Sep 06, 2021
Version 1
Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy V.1
- Vinay V. Eapen1,
- Sharan Swarup1,
- Melissa Hoyer1,
- Harper JW1
- 1Department of Cell Biology, Harvard Medical School, Boston MA 02115
- Harper JW: wade_harper@hms.harvard.edu
External link: https://doi.org/10.7554/eLife.72328
Protocol Citation: Vinay V. Eapen, Sharan Swarup, Melissa Hoyer, Harper JW 2021. Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy. protocols.io https://dx.doi.org/10.17504/protocols.io.bxghpjt6
Manuscript citation:
Eapen VV, Swarup S, Hoyer MJ, Paulo JA, Harper JW, Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy. eLife doi: 10.7554/eLife.72328
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: August 18, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 52457
Keywords: Immunofluorescence, Autophagic cargo receptors, p-TBK1, LAMP1, Lysosomes, Lysophagy, ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
NIH
Grant ID: NS110395
NIH
Grant ID: NS083524
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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.
Materials
Materials:
| A | B | C | |
| REAGENT or RESOURCE | SOURCE | IDENTIFIER | |
| Chemicals | |||
| LLoMe (L-Leucyl-L-Leucine methyl ester (hydrochloride)) | Cayman Chemical | 16008 | |
| Dulbecco’s MEM (DMEM), high glucose, pyruvate | GIBCO / Invitrogen | 11995 | |
| Phosphate Buffered Saline 1X | Corning | 21-031-CV | |
| Fetal Bovine Serum | Fisher | SH3008003 | |
| Bovine Serum Albumin | Gold biotechnology | A-420-250 | |
| paraformaldehyde | Electron Microscopy Sciences | 15710 | |
| Triton-X | Sigma | T8787 | |
| Antibodies | |||
| LAMP1 (D401S) Mouse mAb | Cell Signaling Technology | 15665S | |
| Anti-CALCOCO2 antibody produced in rabbit | Abcam | ab68588 | |
| Anti-OPTN antibody produced in rabbit | Sigma | HPA003279 | |
| Anti-TAX1BP1 antibody produced in rabbit | Sigma | HPA024432 | |
| phospho-TBK1/NAK (Ser172) (D52C2) Rabbit mAb | Cell Signaling Technology | 5483S | |
| Software | |||
| Cell Profiler | CellProfiler v4.0.6 | https://cellprofiler.org/ | |
| Fiji | ImageJ V.2.0.0 | https://imagej.net/software/fiji/ | |
| Metamorph | Metamorph v | https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#gref |
Chemicals:
L-Leucyl-L-Leucine methyl ester (hydrochloride)Cayman Chemical CompanyCatalog #16008
DMEM, high glucose, pyruvateThermo FisherCatalog #11995065
Phosphate Buffered Saline (PBS)CorningCatalog #MT21-031-CV
ParaformaldehydeElectron Microscopy SciencesCatalog #15710
Triton X-100Sigma AldrichCatalog #T8787
Antibodies:
LAMP1 (D4O1S) Mouse mAb #15665Cell Signaling TechnologyCatalog #15665S
Anti-NDP52 antibody (ab68588)AbcamCatalog #ab68588
Anti-OPTN antibody produced in rabbitSigma AldrichCatalog #HPA003279
Anti-TAX1BP1 antibody produced in rabbitSigma AldrichCatalog #HPA024432
Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb #5483Cell Signaling TechnologyCatalog #5483S
Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy
Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy
4h 7m
4h 7m
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.
Note
For HeLa cells, we use Dulbecco’s MEM (DMEM), high glucose (4500 mg/L), pyruvate (100 mg/L) supplemented with 10% fetal bovine serum
Treat the cells with 500 micromolar (µM) – 1 millimolar (mM) of LLoMe for 01:00:00 .
Note
Note: The exact dosage varies with cell line and should be determined empirically depending on the line used. This dose range has been tested extensively in Hela cells, and routinely generated lysophagic flux.
1h
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 00:15:00 at Room temperature .
15m
Remove 4% paraformaldehyde in PBS, wash the cells once with PBS, and then solubilize cells with 0.1% triton-X in PBS for 00:15:00 at Room temperature .
15m
Block the cells for 00:30:00 at Room temperature with sterile filtered blocking buffer (1% bovine serum albumin, 0.1% triton-X in PBS).
30m
Add primary antibodies for relevant cargo adaptors or TBK1 to blocking buffer at 1:300 and then spun down for 00:01:00 at 10000 x g . Remove the blocking buffer completely and then apply the antibody in blocking buffer to the cells (100 µL applied to the center of the well where the glass coverslip is attached) for 01:00:00 at Room temperature .
1h 1m
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 00:01:00 at 10000 x g . Remove the blocking buffer completely and then apply the antibody in blocking buffer to the cells (100 µL applied to the center of the well where the glass coverslip is attached) for 01:00:00 at Room temperature .
1h 1m
Wash the cells 4 times with PBS (00:05:00 for each wash) and left in PBS.
5m
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.
Note
445nm (75mW), 488nm (100mW), 561nm (100mW) & 642nm (100mW) laser lines are controlled by AOTF. Collect all images with a Hamamatsu ORCA-ER cooled CCD camera (6.45 µm2 photodiode) with MetaMorph image acquisition software.
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).
Note
Each field of view for every unique condition is thresholded in the same way. The “identify primary objects” tool is used to find puncta for both the lysosome channel and for the respective receptor or p-TBK1 stain. The “measure colocalization” module is used to compare the fluorescence intensities within the areas defined by the threshold. The MOC with Costes was reported for each field of view..
Each channel z series are brightness and adjust contrast equally and then convert to rgb for publication using FIJI software.