TITLE: Analysis of ER structures in Cultured Induced Neuron axons
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)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.

[STEPS]
SECTION: Genetically modify Ngn2-inducible embryonic stem (ES) cell H9 line using Cas9
1. 1. Genetic editing of Ngn2-inducible ES cells is done using the following protocol “Electroporation of Cas9 protein into human pluripotent stem cells” (dx.doi.org/10.17504/protocols.io.br87m9zn)
SECTION: Differentiation of Stable Cell ES H9 line to induced neurons (iN)
2. 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: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
3. Fixation and immunofluorescence is next done to label the ER, axons and dendrites of the iNs
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
3.1. 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: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
3.2. 2)At day 20, iN are fixed. phosphate buffered saline (1xPBS) with 8% paraformaldehyde is prewarmed to 37C and then slowly added to an equal volume of media on the cells for a resulting 4% paraformaldehyde solution. iN are incubated with this solution at 37C for 15min. 
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
3.3. 3)Fixed iN are washed one time with room temperature 1xPBS 
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
3.4. 4)Fixed iN are solubilized with 0.1% triton-X in PBS for 5 min at RT.
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
3.5. 5)Fixed iN are then blocked for 30 min at RT with sterile filtered blocking buffer (1% bovine serum albumin, 0.1% triton-X in PBS)
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
3.6. 6)Primary antibodies for Endoplasmic reticulum (ER), axons and dendrites are added to blocking buffer at 1:300 and then spun down for 1min at 10,000 x g. The blocking buffer is completely removed and then the antibody in blocking buffer is applied to the cells (100mL applied to the center of the well where the glass coverslip is attached) for 1h at RT.
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
3.7. 7)Cells are washed 4 times with PBS (5min for each wash).
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
3.8. 8)Fluorescently conjugated secondary antibodies are added to blocking buffer at 1:300 and then spun down for 1min at 10,000 x g. The blocking buffer is completely removed and then the antibody in blocking buffer is applied to the cells (100mL applied to the center of the well where the glass coverslip is attached) for 1h at RT.
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
3.9. 9)Cells are washed 4 times with PBS (5min for each wash) and left in 1XPBS. Dapi at 1:10,000 is added in the second 1XPBS wash.
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
4. iN are imaged via confocal fluorescence microscopy and ER, axons and dendrites of the iNs are measured.
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
4.1. 1)Cells are imaged at RT 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 Imaging Elements.
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
4.2. 2)Z series are displayed as maximum z-projections and saved using Fiji software.
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
4.3. 3)The ER positive structures are detected using Cell Profiler with the same pipeline applied for each condition (see attached CellProfiler pipeline for specific thresholding details). Cell nuclei are detected using a “identify primary objects” module with 50-to-125-pixel units min and max and a global minimum cross-entropy thresholding method. Both axon and dendrite area are identified using a “identify primary objects” module with 10-to-40-pixel units min and max using default thresholding method. Each ER structure is identified using a “identify primary objects” module with 5-to-50-pixel units min and max and a global robust background thresholding method. The number of ER structures masked specifically within the axonal regions is related to the number of nuclei in each field of view to yield the number of ER structures in axons per nuclei readout. Object size and shape is measured, and these values are also exported.
SECTION: Analysis of endoplasmic reticulum (ER) in iN via confocal microscopy
4.4. 4)Each channel z series are brightness and contrast adjusted equally and then converted to RGB for publication using FIJI software.