Combinatorial selective ER-phagy remodels the ER during neurogenesis.
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: 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 remodeling, 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 ER and the role of individual ER-phagy receptors is limited. Here, we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodeling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both 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 remodeling and versatile genetic toolkit provides a quantitative framework for understanding contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.
Pubmed ID: 37425907
Research resources used in this publication
- Bio Rad ChemiDoc MP Imaging System (RRID:SCR_019037) (instrument resource)
- Thermo Orbitrap Fusion Tribrid Mass Spectrometer (RRID:SCR_020559) (instrument resource)
- CHOPCHOP (RRID:SCR_015723) (web application)
- NIS-Elements (RRID:SCR_014329) (software resource)
- Image Lab Software (RRID:SCR_014210) (software resource)
- Sony Biotechnology SH800S Cell Sorter (RRID:SCR_018066) (instrument resource)
- Attune Nxt Nxt (RRID:SCR_019590) (instrument resource)
- Fiji (RRID:SCR_002285) (software resource)
- CellProfiler Image Analysis Software (RRID:SCR_007358) (data processing software)
- HEK293 (RRID:CVCL_0045) (cell line)
- FlowJo (RRID:SCR_008520) (software resource)
- GraphPad Prism (RRID:SCR_002798) (software resource)
- WA09 (RRID:CVCL_9773) (cell line)
- Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568 (RRID:AB_143157) (antibody)
- GAPDH (D16H11) XP Rabbit mAb (RRID:AB_10622025) (antibody)
Additional research tools detected in this publication
Antibodies used in this publication
- Goat anti-Rabbit IgG (Heavy Chain), Superclonal Recombinant Secondary Antibody, Alexa Fluor™ 647 (RRID:AB_2536101)
- Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568 (RRID:AB_143157)
- Goat anti-Chicken IgY (H+L) Secondary Antibody, Alexa Fluor™ 488 (RRID:AB_2534096)
- Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (RRID:AB_2534069)
- GAPDH (D16H11) XP Rabbit mAb (RRID:AB_10622025)
- calreticulin antibody (RRID:AB_513777)
- Nogo (C-4) (RRID:AB_10709573)
- Goat Anti-Guinea Pig IgG (H+L) Highly Cross-adsorbed Antibody, Alexa Fluor 647 Conjugated (RRID:AB_141882)
- Anti-MAP 2 (RRID:AB_2138181)
- Goat Anti-Guinea Pig IgG (H+L) Highly Cross-adsorbed Antibody, Alexa Fluor ?? 488 Conjugated (RRID:AB_2534117)
- Mouse Anti-Neurofilament, Heavy, 200 kD Monoclonal Antibody, Unconjugated, Clone NF-01 (RRID:AB_306084)
- anti-Keima monoclonal antibody (RRID:AB_10794910)
- REEP5 antibody (RRID:AB_2178440)
- HSP90 antibody (RRID:AB_2881429)
- hFAB™ Rhodamine Anti-Tubulin Primary Antibody (RRID:AB_2884950)
- CCPG1 (E3C5G) Rabbit mAb (RRID:AB_2935809)
- Anti-FAM134C antibody produced in rabbit (RRID:AB_1853027)
- FAM134B antibody (RRID:AB_2878879)
- Anti-REEP4 antibody produced in rabbit (RRID:AB_2571730)
- Anti-REEP1 antibody produced in rabbit (RRID:AB_2683591)
- Anti-TEX264 antibody produced in rabbit (RRID:AB_1857910)
Associated grants
- Agency: NIGMS NIH HHS, United States
Id: R01 GM132129
Publication data is provided by the National Library of Medicine ® and PubMed ®. Data is retrieved from PubMed ® on a weekly schedule. For terms and conditions see the National Library of Medicine Terms and Conditions.
This is a list of tools and resources that we have found mentioned in this publication.
ATCC (tool)
RRID:SCR_001672
Global nonprofit biological resource center (BRC) and research organization that provides biological products, technical services and educational programs to private industry, government and academic organizations. Its mission is to acquire, authenticate, preserve, develop and distribute biological materials, information, technology, intellectual property and standards for the advancement and application of scientific knowledge. The primary purpose of ATCC is to use its resources and experience as a BRC to become the world leader in standard biological reference materials management, intellectual property resource management and translational research as applied to biomaterial development, standardization and certification. ATCC characterizes cell lines, bacteria, viruses, fungi and protozoa, as well as develops and evaluates assays and techniques for validating research resources and preserving and distributing biological materials to the public and private sector research communities.
