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Multicolour imaging of post-Golgi sorting and trafficking in live cells

Nature Cell Biology volume 3, pages 140–149 (2001)Cite this article

Abstract

The biogenesis and maintenance of asymmetry is crucial to many cellular functions including absorption and secretion, signalling, development and morphogenesis. Here we have directly visualized the segregation and trafficking of apical (glycosyl phosphatidyl inositol-anchored) and basolateral (vesicular stomatitis virus glycoprotein) cargo in living cells using multicolour imaging of green fluorescent protein variants. Apical and basolateral cargo segregate progressively into large domains in Golgi/trans-Golgi network structures, exclude resident proteins, and exit in separate transport containers. These remain distinct and do not merge with endocytic structures suggesting that lateral segregation in the trans-Golgi network is the primary sorting event. Fusion with the plasma membrane was detected by total internal reflection microscopy and reveals differences between apical and basolateral carriers as well as new 'hot spots' for exocytosis.

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Figure 1: Biochemical characterization of fluorescent apical and basolateral cargo in MDCK cells.
Figure 2: Surface distribution in polarized MDCK cells.
Figure 3: Apical and basolateral cargo segregate in the Golgi of polarized MDCK and non-polarized PtK2 cells.
Figure 4: Dynamics of cargo segregation in the Golgi of live cells.
Figure 5: Cargo progresses from the Golgi stack to the TGN before exit.
Figure 6: Apical and basolateral cargo move in separate TCs to the cell periphery.
Figure 7: Basolateral post-Golgi TCs do not intersect with endocytic structures.
Figure 8: Fusion of apical and basolateral TCs at plasma membrane 'hot spots'.

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References

  1. Ikonen, E. & Simons, K. Protein and lipid sorting from the trans-Golgi network to the plasma membrane in polarized cells. Semin. Cell Dev. Biol. 9, 503–509 (1998).

    Article  CAS  Google Scholar 

  2. Yeaman, C., Grindstaff, K. K. & Nelson, W. J. New perspectives on mechanisms involved in generating epithelial cell polarity. Physiol. Rev. 79, 73–98 (1999).

    Article  CAS  Google Scholar 

  3. Simons, K. et al. Biogenesis of cell-surface polarity in epithelial cells and neurons . Cold Spring Harb. Symp. Quant. Biol. 57, 611–619 (1992).

    Article  CAS  Google Scholar 

  4. Winckler, B., Forscher, P. & Mellman, I. A diffusion barrier maintains distribution of membrane proteins in polarized neurons. Nature 397, 698–701 (1999).

    Article  CAS  Google Scholar 

  5. Bretscher, M. S. Moving membrane up to the front of migrating cells. Cell 85, 465–467 (1996).

    Article  CAS  Google Scholar 

  6. Traub, L. M. & Kornfeld, S. The trans-Golgi network: a late secretory sorting station. Curr. Opin. Cell Biol. 9 , 527–533 (1997).

    Article  CAS  Google Scholar 

  7. Mostov, K. E. & Cardone, M. H. Regulation of protein traffic in polarized epithelial cells. BioEssays 17, 129–138 (1995).

    Article  CAS  Google Scholar 

  8. Müsch, A., Xu, H., Shields, D. & Rodriguez-Boulan, E. Transport of vesicular stomatitis virus to the cell surface is signal mediated in polarized and nonpolarized cells. J. Cell Biol. 133, 543–558 (1996).

    Article  Google Scholar 

  9. Yoshimori, T., Keller, P., Roth, M. G. & Simons, K. Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. J. Cell Biol. 133, 247–256 (1996).

    Article  CAS  Google Scholar 

  10. Keller, P. & Simons, K. Post-Golgi biosynthetic trafficking . J. Cell Sci. 110, 3001– 3009 (1997).

    CAS  PubMed  Google Scholar 

  11. Mellman, I. et al. Molecular sorting in polarized and non-polarized cells: common problems, common solutions. J. Cell Sci. 17 (Suppl.), 1–7 (1993).

    Article  CAS  Google Scholar 

  12. Matter, K. & Mellman, I. Mechanisms of cell polarity: sorting and transport in epithelial cells. Curr. Opin. Cell Biol. 6, 545–554 (1994).

    Article  CAS  Google Scholar 

  13. Fölsch, H., Ohno, H., Bonifacino, J. S. & Mellman, I. A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell 99, 189– 198 (1999).

