Analysis of Lysophagic Flux in Cultured Cells using Lyso-Keima
Harper JW, Sharan Swarup, Vinay V. Eapen, Melisa 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 facile method for monitoring lysophagy using the acid sensitive fluorophore mKeima, affixed onto Galectin-3, which allows for the monitoring of lysophagic flux by Flow cytometry, Western blotting or Confocal imaging. This method, which we have termed Lyso-Keima, serves as a facile and quantitative assay for monitoring lysophagy in tissue culture cells.
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Steps
Generation of Stable Cell line expressing mKeima-Galectin-3
Pack mKeima tagged Galectin 3 Lentiviral vector in HEK293T by cotransfection of pPAX2, pMD2 and the vector of interest in a 4:2:1 ratio using polyethelenimine (PEI).
Collect virus containing supernatant 2 days after transfection and filter through a .22 micron syringe filter. Add polybrene at 8mg/mL to the viral supernatant.
After infecting target cells with varying amounts of relevant viruses, select cells in puromycin (1mg/mL for Hela cells, will vary for other cell lines).
Analysis of Lyso-Keima using Fluorescent activated cell sorting (FACS)
Grow cells stably expressing Keima-Galectin-3 to 50-70% confluency in 6-well plates in triplicates.
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.
At the time of harvesting, trypsinize cells, pellet at 1000rpm,0h 0m 0s for 0h 3m 0s, and then resuspend in FACS buffer (1X PBS, 2% FBS). Filter the resuspended cells through cell strainer caps into FACS tubes and place them On ice. Analyze the cells (~10,000 per replicate) by flow cytometry the data was exported into Flowjo.
Gating Strategy
Select the live cells by gating Side Scatter (Area-SSC-A) with Forward Scatter (Area-FSC-A).
Select the singlets by gating SSC-A with SSC-W(width).
Select the Keima positive cells from the singlets by plotting the Acidic Keima (Texas Red A) to the Neutral Keima (BB630-A).
Calculate the ratio of Acidic Keima to Neutral in flowjo by dividing the mean of acidic keima signal to neutral keima signal.
Analysis of Lyso-Keima using Western Blotting of processed Keima
Treat the cells as in section 2 (Analysis of Lyso-Keima using Fluorescent activated cell sorting (FACS)).
For western blotting, collect the cell pellets and resuspend in 8Molarity (M) Urea buffer (8Molarity (M) Urea, 150millimolar (mM) TRIS 7.4, 50millimolar (mM) NaCl) supplemented with Protease and Phosphatase Inhibitors.
Sonicate the resuspended pellets, and spin the lysate at 13000rpm,0h 0m 0s for 0h 10m 0s. Perform the Bradford or BCA assay on clarified lysate and boil the equal amounts of lysate in 1X SDS containing Laemmeli buffer.
Run the lysates on 4-20% Tris Glycine gels (BioRad) and transfer via Wet transfer onto PVDF membranes for immunoblotting with the indicated antibodies. For Anti Keima Immunoblotting use -Keima antibody at 1:1000 dilution in 5% Milk TBST (Tris buffered saline -Tween 1%).
Acquire the images of blots using Enhanced-Chemi luminescence via film or digital imaging.
Analysis of Lyso-Keima using Live Cell Fluorescent Microscopy (LC-FM)
Plate the cells stably expressing Keima-Galectin-3 onto 35mm-glass bottom dishes (No. 1.5, 14mm glass diameter, MatTek) and grow to 50-70% confluency in media (Dulbecco’s MEM (DMEM), high glucose, pyruvate supplemented with 10% fetal bovine serum).
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 and devoid of phenol red (FluoroBrite DMEM supplemented with 10% FBS).
After the indicated washout timepoint, image the cells at 37°C 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. Collect the pairs of images for ratiometric analysis of mKeima fluorescence sequentially using 100 mW 442 nm and 100 mW 561 solid state lasers and collect the emission with a 620/60 nm filter (Chroma Technologies). Collect all images with a Hamamatsu ORCA-ER cooled CCD camera (6.45 µm2 photodiode) with MetaMorph image acquisition software.
Display the Z series as maximum z-projections and save using Fiji software.
Analyze acidic Keima-LGALS3 puncta at 12h washout in CellProfiler using the same pipeline for each condition (see attached CellProfiler pipeline).
Each channel z series are brightness and adjust contrast equally and then convert to rgb for publication using FIJI software.
Apply a “Fire” look up table in Fiji to the exported “image math” image to show the acidic signal (561/442) hotspots and then convert to rgb for publication using Fiji software.
Convert the image of the acidic puncta which is identified (also exported from CellProfiler) to rgb for publication using Fiji software.
