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. 2012 Dec 11;109(50):20485-90.
doi: 10.1073/pnas.1206811109. Epub 2012 Nov 28.

Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34

Bahram Kasmapour  1 , Achim Gronow, Christopher K E Bleck, Wanjin Hong, Maximiliano Gabriel Gutierrez
Affiliations

Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34

Bahram Kasmapour et al. Proc Natl Acad Sci U S A. .

Abstract

Phagosome maturation is an essential part of the innate and adaptive immune response. Although it is well established that several Ras-related proteins in brain (Rab) proteins become associated to phagosomes, little is known about how these phagosomal Rab proteins influence phagosome maturation. Here, we show a specific role for Rab34 and mammalian uncoordinated 13-2 (Munc13-2) in phagolysosome biogenesis and cargo delivery. Rab34 knockdown impaired the fusion of phagosomes with late endosomes/lysosomes and high levels of active Rab34 promoted this process. We demonstrate that Rab34 enhances phagosome maturation independently of Rab7 and coordinates phagolysosome biogenesis through size-selective transfer of late endosomal/lysosomal cargo into phagosomes. More importantly, we show that Rab34 mediates phagosome maturation through the recruitment of the protein Munc13-2. Finally, we report that the alternative maturation pathway controlled by Rab34 is critical for mycobacterial killing because Rab34 silencing resulted in mycobacterial survival, and Rab34 expression led to mycobacterial killing. Altogether, our studies uncover Rab34/Munc13-2 as a critical part of an alternative Rab7-independent phagosome maturation machinery and lysosome-mediated killing of mycobacteria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Rab34 is required for phagosome maturation, (A) Macrophages expressing the different constructs were incubated with fluorescent beads for 30 min of pulse and 30 min of chase and stained for LAMP-1. (B) Quantitative analysis of LAMP-1 fluorescence intensity associated with phagosomes. (C) Association of LAMP-2 to phagosomes as in A. (D) Quantitative analysis of LAMP-2 fluorescence intensity associated with phagosomes as indicated in B. (E) Rab34 was silenced by transfection with the pSIREN vector expressing the Rab34-shRNA oligomers (1, 2, or 3) or the scrambled shRNA as control. Cells were sorted based on DsRed signal and analyzed by Western blot to detect endogenous Rab34. Actin was used as loading control. (F) Quantification of Rab34 levels from three independent experiments. (G) Representative images of Rab34 silenced (oligo 2, pSIREN pseudocolored in green) and control cells stained for LAMP-1. (H) Quantitative analysis of LAMP-1 fluorescence intensity associated to phagosomes. (I) Association of LAMP-2 association to phagosomes as in G. (J) Quantitative analysis of LAMP-2 association to phagosomes as in H. Data show one representative experiment out of three in which at least 45 cells were analyzed. Mean ± SEM; *P ≤ 0.01; **P ≤ 0.001 from two-tailed Student t test. (Scale bar, 10 µm.) (Inset magnification: A and C: ∼2.5×, G and I: ∼2×.)
Fig. 2.
Fig. 2.
Rab34 regulates size-selective cargo delivery into phagosomes. (A) Outline of the protocol followed for lysosomal compartment preloading in transfected macrophages before phagocytosis of beads. (B) Analysis of delivery of preloaded Dex70kDa to phagosomes in Rab34 expressing macrophages during the first hour. The phagosomes in the Insets are indicated with white arrows. (C) Quantitative analysis of the Dex70kDa delivered to phagosomes. The curve shows the average of at least five phagosomes from three independent experiments, plotted with a third-degree polynomial regression curve fitting. (D) Data points from 30 to 60 min in E were pooled. (E) Analysis of delivery of preloaded Dex70kDa to phagosomes in Rab34 silenced and control macrophages (pSIREN pseudocolored green) during the first hour. The phagosomes in the Insets are indicated with white arrows (Movie S1). (F) Quantitative analysis of the Dex70kDa delivered to phagosomes. The curve shows the average of at least five phagosomes from three independent experiments, plotted with a third-degree polynomial regression curve fitting. (G) Data points from 30 to 60 min in F were pooled. (H) Analysis of delivery of preloaded Dex10kDa to phagosomes as in E (Movie S2). (I) Quantitative analysis of the fluorescence signal intensity of Dex10kDa delivered to phagosomes as in F. (J) Quantitative analysis as in G. Data show mean ± SD; †P > 0.01; **P ≤ 0.001 from two-tailed Student t test. (Scale bar, 10 µm.) (Inset magnification: ∼2×.)