    Article  Google Scholar 

  14. Scheiffele, P. & Simons, K. in Epithelial Morphogenesis in Development and Disease (eds Birchmeier, W. & Birchmeier, C.) 41–71 (Harwood Academic, Newark, New Jersey, 1999).

    Google Scholar 

  15. Rodriguez-Boulan, E. & Gonzalez, A. Glycans in post-Golgi apical targeting: sorting signals or structural props? Trends Cell. Biol. 9, 291–294 ( 1999).

    Article  CAS  Google Scholar 

  16. Simons, K. & Ikonen, E. Functional rafts in cell membranes . Nature 387, 569–572 (1997).

    Article  CAS  Google Scholar 

  17. Rindler, M. J., Ivanov, I. E., Plesken, H., Rodriguez-Boulan, E. & Sabatini, D. D. Viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same Golgi apparatus during their intracellular transport in doubly infected Madin–Darby canine kidney cells. J. Cell Biol. 98, 1304 –1319 (1984).

    Article  CAS  Google Scholar 

  18. Fuller, S. D., Bravo, R. & Simons, K. An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments. EMBO J. 4, 297– 307 (1985).

    Article  CAS  Google Scholar 

  19. Griffiths, G. & Simons, K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science 234, 438–443 (1986).

    Article  CAS  Google Scholar 

  20. Hirschberg, K. et al. Kinetic analysis of secretory protein traffic and characterization of Golgi to plasma membrane transport intermediates in living cells. J. Cell Biol. 143, 1485–1503 (1998).

    Article  CAS  Google Scholar 

  21. Nakata, T., Terada, S. & Hirokawa, N. Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons. J. Cell Biol. 140, 659–674 (1998).

    Article  CAS  Google Scholar 

  22. Toomre, D., Keller, P., White, J., Olivo, J. C. & Simons, K. Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells. J. Cell Sci. 112, 21–33 ( 1999).

    CAS  Google Scholar 

  23. Toomre, D., Steyer, J. A., Keller, P., Almers, W. & Simons, K. Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy. J. Cell Biol. 149, 33–40 (2000).

    Article  CAS  Google Scholar 

  24. Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    Article  CAS  Google Scholar 

  25. Röttger, S. et al. Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus. J. Cell Sci. 111, 45 –60 (1998).

    PubMed  Google Scholar 

  26. Storrie, B. et al. Recycling of Golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering. J. Cell Biol. 143, 1505 –1521 (1998).

    Article  CAS  Google Scholar 

  27. Girotti, M. & Banting, G. TGN38-green fluorescent protein hybrid proteins expressed in stably transfected eukaryotic cells provide a tool for the real-time, in vivo study of membrane traffic pathways and suggest a possible role for ratTGN38. J. Cell Sci. 109, 2915–2926 (1996).

    CAS  PubMed  Google Scholar 

  28. Füllekrug, J. et al. Localization and recycling of gp27 (hp24γ3): Complex formation with other p24 family members. Mol. Biol. Cell 10, 1939–1955 (1999).

    Article  Google Scholar 

  29. Leitinger, B., Hille-Rehfeld, A. & Spiess, M. Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes. Proc. Natl Acad. Sci. USA 92, 10109–10113 ( 1995).

    Article  CAS  Google Scholar 

  30. Futter, C. E., Connolly, C. N., Cutler, D. F. & Hopkins, C. R. Newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell surface. J. Biol. Chem. 270 , 10999–11003 (1995).

    Article  CAS  Google Scholar 

  31. Axelrod, D. Cell–substrate contacts illuminated by total internal reflection fluorescence . J. Cell Biol. 89, 141– 145 (1981).

    Article  CAS  Google Scholar 

  32. Schmoranzer, J., Goulian, M., Axelrod, D. & Simon, S. M. Imaging constitutive exocytosis with total internal reflection fluorescence microscopy. J. Cell Biol. 149, 23–31 (2000).

    Article  CAS  Google Scholar 

  33. Harder, T., Scheiffele, P., Verkade, P. & Simons, K. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol. 141, 929– 942 (1998).

    Article  CAS  Google Scholar 

  34. Benting, J. H., Rietveld, A. G. & Simons, K. N–Glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin–Darby canine kidney cells. J. Cell Biol. 146, 313– 320 (1999).

