Oct 14, 2024

Public workspaceEndo-IP and Lyso-IP in hESCs and iNeurons 

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg32zoev25/v1
  • Frances V Hundley1,2,
  • Miguel A. Gonzalez-Lozano1,2,
  • Harper JW1,2
  • 1Harvard Medical School;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Frances V Hundley
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg32zoev25/v1
Protocol Citation: Frances V Hundley, Miguel A. Gonzalez-Lozano, Harper JW 2024. Endo-IP and Lyso-IP in hESCs and iNeurons . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32zoev25/v1
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: October 01, 2024
Last Modified: October 14, 2024
Protocol Integer ID: 108744
Keywords: ASAPCRN, positive endosome isolation technique, capturing endosome, endosome, downstream proteomic analysis of the endoslysomal system, hundreds of resident endolysosomal protein, resident endolysosomal protein, endolysosomal protein, positive lysosome, endolysosomal system, lysosome, endoslysomal system, downstream proteomic analysis, endo, human embyronic stem cell, ineuron
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-025160
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Abstract
Building off of our previously published EEA1-positive endosome isolation technique (Endo-IP), we describe an approach for capturing endosomes and TMEM192-positive lysosomes (Lyso-IP) from human embyronic stem cells (hESCs) and induced cortical-like neurons (iNeurons) providing a relatively rapid means by which to examine the endolysosomal system. This approach enriches hundreds of resident endolysosomal proteins and cargo, and enables downstream proteomic analysis of the endoslysomal system, along with other applications.




Troubleshooting
Endosomal immunoprecipitation (Endo-IP) from hESCs and iNeurons for immunoblotting and proteomics
Seed cells on 3x15-cm Matrigel-coated dishes per replicate.
Note
Note that untagged control cells must be included in each experiment to assess the IP efficiency.

Critical
For hESCs, seed cells 1-3 days before performing the Endo-IP such that they will be ~70% confluent on the day of the Endo-IP.
For iNeurons, seed cells on day 4, 5, or 6 of the differentiation, aiming for ~60% confluency, and perform the Endo-IP on day 21 of the differentiation.
Note
Endo-IP can be performed at any desired stage of differentiation.

Harvest cells.
Place each 15-cm on ice, gently pour media into a waste container, and gently aspirate remaining media.
Wash once on-plate by gently adding Amount2 mL ice-cold PBS then gently aspirating PBS.

Add Amount1 mL ice-cold PBS supplemented with protease inhibitors (Roche), gently detach cells with a cell scraper, and transfer cells to tubes on ice using a wide-bore transfer pipette.

To recover residual cells, add an additional Amount1 mL of ice-cold PBS supplemented with protease inhibitors to each plate, and transfer cells to corresponding tubes using a wide-bore transfer pipette.

Pellet cells at 500xg for Duration00:04:00 at Temperature4 °C . Remove supernatant.

4m
Gently resuspend cells using a wide-bore transfer pipette withAmount950 µL KPBS (100mM potassium phosphate, 25mM KCl, pH 7.2) with protease inhibitors (KPBS+i).

Lyse cells.
Lyse cells on ice with 30 strokes of a 2mL Dounce homogenizer (DWK Life Sciences) and B type pestle.
Note
Cell lysis is a critical step that can affect the efficiency of the Endo-IP. Dounce cells gently but thoroughly, and ensure that bubbles do not form during douncing. Cell lysis efficiency can be gauged under a light microscope using trypan blue. Lysis of ~50-70% of the cells is ideal as higher percent lysed usually results in lower efficiency enrichment of EEA1-positive endosomes, possibly due to rupture of organelle membranes.


Critical
Pellet lysate at 1,000xg for Duration00:05:00 at Temperature4 °C . Transfer supernatant to new tube on ice using wide-bore transfer pipette, and spin lysate again at 1,000xg for Duration00:05:00 at Temperature4 °C .

