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* These authors contributed equally
A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.
Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons.
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.
Lysosomes are catabolic organelles that degrade macromolecules via the lysosomal-autophagy pathway1. Besides degradation, lysosomes are involved in diverse cellular functions such as signaling transduction, nutrient sensing, and secretion2,3,4. Perturbations in lysosomal function have been implicated in lysosomal storage disorders, cancer, aging, and neurodegeneration3,5,6,7. For postmitotic and highly polarized neurons, lyso....
All procedures were approved by the George Washington University biosafety and ethics committee. The compositions of media and buffers used in this protocol are provided in Table 1. The commercial product information used here is provided in the Table of Materials.
1. Human iPSC-derived neuron culture
This lysosome proximity labeling proteomics study was conducted in human iPSC-derived neurons to capture the dynamic lysosomal microenvironment in situ in live neurons. Cell morphologies of hiPSCs and hiPSC-derived neurons at different time points are illustrated in Figure 2A. Human iPSCs grow in colonies in E8 medium. Differentiation is initiated by plating iPSCs into doxycycline-containing neuron induction medium. Neurite extensions become more visible each day during the 3 day di.......
Using this LAMP1-APEX probe, proteins on and near the lysosomal membrane are biotinylated and enriched. Given the typical lysosome diameter of 100-1,200 nm, this method provides excellent intracellular resolution with a 10-20 nm labeling radius. LAMP1 is an abundant lysosomal membrane protein and a classical marker for lysosomes, serving as an excellent bait protein for lysosomal APEX labeling at the endogenous expression level. However, limitations also exist when using LAMP1 to target lysosomes, as LAMP1 is also presen.......
This study is supported by the NIH grant (R01NS121608). A.M.F. acknowledges the ARCS-Metro Washington Chapter Scholarship and the Bourbon F. Scribner Endowment Fellowship. We thank the Michael Ward lab at the National Institute for Neurological Disorders and Stroke (NINDS) for molecular biology support and the i3Neuron technology development.
....Name | Company | Catalog Number | Comments |
10% (w/v) Saponin solution | Acros Organics | 419231000 | Flourescent Microscopy |
Accutase | Life Technologies | A1110501 | cell detachment solution, Cell Culture |
B27 Supplement | Fisher Scientific | 17504044 | Cell Culture, Cortical Neuron Medium |
BDNF | PeproTech | 450-02 | Cell Culture, Cortical Neuron Medium |
Boric acid | Sigma-Aldrich | B6768 | Cell Culture, Borate Buffer |
Bovine Serum Albumin | Millipore Sigma | A8806 | To make standard solutions to measure total protein concentrations |
Brainphys neuronal medium | STEMCELL Technologies | 5790 | Cell Culture, Cortical Neuron Medium |
CD45R (B220) Antibody Alexa Fluor 561 | Thermo Fisher Scientific | 505-0452-82 | Flourescent Microscopy |
Chroman1 ROCK inhibitor | Tocris | 716310 | Cell Culture |
cOmplete mini Protease Inhibitor | Roche | 4693123001 | cocktail inhibitor in Lysis Buffer |
DC Protein Assay Kit II | Bio-Rad | 5000112 | To determine total protein concentrations of cell lysate |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D8418 | Proximity-labeling Reaction |
DMEM/F12 medium | Thermo Fisher Scientific | 11320082 | Cell Culture, Dish Coating |
DMEM/F12 medium with HEPES | Thermo Fisher Scientific | 11330057 | Cell Culture, Induction Medium |
Donkey serum | Sigma-Aldrich | D9663 | Flourescent Microscopy |
Doxycycline hyclate, ≥98% (HPLC) | Sigma-Aldrich | D9891-1G | Cell Culture, Induction Medium |
Essential 8 Medium | Thermo Fisher Scientific | A1517001 | Cell Culture |
Essential 8 Supplement (50x) | Thermo Fisher Scientific | A1517101 | Cell Culture |
Extraction plate vacuum manifold kit | Waters | WAT097944 | For Peptide desalting |
