TITLE: Characterizing spatial and temporal properties of ER-phagy receptors
AUTHORS: Melissa Hoyer, Harper JW

[DESCRIPTION]
The endoplasmic reticulum (ER) has a vast proteomic landscape to preform many diverse functions including protein and lipid synthesis, calcium ion flux, and inter-organelle communication. The ER proteome is remodeled in part through membrane-embedded receptors linking ER to degradative autophagy machinery (selective ER-phagy). A refined tubular ER network is formed in neurons within highly polarized dendrites and axons. Autophagy-deficient neurons in vivodisplay axonal ER accumulation within synaptic ER boutons, and the ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, mechanisms and receptor selectivity underlying ER remodeling by autophagy in neurons is limited. Here, we combine a genetically tractable induced neuron (iNeuron) system for monitoring extensive ER remodeling during differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy.Using spatial sensors or ER-phagy we demonstrate receptor-specific autophagic capture of ER in axons.

[STEPS]
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
1. Electroporation of PB vectors. Use ThermoFisher kit and ThermoFisher Neon Electroporator to electroporate ES cells with PB vector and PB helper vector.
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
2. Add 10microliter buffer R to a sterile 1.5ml tube. Add 1.0mg of pAC150- TEX264-GFP, pAC150-TEX264deltaLIR-GFP, or pAC150- FAM134C-GFP vectors and 1.0mg of pCMV-hyPBase hyperactive piggyBac vector. Pipet up and down to mix. Let it sit at RT for 10min. This is enough for 2 transfections (== one 6 well). 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
3. Individualize cells with Accutase. Neutralize Accutase with 5 times volume E8 with Rock inhibitor. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
4. Count cells. You will need 2x105 for each transfection.
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
5. Spin down cells. Let it sit for a while so all the residue media can go down to the bottom of the tube. If the residue media is too much, take it out with a P200 pipet. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
6. Resuspend cells to a concentration of 2x105 per 5 microliter (ie 4x107 per ml) using buffer R. You don’t have to take all the residue media off but you will need to take into account the volume of residue media so you are not too much off. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
7. Prepare a 6 well matrigel coated plate. Add 2mL of E8+ rock inhibitor (1:1000) to the wells you will use. Two transfections go into one well. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
8. Wipe the Neon pipet station with EtOH and place it inside the hood. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
9. Add 3ml of electrolytic buffer (buffer E) to the neon tube. Place the tube inside the station. You should feel a click before the tube is securely seated in the station. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
10. Use program 13 from the optimization tab for electroporation parameter (Voltage: 1100. Pulse width: 20 Pulse number: 2 ). Program 9 should also work. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
11. When everything is ready, mix 10-11microliters of resuspended cells with the plasmid containing R buffer. The final volume should be in the range of 21-22microliters. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
12. Take up a neon tip, pipet 10microliters of the cell protein mix and electroporate with program 13. It is important to pipet slowly to avoid air bubble formation. It is also important to insert the pipet slowly into the station, especially during the end of the insertion when you will feel a click. Help the pipet down slowly during the clicking so there is no sudden movement of the tip, which might create tiny air bubbles. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
13. If you see air bubble in the tip, take it out, push everything out of the tip and re-pipet the mixture. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
14. If you see sparking during the electroporation, your efficiency will reduce significantly. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
15. Once electroporation is complete, push everything into one well of a 6 well plate. Do not pipet up and down with Neon tip. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
16. Repeat the same procedure with the same tip and the left over cell mixture. Place the second electroporated mixture into the same well. 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
17. Disperse cells evenly in the well and place cells in a low O2 incubator. 17. Put electroporated cells into low oxygen incubator for 2 days 
SECTION: Generation of Stable Cell ES H9 Ngn2 line expressing ER receptors
18. Start selection of cells with 50mg/mL hygromycin B 4 days post-electroporation. Grow cells in selection medium for 7-10 days until there is no longer any cell death and every cell has integration. Cells can also be sorted for equal amount of GFP expression.
SECTION: Differentiation of Stable Cell ES to induced neurons (iN) for imaging
19. Differentiation to induced neurons (iN) is done by following the protocol “Neural differentiation of AAVS1-TREG3-NGN2 pluripotent stem cells (dx.doi.org/10.17504/protocols.io.br9em93e)
SECTION: Differentiation of Stable Cell ES to induced neurons (iN) for imaging
19.1. To help get spread out iNeurons for imaging, when cells get 90 percent confluent at any point in the day 5-7 range, cells are plated into onto 35 mm-glass bottom dishes (No. 1.5, 14 mm glass diameter, MatTek) at a low confluency (approximately 1x105 cells per 2mL dish).
SECTION: Lentiviral iN transduction
20. LC3B tagged lentiviral pHAGE constructs (or if not using a stable piggyBac ER-phagy receptor line, co transduced with ER-phagy receptor lentiviral constructs) were packaged in HEK293T by cotransfection of pPAX2, pMD2 and the vector of interest in a 4:2:1 ratio using polyethelenimine (PEI). Normally a 6cm dish of 70-80% confluent HEK293T was transfected.
SECTION: Lentiviral iN transduction
21. The next day the media on the transfected HEK293T cells is switched to ND2 no doxycycline media. If 6cm of HEK293T cells was transfected, 5mL of the ND2 no DOX media is added.
SECTION: Lentiviral iN transduction
22. The following day, virus containing supernatant (ND2 media with virus) was and filtered through a .22 micron syringe filter, aliquoted into epi tubes and frozen at -80C. 
SECTION: Lentiviral iN transduction
23. Target induced neurons are cultured to day10. After routine ND2 no doxycycline feeding cells were treated with varying amounts of the virus-containing media (for 20-40 percent iN confluent 2mL imaging dish, 300-500 microliters of ER-phagy receptor lentivirus media was added and 100-200 microliters of mCh-LC3B lentivirus media was added). 
SECTION: Analysis of autophagosomes and ER-phagy receptors in iN via confocal microscopy
24. Cells are imaged at various days in the differentiation live at 37C using a Yokogawa CSU-X1 spinning disk confocal on a Nikon Ti-E inverted microscope at the Nikon Imaging Center in Harvard Medical School. Nikon Perfect Focus System was used to maintain cell focus over time. The microscope is equipped with a Nikon Plan Apo 40x/1.30 N.A or 100x/1.40 N.A objective lens. 488nm (100mW) and 561nm (100mW) laser lines are controlled by AOTF.All images are collected with a Hamamatsu ORCA-ER cooled CCD camera (6.45 µm2 photodiode) with Nikon Elements image acquisition software. Timelapses with various lengths and intervals were acquired. 
SECTION: Analysis of autophagosomes and ER-phagy receptors in iN via confocal microscopy
25. Each channel time series are brightness and contrast adjusted equally and then converted to RGB for publication using FIJI software.