10m
Transfer final post-nuclear supernatant (PNS) to a new tube on ice using a wide-bore transfer pipette, and estimate the total final volume of PNS.
Quantify total protein in all PNS samples using a Bradford protein assay or similar. Normalize samples based on protein quantification result such that each sample has the same total protein and same final volume, using KPBS+i to normalize volume.
For later immunoblotting, set aside Amount10 µL of each PNS and combine with Amount30 µL 1xRIPA buffer or similar and Amount10 µL 5xLDS buffer with DTT.

Endo-IP.
Prepare Amount70 µL anti-FLAG M2 magnetic bead slurry (Sigma-Aldrich) per sample, plus extra to account for a small amount of loss during washes. Wash beads three times with KPBS+i using a magnetic stand and resuspend in Amountx µL KPBS+i, where x = (0.75)*(total original volume of slurry transferred from stock bottle of beads).

Incubate each normalized PNS sample with Amount70 µL washed anti-FLAG M2 magnetic bead slurry at Temperature4 °C for Duration00:50:00 with gentle rotation.

50m
Wash beads & Elute.
After incubation, briefly (~Duration00:00:01 ) spin down tube, place tube on magnetic rack, and set aside Amount10 µL of the flow through and combine with Amount30 µL 1xRIPA buffer or similar and Amount10 µL 5xLDS buffer with DTT for later immunoblotting.

1s
Remove the remainder of the flow through, and wash beads three times with Amount1000 µL KPBS+i.
Note
To ensure optimal enrichment and minimal background, washes must be performed quickly and thoroughly. Ensure beads stuck in the lid after incubation are washed along with the beads in the rest of the tube. To reduce background, sample can be transferred to a fresh tube on the final wash and/or protein low-bind tubes can be used starting at douncing step through final elution step.


Critical
Elute each sample with Amount120 µL 0.5% NP-40 in KPBS+i at Temperature4 °C for Duration00:30:00 with gentle rotation.

30m
Briefly (~Duration00:00:01 ) spin down tube after elution incubation, place tube on magnetic rack, and set aside Amount20 µL of eluate and combine with Amount6 µL 5xLDS and DTT for later immunoblotting. The remaining Amount100 µL of eluate can be further processed for proteomics or stored at Temperature-80 °C .

1s
Lysosomal immunoprecipitation (Lyso-IP) from hESCs and iNeurons for immunoblotting and proteomics
4m
Seed cells on 2x15-cm Matrigel-coated dishes per replicate.
Note
Note that untagged control cells must be included in each experiment to assess the IP efficiency.

Critical
For hESCs, seed cells 1-3 days before performing the Lyso-IP such that they will be ~70% confluent on the day of the Lyso-IP.
For iNeurons, seed cells on day 4, 5, or 6 of the differentiation, aiming for ~60% confluency, and perform the Lyso-IP on day 21 of the differentiation.
Note
Lyso-IP can be performed at any desired stage of differentiation.

Harvest cells.
Place each 15-cm on ice, gently pour media into a waste container, and gently aspirate remaining media.
Wash once on-plate by gently adding Amount2 mL ice-cold PBS then gently aspirating PBS.
Add Amount1 mL ice-cold PBS supplemented with protease inhibitors (Roche), gently detach cells with a cell scraper, and transfer cells to tubes on ice using a wide-bore transfer pipette.
To recover residual cells, add an additional Amount1 mL of ice-cold PBS supplemented with protease inhibitors to each plate, and transfer cells to corresponding tubes using a wide-bore transfer pipette.
Pellet cells at 500xg for Duration00:04:00 at Temperature4 °C . Remove supernatant.
4m
Gently resuspend cells using a wide-bore transfer pipette withAmount950 µL KPBS (100mM potassium phosphate, 25mM KCl, pH 7.2) with protease inhibitors (KPBS+i).
Lyse cells.
Lyse cells on ice with 30 strokes of a 2mL Dounce homogenizer (DWK Life Sciences) and B type pestle.
Note
Cell lysis is a critical step that can affect the efficiency of the Lyso-IP. Dounce cells gently but thoroughly, and ensure that bubbles do not form during douncing. Cell lysis efficiency can be gauged under a light microscope using trypan blue. Lysis of ~50-70% of the cells is ideal as higher percent lysed usually results in lower efficiency enrichment of TMEM192-positive lysosomes possibly due to rupture of organelle membranes.

