Journal: bioRxiv

Article Title: Depletion of endomembrane reservoirs drives phagocytic appetite exhaustion in macrophages

doi: 10.1101/2024.07.31.605905

Figure Lengend Snippet: A. Reconstructed micrographs of LAMP1 staining (grayscale) in resting and macrophages that engulfed filamentous Legionella (red). Phagosome-associated LAMP1 was defined as LAMP1 that colocalized onto and to the periphery of Legionella . Dashed lines contours the cell. Free lysosomes were interpreted as LAMP1 puncta not proximal to phagosomes. Scale bar = 10 µm. B. Quantification of LAMP1-puncta per cell as described in A. C, H. Macrophages were given no particles (Mock phagocytosis, lanes 1-4) or allowed to engulf BSA-anti-BSA-opsonized magnetic beads (Magnetic beads, lanes 5-8) for 2 h, followed by 1 h incubation to elicit maturation. Phagosomes were then magnetically isolated, washed with PBS, and analyzed with Western Blot. LAMP1 (C) and VAMP3 (H) abundance were probed as a proxy for lysosome and endosome content, respectively. GAPDH was used as a loading control in whole cell lysates (WCL). Post-magnetic lysate (PML) is content remaining in lysate after magnetic separation; Wash fractions (W); Magnetic phagosome fraction is represented by M. D, I : Quantification of percent LAMP1 (D) and VAMP3 (I) remaining in lysate after magnetic separation. Percent remaining protein is defined as the percent ratio of GAPDH-normalized protein content in the PML fraction (lanes 2 and 6 for mock phagocytosis and magnetic beads, respectively) to GAPDH-normalized protein content in the WCL fraction (lanes 1 and 5 for mock phagocytosis and magnetic beads, respectively). E, J : Analysis in C and H, respectively, but normalized to mock phagocytosis to account for experimental variation in absolute values. Data represented as mean ± STD of N=4 independent experiments. Sample means were compared using two-tailed, paired Student’s t-test. *: p<0.05; **: p<0.01 (D, I) or one sample t and Wilcoxon test (E, J). F. Macrophages were transfected to express VAMP3-GFP (magenta) and then were given no particles (resting) or allowed to engulf IgG-opsonized beads for 2 h. Cytosol was demarcated with CFSE staining (green). Phagosome-associated VAMP3 was defined as VAMP3 that colocalized to the phagosome periphery as defined by black void in CFSE resulting from the bead contour. Free endosomes were interpreted as small, VAMP3 puncta not proximal to phagosomes. Scale bar = 10 µm. G. Quantification of VAMP3-puncta per cell as described in E. Data represented as mean ± SEM of N=3 independent experiments using 24-40 individual cells per condition per replicate. Data was analysed two-tailed, paired Student’s t-test.

Article Snippet: Membranes were blocked with 5% BSA in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature, then incubated overnight at 4°C with the following primary antibodies diluted in 1% BSA in TBS-T: 1:1000 rabbit anti-Na + /K + ATPase monoclonal antibodies (clone number: EP1845Y; Abcam), 1:1000 rat anti-LAMP1 monoclonal antibodies (clone number: 1D4B, Santa Cruz Biotechnology), 1:400 rabbit anti-Cellubrevin/VAMP3 polyclonal antibodies (104103(SY), Synaptic Systems), 1:1000 rabbit anti-CD16A/B polyclonal antibodies (BS-6028R, ThermoFisher Scientific), 1:1000 rabbit anti-CD64 polyclonal antibodies (PA5-116539, ThermoFisher Scientific), 1:1000 rabbit anti-GAPDH antibodies (clone number: 14C10; Cell Signaling Technology).

Techniques: Staining, Magnetic Beads, Incubation, Isolation, Western Blot, Control, Two Tailed Test, Transfection