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Bioquímica

Análise de absorção endocítica e transporte retrógrado à rede Trans-Golgi usando funcionalizados Nanobodies em culturas de células

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

Summary

Transporte retrógrado de proteínas de superfície celular para o Golgi é essencial para manter a homeostase de membrana. Aqui, descrevemos um método para analisar bioquimicamente transporte de superfície-para-Golgi celular de proteínas recombinantes usando funcionalizados nanobodies em células HeLa.

Abstract

Transporte de proteínas e membranas de superfície celular para o Golgi e além é essencial para a homeostase, organela identidade e fisiologia. Para estudar o tráfego de proteína retrógrada, recentemente desenvolvemos um versátil baseado em nanobody toolkit para analisar o transporte de superfície celular para o complexo de Golgi, quer por células fixas e ao vivo de imagens, por microscopia electrónica, ou bioquimicamente. Nós produzimos funcionalizados antiproteína verde fluorescente (GFP) nanobodies — ligantes pequenos, monomérico, alta afinidade da proteína — que pode ser aplicado a linha celular expressam proteínas de membrana de interesse com um moiety extracelular de GFP. Nanobodies pyrazole vinculados para os repórteres de GFP especificamente são internalizados e transportado às costas ao longo das rotas de classificação dos repórteres. Nanobodies foram acrescidas com fluorophores siga transporte retrógrado por microscopia de fluorescência e viver de imagem, com peroxidase de ascorbato 2 (APEX2) para investigar a localização ultra-estrutural do repórter-nanobody complexos por elétrons microscopia e com motivos de tirosina sulfatação (TS) para avaliar a cinética da chegada de rede (TGN) trans-Golgi. Neste artigo metodológico, descrevem o procedimento geral para o modo express e purificar nanobodies funcionalizados. Ilustramos o uso poderoso de nossa ferramenta usando o nanobodies mCherry – e TS-modificado para analisar a captação endocítica e TGN chegada de proteínas de carga.

Introduction

Trânsito retrógrado de proteínas e lipídios de superfície celular para vários compartimentos intracelulares é crucial para a manutenção da homeostase de membrana para contrabalançar a secreção e reciclar componentes de anterógrada transporte machineries1 , 2. após internalização através de endocitose dependente de Clatrina ou – independente, carga de proteínas e lipídios primeiro preencher cedo endossomos de onde eles são mais Redirecionado ou ao longo do sistema endo-lisossomal, reciclado para a membrana plasmática, ou direcionados à rede trans-Golgi (TGN). Reciclagem de endossomos e/ou a superfície da célula para o TGN é parte do ciclo funcional de um número de receptores de carga do transmembrane anterógrada, tais como os receptores de manose-6-fosfato de cação-dependente e independente de cátion (CDMPR e CIMPR) entregando recém sintetizado hidrolases lisossômicas do TGN para tarde endossomos lisossomos3,4,5, sortilin e SorLA6,7e Wntless (WLS) transportando Wnt ligantes à superfície da célula 8 , 9 , 10 , 11. outras proteínas obtidas para o TGN são TGN46 e suas isoformas relacionados12,13,14, SNAREs (solúvel em N– proteà sensíveis à fusão fator acessório receptores) 15 , 16 , 17, precursora amiloide (APP) de proteína18,19, anquilose progressiva (ANK) proteína20transportadores metálicos tais como ATP7A/B ou DMT121,22e do transmembrane processamento de enzimas, incluindo a carboxipeptidase D, furina ou BACE123,24,25. Para além destas proteínas endógenas, toxinas bacterianas e vegetais (por exemplo, toxina Shiga e cólera, ricina e abrin) sequestrar máquinas de transporte retrógrado para alcançar a ER para retrotranslocation para o citosol26,27, 28,29.

