Feb 08, 2024

Public workspaceCombinatorial selective ER-phagy remodels the ER during neurogenesis V.3

DOI
dx.doi.org/10.17504/protocols.io.81wgbx13nlpk/v3
  • 1Harvard Medical School;
  • 2Max Planck Institute of Biochemistry;
  • 3Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry
  • Cristina Capitanio: *Joint second author;
  • Ian R. Smith: *Joint second author;
Melissa Hoyer
Open access
DOI: dx.doi.org/10.17504/protocols.io.81wgbx13nlpk/v3
Collection Citation: Melissa Hoyer, Cristina Capitanio, Ian R. Smith, Julia C. Paoli, Anna Bieber, Yizhi Jiang, Joao A. Paulo, Florian Wilfling, Brenda A. Schulman, Harper JW 2024. Combinatorial selective ER-phagy remodels the ER during neurogenesis. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbx13nlpk/v3Version created by Melissa Hoyer
Manuscript citation:
Hoyer MJ, Capitanio, C, Smith IR*, Paoli, JC, Bieber, A, Jiang, Y, Paulo JA, Gonzalez-Lozano, MA, Wilfling, F, Schulman, BA, and Harper JW (2023). Combinatorial selective ER-phagy remodels the ER during neurogenesis.
*, equal contribution
License: This is an open access collection 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 collection and it's working
Created: February 08, 2024
Last Modified: May 31, 2024
Collection Integer ID: 94913
Keywords: ASAPCRN, ER-phagy, induced neurons, axons, whole cell proteomics, differentiation, live cell fluorescence microscopy, Keima flux, autophagy
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000282
Abstract
The endoplasmic reticulum (ER) employs a diverse proteome landscape
to orchestrate many cellular functions, ranging from protein and lipid
synthesis to calcium ion flux and inter-organelle communication. A case
in point concerns the process of neurogenesis, where a refined tubular
ER network is assembled via ER shaping proteins into the newly formed
neuronal projections to create highly polarized dendrites and axons.
Previous studies have suggested a role for autophagy in ER remodelling,
as autophagy-deficient neurons in vivo display axonal ER accumulation
within synaptic boutons, and the membrane-embedded ER-phagy receptor
FAM134B has been genetically linked with human sensory and autonomic
neuropathy. However, our understanding of the mechanisms underlying
selective removal of the ER and the role of individual ER-phagy receptors is
limited. Here we combine a genetically tractable induced neuron (iNeuron)
system for monitoring ER remodelling during in vitro differentiation with
proteomic and computational tools to create a quantitative landscape of ER
proteome remodelling via selective autophagy. Through analysis of single
and combinatorial ER-phagy receptor mutants, we delineate the extent to
which each receptor contributes to both the magnitude and selectivity of
ER protein clearance. We define specific subsets of ER membrane or lumenal
proteins as preferred clients for distinct receptors. Using spatial sensors and
flux reporters, we demonstrate receptor-specific autophagic capture of ER
in axons, and directly visualize tubular ER membranes within
autophagosomes in neuronal projections by cryo-electron tomography. This molecular
inventory of ER proteome remodelling and versatile genetic toolkit provide
a quantitative framework for understanding the contributions of individual
ER-phagy receptors for reshaping ER during cell state transitions.
Materials
REAGENT or RESOURCE SOURCE IDENTIFIER RRID
Antibodies
FAM134B Rabbit Polyclonal Antibody Proteintech 21537-1-AP RRID:AB_2878879
FAM134C Rabbit Polyclonal Antibody Sigma-Aldrich HPA016492 RRID:AB_1853027
CCPG1 Rabbit Polyclonal Antibody Cell Signaling Technology 80158 RRID:AB_2935809
TEX264 Rabbit Polyclonal Antibody Sigma-Aldrich HPA017739 RRID:AB_1857910
REEP1 Rabbit Polyclonal Antibody Sigma-Aldrich HPA058061 RRID:AB_2683591
REEP4 Rabbit Polyclonal Antibody Sigma-Aldrich HPA042683 RRID:AB_2571730
REEP5 Rabbit Polyclonal Antibody Proteintech 14643-1-AP RRID:AB_2178440
hFABâ„¢ Rhodamine Anti-Tubulin Antibody BioRad 12004166 RRID:AB_2884950