Formic Acid (FA) | Fisher Scientific | A11750 | For LC-MS analysis |
GDNF | PeproTech | 450-10 | Cell Culture, Cortical Neuron Medium |
Hoechst dye | Thermo Fisher Scientific | 62239 | Flourescent Microscopy |
HPLC grade methanol | Fisher Scientific | A452 | For Peptide desalting |
HPLC grade water | Fisher Scientific | W5 | For Peptide desalting |
Human induced pluripotent stem cells | Corriell Institute | GM25256 | Cell Culture |
Hydrogen peroxide, ACS, 29-32% w/w aq. soln., stab. | Thermo Fisher Scientific | AA33323AD | Proximity-labeling Reaction |
Iodoacetamide (IAA) | Millipore Sigma | I6125 | For Protein Digestion |
Laminin | Fisher Scientific | 23017015 | Cell Culture, Cortical Neuron Medium |
LC-MS grade Acetonitrile | Fisher Scientific | A955 | For LC-MS analysis |
LC-MS grade water | Fisher Scientific | W64 | For LC-MS analysis |
L-glutamine | Fisher Scientific | 25-030-081 | Cell Culture, Induction Medium |
Matrigel | Thermo Fisher Scientific | 08-774-552 | basement membrane matrix, Cell Culture, Dish Coating |
Mouse anti-human LAMP1 monoclonal antibody | Developmental Studies Hybridoma Bank | h4a3 | Flourescent Microscopy |
N-2 Supplement (100x) | Fisher Scientific | 17-502-048 | Cell Culture, Induction Medium |
Nitrocellulose Membrane, Precut, 0.45 µm, 7 x 8.5 cm | Bio-Rad | 1620145 | To conduct dot blot assay for bead titration |
Non-essential amino acids (NEAA) | Fisher Scientific | 11-140-050 | Cell Culture, Induction Medium |
NT-3 | PeproTech | 450-03 | Cell Culture, Cortical Neuron Medium |
Oasis HLB 96-well solid phase extraction plate | Waters | 186000309 | For Peptide desalting |
Odyssey Blocking Buffer (TBS) | LI-COR Biosciences | 927-50000 | To conduct dot blot assay for bead titration |
Paraformaldehyde | Electron Microscopy Sciences | 15710 | Flourescent Microscopy |
Phenol Biotin (1,000x stock) | Adipogen | 41994-02-9 | Proximity-labeling Reaction |
Phosphate-buffered saline (PBS) without calcium or magnesium | Gibco | 10010049 | Cell Culture, Proximity-labeling Reaction, Flourescent Microscopy |
Pierce Quantitative Colorimetric Peptide Assay | Thermo Fisher | 23275 | Peptide Concentration Assay |
Poly-L-Ornithine (PLO) | Millipore Sigma | P3655 | Cell Culture, Dish Coating |
Sodium Ascorbate | Sigma-Aldrich | A4034 | Proximity-Labeling Quench Buffer, Lysis Buffer |
Sodium azide | Sigma-Aldrich | S8032 | Proximity-Labeling Quench Buffer, Lysis Buffer, Flourescent Microscopy |
Sodium chloride | Thermo Fisher Scientific | S271500 | Cell Culture, Borate Buffer |
Sodium dodecyl sulfate (SDS) | Thermo Fisher Scientific | BP1311220 | Lysis Buffer, Dot blot assay buffer, Beads wash buffer |
Sodium hydroxide | Sigma-Aldrich | 415413 | Cell Culture, Borate Buffer |
Sodium tetraborate | Sigma-Aldrich | 221732 | Cell Culture, Borate Buffer |
SpeedVac concentrator | vacuum concentrator | ||
Streptavidin Magnetic Sepharose Beads | Cytiva (formal GE) | 28-9857-99 | Enrich biotinylated proteins |
Streptavidin, Alexa Fluor 680 Conjugate | Thermo Fisher Scientific | S32358 | To conduct dot blot assay for bead titration |
Thermomixer | temperature-controlled mixer | ||
Trifluoacetic acid (TFA) | Millipore Sigma | 302031 | For Peptide desalting |
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) | Millipore Sigma | C4706 | For Protein Digestion |
Tris-HCl | Thermo Fisher Scientific | BP152500 | Lysis Buffer, Dot blot assay buffer, Beads wash buffer |
Triton-X | Thermo Fisher Scientific | BP151500 | Beads wash buffer |
TROLOX | Sigma-Aldrich | 648471 | Proximity-Labeling Quench Buffer, Lysis Buffer |
Trypsin/Lys-C Mix, Mass Spec Grade | Promega | V5073 | For Protein Digestion |
TWEENÂ 20 | Millipore Sigma | P1379 | Dot blot assay buffer |
Urea | Thermo Fisher Scientific | BP169500 | Beads wash and On-Beads Digestion Buffer |
Vitronectin | STEMCELL Technologies | 7180 | Cell Culture, Dish Coating |
Y-27632 ROCK inhibitor | Selleck | S1049 | Cell Culture |
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