Arl8b Decreases CD1d Antigen Presentation (A) Composite data from two sample screening plates. The number of U937 cells surviving drug selection (Alamar Blue) was plotted versus the response of NKT cells (IFN-γ). A best fit line was drawn for each batch of screening plates and shRNA constructs compared to the line to measure the effect on CD1d presentation. Experimental shRNAs (red squares), shRNAs targeting Arl8b (blue triangles), and control shRNAs included on each screening plate targeting GFP, RFP, and LacZ (green diamonds) are shown. (B) Arl8b silenced and control DCs were plated with αGal-αGalCer in the indicated doses along with NKT-cell clone BM2a.3. Following overnight incubation NKT-cell stimulation was assessed by IFN-γ ELISA. (C) Arl8b silenced and control murine RAW macrophages were plated with αGal-αGalCer along with NKT-cell hyrbidoma DN32. Following overnight incubation DN32 stimulation was assessed by IL-2 ELISA. (D and E) Immunoblots of U937 (D) or RAW (E) lysates made in 0.5% Triton blotted with Arl8b antisera (upper panel) or stripped and reprobed for loading controls (lower panel). The arrow indicates the position of the dominant band inferred to be Arl8b, with a Mr of 19.5 kDa based on standards. The lighter arrow indicates the inferred position of Arl8a. (F) HeLa CD1d-expressing cells were stably transduced with control or shArl8b-407 targeting shRNA and subsequently transfected with the construct used in F-containing silent mutations in the shRNA target sequence, termed “rescue.” αGal-αGalCer lipid was then added at 1 ng/mL along with BM2a.3 the response measured by IFN-γ ELISA. All error bars indicate standard error of the mean. See also Figure S1.

Figure 2

Arl8b Silencing Results in Delayed Delivery of Dextran and LDL to Lysosomal Compartments (A) Lysosomes of Arl8b-silenced and control RAW cells were labeled with dextran (Alexa Fluor 546, red) pulsed for 60 min and then underwent a >6 hr chase to allow accumulation in lysosomes (left). Cells were then incubated with a second dextran (Alexa Fluor 488, green) for various times (10 min, 30 min, 60 min) and colocalization of both dextrans was assessed (right). Alternatively, cells were prelabeled for 30 min with transferrin (Alexa Fluor 546) to mark early endocytic compartments and were subsequently pulsed with dextran (Alexa Fluor 488) in the continued presence of transferrin for short time points (5 min, 10 min, 30 min). Shown are representative images for 30 min of dextran → dextran pulse (left panels) and 10 min of dextran → transferrin pulse (right panels). (B) Colocalization quantification for >20 cells for each condition at each time point are shown for dextran → dextran pulse (top panel) at 10 min, 30 min, and 60 min and dextran → transferrin pulse (bottom panel) measured at 10 min. (C) Lysosomes of Arl8b silenced and control cells were prelabeled with dextran (Alexa Fluor 488, 1 hr pulse followed by 6 hr chase). Cells were subsequently starved in serum-free medium for 2 hr and then incubated with DiI-LDL (red) for up to 30 min. The 60 min time point represents 30 min of pulse followed by 30 min of chase in serum-free medium. Representative images from 60 min are shown. (D) Colocalization quantification of DiI-LDL with dextran in Arl8b-silenced and control cells. All error bars indicate standard error of the mean. See also Figure S2.

Figure 3

Arl8b Silencing Does Not Alter the Recruitment of Rab7 or Its Effectors to Lysosomes Control or Arl8b-silenced HeLa cells were transfected with the indicated constructs (left panels), plated on glass coverslips, fixed, permeabilized, and stained for LAMP1 (middle panels). Merged images are shown (right panels). (A) Colocalization between Rab7-GFP and LAMP1 in control cells (top row) and Arl8b-silenced cells (bottom row). (B) Colocalization between RILP-GFP and LAMP1 in control cells (top row) and Arl8b-silenced cells (bottom row). (C) Colocalization between GFP-ORLP1 and LAMP1 in control cells (top row) and Arl8b-silenced cells (bottom row). (D) Distribution of the dynactin motor subunit p150 in control cells (left) and Arl8b-silenced cells (right).

Figure 4

Arl8b Recruits the HOPS Complex Member VPS41 to Lysosomes (A) Adapted from Nickerson et al. (2009). Proposed subunits of the mammalian HOPS complex based on orthology to S. cerevisiae. The VPS-C core is composed of hVPS11, hVPS18, hVPS16, and hVPS33. In yeast, VPS39 and VPS41 combine with VPS-C to form HOPS. The HOPS complex regulates trafficking to lysosomes in S. cerevisiae. (B) Lysates from HeLa cells transfected with HA-VPS41 were run over glutathione-conjugated beads bound to GST (lane 2), GST-Arl8b (WT, lane 3), GST-Arl8b-Q75L (dominant-active mutant, lane 4), GST-Arl8b-T34N (dominant negative mutant, lane 5) and immunoblotted anti-HA. Equal amounts of lysate were run on a separate SDS-PAGE gel stained with Coomassie Brilliant Blue for total protein detection. (C) Yeast two-hybrid analysis. Arl8b, dominant-active Arl8b Q75L, and p53 were cloned into the DNA-binding domain vector (Matchmaker, Clonetech). VPS41, SV40-T, VPS39, and VPS18 were cloned into the activation domain vector. Yeast were plated on nonselective medium (+His) to confirm viability and plated on selective medium (−His) to detect interactions. (D) Interaction of Arl8b with VPS41 in two-protein system. Purified Histadine-tagged Arl8b (shown by Coomassie stain, middle) was immobilized on a cobalt-resin column and exposed to purified GST (lane 1), GST-VPS41 (lane 2), or GST-RILP (lane 3) (shown by silver stain, bottom). Eluates were run on SDS-PAGE and blotted anti-GST. (E) Control (top row), Arl8b-silenced (middle row), and Arl8b-overexpressing (bottom row) HeLa cells were transfected with HA-VPS41 and analyzed in confocal microscopy for the distribution of VPS41 to LAMP1+ compartments. For a list of GST-Arl8b interacting proteins identified, see also Figure S4.

