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Neuroscienze

Karakterisering af neuronalt lysosominteractom med nærhedsmærkning proteomics

Published: June 23, 2022
doi:
10.3791/64132
, ,
1Department of Chemistry,The George Washington University

Summary

En neuronal lysosomnærhed mærkning proteomics protokol er beskrevet her for at karakterisere det dynamiske lysosomale mikromiljø i humant inducerede pluripotente stamcelleafledte neuroner. Lysosomale membranproteiner og proteiner, der interagerer med lysosomer (stabilt eller forbigående), kan nøjagtigt kvantificeres i denne metode med fremragende intracellulær rumlig opløsning i levende humane neuroner.

Abstract

Lysosomer kommunikerer ofte med en række biomolekyler for at opnå nedbrydning og andre forskellige cellulære funktioner. Lysosomer er kritiske for menneskets hjernefunktion, da neuroner er postmitotiske og er stærkt afhængige af autofagi-lysosomvejen for at opretholde cellulær homeostase. På trods af fremskridt i forståelsen af forskellige lysosomale funktioner er det teknisk udfordrende at fange den meget dynamiske kommunikation mellem lysosomer og andre cellulære komponenter, især på en måde med høj kapacitet. Her leveres en detaljeret protokol for den nyligt offentliggjorte endogene (knock-in) lysosomnærhedsmærkning proteomiske metode i humaninducerede pluripotente stamcelle (hiPSC) -afledte neuroner.

Både lysosomale membranproteiner og proteiner, der omgiver lysosomer inden for en radius på 10-20 nm, kan med sikkerhed identificeres og kvantificeres nøjagtigt i levende humane neuroner. Hvert trin i protokollen er beskrevet detaljeret, dvs. hiPSC-neuronkultur, nærhedsmærkning, neuronhøst, fluorescensmikroskopi, biotinyleret proteinberigelse, proteinfordøjelse, LC-MS-analyse og dataanalyse. Sammenfattende giver denne unikke endogene lysosomale nærhedsmærkningsproteomics-metode et højt gennemstrømnings- og robust analytisk værktøj til at studere de meget dynamiske lysosomale aktiviteter i levende menneskelige neuroner.

Introduction

Lysosomer er kataboliske organeller, der nedbryder makromolekyler via lysosomal-autofagivejen1. Udover nedbrydning er lysosomer involveret i forskellige cellulære funktioner såsom signaltransduktion, næringsstofføling og sekretion 2,3,4. Forstyrrelser i lysosomal funktion har været impliceret i lysosomale opbevaringsforstyrrelser, kræft, aldring og neurodegeneration 3,5,6,7. For postmitotiske og stærkt polariserede neuroner spiller lysosomer kritiske roller i neuronal cellulær homeostase, frigivelse af neurotransmitter og langdistancetransport langs axonerne 8,9,10,11. Imidlertid har undersøgelse af lysosomer i humane neuroner været en udfordrende opgave. Nylige fremskridt inden for inducerede pluripotente stamcelle (iPSC) -afledte neuronteknologier har gjort det muligt for kulturen af levende menneskelige neuroner, der tidligere var utilgængelige, og bygger bro over kløften mellem dyremodeller og menneskelige patienter til at studere den menneskelige hjerne12,13. Især integrerer den avancerede i3Neuron-teknologi stabilt neurogenin-2-transkriptionsfaktoren i iPSC-genomet under en doxycyclin-inducerbar promotor, hvilket får iPSC’er til at differentiere sig til rene kortikale neuroner i 2 uger14,15.

På grund af den meget dynamiske lysosomale aktivitet er det teknisk udfordrende at fange lysosomale interaktioner med andre cellulære komponenter, især på en måde med høj kapacitet. Nærhedsmærkningsteknologi er velegnet til at studere disse dynamiske interaktioner på grund af dens evne til at fange både stabile og forbigående / svage proteininteraktioner med enestående rumlig specificitet16,17. Konstrueret peroxidase eller biotin ligase kan genetisk smeltes sammen med agnproteinet. Ved aktivering produceres stærkt reaktive biotinradikaler for kovalent at mærke naboproteiner, som derefter kan beriges med streptavidinbelagte perler til nedstrøms bottom-up proteomics via væskekromatografi-massespektrometri (LC-MS) platforme 17,18,19,20,21.