View all literature mentionsAddgene (tool)
RRID:SCR_002037
Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
View all literature mentionsThermo Fisher Scientific (tool)
RRID:SCR_008452
Commercial vendor and service provider of laboratory reagents and antibodies. Supplier of scientific instrumentation, reagents and consumables, and software services.
View all literature mentionsAdobe Illustrator (tool)
RRID:SCR_010279
Vector graphics software to create digital graphics, illustrations, and typography for several types of media: print, web, interactive, video, and mobile.
View all literature mentionsggplot2 (tool)
RRID:SCR_014601
Open source software package for statistical programming language R to create plots based on grammar of graphics. Used for data visualization to break up graphs into semantic components such as scales and layers.
View all literature mentionsThermoFisher Scientific EVOS M5000 Imaging System (tool)
RRID:SCR_023650
Microsoft system includes integrated digital inverted benchtop microscope for four-color fluorescence, transmitted-light, and color imaging. Provides interchangeable optics with autofocus, single-click multi-channel image acquisition.3.2 Megapixels, monochrome, CMOS camera. Offers software for acquisition, analysis, and automated cell counting.
View all literature mentionsGoat anti-Rabbit IgG (Heavy Chain), Superclonal Recombinant Secondary Antibody, Alexa Fluor™ 647 (antibody)
RRID:AB_2536101
This recombinant polyclonal secondary targets IgG (Heavy Chain)
View all literature mentionsGoat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568 (antibody)
RRID:AB_143157
This polyclonal secondary targets IgG (H+L)
View all literature mentionsGoat anti-Chicken IgY (H+L) Secondary Antibody, Alexa Fluor™ 488 (antibody)
RRID:AB_2534096
This polyclonal secondary targets IgY (H+L)
View all literature mentionsGoat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (antibody)
RRID:AB_2534069
This polyclonal secondary targets IgG (H+L)
View all literature mentionsGAPDH (D16H11) XP Rabbit mAb (antibody)
RRID:AB_10622025
This monoclonal targets GAPDH
View all literature mentionscalreticulin antibody (antibody)
RRID:AB_513777
This polyclonal targets calreticulin
View all literature mentionsNogo (C-4) (antibody)
RRID:AB_10709573
This monoclonal targets Nogo (C-4)
View all literature mentionsGoat Anti-Guinea Pig IgG (H+L) Highly Cross-adsorbed Antibody, Alexa Fluor 647 Conjugated (antibody)
RRID:AB_141882
This polyclonal secondary targets IgG (H+L)
View all literature mentionsGoat Anti-Guinea Pig IgG (H+L) Highly Cross-adsorbed Antibody, Alexa Fluor ?? 488 Conjugated (antibody)
RRID:AB_2534117
This unknown targets Guinea Pig IgG (H+L)
View all literature mentionsMouse Anti-Neurofilament, Heavy, 200 kD Monoclonal Antibody, Unconjugated, Clone NF-01 (antibody)
RRID:AB_306084
This monoclonal targets 200 kD Neurofilament Heavy
View all literature mentionsanti-Keima monoclonal antibody (antibody)
RRID:AB_10794910
This unknown targets anti-Keima monoclonal antibody
View all literature mentionsREEP5 antibody (antibody)
RRID:AB_2178440
This polyclonal targets REEP5
View all literature mentionsHSP90 antibody (antibody)
RRID:AB_2881429
This monoclonal targets HSP90
View all literature mentionshFAB™ Rhodamine Anti-Tubulin Primary Antibody (antibody)
RRID:AB_2884950
This unknown targets Tubulin
View all literature mentionsCCPG1 (E3C5G) Rabbit mAb (antibody)
RRID:AB_2935809
This monoclonal targets CCPG1
View all literature mentionsAnti-FAM134C antibody produced in rabbit (antibody)
RRID:AB_1853027
This unknown targets Human FAM134C
View all literature mentionsFAM134B antibody (antibody)
RRID:AB_2878879
This polyclonal targets FAM134B
View all literature mentionsAnti-REEP4 antibody produced in rabbit (antibody)
RRID:AB_2571730
This polyclonal targets
View all literature mentionsAnti-REEP1 antibody produced in rabbit (antibody)
RRID:AB_2683591
This polyclonal targets REEP1
View all literature mentionsAnti-TEX264 antibody produced in rabbit (antibody)
RRID:AB_1857910
This unknown targets Human TEX264
View all literature mentionsBio Rad ChemiDoc MP Imaging System (instrument resource)
RRID:SCR_019037
Instrument for imaging and analyzing gels and western blots by Bio Rad. Designed to address multiplex fluorescent western blotting, chemiluminescence detection, general gel documentation applications, and stain-free technology imaging needs.
