Fig. 1: Validation of TRPML1-mNeonGreen-1D4- and TMEM192-mCherry-1D4-positive lysosomes.
From: Visualization of lysosomal membrane proteins by cryo electron tomography
a Schematic of TRPML1-mNeonGreen-1D4 and TMEM192-mCherry-1D4 lysosomes immunopurified from HEK 293 cells, eluted, and plunge frozen on cryoEM grids for cryoET and TRPML1-mNeonGreen-1D4 and TMEM192-mCherry-1D4 HEK 293 cells cultured and plunge frozen on cryoEM grids for cryo confocal microscopy, cryoFIB-SEM, and cryoET. Created in BioRender. McVeigh, B. (https://BioRender.com/ns2gzr5). b Representative fluorescence microscopy image of HEK 293 cells stably overexpressing TRPML1-mNeonGreen-1D4 (green) and a lysosomal marker, LAMP1, (magenta). Scale bar is 10 μm and is 5 μm in MERGE image (n = 3). Pearson’s correlation coefficient measured for TRPML1-mNeonGreen-1D4 and LAMP1 colocalization. Data are presented as a violin plot (n = 5, cells examined over one experiment). Source data are provided as a Source Data file. c Representative fluorescence microscopy image of HEK 293 cells stably overexpressing TMEM192-mCherry-1D4 (green and a lysosomal marker, LAMP1, (magenta). Scale bar is 10 μm and is 5 μm in MERGE image (n = 3). Pearson’s correlation coefficient measured for TMEM192-mCherry-1D4 and LAMP1 colocalization. Data are presented as a violin plot (n = 5, cells examined over one experiment). Source data are provided as a Source Data file. d Fluorescence signal was aligned with and overlaid on a TEM overview of the lamellae using mitochondria (M) as fiducials and a virtual slice through reconstructed tomogram of lysosome/ autolysosome (L/AL) in non-transfected HEK 293 cells (green, LysoTracker Red) and mitochondria (magenta, MitoTracker DR). e TRPML1-mNeonGreen-1D4 HEK 293 cells (green, TRPML1-mNeonGreen-1D4-positive lysosomes) and mitochondria (magenta, MitoTracker DR). f TMEM192-mCherry-1D4 HEK 293 cells (green, TMEM192-mCherry-1D4-positive lysosomes) and mitochondria (magenta, MitoTracker DR). Scale bar of aligned TEM image with fluorescence signal is 500 nm and of virtual slice of reconstructed tomogram is 100 nm. Experiment was performed one time.
