Molecular Cloning- Gibson and LR reactions v1

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)1,2. A refined tubular ER network3,4 is formed in neurons within highly polarized dendrites and axons5,6. Autophagy-deficient neurons in vivodisplay axonal ER accumulation within synaptic ER boutons,7 and the ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy8,9. 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 this system, we imaged fixed iNeuron cultures, imaged these via confocal fluorescence microscopy, and quantified ER structures in axons.