Figure 5

Other Components of the HOPS Complex Are Recruited to Lysosomes by Arl8b and hVPS41 (A) Control HeLa cells (top row) were transfected with hVPS18 and Arl8b, fixed, and stained for hVPS18 (left) and Arl8b (middle). In the middle row, cells were transfected with hVPS41 (middle, Arl8b staining at right) in addition to hVPS18. In the bottom row, Arl8b-silenced HeLa cells were transfected with hVPS18 (left), hVPS41 (middle) and Arl8b (right), fixed, and stained. Similar data were obtained with VPS11 and VPS16 (Figure S5). (B) Similar to Figure 2C, lysosomes of hVPS41 silenced cells were prelabeled with dextran conjugated to Alexa Fluor 488 (green). Cells were then starved to allow LDL-R to accumulate, pulsed with DiI-LDL (red) for 15 min, and chased in DiI-LDL free medium for varying times, and the trafficking of LDL to lysosomes was assessed by colocalization with dextran. Shown are representative images of LDL trafficking to lysosomes after 90 min of chase time in control cells (top row) and hVPS41-silenced cells (bottom row). (C) Quantification of the colocalization between dextran (green) and LDL in control and hVPS41-silenced cells was performed for >30 cells at each indicated time point. (D) Indicated HOPS complex members were stably silenced by shRNA treatment in CD1d+ U937 cells as described (see Experimental Procedures). Cells were then pulsed with 70 ng/mL αGal-αGalCer, 50,000 NKT cells (J3N.5) added, and the combination was incubated overnight. Stimulation of NKT-cells was assessed by IFN-γ cytokine Elisa. A list of targeting shRNA sequences is given in the Supplemental Information. All error bars indicate standard error of the mean. See also Figure S5.

Figure 6

Arl8b-Silenced Cells Show Delayed Formation of CD1d⋅αGalCer Complexes (A) Arl8b-silenced and control RAW cells were cocultured with 500 ng/mL αGal-αGalCer for 24 hr, costained with L363 (CD1d⋅αGalCer complex specific) and LAMP1 mAbs, and analyzed by confocal microscopy. (B) Cells were pulsed with 2.5 μg/mL αGal-αGalCer for 2 hr and then underwent a washout and replacement with fresh medium (“chase”). Representative images are shown for 1 hr of chase. (C) Quantification of CD1d⋅αGalCer staining for >30 Arl8b-silenced and control cells at the indicated time points. The average pixel intensity for CD1d⋅αGalCer staining per cell was divided by the average LAMP1 pixel intensity for the same cell to normalize. Error bars indicate the standard error of the mean. (D) Arl8b-silenced and control cells were cocultured with 500 ng/mL αGal-αGalCer for 0 (left panel) or 12 (right panel) hr and CD1d⋅αGalCer complexes at the cell surface were analyzed by flow cytometric staining with mAb L363. (E) Arl8b-silenced and control cells were cocultured either with 500 ng/mL αGal-αGalCer for increasing amounts of time (upper panel) or for 12 hr with increasing concentrations of αGal-αGalCer (lower panel), and CD1d⋅αGalCer complexes at the cell surface were analyzed. Mean fluorescence intensities (MFIs) relative to time 0 were plotted for staining with L363 mAb. (F) Instead of continuous coculture, Arl8b-silenced and control cells were pulsed with 2.5 μg/mL αGal-αGalCer for 2 hr and then chased in complete media for 1 hr, 4 hr, and 9 hr. The appearance of CD1d⋅αGalCer complexes at the cell surface was monitored by L363 flow cytometric staining and MFIs plotted.

Figure 7

Arl8b Silencing Delays Fusion of Phagosomes with Lysosomes Leading to a Defect in Microbial Clearance (A) Control or Arl8b silenced RAW cells were plated on glass coverslips and then centrifuged with 3 μm IgG-coated latex beads for 0 min, 15 min, 30 min, 60 min, and 120 min. Cells were fixed and stained for LAMP1 (red) or IgG (green). Representative images for 60 min are shown. Higher-power magnification (inset) is shown for clarity. (B) Arl8b-silenced and control cells were plated as in (A) but without coverslips. Cells were disrupted by repeated passage through a small-gauge needle in a hypotonic lysis buffer, and latex bead-containing phagosomes recovered. These were then stained with LAMP1 antibody, analyzed via flow cytometry, and LAMP1 MFI plotted against time of bead incubation. (C) Similar to (A), only cells were infected with E. coli expressing a GFP plasmid at an MOI of 20. Shown are representative images taken from 1 hr after infection. On the right are high-magnification images of intracellular E. coli from a control cell (upper-right panel) and Arl8b-silenced cell (lower-right panel). (D) Similar to (C), however instead of fixation and analysis by microscopy, RAW cells were lysed at 30 min and 90 min of chase time in gentamycin, and live E. coli recovered through assaying CFU on agar plates. Error bars indicate the standard error of the mean. See also Figure S6.

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Lysosomal trafficking, antigen presentation, and microbial killing are controlled by the Arf-like GTPase Arl8b - PubMed (original) (raw)