View all literature mentionsThermo Orbitrap Fusion Tribrid Mass Spectrometer (instrument resource)
RRID:SCR_020559
Thermo Orbitrap Fusion Tribrid mass spectrometer and Waters M-Class Acquity nanoUPLC is a versatile platform. This platform features sequential acquisition capabilities and multiple fragmentation types, enabling complex, in depth proteomic experiments.
View all literature mentionsCHOPCHOP (web application)
RRID:SCR_015723
Web application for designing gRNAs for CRISPR/Cas9 experiments. It selects target sites for CRISPR/Cas9, CRISPR/Cpf1 or TALEN-directed mutagenesis.
View all literature mentionsNIS-Elements (software resource)
RRID:SCR_014329
Microscope imaging software suite used with Nikon products. NIS-Elements includes software applications for advanced and standard research, documentation, confocal microscopy, and high-content analysis.
View all literature mentionsImage Lab Software (software resource)
RRID:SCR_014210
Imaging software used to acquire and analyze images from specific Bio-Rad imaging systems. Users can analyze gel or blot features, capture optimized image data, and generate a report of the data. Image Lab software exclusively runs on the Gel Doc EZ imager, Gel Doc XR+ imaging system, ChemiDo MP, ChemiDoc XRS+ imaging systems, Criterion Stain Free imager, and the GS-900calibrated densitometer.
View all literature mentionsSony Biotechnology SH800S Cell Sorter (instrument resource)
RRID:SCR_018066
Benchtop cell sorter that sorts wide range of cell sizes for many applications using microfluidic sorting chips. Integrated with comprehensive fluidics controls and advanced automation for set-up, acquisition, sort and analysis. System software supports sorting into tubes and 96- and 384-well plates. Software generates FCS 3.0 and FCS 3.1 files that can be exported to third party analysis tools. For non-clinical research use only. For applications in which cross-contamination is concern, optional e-beam sterilized consumables can be used to replace sample line and sorting chip. Optional class A2 Level II biosafety cabinet provides protection for personnel and products. Biosafety cabinet meets international standards.Optical design offers up to four collinear excitation lasers (488 nm, 405nm, 561nm and 638nm) and six fluorescence detectors. The six free-form PMTs enable detection of fluorescence signals from any laser based on filter selection.
View all literature mentionsAttune Nxt Nxt (instrument resource)
RRID:SCR_019590
2020 Attune NxT Flow Cytometer makes multiparametric flow cytometry available to both new and experienced researchers.
View all literature mentionsFiji (software resource)
RRID:SCR_002285
Software package as distribution of ImageJ and ImageJ2 together with Java, Java3D and plugins organized into coherent menu structure. Used to assist research in life sciences.
View all literature mentionsCellProfiler Image Analysis Software (data processing software)
RRID:SCR_007358
Software tool to enable biologists without training in computer vision or programming to quantitatively measure phenotypes from thousands of images automatically. It counts cells and also measures the size, shape, intensity and texture of every cell (and every labeled subcellular compartment) in every image. It was designed for high throughput screening but can perform automated image analysis for images from time-lapse movies and low-throughput experiments. CellProfiler has an increasing number of algorithms to identify and measure properties of neuronal cell types.
View all literature mentionsHEK293 (cell line)
RRID:CVCL_0045
Cell line HEK293 is a Transformed cell line with a species of origin Homo sapiens (Human)
View all literature mentionsFlowJo (software resource)
RRID:SCR_008520
Software for single-cell flow cytometry analysis. Its functions include management, display, manipulation, analysis and publication of the data stream produced by flow and mass cytometers.
View all literature mentionsGraphPad Prism (software resource)
RRID:SCR_002798
Statistical analysis software that combines scientific graphing, comprehensive curve fitting (nonlinear regression), understandable statistics, and data organization. Designed for biological research applications in pharmacology, physiology, and other biological fields for data analysis, hypothesis testing, and modeling.
View all literature mentionsWA09 (cell line)
RRID:CVCL_9773
Cell line WA09 is a Embryonic stem cell with a species of origin Homo sapiens (Human)
View all literature mentionsGoat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568 (antibody)
RRID:AB_143157
This polyclonal secondary targets IgG (H+L)
View all literature mentionsGAPDH (D16H11) XP Rabbit mAb (antibody)
RRID:AB_10622025
This monoclonal targets GAPDH
View all literature mentions