En endogen lysosomal nærhedsmærkning proteomics metode blev for nylig udviklet til at fange det dynamiske lysosomale mikromiljø ii 3neuroner22. Konstrueret ascorbatperoxidase (APEX2) blev banket ind på C-terminalen af det lysosomale associerede membranprotein 1 (LAMP1) i iPSC’er, som derefter kan differentieres til kortikale neuroner. LAMP1 er et rigeligt lysosomalt membranprotein og en klassisk lysosomal markør23. LAMP1 udtrykkes også i sene endosomer, som modnes til lysosomer; Disse sene endosom-lysosomer og ikke-nedbrydende lysosomer omtales alle som lysosomer i denne protokol. Denne endogene LAMP1-APEX-sonde, udtrykt på det fysiologiske niveau, kan reducere LAMP1-fejllokalisering og overudtryksartefakter. Hundredvis af lysosomale membranproteiner og lysosomale interactors kan identificeres og kvantificeres med fremragende rumlig opløsning i levende menneskelige neuroner.

Her beskrives en detaljeret protokol for lysosomnærhedsmærkning af proteomics i humane iPSC-afledte neuroner med yderligere forbedringer fra den nyligt offentliggjorte metode22. Den overordnede arbejdsgang er illustreret i figur 1. Protokollen inkluderer hiPSC-afledt neuronkultur, nærhedsmærkningsaktivering i neuroner, validering af APEX-aktivitet ved fluorescensmikroskopi, bestemmelse af et optimalt streptavidin-perle-til-input-proteinforhold, berigelse af biotinylerede proteiner, proteinfordøjelse på perler, peptidafsaltning og kvantificering, LC-MS-analyse og proteomics-dataanalyse. Retningslinjer for fejlfinding og eksperimentelle optimeringer diskuteres også for at forbedre kvalitetskontrol og ydeevne for nærhedsmærkning.

Protocol

Alle procedurer blev godkendt af George Washington University biosikkerhed og etik komité. Sammensætningerne af medier og buffere, der anvendes i denne protokol, er angivet i tabel 1. De kommercielle produktoplysninger, der bruges her, findes i materialetabellen. 1. Human iPSC-afledt neuronkultur Human iPSC-kultur og LAMP1-APEX-sondeintegration (7 dage)Matrigel stamopløsning optøes i en isspand ved 4 °C natten over, aliq…

Representative Results

Denne lysosom-nærhedsmærkning proteomics-undersøgelse blev udført i humane iPSC-afledte neuroner for at fange det dynamiske lysosomale mikromiljø in situ i levende neuroner. Cellemorfologier af hiPSC’er og hiPSC-afledte neuroner på forskellige tidspunkter er illustreret i figur 2A. Humane iPSC’er vokser i kolonier i E8-medium. Differentiering initieres ved at plettere iPSC’er i doxycyclinholdigt neuroninduktionsmedium. Neurite-udvidelser bliver mere synlige hver dag i løbet a…

Discussion

Ved hjælp af denne LAMP1-APEX-sonde biotinyleres proteiner på og nær lysosomalmembranen og beriges. I betragtning af den typiske lysosomdiameter på 100-1.200 nm giver denne metode fremragende intracellulær opløsning med en mærkningsradius på 10-20 nm. LAMP1 er et rigeligt lysosomalt membranprotein og en klassisk markør for lysosomer, der tjener som et fremragende agnprotein til lysosomal APEX-mærkning på det endogene ekspressionsniveau. Der findes dog også begrænsninger, når man bruger LAMP1 til at målrett…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Denne undersøgelse understøttes af NIH-bevillingen (R01NS121608). A.M.F. anerkender ARCS-Metro Washington Chapter Scholarship og Bourbon F. Scribner Endowment Fellowship. Vi takker Michael Ward-laboratoriet ved National Institute for Neurological Disorders and Stroke (NINDS) for molekylærbiologisk støtte og i3Neuron-teknologiudviklingen.

Materials

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
TWEEN 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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Tags

Proximity Labeling ProteomicsLysosomal MicroenvironmentIPSC-derived NeuronsLysosomal DysfunctionBrain DiseasesBiotin PhenolHydrogen PeroxideAcetone PrecipitationProtein PelletDCA Assay
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Frankenfield, A., Ni, J., Hao, L. Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics. J. Vis. Exp. (184), e64132, doi:10.3791/64132 (2022).
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