A fim de analisar diretamente trânsito retrógrado, anteriormente desenvolvemos um conjunto de ferramentas baseadas em nanobody para rotular e siga proteínas de carga de superfície celular para compartimentos intracelulares30. Nanobodies representam uma nova família de proteínas ligantes derivados de homodimeric cadeia pesada-somente anticorpos (hcAbs) que ocorrem naturalmente em camelídeos e peixes cartilagíneos31,32. Eles constituem o domínio variável de cadeia pesada (VHH) de hcAbs e têm muitas vantagens sobre anticorpos convencionais (por exemplo, IgGs): eles são monoméricos, pequeno (~ 15 kDa), altamente solúvel, desprovida de ligações de bissulfeto, pode ser bacteriana expressos e selecionado para ligação de alta afinidade33,34,35,36. Para tornar a nossa ferramenta de nanobody versátil e amplamente aplicável, utilizamos funcionalizados anti-GFP nanobodies às proteínas de superfície-rótulo e faixa com GFP no seu domínio extracelular/lumenal. Por functionalization de nanobodies com mCherry, peroxidase de ascorbato 2 (APEX2)37, ou sequências de sulfatação (TS) de tirosina, transporte retrógrado de proteínas transmembranares carga de bonafide pode ser analisado por qualquer fixo e vive de imagem latente da pilha, por microscopia eletrônica de varredura, ou bioquimicamente. Desde sulfatação tirosina mediada por sulfotransferases tyrosylprotein (TPST1 e TPST2) é uma modificação pós-traducional restringida para o trans-Golgi/TGN, podemos diretamente estudar transporte e cinética de proteínas de interesse da superfície da pilha para isso intracelulares Golgi compartimento38,39,40.

Neste artigo de métodos, descrevemos a facilidade de produção de nanobodies funcionalizados (VHH-2xTS, – APEX2, – mCherry e derivados) adequado para uma série de aplicativos para analisar o transporte retrógrado em células de mamíferos,30. Focamos principalmente o uso de TS site-modificado nanobody para análise de tráfego intracelular de superfície celular para o compartimento de sulfatação.

Protocol

1. bacteriana transformação com funcionalizados Nanobodies Nota: Este protocolo foi otimizado para a expressão, purificação e análise de nanobodies anti-GFP funcionalizados como descrito anteriormente,30. Derivatização com outras partes da proteína pode requerer a modificação do presente protocolo padrão. Descongelar as bactérias chemocompetent (~ 100 µ l) adequadas para a expressão da proteína (por exemplo, Escherichia col…

Representative Results

Para investigar o transporte retrógrado proteína para vários destinos intracelulares, nós temos estabelecido recentemente uma ferramenta baseada em nanobody de anti-GFP para rotular e siga as proteínas recombinantes de fusão entre a superfície celular30. Aqui, demonstramos a produção bacteriana de tais derivatizado nanobodies e demonstrar sua aplicação para estudar endocítica absorção por microscopia de fluorescência e immunoblotting, bem como a sua …

Discussion

Nanobodies representam uma classe emergente de andaimes de proteína ligante com muitas vantagens sobre anticorpos convencionais: eles são pequenos, estável, monomérico, podem ser selecionados para alta afinidade e falta dissulfeto títulos33,35, 44 , 45. são usados em muitas aplicações, tais como nos sistemas de cultura de células e organismos em biologia do desenvolvimento<sup class="x…

Declarações

The authors have nothing to disclose.

Acknowledgements

Este trabalho foi apoiado por Grant 31003A-162643 pela Fundação de ciência nacional suíço. Agradecemos a Nicole Beuret e o Biozentrum Imaging Core Facility (IMCF) pelo apoio.

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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Referências

