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Biochemistry

Endositik alımını ve kültürlü hücrelerde Functionalized Nanobodies kullanarak Trans-Golgi ağına retrograd taşıma analizi

Published: February 21, 2019
doi:
10.3791/59111
,
1Biozentrum,University of Basel

Summary

Golgi için hücre yüzeyinden proteinlerin retrograd taşıma membran homeostazı korumak için önemlidir. Burada, biyokimyasal olarak hücre yüzey Golgi taşıma rekombinant proteinlerin functionalized nanobodies HeLa hücreleri kullanarak analiz etmek için bir yöntem açıklanmaktadır.

Abstract

Proteinler ve membran hücre yüzeyinden Golgi ve ötesine taşıma Homeostazı, organel kimlik ve Fizyoloji için esastır. Retrograd protein Rating çalışmaya, biz son zamanlarda hücre yüzeyinden ışınlamaya Golgi kompleksi, ya sabit ve canlı hücre Imaging, elektron mikroskobu, ya da biyokimyasal olarak analiz etmek için çok yönlü bir nanobody tabanlı araç geliştirdik. Functionalized Anti-yeşil flüoresan protein (GFP) nanobodies mühendislik — küçük, monomeric, yüksek-benzeşme protein bağlayıcı — membran proteinlerinin bir hücre dışı GFP yan ile ilgi ifade hücre hatları için uygulanabilir. Derivatized nanobodies GFP gazetecilere bağlı özellikle içselleştirilmiş ve gazetecilere sıralama güzergah boyunca sırtlama nakletti. Nanobodies retrograd taşıma Floresans mikroskobu tarafından takip etmek ve askorbat peroksidaz 2 elektron tarafından muhabir-nanobody kompleksleri ultrastructural lokalizasyonu araştırmak için (APEX2) ile görüntülemede canlı fluorophores ile functionalized mikroskobu ve Kinetik trans-Golgi ağ (TGN) varış değerlendirmek için tirozin sulfation (TS) motiflerle. Bu yöntembilimsel makalede, biz bacterially hızlı ve functionalized nanobodies arındırmak için genel yordamı anahat. Biz bizim aracı endositik alımı ve kargo proteinler varış TGN analiz etmek için mCherry ve TS modifiye nanobodies kullanarak güçlü kullanımını göstermektedir.

Introduction

Proteinler ve çeşitli hücre içi bölmeleri için hücre yüzeyinden lipidler retrograd Rating membran homeostazı salgı dengelemek için ve bileşenleri anterograd taşıma makineleri1 geri dönüşüm için bakımı için önemlidir , 2. clathrin bağlı veya – bağımsız endositoz ile içselleştirilmesi, protein ve lipit kargo ilk doldurmak erken endosomes daha fazla nerede üzerinden ya plazma zarı için geri dönüşümlü endo lizozomal sistemi boyunca yönlendirildi veya hedeflenen trans-Golgi ağa (TGN). Endosomes ve/veya hücre yüzeyine gelen TGN için geri dönüşüm anterograd transmembran kargo reseptörleri ba * bağlı ve katyon bağımsız mannoz-6-fosfat reseptörleri (CDMPR ve CIMPR) gibi bir dizi fonksiyonel döngüsünün bir parçası olduğunu teslim etmek Yeni sentezlenen lizozomal TGN geç endosomes ve organellerin3,4,5, sortilin ve SorLA6,7ve Wntless (WLS) Wnt ligandlar hücre yüzeyine taşınması için hidrolazlar 8 , 9 , 10 , 11. TGN46 ve onun ilgili izoformlarının12,13,14, tuzaklarına (çözünür N– ethylmaleimide-duyarlı füzyon faktör eki reseptörleri) geri TGN için alındı diğer proteinler vardır 15 , 16 , 17, amiloid precursor protein (APP)18,19, ilerleyici eklem (ANK) protein20, ATP7A/B veya DMT121,22gibi ve transmembran metal taşıyıcı enzimler Karboksipeptidaz D, furin veya BACE123,24,25gibi işleniyor. Endojen bu proteinler dışında bakteriyel ve bitki toksinler (örneğin, Shiga ve kolera toksini, risin ve abrin) retrotranslocation için ER sitozol26,27ulaşmak için retrograd taşıma makineleri kaçırmak, 28,29.

