A Rab3a-dependent complex essential for lysosome positioning and plasma membrane repair

Abstract

Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis.

Figure 1.

Rab3a and Rab10 are required for Ca²⁺-dependent lysosomal exocytosis. (A) Scheme showing the different steps of the screening procedure (see Materials and methods). (B) A representative histogram of three independent experiments, with numbers indicating the LAMP1 MFI of the selected cell population. (C) Table with Rab-positive hits obtained from three independent experiments. (D) Scheme of the different steps and readouts used in the secondary screen. (E) Flow cytometry analysis of nontransduced THP-1 cells treated with SLO (see Materials and methods). Numbers in histograms indicate MFI of PI-positive cells. (F) Graph showing percent β-hex release upon SLO treatment with and without Ca²⁺. Error bars represent SD from five independent experiments (n = 5). (G) Graph showing the percentage PMR in Rab-silenced cells treated with SLO. Error bars represent SD from two to five independent experiments. (H) Graph showing percentage of β-hex release in the supernatant from Rab-silenced cells treated with SLO. Results were normalized to the negative control (Ct). Error bars represent SD from three independent experiments (n = 3). In G and H, only the best two shRNAs were used. Rab3a and Rab10 shRNA are marked as black bars in the graphs.

Figure 2.

Rab3a silencing induces lysosome clustering in the perinuclear region. (A) Percentage of PMR in HeLa cells silenced for Syt VII, Rab10 or Rab3a and control shRNA and challenged with SLO. (B) Representative confocal images of HeLa cells silenced for KIF5B, Rab10, or Rab3a stained for lysosomes, with LAMP1 antibodies (in red) and nuclei, with DAPI (in blue). Control shRNA and KIF5B were used as negative and positive control, respectively. Bars, 10 µm. (C) Quantification of the number of cells with lysosome clustering. This plot also includes the rescue of lysosome clustering in Rab3a-silenced cells infected with adenoviruses expressing the murine Rab3a. In A and C, error bars represent SD from three to four independent experiments. **, P < 0.01; ***, P < 0.001, comparing differences between control and Rab3- or Rab10-silenced cells. (D) Representative confocal images of Rab3a-silenced HeLa cells, infected by adenovirus expressing the murine Rab3a tagged with GFP and then immunostained for LAMP1. Bar, 10 μm. (E) Western blot showing endogenous and ectopical murine Rab3a levels in different experimental conditions. NT, nontransduced HeLa cells. GAPDH was used as loading control. (F) Percentage of necrotic cells in control and Rab3a-silenced primary human macrophages infected with H37Ra Mtb. The plot represents the mean ± SD of two independent experiments. **, P < 0.01, comparing differences between control and Rab3-silenced cells.

Figure 3.

GFP-Rab3a associates with lysosomes near the PM. (A and B) Representative confocal images of HeLa cells (A) and Melan-ink4a melanocytes (B) overexpressing GFP-Rab3a or FLAG-Rab3a, respectively, and imunostained for the Golgi marker COPI. (C) Live-cell imaging of HeLa cells expressing GFP-Rab3a and stained with Lysotracker (red). The region outlined with a square was zoomed in and the channels were split. The zoomed-in region shows the colocalization of GFP-Rab3a and Lysotracker-loaded vesicles (visualized in yellow in the merge). (D) Representative image of TIRF microscopy of HeLa cells loaded with dextran 647 to stain lysosomes and transfected with GFP-Rab3a. The region outlined with a rectangle was zoomed in and the channels were split. The zoomed in region shows Rab3-positive lysosomes underneath the PM. Bars, 10 µm. WT, wild type.

Figure 4.

Silencing of the Rab3a effector Slp4-a induces lysosome clustering. (A) Representative images of HeLa cells silenced for Rab3a effectors Slp4-a, Rim2, Noc2, or MyoVa or transduced with control shRNA and then immunostained for the lysosomal marker LAMP1 (red) and DAPI (blue). (B) Quantification of the number of cells showing lysosome clustering (in percentage). More than 300 cells were analyzed per condition. (C) Effect of Slp4-a silencing in PMR in HeLa cells treated with SLO. In B and C, plots represent the mean ± SD of three independent experiments. ***, P < 0.001, comparing differences between control and Rab3-silenced cells. (D) Lysates from HeLa cells cotransfected with constitutively-active FLAG-Rab3a Q81L and GFP-Slp4-a-SHD and immunoprecipitated with GFP-Trap-A beads. Immunoprecipitation products were analyzed by immunoblotting using the indicated antibodies. NT, non-transfected; FT, flow-through. (E) Representative image of confocal microscopy of HeLa cells cotransfected with GFP-Rab3a and mCherry-Slp4-a-FL. The region outlined with a square was zoomed in and channels were split. The zoomed-in region shows colocalization of Rab3a and Slp4-a–positive vesicles (visualized in yellow in the merge). Bars: 10 µm; (zoomed region) 5 µm.

Figure 5.

NMHCIIA is required for lysosome positioning. (A) Lysates from HeLa cells transfected with GFP-Rab3a were immunoprecipitated with GFP-Trap-A beads in the presence of nonhydrolysable GTP (GTPγS) or GDP. Bands visualized with Coomassie brilliant blue staining were excised and identified by mass spectrometry. (B) Validation of the NMHC IIA and GFP-Rab3a interaction by immunoprecipitation of HeLa cell lysates with GFP-Trap-A beads in the presence of GTPγS or GDP. Immunoprecipitation products were analyzed by immunoblotting using the indicated antibodies. (C) HeLa cells transfected with GFP-Rab3 and immunostained for the endogenous NMHC IIA. The region outlined with a square was zoomed in, and channels were split. (D) Lysates from HeLa cells transfected with GFP-Slp4-a-SHD were immunoprecipitated with GFP-Trap-A beads in the presence of nonhydrolysable GTP (GTPγS) or GDP. Immunoprecipitation products were analyzed by immunoblotting using the indicated antibodies. (E) Lysates from control shRNA and Slp4-a silenced HeLa cells transfected with GFP-Rab3a were immunoprecipitated with GFP-Trap-A beads in the presence of nonhydrolysable GTP (GTPγS) or GDP. Immunoprecipitation products were analyzed by immunoblotting using the indicated antibodies. (F) HeLa cells transfected with control siRNA or siRNA targeting NMHC IIA were fixed and stained with LAMP1 antibodies and DAPI to visualize lysosomes and the nucleus, respectively. Representative images for both are shown. Bars, 10 µm. (G) Quantification of the number of cells showing lysosome clustering (in percentage) in NMHC IIA–silenced and control cells. (H) Effect of NMHC IIA silencing in lysosomes exocytosis in cells challenged with ionomycin. Plots represent the mean ± SD of three independent experiments. *, P < 0.1; ***, P < 0.001, comparing differences between control and NMHC IIA–silenced cells. (I) Our working model postulates that lysosomes positive for Rab3a can recruit NMHCIIA when it is bound to GTP. This myosin motor is responsible for the positioning of lysosomes to the periphery of the cell. Slp4-a, another Rab3 effector, although not necessary for the recruitment of NMHC IIA, seems to be involved in peripheral lysosome positioning.