  1. Johannes, L., Popoff, V. Tracing the retrograde route in protein trafficking. Cell. 135 (7), 1175-1187 (2008).
  2. Bonifacino, J. S., Rojas, R. Retrograde transport from endosomes to the trans-Golgi network. Nature Reviews. Molecular Cell Biology. 7 (8), 568-579 (2006).
  3. Duncan, J. R., Kornfeld, S. Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus. Journal of Cell Biology. 106 (3), 617-628 (1988).
  4. Ghosh, P., Dahms, N. M., Kornfeld, S. Mannose 6-phosphate receptors: new twists in the tale. Nature Reviews. Molecular Cell Biology. 4 (3), 202-212 (2003).
  5. Doray, B., Ghosh, P., Griffith, J., Geuze, H. J., Kornfeld, S. Cooperation of GGAs and AP-1 in packaging MPRs at the trans-Golgi network. Science. 297 (5587), 1700-1703 (2002).
  6. Pallesen, L. T., Vaegter, C. B. Sortilin and SorLA regulate neuronal sorting of trophic and dementia-linked proteins. Molecular Neurobiology. 45 (2), 379-387 (2012).
  7. Gustafsen, C., et al. Sortilin and SorLA display distinct roles in processing and trafficking of amyloid precursor protein. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 33 (1), 64-71 (2013).
  8. Yu, J., et al. WLS retrograde transport to the endoplasmic reticulum during Wnt secretion. Developmental Cell. 29 (3), 277-291 (2014).
  9. Harterink, M., et al. A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion. Nature Cell Biology. 13 (8), 914-923 (2011).
  10. Port, F., et al. Wingless secretion promotes and requires retromer-dependent cycling of Wntless. Nature Cell Biology. 10 (2), 178-185 (2008).
  11. McGough, I. J., et al. SNX3-retromer requires an evolutionary conserved MON2:DOPEY2:ATP9A complex to mediate Wntless sorting and Wnt secretion. Nature Communications. 9 (1), 3737 (2018).
  12. Banting, G., Ponnambalam, S. TGN38 and its orthologues: roles in post-TGN vesicle formation and maintenance of TGN morphology. Biochimica et Biophysica Acta. 1355 (3), 209-217 (1997).
  13. Banting, G., Maile, R., Roquemore, E. P. The steady state distribution of humTGN46 is not significantly altered in cells defective in clathrin-mediated endocytosis. Journal of Cell Science. 111 (Pt 23), 3451-3458 (1998).
  14. Ponnambalam, S., Rabouille, C., Luzio, J. P., Nilsson, T., Warren, G. The TGN38 glycoprotein contains two non-overlapping signals that mediate localization to the trans-Golgi network. The Journal of Cell Biology. 125 (2), 253-268 (1994).
  15. Mallard, F., et al. Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform. The Journal of Cell Biology. 156 (4), 653-664 (2002).
  16. Lewis, M. J., Nichols, B. J., Prescianotto-Baschong, C., Riezman, H., Pelham, H. R. Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes. Molecular Biology of the Cell. 11 (1), 23-38 (2000).
  17. Hirst, J., et al. Distinct and overlapping roles for AP-1 and GGAs revealed by the "knocksideways" system. Current biology. 22 (18), 1711-1716 (2012).
  18. Burgos, P. V., et al. Sorting of the Alzheimer’s disease amyloid precursor protein mediated by the AP-4 complex. Developmental Cell. 18 (3), 425-436 (2010).
  19. Choy, R. W., Cheng, Z., Schekman, R. Amyloid precursor protein (APP) traffics from the cell surface via endosomes for amyloid beta (Abeta) production in the trans-Golgi network. Proceedings of the National Academy of Sciences of the United States. 109 (30), E2077-E2082 (2012).
  20. Seifert, W., et al. The progressive ankylosis protein ANK facilitates clathrin- and adaptor-mediated membrane traffic at the trans-Golgi network-to-endosome interface. Human Molecular Genetics. 25 (17), 3836-3848 (2016).
  21. Tabuchi, M., Yanatori, I., Kawai, Y., Kishi, F. Retromer-mediated direct sorting is required for proper endosomal recycling of the mammalian iron transporter DMT1. Journal of Cell Science. 123 (Pt 5), 756-766 (2010).
  22. La Fontaine, S., Mercer, J. F. Trafficking of the copper-ATPases, ATP7A and ATP7B: role in copper homeostasis. Archives of Biochemistry and Biophysics. 463 (2), 149-167 (2007).
  23. Burd, C. G. Physiology and pathology of endosome-to-Golgi retrograde sorting. Traffic. 12 (8), 948-955 (2011).
