Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy
Harper JW, Sharan Swarup, Vinay V. Eapen, Melissa Hoyer
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Abstract
Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe a method for monitoring protein recruitment to damaged lysosomes via immunofluorescence and confocal imaging.
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Steps
Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy
Plate the cells (to be selected by the investigator) into 12 well glass bottom dishes (No. 1.5, 14 mm glass diameter, MatTek) are grown to 50-70% confluency in media.
Treat the cells with 500micromolar (µM) – 1millimolar (mM) of LLoMe for 1h 0m 0s.
Remove the LLoMe containing media from the cells and replace with fresh media not containing LLoMe.
After the indicated washout timepoint (4h for optimal receptor recruitment), wash the cells one time with phosphate buffered saline (PBS) and then fix with 4% paraformaldehyde in PBS for 0h 15m 0s at Room temperature.
Remove 4% paraformaldehyde in PBS, wash the cells once with PBS, and then solubilize cells with 0.1% triton-X in PBS for 0h 15m 0s at Room temperature.
Block the cells for 0h 30m 0s at Room temperature with sterile filtered blocking buffer (1% bovine serum albumin, 0.1% triton-X in PBS).
Add primary antibodies for relevant cargo adaptors or TBK1 to blocking buffer at 1:300 and then spun down for 0h 1m 0s at 10000x g,0h 0m 0s. Remove the blocking buffer completely and then apply the antibody in blocking buffer to the cells (100µL applied to the center of the well where the glass coverslip is attached) for 1h 0m 0s at Room temperature.
Wash the cells 4 times with PBS (5min for each wash).
Add fluorescently conjugated secondary antibodies to blocking buffer at 1:300 and then spun down for 0h 1m 0s at 10000x g,0h 0m 0s. Remove the blocking buffer completely and then apply the antibody in blocking buffer to the cells (100µL applied to the center of the well where the glass coverslip is attached) for 1h 0m 0s at Room temperature.
Wash the cells 4 times with PBS (0h 5m 0s for each wash) and left in PBS.
Image the cells at Room temperature using a Yokogawa CSU-X1 spinning disk confocal on a Nikon Ti-E inverted microscope at the Nikon Imaging Center in Harvard Medical School. Use the Nikon Perfect Focus System to maintain cell focus over time. Equip the microscope with a Nikon Plan Apo 40x/1.30 N.A or 100x/1.40 N.A objective lens.
Display Z series as maximum z-projections and save using Fiji software.
Perform the Mander’s Overlap Correlation (MOC) in lysosomes in CellProfiler (see attached CellProfiler pipeline file).
Each channel z series are brightness and adjust contrast equally and then convert to rgb for publication using FIJI software.
