imaging-based methods MARINO ZERIAL Max Planck Institute of - - PowerPoint PPT Presentation

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imaging based methods

imaging-based methods MARINO ZERIAL Max Planck Institute of - - PowerPoint PPT Presentation

Nov 16, 2022 42 likes •389 views

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

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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

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Self-Organization Propagated over Scales

Organism (Mouse, Human)

Understanding biological systems from the molecular to organ scale

Molecular (Endosomal effectors) Sub-Cellular (endosomes) Cellular (Hepatocytes) Tissue (Liver lobule) Organ (Liver)

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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

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ENDOSOMES AND CARGO SORTING

EGF Tfn TfnR EGFR

Early endosome Multivesicular body (MVB)

r Endocytic carrier vesicle (ECV)

Clathrin-coated vesicle

Urska Repnik

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Early Endosome

GDI

Rabenosyn-5 Vps45 EEA1 PRA-1

PI3-K

VPS34

Syntaxin13 Syntaxin6 Vti1a Vamp4

Rabankyrin-5

RAB5 AS MEMBRANE ORGANIZER

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6

EGF EEA1 APPL1

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RAB5 RAB7 RAB5

CCVS CONVERSION

ENDOSOME FUSION → ENDOSOME FISSION →

Funnel Model of the Early Endosomal Network

Rink et al. 2005

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RAB5 RAB7 RAB5

CCVS CONVERSION

ENDOSOME FUSION → ENDOSOME FISSION →

Funnel Model of the Early Endosomal Network

Rink et al. 2005

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scalebar 2μm

confocal multicolor images J Cell l Biol.

l. 2000

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Super-

Resolution

Imaging

Post-Processing
Quantification
EM
Post-Processing
Quantification
Genome-edited

cells

High-Resolution

Imaging LLSM & SIM CORRELATIVE

Christian Franke Sandra Segeletz Urska Repnik

MULTI-COLOR SINGLE MOLECULE LOCALIZATION MICROSCOPY (SMLM) OF EARL Y ENDOSOMES

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250 nm

EGF (Alexa647) Tfn (Alexa568) Ra Rab5 (Dronpa) 250 nm

Confocal (projection)

MULTI-COLOR SINGLE MOLECULE LOCALIZATION MICROSCOPY (SMLM) OF EARL Y ENDOSOMES

Franke C, Repnik U, Segeletz S, Brouilly N

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250 nm

EGF (Alexa647) Tfn (Alexa568) Ra Rab5 (Dronpa) 250 nm

super-resolved SMLM

MULTI-COLOR SINGLE MOLECULE LOCALIZATION MICROSCOPY (SMLM) OF EARL Y ENDOSOMES

Franke C, Repnik U, Segeletz S, Brouilly N

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250 nm

250 nm

EGF (Alexa647) Tfn (Alexa568) Rab5c (Dronpa) merge

MULTI-COLOR SINGLE MOLECULE LOCALIZATION MICROSCOPY (SMLM) OF EARL Y ENDOSOMES

Franke C, Repnik U, Segeletz S, Brouilly N

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z1 z2 z3

Z – stack of Tomogram slides

250 nm 250 nm 250 nm

CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY

Franke C, Repnik U, Segeletz S, Brouilly N

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250 nm

CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY

Franke C, Repnik U, Segeletz S, Brouilly N

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250 nm

Multi-Color superCLEM - EGF – intraluminar vesicles

CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY

Franke C, Repnik U, Segeletz S, Brouilly N

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250 nm

Multi-Color superCLEM - EGF – intraluminar vesicles

CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY

Franke C, Repnik U, Segeletz S, Brouilly N

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250 nm

Multi-Color superCLEM - EGF

CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY

Franke C, Repnik U, Segeletz S, Brouilly N

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250 nm

Multi-Color superCLEM - EGF – sorting micro-domain

CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY

Franke C, Repnik U, Segeletz S, Brouilly N

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250 nm

Multi-Color superCLEM - EGF – stages of sorting

CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY

Franke C, Repnik U, Segeletz S, Brouilly N

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250 nm

Multi-Color superCLEM - Transferrin tubules

CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY

Franke C, Repnik U, Segeletz S, Brouilly N

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250 nm

Multi-Color superCLEM - Rab5c - nanodomains

CORRELATIVE MULTI-COLOR SMLM & EM-TOMOGRAPHY

Franke C, Repnik U, Segeletz S, Brouilly N

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ENDOSOME DYNAMICS IN NEURONS

Heerssen and Segal, 2002

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Central vein Bile duct Portal vein

Liver lobule

Bile

Bile Canalicular Network

Blood

Sinusoidal Network

LIVER FUNCTION DEPENDS ON THE BLOOD AND BILE NETWORKS

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BILE FLOW BY INTRA VITAL MICROSCOPY

Two-photon microscopy (Leica SP8 MPI-CBG), 880 nm laser, 40x1.10 NA water

6-CF Dextran Merged Sophie Nehring

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LIVER TISSUE IMAGED WITH CARE

Florian Jug

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LIVER ARCHITECTURE DEPENDS ON HEPATOCYTE POLARITY

BILE CANALICULUS APICAL SURFACE BASAL SURFACE ENDOTHELIUM BILE CANALI- CULUS BILE CANALI- CULUS

Apical Surface

BASAL SURFACE

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APICAL RECYCLING ENDOSOMES IN HEPA TOCYTES

Basolateral Endocytosis Apical Endocytosis

Rab11

Bile canaliculus Lysosomal degradation Lysosomal degradation

Apical recycling endosome Apical recycling endosome

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JUNCTION FORMA TION PRECEDES POLARIZED TRAFFICKING IN MAMMALIAN EPITHELIAL CELLS

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.

Lumen formation Junction formation Polarized trafficking

Microtubules Chromatin Tight junctions Apical Surface markers Recycling endosomes

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APICAL RECYCLINGENDOSOMES CLUSTER AROUND CENTROSOMES Phalloidin (Cell border)

CD13 (apical) Pericentrin (Centrosome)

10 µm

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ULTRASTRUCTURE OF APICAL RECYCLING ENDOSOMES CD13

100 nm 300 nm 5 µm

Urska Repnik

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HEPATOCYTE CELL POLARITY

Apical Surface

BASAL SURFACE BILE CANALICULUS APICAL SURFACE BASAL SURFACE ENDOTHELIUM BILE CANALI- CULUS BILE CANALI- CULUS

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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)

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zerial.mpi-cbg.de

ACKNOWLEDGEMENTS

Sandra Segeletz Christian Franke Urska Repnik Yannis Kalaidzidis Sophie Nehring