Combinatorial selective ER-phagy remodels the ER during neurogenesis

Harper JW, Melissa Hoyer, Cristina Capitanio, Anna Bieber, Florian Wilfling, Brenda A. Schulman, Ian R. Smith, Julia C. Paoli, Yizhi Jiang, Joao A. Paulo

Published: 2024-02-09 DOI: 10.17504/protocols.io.81wgbx13nlpk/v3

ASAPCRN

ER-phagy

induced neurons

axons

whole cell proteomics

differentiation

live cell fluorescence microscopy

Keima flux

autophagy

AI 解读

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.

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Combinatorial selective ER-phagy remodels the ER during neurogenesis

Abstract

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