    Article  CAS  Google Scholar 

  35. Füllekrug, J., Scheiffele, P. & Simons, K. VIP36 localisation to the early secretory pathway. J. Cell Sci. 112, 2813–2821 (1999).

    PubMed  Google Scholar 

  36. Dell'Angelica, E. C., Mullins, C. & Bonifacino, J. S. AP-4, a novel protein complex related to clathrin adaptors. J. Biol. Chem. 274, 7278– 7285 (1999).

    Article  CAS  Google Scholar 

  37. Ladinsky, M. S., Kremer, J. R., Furcinitti, P. S., McIntosh, J. R. & Howell, K. E. HVEM tomography of the trans-Golgi network: structural insights and identification of a lace-like vesicle coat. J. Cell Biol. 127, 29– 38 (1994).

    Article  CAS  Google Scholar 

  38. Ladinsky, M. S., Mastronarde, D. N., McIntosh, J. R., Howell, K. E. & Staehelin, L. A. Golgi structure in three dimensions: functional insights from the normal rat kidney cell. J. Cell Biol. 144, 1135–1149 ( 1999).

    Article  CAS  Google Scholar 

  39. Hedman, K., Goldenthal, K. L., Rutherford, A. V., Pastan, I. & Willingham, M. C. Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry. J. Histochem. Cytochem. 35, 233–243 (1987).

    Article  CAS  Google Scholar 

  40. Grindstaff, K. K. et al. Sec6/8 complex is recruited to cell–cell contacts and specifies transport vesicle delivery to the basal–lateral membrane in epithelial cells. Cell 93, 731– 740 (1998).

    Article  CAS  Google Scholar 

  41. Mantei, N. et al. Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme. EMBO J. 7, 2705– 2713 (1988).

    Article  CAS  Google Scholar 

  42. Seed, B. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature 329, 840– 842 (1987).

    Article  CAS  Google Scholar 

  43. Harrison, P. T., Hutchinson, M. J. & Allen, J. M. A convenient method for the construction and expression of GPI-anchored proteins. Nucleic Acids Res. 22, 3813–3814 (1994).

    Article  CAS  Google Scholar 

  44. He, T. C. et al. A simplified system for generating recombinant adenoviruses. Proc. Natl Acad. Sci. USA 95, 2509– 2514 (1998).

    Article  CAS  Google Scholar 

  45. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    Article  CAS  Google Scholar 

  46. White, J. et al. Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells. J. Cell Biol. 147, 743– 759 (1999).

    Article  CAS  Google Scholar 

  47. Diggle, P. J. Statistical Snalysis of Spatial Point Patterns (Academic, London, 1983).

    Google Scholar 

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Acknowledgements

We acknowledge Olympus and TILL Photonics for providing equipment and support to the Advanced Light Microscope Facility at EMBL; J. Rietdorf and R. Pepperkok for technical support; R. Tsien and G. Banting for ECFP and TGN38–EGFP cDNA, respectively; and N. LeBot and J. Füllekrug for GFP and gp27 antibodies, respectively. J. Ellenberg, G. Griffiths and R. Pepperkok are acknowledged for critical comments on the manuscript. P.K. was supported by a grant from the Max Plank Gesellschaft, D.T. by a Marie Curie Research grant, E.D. by a grant from the Secretaría de Estado de Educación, Universidades, Investigación y Desarollo, and K.S. by an EU network grant and a grant from the Deutsche Forschungsgemeinschaft. Supplementary Information is available on http://www.mpi-cbg.de/content.php3?lang=en&aktID=videos8.

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Author notes

  1. Jamie White

    Present address: Massachusetts General Hospital Cancer Center, 149-7202 13th Street, Charlestown, Massachusetts, 02129, USA

  2. Patrick Keller and Derek Toomre: These authors contributed equally to this work.

Authors and Affiliations

  1. Cell Biology/Biophysics Programme, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, Heidelberg, D-69117, Germany

    Patrick Keller, Derek Toomre, Elena Díaz, Jamie White & Kai Simons

  2. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, D-01307, Germany

    Patrick Keller & Kai Simons

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Correspondence to Kai Simons.

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Keller, P., Toomre, D., Díaz, E. et al. Multicolour imaging of post-Golgi sorting and trafficking in live cells . Nat Cell Biol 3, 140–149 (2001). https://doi.org/10.1038/35055042

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