Doğrudan retrograd trafiğini analiz için biz daha önce etiket ve kargo proteinler hücre yüzeyinden hücre içi bölmeleri30‘ a takip nanobody tabanlı bir araç geliştirdik. Nanobodies protein bağlayıcı Devegiller ve kıkırdaklı balıklar31,32içinde doğal olarak oluşan homodimeric ağır zinciri yalnızca antikorlar (hcAbs) türetilen yeni bir aile temsil eder. HcAbs değişken ağır zincir etki alanını (VHH) teşkil ve geleneksel antikorlar (örneğin, IgGs) üzerinde pek çok avantajı var: onlar monomeric, küçük (~ 15 kDa), yüksek oranda çözünür, disülfür bağları, yoksun bacterially ifade ve seçilmiş yüksek-benzeşme bağlama33,34,35,36. Bizim nanobody araç çok yönlü ve geniş uygun yapmak için functionalized anti-GFP nanobodies tagged GFP onların hücre dışı/lumenal etki ile yüzey-etiket ve parça proteinlere çalıştırmaya başladık. Nanobodies (APEX2) mCherry, askorbat peroksidaz 2 ile functionalization tarafından37veya tirozin sulfation (TS) dizileri, retrograd taşıma bonafide transmembran kargo proteinlerin sabit ya da-ebilmek var olmak çözümlemek ve hücre görüntüleme, yaşamak elektron mikroskobu, ya da biyokimyasal olarak. Tyrosylprotein sulfotransferases (TPST1 ve TPST2) aracılı tirozin sulfation ardından bir değişiklik için trans-Golgi sınırlı olduğundan/TGN, biz doğrudan çalışma taşıma ve Kinetik bu hücre yüzeyinden ilgi proteinlerin Hücre içi Golgi yuvası38,39,40.

Bu yöntemleri makalede, biz functionalized nanobodies (VHH-2xTS, – APEX2, – mCherry ve türevleri) memeli hücreleri30retrograd Ulaştırma analiz etmek için uygulamalar için uygun üretim kolaylığı tanımlamak. Biz esas olarak TS siteyi değiştiren nanobody kullanım için sulfation yuvası hücre yüzeyinden hücre içi trafik analiz odaklanın.

Protocol

1. Functionalized Nanobodies ile bakteriyel dönüştürme Not: Bu iletişim kuralı yukarıda açıklanan30ifade, arıtma ve functionalized anti-GFP nanobodies analizi için optimize edilmiştir. Derivatization diğer protein moieties ile bu standart protokol değişikliği gerektirebilir. Chemocompetent bakteri (~ 100 µL) protein ifade için uygun çözülme (örneğin, Escherichia coli Rosetta BL21 (DE3) hücreleri) buz yerleştir…

Representative Results

Retrograd protein taşıma çeşitli hücre içi hedeflere araştırmak için biz son zamanlarda etiket ve rekombinant füzyon proteinler hücre yüzey30takip nanobody tabanlı bir anti-GFP aracı kurduk. Burada, biz böyle bakteri üretimi nanobodies derivatized ve endositik alımını Floresans mikroskobu ve immunoblotting yanı sıra TGN varış göre sulfation analizi araştırmak için bunların kullanımı eğitim için kendi uygulama göstermek göstermek. ?…

Discussion

Nanobodies protein bağlayıcı iskele geleneksel antikorlar üzerinden pek çok avantajları ile ortaya çıkan bir sınıfı temsil: Bunlar küçük, istikrarlı, monomeric,33,35, yüksek benzeşme ve eksikliği disülfür bağları için seçilen 44 , 45. hücre kültür sistemleri ve gelişim biyolojisi46,47,organizmalard…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Bu eser Grant 31003A-162643 tarafından İsviçre Ulusal Bilim Vakfı tarafından desteklenmiştir. Nicole Beuret ve Biozentrum Imaging Core tesisi (IMCF) destek için teşekkür ederiz.