Fig. 3.
Fig. 3.
Rab34 size-selective cargo delivery to phagosomes is independent of Rab7, (A) Phagosome (asterisk) maturation in macrophages coexpressing EGFP-Rab7 and mCherry-Rab34 (Movie S3). (B) Rab7-positive, Rab34-negative tubule extended from the phagosome (white arrows). (C) Expressed Rab7 and Rab34 partially colocalized in phagosomes and endocytic vesicles (white arrows). (D) Extended tubular vesicle, positive for Rab7 and negative for Rab34. (E) Endocytic vesicle with strong EGFP-Rab7, but no mCherry-Rab34 association. (Scale bars, 10 µm in A, 3 µm in B–D, and 2 µm in E.) Time stamp refers to the event in Movie S3. (F) Quantification of LAMP-2 fluorescence signal intensity associated to phagosomes in expressing or coexpressing cells, relative to nonexpressing cells in the same field as the internal control. (G) Quantification of Dex70kDa fluorescence signal intensity associated to phagosomes quantification as indicated in F. (H) Quantification of Dex10kDa fluorescence signal intensity associated to phagosomes as indicated in G. Data show mean ± SEM from four independent experiments, with at least 30 cells evaluated in each. †P > 0.01; *P ≤ 0.01; **P ≤ 0.001 from two-tailed Student t test.
Fig. 4.
Fig. 4.
Role of Munc13-2 in the Rab34-dependent regulation of phago-lysosome fusion, (A) Quantitative analysis of EGFP-Munc13-2 association to phagosomes under the different conditions. Data show mean ± SD from one representative experiment out of two with at least 50 cells analyzed in each. (B) Quantitative analysis of endogenous Munc13-2 association to phagosomes in macrophages expressing the indicated constructs. Data show mean ± SD from one representative experiment in which at least 50 cells were analyzed. (C) Association of LAMP-1 to phagosomes in Munc13-2 WT and KO mouse bone marrow–derived macrophages (BMMs). (Right) Quantitative analysis of LAMP-1 fluorescence intensity association to bead phagosomes. (D) Delivery of Dex70kDa to phagosomes in BMMs preloaded with Dex70kD. (Right) Quantitative analysis of Dex70kDa delivery to phagosomes. Mean ± SD from three experiments; at least 100 cells were analyzed. *P ≤ 0.01, **P ≤ 0.001, from two-tailed Student t test. (Scale bar, 10 µm.) (Inset magnification: ∼1.75×.)
Fig. 5.
Fig. 5.
Rab34 is required for the lysosome-mediated killing of mycobacteria, (A) Analysis of the association of EGFP-Rab34 to M. bovis bacillus Calmette–Guérin-DsRed containing phagosomes during the early time points after phagocytosis (Movie S4). (B) Association of EGFP-Rab34 to mycobacterial phagosome (white arrows) (C) 3D surface plot at 14:00 s after uptake of mycobacteria. (Scale bars, 10 µm in A and 2 µm in B). (D) Macrophages transfected with the indicated constructs were infected with M. bovis bacillus Calmette–Guérin-GFP, and cells were fixed and stained for LAMP-2. (E) Analysis of number of mycobacterial phagosomes positive for LAMP-2. Mean ± SD of three independent experiments; at least 100 cells were evaluated in each. (F) Macrophages selected for the expression of EGFP, EGFP-Rab34, or EGFP-Rab34-Q111L (pseudocolored blue) were infected with bacillus Calmette–Guérin-DsRed. After 24 h, cells were lysed, and cfu determined. Mean ± SEM of the percentage of bacteria recovered compared with control cells from two independent experiments. (G) Rab34 silenced and control cells (pseudocolored blue) were infected with bacillus Calmette–Guérin-GFP as described in D and stained for LAMP-2. (H) Analysis of number of mycobacterial phagosomes positive for LAMP-2 as in E. (I) Rab34 silenced and control cells were sorted and infected with bacillus Calmette–Guérin-GFP. After 1 and 24 h of infection, macrophages were lysed and cfu determined. Data show mean ± SEM of the percentage of bacteria recovered compared with control cells from two independent experiments. †P > 0.01; *P ≤ 0.01; **P ≤ 0.001 from two-tailed or two-paired Student t test. (Inset magnification: ∼1.75×.)
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