Generation of a homozygous knock-in human embryonic stem cell line expressing SNAP-tagged SOD1

Huang, Pei-San; Wen, Meng-Hsuan; Xie, Xihong; Xu, An; Lee, Dung‐Fang; Chen, Tai-Yen

doi:10.1016/j.scr.2021.102415

Cited by 5 publications

(4 citation statements)

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“…Recent progress of human embryonic stem cells (hESCs)/inducible pluripotent stem cell (iPSC)-derived neurons have shown that they can faithfully recapitulate an individual's idiosyncratic neural development. Generations of knock-in stem cell lines expressing fluorophore-tagged Cu-binding proteins could provide an ideal platform for studying the causality of the mutations of diseases [ 236 , 237 ]. Furthermore, the recent advance in super-resolution microscopy enables researchers to approach biophysical problems like protein kinetics and oligomeric states from a single-molecule perspective.…”

Section: Conclusion Remarks and Perspectivesmentioning

confidence: 99%

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system

et al. 2021

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Imbalanced copper homeostasis and perturbation of membrane trafficking are two common symptoms that have been associated with the pathogenesis of neurodegenerative and neurodevelopmental diseases. Accumulating evidence from biophysical, cellular and in vivo studies suggest that membrane trafficking orchestrates both copper homeostasis and neural functions—however, a systematic review of how copper homeostasis and membrane trafficking interplays in neurons remains lacking. Here, we summarize current knowledge of the general trafficking itineraries for copper transporters and highlight several critical membrane trafficking regulators in maintaining copper homeostasis. We discuss how membrane trafficking regulators may alter copper transporter distribution in different membrane compartments to regulate intracellular copper homeostasis. Using Parkinson's disease and MEDNIK as examples, we further elaborate how misregulated trafficking regulators may interplay parallelly or synergistically with copper dyshomeostasis in devastating pathogenesis in neurodegenerative diseases. Finally, we explore multiple unsolved questions and highlight the existing challenges to understand how copper homeostasis is modulated through membrane trafficking.

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Section: Conclusion Remarks and Perspectivesmentioning

confidence: 99%

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system

et al. 2021

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“…(ii) What key redox-sensitive residues induce structural changes leading to different SOD1 oligomeric stoichiometries? One may address these questions by combing super-resolution microscopy, organelle-specific sensor, and non-targeted proteomics to investigate SOD1 behaviors in homozygous knocked-in lines . This will provide physiologically relevant information on where SOD1 modulates its oligomeric state, what local oxidative stress is, and how post-translational modifications may be responsible for the shifts between different SOD1 oligomers.…”

Section: Discussionmentioning

confidence: 99%

Probing Oxidant Effects on Superoxide Dismutase 1 Oligomeric States in Live Cells Using Single-Molecule Fluorescence Anisotropy

Chen

2023

Chemical & Biomedical Imaging

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The protein Cu/Zn superoxide dismutase (SOD1) is known to function as a dimer, but its concentration in cells (∼50 μM) and the dimerization constant (K d of 500 μM) results suggest that it exists in a monomer-dimer equilibrium. It is unclear how the oligomeric state of SOD1 changes when cells are initially exposed to high levels of extracellular oxidative stress. To address this problem, we introduced the single-molecule fluorescence anisotropy (smFA) assay to explore SOD1 oligomeric states in live COS7 cells. smFA specifically probes the fluorescence polarization changes caused by molecular rotations where the fast-rotating molecules (either due to smaller hydrodynamic volume or less viscous environments) deteriorate the emission polarization and thus lower the anisotropy. After validating that smFA is effective in distinguishing monomeric and dimeric fluorescence proteins, we overexpressed SOD1 in live COS7 cells and investigated how its oligomeric state changes under basal, 2 h, and 24 h 100 μM H 2 O 2 treatments. We found that treating cells with H 2 O 2 promotes SOD1 dimerization and decreases cellular viscosity in 2 h. Interestingly, prolonged H 2 O 2 treatments show similar results as the basal conditions, indicating that cells return to a steady state similar to the basal state after 24 h, despite the presence of H 2 O 2 . Our results demonstrate that SOD1 changes its oligomeric state equilibrium in response to extracellular oxidative stresses. smFA will open new opportunities to explore the relationship between the SOD1 oligomer state and its H 2 O 2 -based signaling and transcription regulation roles.

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“…H1 hESCs were cultured as previously described ( Huang et al, 2021 ; Wen et al, 2019 ). Accutase was used for cell detachment, and ROCK-inhibitor was added on the day of cell attachment.…”

Section: Methodsmentioning

confidence: 99%

Generation of a homozygous knock-in human embryonic stem cell line expressing mEos4b-tagged CTR1

et al. 2022

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Generation of a homozygous knock-in human embryonic stem cell line expressing SNAP-tagged SOD1

Cited by 5 publications

References 5 publications

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system

Crossroads between membrane trafficking machinery and copper homeostasis in the nerve system

Probing Oxidant Effects on Superoxide Dismutase 1 Oligomeric States in Live Cells Using Single-Molecule Fluorescence Anisotropy

Generation of a homozygous knock-in human embryonic stem cell line expressing mEos4b-tagged CTR1

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