  24. Chia, P. Z., Gasnereau, I., Lieu, Z. Z., Gleeson, P. A. Rab9-dependent retrograde transport and endosomal sorting of the endopeptidase furin. Journal of Cell Science. 124 (Pt 14), 2401-2413 (2011).
  25. Wahle, T., et al. GGA proteins regulate retrograde transport of BACE1 from endosomes to the trans-Golgi network. Molecular and Cellular Neurosciences. 29 (3), 453-461 (2005).
  26. Johannes, L., Goud, B. Surfing on a retrograde wave: how does Shiga toxin reach the endoplasmic reticulum. Trends in Cell Biology. 8 (4), 158-162 (1998).
  27. van Deurs, B., Tonnessen, T. I., Petersen, O. W., Sandvig, K., Olsnes, S. Routing of internalized ricin and ricin conjugates to the Golgi complex. Journal of Cell Biology. 102 (1), 37-47 (1986).
  28. Sandvig, K., van Deurs, B. Membrane traffic exploited by protein toxins. Annual Review of Cell and Developmental Biology. 18, 1-24 (2002).
  29. Sandvig, K., et al. Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum. Nature. 358 (6386), 510-512 (1992).
  30. Buser, D. P., Schleicher, K. D., Prescianotto-Baschong, C., Spiess, M. A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface. Proceedings of the National Academy of Sciences of the United States. 115 (27), E6227-E6236 (2018).
  31. Hamers-Casterman, C., et al. Naturally occurring antibodies devoid of light chains. Nature. 363 (6428), 446-448 (1993).
  32. Greenberg, A. S., et al. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature. 374 (6518), 168-173 (1995).
  33. Bieli, D., et al. Development and Application of Functionalized Protein Binders in Multicellular Organisms. International Review of Cell and Molecular Biology. 325, 181-213 (2016).
  34. Muyldermans, S. Nanobodies: natural single-domain antibodies. Annual Review of Biochemistry. 82, 775-797 (2013).
  35. Helma, J., Cardoso, M. C., Muyldermans, S., Leonhardt, H. Nanobodies and recombinant binders in cell biology. Journal of Cell Biology. 209 (5), 633-644 (2015).
  36. Harmansa, S., Affolter, M. Protein binders and their applications in developmental biology. Development. 145 (2), (2018).
  37. Lam, S. S., et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nature Methods. 12 (1), 51-54 (2015).
  38. Huttner, W. B. Tyrosine sulfation and the secretory pathway. Annual Review of Physiology. 50, 363-376 (1988).
  39. Baeuerle, P. A., Huttner, W. B. Tyrosine sulfation is a trans-Golgi-specific protein modification. The Journal of Cell Biology. 105 (6 Pt 1), 2655-2664 (1987).
  40. Stone, M. J., Chuang, S., Hou, X., Shoham, M., Zhu, J. Z. Tyrosine sulfation: an increasingly recognised post-translational modification of secreted proteins. New Biotechnology. 25 (5), 299-317 (2009).
  41. Leitinger, B., Brown, J. L., Spiess, M. Tagging secretory and membrane proteins with a tyrosine sulfation site. Tyrosine sulfation precedes galactosylation and sialylation in COS-7 cells. The Journal of Biological Chemistry. 269 (11), 8115-8121 (1994).
  42. Snider, M. D., Rogers, O. C. Intracellular movement of cell surface receptors after endocytosis: resialylation of asialo-transferrin receptor in human erythroleukemia cells. Journal of Cell Biology. 100 (3), 826-834 (1985).
  43. Shi, G., et al. SNAP-tag based proteomics approach for the study of the retrograde route. Traffic. 13 (7), 914-925 (2012).
  44. Kaiser, P. D., Maier, J., Traenkle, B., Emele, F., Rothbauer, U. Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells. Biochimica et Biophysica Acta. 1844 (11), 1933-1942 (2014).
  45. Dmitriev, O. Y., Lutsenko, S., Muyldermans, S. Nanobodies as Probes for Protein Dynamics in Vitro and in Cells. The Journal of Biological Chemistry. 291 (8), 3767-3775 (2016).
  46. Harmansa, S., Hamaratoglu, F., Affolter, M., Caussinus, E. Dpp spreading is required for medial but not for lateral wing disc growth. Nature. 527 (7578), 317-322 (2015).
  47. Harmansa, S., Alborelli, I., Bieli, D., Caussinus, E., Affolter, M. A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila. eLife. 6, (2017).
  48. Caussinus, E., Kanca, O., Affolter, M. Fluorescent fusion protein knockout mediated by anti-GFP nanobody. Nature Structural & Molecular Biology. 19 (1), 117-121 (2012).
