Figure 4. Endogenous Perforin-2 is located in intracellular sites allowing for rapid translocation to bacteria.

(A) Schematic demonstrating proposed orientation of Perforin-2 in vesicles. (B) Fractionation results of endogenous Perforin-2 from human macrophages. (Lane L) is a post-nuclear lysate control, (Lane 1–8) are individual fractions corresponding with specific indicated organelles. (C) Overexpression of murine Perforin-2-GFP in murine BV2 microglial cells. (D–F) Confocal images taken 5 min after S. typhimurium infection in Perforin-2-GFP + Perforin-2 siRNA transfected BV2 cells. White arrows denote extracellular S. typhimurium, red arrows highlight a DNA cloud corresponding with S. typhimurium (D) DAPI only, (E) Perforin-2-GFP only, (F) Merge of DAPI and Perforin-2-GFP. (G–I) Confocal images taken 5 min after Escherichia coli-GFP infection in Perforin-2-RFP + Perforin-2 siRNA transfected BV2 cells. Arrows point to extracellular E. coli-GFP that has made contact but is still extracellular with normal bacilli morphology maintained. (G) E. coli-GFP only, (H) Perforin-2-RFP only, and (I) merge E. coli-GFP and Perforin-2-RFP. Fractions in B were probed as follows: Cytoplasm—MEK1/2; Early Endosome—EEA1; Lysosome—Lamp1; ER—calreticulin; Golgi—Golgin-97; Mitochondria—Prohibitin; Peroxisome—Catalase; Plasma Membrane—Cadherin.

DOI: http://dx.doi.org/10.7554/eLife.06508.016

Figure 4—figure supplement 1. Perforin-2-RFP colocalizes with ER, Golgi, and early endosomes.

(A-L) We used RAW264.7 macrophages which constitutively express Perforin-2 and studied the subcellular localization of fluorescent Perforin-2. RAW264.7 macrophages were transfected with Perforin-2-RFP and stimulated with IFN-γ and LPS for 14 hr to induce a shift towards M1 macrophages. Cells were fixed and stained as indicated. These images are representative of 3 separate experiments.

DOI: http://dx.doi.org/10.7554/eLife.06508.017

Figure 4—figure supplement 2. S. typhimurium infection of macrophages with Perforin-2 and DAPI localization through the cell (multiple Z-sections).

(A-F) BV2 cell line overexpressing Perforin-2-GFP infected with S. typhimurium. Images are collected 5 min post-infection. Arrows indicate extracellular S. typhimurium that has maintained the normal shape of S. typhimurium-likely attributed to not being surrounded by the bactericidal Perforin-2-GFP.

DOI: http://dx.doi.org/10.7554/eLife.06508.018

Figure 4—figure supplement 3. Perforin-2-RFP colocalizes with E. coli-GFP within minutes of infection.

BV2 microglia were transfected with Perforin-2-RFP and stimulated overnight with IFN-γ. Transfected cells were infected with E. coli-GFP for several minutes upon which the cells were fixed and imaged. Arrows point to extracellular E. coli-GFP. (A) Perforin-2-RFP only, (B) E. coli-GFP only, (C) Merge of Perforin-2-RFP and E. Coli-GFP. Yellow in (C) corresponds with colocalization of Perforin-2-RFP with E. coli-GFP.

DOI: http://dx.doi.org/10.7554/eLife.06508.019