Materials

Anti-GFP antibody Sigma-Aldrich 118144600001 Product is distributed by Sigma-Aldrich, but manufactured by Roche
Anti-His6 antibody Bethyl Laboratories A190-114A
Anti-actin antibody EMD Millipore MAB1501
Goat anti-rabbit HRP Sigma-Aldrich A-0545
Goat anti-mouse HRP Sigma-Aldrich A-0168
4',6-diamidino-2-phenylindole (DAPI) Sigma-Aldrich D9542 dissolved in 1 x PBS/1%BSA
Dimethyl sulfoxide (DMSO) Applichem A3672
D-biotin Sigma-Aldrich B4501 dissolved in sterile 500 mM NaH2PO4 or DMSO
5-aminolevuilnic acid (dALA) hydrochloride Sigma-Aldrich A3785 dissolved in sterile water
DNase I Applichem A3778 dissolved in sterile water
Lysozyme Sigma-Aldrich 18037059001 Product is distributed by Sigma-Aldrich, but manufactured by Roche
Brefeldin A (BFA) Sigma-Aldrich B5936
Puromycin Invivogen ant-pr-1
Penicillin/Streptomycin Bioconcept 4-01F00-H
L-glutamine Applichem A3704
Dulbecco’s modified Eagle’s medium (DMEM) Sigma-Aldrich D5796
Fetal calf serum (FCS) Biowest S181B-500
Sulfur-35 as sodium sulfate Hartmann Analytics ARS0105 Product contains 5 mCi
Earle's balanced salts Sigma-Aldrich E6267
MEM amino acids (50 x) solution Sigma-Aldrich M5550
MEM vitamin solution (100 x) Sigma-Aldrich M6895
cOmplete, Mini Protease inhibitor cocktail Sigma-Aldrich 11836153001 Product is distributed by Sigma-Aldrich, but manufactured by Roche
Isopropyl-β-D-thiogalactopyranosid (IPTG) Applichem A1008 dissolved in sterile water, stock is 1 M
Carbenicillin disodium salt Applichem A1491 dissolved in sterile water, stock is 100 mg/mL
Kanamycin sulfate Applichem A1493 dissolved in sterile water, stock is 100 mg/mL
Coomassie-R (Brilliant Blue) Sigma-Aldrich B-0149
Paraformaldehyde (PFA) Applichem A3813
Bovine serum albumin (BSA) Sigma-Aldrich A2153
Fluoromount-G Southern Biotech 0100-01
Ni Sepharose High Performance GE Healthcare 17-5268-01
His GraviTrap columns GE Healthcare GE11-0033-99
His buffer kit GE Healthcare GE11-0034-00
Disposable PD10 desalting columns GE Healthcare GE17-0851-01
Mini-Protean TGX gels, 4-20%, 15-well Bio-Rad 456-1096
Dulbecco’s phosphate buffered saline (DPBS) w/o Ca2+/Mg2+ Sigma-Aldrich D8537
35-mm dishes Falcon 353001
6-well plates TPP 92406
Glass coverslips (No. 1.5H) VWR 631-0153
Phenylmethylsulfonyl fluoride (PMSF) Applichem A0999.0025 dissolved in 40% DMSO 60% isopropanol, stock in 500 mM
Tryptone Applichem A1553
Yeast extract Applichem A1552
Magnesium chloride hexahydrate Merck Millipore 105833 dissolved in sterile water, stock is 1 M
Calcium chloride dihydrate Merck Millipore 102382 dissolved in sterile water, stock is 1 M
Sodium chloride Merck Millipore 106404 dissolved in sterile water, stock is 5 M
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Tags

NanobodyEndocytic UptakeRetrograde TransportTrans-Golgi NetworkCell Surface ReceptorsFluorescence MicroscopyElectron MicroscopyTyrosine Sulfation

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Buser, D. P., Spiess, M. Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells. J. Vis. Exp. (144), e59111, doi:10.3791/59111 (2019).
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