HSP90 mouse monoclonal Antibody Proteintech 60318 RRID:AB_2881429
Anti-Keima-Red mAb MBL international M182-3M RRID:AB_10794910
Neurofilament heavy polypeptide antibody Abcam ab7795 RRID:AB_306084
MAP2 Guinea Pig Polyclonal Antibody Synaptic systems 188004 RRID:AB_2138181
Nogo-A (C-4) Mouse Monoclonal Antibody Santa Cruz sc-271878 RRID:AB_10709573
Calreticulin Rabbit Polyclonal Antibody Proteintech 10292-1-AP RRID:AB_513777
GAPDH (D16H11) XP Rabbit Monoclonal Antibody Cell Signaling Technology 5174 RRID:AB_10622025
Goat anti-mouse Alexa488 Thermo Fisher Scientific A-11001 RRID:AB_2534069
Goat anti-chicken Alexa488 Thermo Fisher Scientific A11039 RRID:AB_2534096
Goat anti-rabbit Alexa568 Thermo Fisher Scientific A-11011 RRID:AB_143157
Goat anti-rabbit Alexa647 Thermo Fisher Scientific A27040 RRID:AB_2536101
Goat anti-guinea pig Alexa488 Thermo Fisher Scientific A-11073 RRID:AB_2534117
Goat anti-guinea pig Alexa647 Thermo Fisher Scientific A-21450 RRID:AB_141882
Bacterial and virus strains
DH5 alpha E. coli competent cells Homemade
T1R E. coli Competent cells Homemade
Chemicals, peptides, and recombinant proteins
DAPI Thermo Fisher Scientific D1306
TMTproâ„¢ 16plex Label Reagent Set Thermo Scientific A44520
Q5 Hot Start High-Fidelity DNA Polymerase New England BioLabs M0493
QuikChange II Site-Directed Mutagenesis Kit Agilent 200523
MiSeq Reagent Nano Kit v2 (300 cycles) Illumina MS-103-1001
Bafilomycin A1 Cayman Chemical 88899-55-2
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) Thermo Fisher Scientific D1306
16% Paraformaldehyde, Electron-Microscopy Grade Electron Microscopy Science 15710
PhosSTOP Sigma-Aldrich T10282
Protease inhibitor cocktail Roche 4906845001
TCEP Gold Biotechnology TCEP2
Formic Acid Sigma-Aldrich 94318
Trypsin Promega V511C
Lys-C Wako Chemicals 129-02541
Urea Sigma U5378
EPPS Sigma-Aldrich E9502
2-Chloroacetamide Sigma-Aldrich C0267
Trypan Blue Stain Thermo Fisher Scientific Wako Chemicals 129-02541w
Bio-Rad Protein Assay Dye Reagent Concentrate Bio-Rad 5000006
Urea Sigma U5378
EPPS Sigma-Aldrich E9502
2-Chloroacetamide Sigma-Aldrich C0267
Empore SPE Disks C18 3M Sigma-Aldrich 66883-U
Pierce Quantitative Colorimetric Peptide Assay Thermo Fisher Scientific 23275
GeneArt Precision gRNA Synthesis Kit Thermo Fisher Scientific A29377
12 Well glass bottom plate with high performance #1.5 cover glass Cellvis P12-1.5H-N
Nunc Cell-Culture Nunclon Delta Treated 6-well Thermo Fisher Scientific 140685
Nunc Cell-Culture Nunclon Delta Treated 12-well Thermo Fisher Scientific 150628
100x21mm Dish, Nunclon Delta Thermo Fisher Scientific 172931
Corning Matrigel Matrix, Growth Factor Reduced Corning 354230
DMEM/F12 Thermo Fisher Scientific 11330057
Neurobasal Thermo Fisher Scientific 21103049
NEAA Life Technologies 11140050
GlutaMax Life Technologies 35050061
N-2 Supplement Thermo Fisher Scientific 17502048
Neurotrophin-3 (NT-3) Peprotech 450-03
Brain-derived neurotrophic factor (BDNF) Peprotech 450-02
B27 Thermo Fisher Scientific 17504001
Y-27632 Dihydrochloride (ROCK inhibitor) PeproTech 1293823
Cultrex 3D Culture Matrix Laminin I R&D Systems 3446-005-01
Accutase StemCell 7920
FGF3 In-house N/A
Insulin Human Sigma-Aldrich I9278-5ML
TGF-beta PeproTech 100-21C
holo-Transferrin human Sigma-Aldrich T0665
Sodium Bicarbonate Sigma-Aldrich S5761-500G
Sodium selenite Sigma-Aldrich S5261-10G
Doxycycline Sigma-Aldrich D9891
Recombinant SpCas9 Zuris et al., 2015; Orderu
Hygromycin B Thermo Fisher Scientific 10687010
UltraPure 0.5M EDTA, pH 8.0 Thermo Fisher Scientific 15575020
GlutaMAX Thermo Fisher Scientific 35050061
Dulbecco’s MEM (DMEM), high glucose, pyruvate GIBCO / Invitrogen 11995
Lipofectamine 3000 Invitrogen L3000008
Experimental models: Cell lines
HEK293T ATCC CRL-1573 CVCL_0045
H9 Wicell WA9 CVCL_9773
Recombinant DNA
pAC150-Keima-RAMP4 This paper Addgene 201929
pAC150-Keima-REEP5 This paper Addgene 201928
pAC150- FAM134C-GFP This paper Addgene 201932
pAC150- TEX264-GFP This paper Addgene 201931