  49. Rothbauer, U., et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nature Methods. 3 (11), 887-889 (2006).
  50. Pardon, E., et al. A general protocol for the generation of Nanobodies for structural biology. Nature Protocols. 9 (3), 674-693 (2014).
  51. Steyaert, J., Kobilka, B. K. Nanobody stabilization of G protein-coupled receptor conformational states. Current Opinion in Structural Biology. 21 (4), 567-572 (2011).
  52. Manglik, A., Kobilka, B. K., Steyaert, J. Nanobodies to Study G Protein-Coupled Receptor Structure and Function. Annual Review of Pharmacology and Toxicology. 57, 19-37 (2017).
  53. Zimmermann, I., et al. Synthetic single domain antibodies for the conformational trapping of membrane proteins. eLife. 7, (2018).
  54. De Genst, E., et al. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proceedings of the National Academy of Sciences of the United States. 103 (12), 4586-4591 (2006).
  55. Truttmann, M. C., et al. HypE-specific nanobodies as tools to modulate HypE-mediated target AMPylation. The Journal of Biological Chemistry. 290 (14), 9087-9100 (2015).
  56. Ashour, J., et al. Intracellular expression of camelid single-domain antibodies specific for influenza virus nucleoprotein uncovers distinct features of its nuclear localization. Journal of Virology. 89 (5), 2792-2800 (2015).
  57. Yamagata, M., Sanes, J. R. Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents. Proceedings of the National Academy of Sciences of the United States. 115 (9), 2126-2131 (2018).
  58. Pleiner, T., Bates, M., Gorlich, D. A toolbox of anti-mouse and anti-rabbit IgG secondary nanobodies. Journal of Cell Biology. 217 (3), 1143-1154 (2018).
  59. Fridy, P. C., et al. A robust pipeline for rapid production of versatile nanobody repertoires. Nature Methods. 11 (12), 1253-1260 (2014).
  60. Robinson, M. S., Sahlender, D. A., Foster, S. D. Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria. Developmental Cell. 18 (2), 324-331 (2010).
  61. Meyer, C., et al. mu1A-adaptin-deficient mice: lethality, loss of AP-1 binding and rerouting of mannose 6-phosphate receptors. The EMBO Journal. 19 (10), 2193-2203 (2000).
  62. Utskarpen, A., Slagsvold, H. H., Iversen, T. G., Walchli, S., Sandvig, K. Transport of ricin from endosomes to the Golgi apparatus is regulated by Rab6A and Rab6A. Traffic. 7 (6), 663-672 (2006).
  63. Mallard, F., Johannes, L. Shiga toxin B-subunit as a tool to study retrograde transport. Methods in Molecular Medicine. 73, 209-220 (2003).
  64. Mallard, F., et al. Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport. Journal of Cell Biology. 143 (4), 973-990 (1998).
  65. Plaut, R. D., Carbonetti, N. H. Retrograde transport of pertussis toxin in the mammalian cell. Cellular Microbiology. 10 (5), 1130-1139 (2008).
  66. Johannes, L., Tenza, D., Antony, C., Goud, B. Retrograde transport of KDEL-bearing B-fragment of Shiga toxin. The Journal of Biological Chemistry. 272 (31), 19554-19561 (1997).
  67. Saint-Pol, A., et al. Clathrin adaptor epsinR is required for retrograde sorting on early endosomal membranes. Developmental Cell. 6 (4), 525-538 (2004).
  68. Amessou, M., Popoff, V., Yelamos, B., Saint-Pol, A., Johannes, L. Measuring retrograde transport to the trans-Golgi network. Current Protocols in Cell Biology. , (2006).
  69. Niewoehner, J., et al. Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle. Neuron. 81 (1), 49-60 (2014).
  70. Villasenor, R., Schilling, M., Sundaresan, J., Lutz, Y., Collin, L. Sorting Tubules Regulate Blood-Brain Barrier Transcytosis. Cell Reports. 21 (11), 3256-3270 (2017).
  71. Dick, G., Grondahl, F., Prydz, K. Overexpression of the 3′-phosphoadenosine 5′-phosphosulfate (PAPS) transporter 1 increases sulfation of chondroitin sulfate in the apical pathway of MDCK II cells. Glycobiology. 18 (1), 53-65 (2008).
  72. Fruholz, S., Fassler, F., Kolukisaoglu, U., Pimpl, P. Nanobody-triggered lockdown of VSRs reveals ligand reloading in the Golgi. Nature Communications. 9 (1), 643 (2018).

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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