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Structure and dynamics of endosomes analyzed by imaging-based methods MARINO ZERIAL Max Planck Institute of Molecular Cell Biology and Genetics Dresden, Germany QBI 2019 9 - 11 Jan 2019, Rennes Self-Organization Propagated over Scales

  1. Structure and dynamics of endosomes analyzed by imaging-based methods MARINO ZERIAL Max Planck Institute of Molecular Cell Biology and Genetics Dresden, Germany QBI 2019 9 - 11 Jan 2019, Rennes

  2. Self-Organization Propagated over Scales Organism (Mouse, Human) Organ (Liver) Tissue (Liver lobule) Cellular (Hepatocytes) Sub-Cellular (endosomes) Molecular (Endosomal effectors) Understanding biological systems from the molecular to organ scale

  3. Topics Endocytosis and the endosomal network Endosomes structure and function by Multi- Color SMLM CLEM Endosomes dynamics in neurons (Yannis Kalaidzidis) Imaging liver tissue down to sub-cellular resolution

  4. ENDOSOMES AND CARGO SORTING Tfn EGF EGFR TfnR Clathrin-coated vesicle Early endosome Multivesicular body (MVB) or Endocytic carrier vesicle (ECV) Urska Repnik

  5. RAB5 AS MEMBRANE ORGANIZER GDI PRA-1 PI3-K VPS34 Rabankyrin-5 Early EEA1 Endosome Rabenosyn-5 Vps45 Syntaxin13 Vamp4 Syntaxin6 Vti1a

  6. EGF EEA1 APPL1 6

  7. Funnel Model of the Early Endosomal Network CCVS RAB5 Rink et al. 2005 CONVERSION ENDOSOME FUSION → ENDOSOME FISSION → RAB5 RAB7

  8. Funnel Model of the Early Endosomal Network CCVS RAB5 Rink et al. 2005 CONVERSION ENDOSOME FUSION → ENDOSOME FISSION → RAB5 RAB7

  9. confocal multicolor J Cell l Biol. ol. 2000 images scalebar 2μm

  10. MULTI-COLOR SINGLE MOLECULE LOCALIZATION MICROSCOPY (SMLM) OF EARL Y ENDOSOMES • Super- Resolution Imaging • Post-Processing • Quantification CORRELATIVE Sandra Segeletz Christian Franke • Genome-edited cells • High-Resolution • EM Imaging • Post-Processing LLSM & SIM • Quantification Urska Repnik

  11. MULTI-COLOR SINGLE MOLECULE LOCALIZATION MICROSCOPY (SMLM) OF EARL Y ENDOSOMES Confocal EGF (Alexa647) (projection) Tfn (Alexa568) Ra Rab5 (Dronpa) 250 nm 250 nm Franke C, Repnik U, Segeletz S, Brouilly N

  12. MULTI-COLOR SINGLE MOLECULE LOCALIZATION MICROSCOPY (SMLM) OF EARL Y ENDOSOMES super-resolved EGF (Alexa647) SMLM Tfn (Alexa568) Ra Rab5 (Dronpa) 250 nm 250 nm Franke C, Repnik U, Segeletz S, Brouilly N

  13. MULTI-COLOR SINGLE MOLECULE LOCALIZATION MICROSCOPY (SMLM) OF EARL Y ENDOSOMES EGF (Alexa647) Tfn (Alexa568) Rab5c (Dronpa) merge 250 nm 250 nm Franke C, Repnik U, Segeletz S, Brouilly N

  14. CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY Z – stack of Tomogram slides 250 nm 250 nm 250 nm z 1 z 2 z 3 Franke C, Repnik U, Segeletz S, Brouilly N

  15. CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY 250 nm Franke C, Repnik U, Segeletz S, Brouilly N

  16. CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY Multi-Color superCLEM - EGF – intraluminar vesicles 250 nm Franke C, Repnik U, Segeletz S, Brouilly N

  17. CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY Multi-Color superCLEM - EGF – intraluminar vesicles 250 nm Franke C, Repnik U, Segeletz S, Brouilly N

  18. CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY Multi-Color superCLEM - EGF 250 nm Franke C, Repnik U, Segeletz S, Brouilly N

  19. CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY Multi-Color superCLEM - EGF – sorting micro-domain 250 nm Franke C, Repnik U, Segeletz S, Brouilly N

  20. CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY Multi-Color superCLEM - EGF – stages of sorting 250 nm Franke C, Repnik U, Segeletz S, Brouilly N

  21. CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY Multi-Color superCLEM - Transferrin tubules 250 nm Franke C, Repnik U, Segeletz S, Brouilly N

  22. CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY Multi-Color superCLEM - Rab5c - nanodomains 250 nm Franke C, Repnik U, Segeletz S, Brouilly N

  23. ENDOSOME DYNAMICS IN NEURONS Heerssen and Segal, 2002

  24. LIVER FUNCTION DEPENDS ON THE BLOOD AND BILE NETWORKS Central vein Portal vein Blood Bile duct Sinusoidal Bile Canalicular Network Network Bile Liver lobule

  25. BILE FLOW BY INTRA VITAL MICROSCOPY Sophie Nehring 6-CF Dextran Merged Two-photon microscopy (Leica SP8 MPI-CBG), 880 nm laser, 40x1.10 NA water

  26. LIVER TISSUE IMAGED WITH CARE Florian Jug

  27. LIVER ARCHITECTURE DEPENDS ON HEPATOCYTE POLARITY ENDOTHELIUM BASAL SURFACE APICAL SURFACE BILE CANALI- BILE BILE CULUS CANALICULUS CANALI- CULUS BASAL SURFACE Apical Surface

  28. APICAL RECYCLING ENDOSOMES IN HEPA TOCYTES Basolateral Endocytosis Lysosomal degradation Apical Apical recycling recycling endosome endosome Bile canaliculus Rab11 Apical Endocytosis Lysosomal degradation

  29. JUNCTION FORMA TION PRECEDES POLARIZED TRAFFICKING IN MAMMALIAN EPITHELIAL CELLS Microtubules Apical Surface Chromatin markers Tight junctions Recycling endosomes Junction Polarized Lumen formation trafficking formation Bryant, D.M. et al. (2010). A molecular network for de novo generation of the apical surface and lumen. Nat. Cell Biol. 12, 1035 – 1045.

  30. APICAL RECYCLINGENDOSOMES CLUSTER AROUND CENTROSOMES CD13 Pericentrin (apical) (Centrosome) Phalloidin (Cell border) 10 µm

  31. ULTRASTRUCTURE OF APICAL RECYCLING ENDOSOMES 5 µm CD13 100 nm 300 nm Urska Repnik

  32. HEPATOCYTE CELL POLARITY ENDOTHELIUM BASAL SURFACE APICAL SURFACE BILE CANALI- BILE BILE CULUS CANALICULUS CANALI- CULUS BASAL SURFACE Apical Surface

  33. CONCLUSIONS Mapping molecules to the ultra-structure of cellular organelles: SMLM-CLEM - SMLM-CLEM applied to reconstituted systems Development of software for cryo-EM tomography (Florian Jug & Gaia Pigino) Multi-scale imaging of cells and tissue: Challenges - Phototoxicity, low SNR (Yannis Kalaidzidis) - Super-resolution in living tissue (IVM)

  34. ACKNOWLEDGEMENTS Christian Urska Sandra Franke Segeletz Repnik Sophie Nehring Yannis Kalaidzidis zerial.mpi-cbg.de

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