Critical
Pellet lysate at 1,000xg for Duration00:05:00 at Temperature4 °C . Transfer supernatant to new tube on ice using wide-bore transfer pipette, and spin lysate again at 1,000xg for Duration00:05:00 at Temperature4 °C .
10m
Transfer final post-nuclear supernatant (PNS) to a new tube on ice using a wide-bore transfer pipette, and estimate the total final volume of PNS.
Quantify total protein in all PNS samples using a Bradford protein assay or similar. Normalize samples based on protein quantification result such that each sample has the same total protein and same final volume, using KPBS+i to normalize volume.
For later immunoblotting, set aside Amount10 µL of each PNS and combine with Amount30 µL 1xRIPA buffer or similar and Amount10 µL 5xLDS buffer with DTT.
Lyso-IP.
Prepare Amount65 µL anti-HA magnetic bead slurry (Pierce) per sample, plus extra to account for a small amount of loss during washes. Wash beads three times with KPBS+i using a magnetic stand and resuspend in Amounty µL KPBS+i, where y = total original volume of slurry transferred from stock bottle of beads.

Incubate each normalized PNS sample with Amount65 µL washed anti-HA magnetic bead slurry at Temperature4 °C for Duration00:30:00 with gentle rotation.
30m
Wash beads & Elute.
After incubation, briefly (~Duration00:00:01 ) spin down tube, place tube on magnetic rack, and set aside Amount10 µL of the flow through and combine with Amount30 µL 1xRIPA buffer or similar and Amount10 µL 5xLDS buffer with DTT for later immunoblotting.
1s
Remove the remainder of the flow through, and wash beads twice with Amount1000 µL high salt KPBS+i (supplemented with 150mM NaCl) and wash once with Amount1000 µL KPBS+i.
Note
To ensure optimal enrichment and minimal background, washes must be performed quickly and thoroughly. Ensure beads stuck in the lid after incubation are washed along with the beads in the rest of the tube. To reduce background, sample can be transferred to a fresh tube on the final wash and/or protein low-bind tubes can be used starting at douncing step through final elution step.

Critical
Elute each sample with Amount120 µL 0.5% NP-40 in KPBS+i at Temperature4 °C for Duration00:30:00 with gentle rotation.
30m
Briefly (~Duration00:00:01 ) spin down tube after elution incubation, place tube on magnetic rack, and set aside Amount20 µL of eluate and combine with Amount6 µL 5xLDS and DTT for later immunoblotting. The remaining Amount100 µL of eluate can be further processed for proteomics or stored at Temperature-80 °C
1s
Protocol references
Frances V. Hundley, Miguel A. Gonzalez-Lozano, Lena M. Gottschalk, Aslan N. K. Cook, Jiuchun Zhang, Joao A. Paulo, J. Wade Harper. Endo-IP and Lyso-IP Toolkit for Endolysosomal Profiling of Human Induced Neurons
bioRxiv 2024.09.24.614704; doi: https://doi.org/10.1101/2024.09.24.614704

Park, H., Hundley, F.V., Yu, Q. et al. Spatial snapshots of amyloid precursor protein intramembrane processing via early endosome proteomics. Nat Commun 13, 6112 (2022). https://doi.org/10.1038/s41467-022-33881-x

Abu-Remaileh, M., Wyant, G.A. et al. Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes.Science 358,807-813(2017).DOI:10.1126/science.aan6298