pAC150- TEX264(deltaLIR, F273A)-GFP This paper Addgene 201930
pHAGE-FAM134C-GFP This paper Addgene 201927
pHAGE-TEX264-GFP An et al 2019 Addgene 201925
pHAGE-TEX264(deltaLIR,F273A)-GFP An et al 2019 Addgene 201926
pHAGE-mCherry-LC3B An et al 2019 Addgene 201924
Software and algorithms
Prism GraphPad, V9 https://www.graphpad.com/scientificsoftware/ prism/ SCR_002798
SEQUEST Eng et al., 1994 N/A
Flowjo Flowjo, v10.7 https://www.flowjo.com SCR_008520
Perseus Perseus v1.6.15.0 Tyanova et al. (2016) https://maxquant.org/perseus/ SCR_007358
Fiji ImageJ V.2.0.0 https://imagej.net/software/fiji/ SCR_002285
Imagelab Biorad, v6.0.1 https://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z&source_wt=imagelabsoftware_surl SCR_014210
Cell Profiler CellProfiler v4.0.6 https://cellprofiler.org/ SCR_007358
Nikon Imaging Software Elements 5.21.3 (Build 1489) SCR_014329
outknocker.org http://www.outknocker.org/outknocker2.htm
ChopChop https://chopchop.cbu.uib.no/ SCR_015723
Instruments
Orbitrap Fusion Lumos Tribrid Mass Spectrometer Thermo Fisher Scientific IQLAAEGAAPFADBMBHQ CR_020562
Orbitrap Eclipse Tribrid Mass Spectrometer Thermo Fisher Scientific FSN04-10000 SCR_020559
Attune NxT Thermo Fisher Scientific SCR_019590
Sony Biotechnology SH800S Cell Sorter Sony Biotechnology SH800S SCR_018066
Neonâ„¢ Transfection System Thermo Fisher Scientific MPK5000 N/A
ChemiDoc MP imaging system BioRad 12003154 SCR_019037
Yokogawa CSU-X1 spinning disk confocal on a Nikon Ti-E inverted microscope Yokogawa/ Nikon
Files
Protocol
Characterizing spatial and temporal properties of ER-phagy receptors
Name
Characterizing spatial and temporal properties of ER-phagy receptors
Version 1
Melissa Hoyer
Melissa HoyerHarvard Medical School
Protocol
Analysis of ER structures in Cultured Induced Neuron axons
Name
Analysis of ER structures in Cultured Induced Neuron axons
Version 1
Melissa Hoyer
Melissa HoyerHarvard Medical School
Protocol
Analysis of ER Flux in Cultured Induced Neurons using Keima ER reporters
Name
ForkAnalysis of ER Flux in Cultured Induced Neurons using Keima ER reporters
Version 1
Melissa Hoyer
Melissa HoyerHarvard Medical School
Protocol
Electroporation of Cas9 protein into human pluripotent stem cells
Name
ForkElectroporation of Cas9 protein into human pluripotent stem cells
Version 1
Melissa Hoyer
Melissa HoyerHarvard Medical School
Protocol
Neural differentiation of AAVS1-TRE3G-NGN2 pluripotent stem cells
Name
ForkNeural differentiation of AAVS1-TRE3G-NGN2 pluripotent stem cells
Version 1
Melissa Hoyer
Melissa HoyerHarvard Medical School
Protocol
Whole-cell proteomics and Analysis by Tandem Mass Tagging-based proteomics
Name
ForkWhole-cell proteomics and Analysis by Tandem Mass Tagging-based proteomics
Version 1
Melissa Hoyer
Melissa HoyerHarvard Medical School
Protocol
Testing ER stress induction in Cultured Induced Neurons via measuring ATF4 protein level or XBP-1 mRNA splicing
Name
Testing ER stress induction in Cultured Induced Neurons via measuring ATF4 protein level or XBP-1 mRNA splicing
Version 1
Melissa Hoyer
Melissa HoyerHarvard Medical School
Protocol
Neural differentiation on EM grids - iNeurons sample preparation for cryo-ET and CLEM
Name
Neural differentiation on EM grids - iNeurons sample preparation for cryo-ET and CLEM
Version 2
Cristina Capitanio
Cristina CapitanioMax Planck Institute of Biochemistry
Protocol
Analysis of nuclei integrity in cultured induced neurons by fluorescence microscopy
Name
Analysis of nuclei integrity in cultured induced neurons by fluorescence microscopy
Version 1
Melissa Hoyer
Melissa HoyerHarvard Medical School
Protocol
Molecular Cloning- Gibson and LR reactions
Name
Molecular Cloning- Gibson and LR reactions
Version c8igzubw
Melissa Hoyer
Melissa HoyerHarvard Medical School
Protocol
Human pluripotent stem cell culture
Name
ForkHuman pluripotent stem cell culture
Version 1
Melissa Hoyer
Melissa HoyerHarvard Medical School
Protocol references
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