Advanced imaging of

cellular processes across scales

Jan Ellenberg
Head, Cell Biology and Biophysics Unit
Coordinator, Euro-BioImaging Preparatory Phases
European Molecular Biology Laboratory (EMBL)
Meyerhofstr. 1, D-69117 Heidelberg, Germany
Imaging technologies are changing life science

1890 1940 1990 2040

Biochemistry

Molecular Biology

„Omics“

Imaging
The “resolution revolution” in light and electron microscopy

1 mm 100 µm 10 µm 1 µm 100 nm 10 nm 1 nm 0,1nm

Light Sheet Super-Resolution Cryo-Electron

Nobel prize 2014 Nobel prize 2017

Betzig, Hell, Moerner Dubochet, Henderson, Frank
Crossing scales in biology
molecular mechanism in cells and organisms

Molecular
Molecules Cells Organisms
Machines

Understanding development and

Structural analysis Function of machines and their networks
disease
Crossing scales with advanced imaging technologies

Super-resolution
Microscopy High throughput Light-sheet
Electron Microscopy Microscopy
Microscopy

Ø Correlate technologies
Ø Integrate and analyse data
Structural dynamics of the NPC
The Nuclear Pore Complex (NPC)

Maimon et al, 2012

Structure 6:998

• essential in all eukaryotes

• mediator of nucleocytoplasmic transport
• mechanical link and signaling hub cytoplasm <-> nucleus
• largest non-polymeric protein assembly:
> 500 proteins / > 100 MDa in vertebrates
Von Appen et al, 2014
• ~4000 NPCs / nucleus of a human cell Nature 526:140
Reconstructing the NPC by SRM

Confocal image Raw GSDIM data Super-resolved image

Szymborska et al., 2013, Science 341:655

 Towards a 3D molecular map of the NPC by astigmatic SMLM

Cytoplasm
Astigmatic SMLM + spline fitter

Nucleoplasm

I. Scaffold Nups
Seh1
Nup107
Nup133
ELYS

II. Flexible Nups

Nup358 from Huang B et al., Science 2008
Tpr

In collaboration with Jonas Ries and Philipp Hoess, EMBL Y Li et al, Nature Meth 2018; doi: 10.1038/nmeth.4661
How do NPCs assembly across the cell cycle?

Nuclear Envelope
Reassembly

Nuclear Growth Otsuka S & Ellenberg J, 2018, FEBS Lett,

doi: 10.1002/1873-3468.12905
 Correlative light and electron microscopy captures
NPC assembly
Live cell imaging Cell relocation 3D EM

Otsuka et al., 2018, Nat Struct Mol Biol,

doi: 10.1038/s41594-017-0001-9

Semi-thin TEM EM tomography

Otsuka et al., 2016, eLife,

doi: 10.7554/eLife.19071.001
Collaboration with Anna Steyer and Yannick Schwab, EMBL
Postmitotic and interphase assembly are kinetically
and structurally distinct

Postmitotic assembly ~5 min

Int. phase

Otsuka et al., 2018, Nat Struct Mol Biol,

doi: 10.1038/s41594-017-0001-9

Interphase assembly ~45 min

Int. phase

Otsuka et al., 2016, eLife,

doi: 10.7554/eLife.19071.001
Towards a structural understanding of
mitotic chromosome formation
How do condensins shape mitotic chromosomes?

Interphase Prophase Prometaphase Metaphase

hinge
coiled-coil arms
Condensin I/II
H/H2

(ATPase
Head

)
HEAT
HEAT

Kschonsak et al., 2015

4D single-cell proteomics by FCS-calibrated imaging

Cai, Hossain et al., 2018, Nature, doi: 10.1038/s41586-018-0518-z

Politi et al., 2018, Nat Protoc, doi: 10.1038/nprot.2018040
175 000 Consdensin complexes organise the mitotic genome

HeLa
metaphase genome
(2x 7.9 Gb / ~1200 µm):
~175,000 Condensins

metaphase chromatid
(123 Mb / ~10 µm):
~1,400 Condensins
~1 Condensin per ~90 kb

N
number of molecules

Walther et al., 2018, J Cell Biol, doi: 10.1038/jcb.201801048

Condensin I/II organise different parts of the chromosome

NCAPH-Halo [I] (CA-SiR)

NCAPH2-mEGFP [II] (anti-GFP-AB, anti-rabbit-STAR580)
3D single chromatid STED imaging

Chromatid surface Chromatid

Condensin cross section

Condensin I Condensin II
(CAP-H) (CAP-H2)

Walther et al., 2018, J Cell Biol, doi: 10.1038/jcb.201801048

A few numbers to consider about human mitotic
chromosomes

Physical length Genomic length Number of Condensins

(I + II)
Genome 1162.5 ± 190.4 µm 2x 7.9 Gb ~175,000
Chromosome ~18.2 µm 2x 123.4 Mb ~2,800
Chromatid ~9.1 µm 123.4 Mb ~1,400
1 µm chromatid 1.0 µm ~13.6 Mb ~250
1 Mb DNA ~73.6 nm 1.0 Mb ~15
Condensin spacing ~6.6 nm ~90.0 kb
Metaphase

SiR-Hoechst
SMC4-mEGFP

Walther et al., 2018, J Cell Biol, doi: 10.1038/jcb.201801048

Three-step hierarchical loop model of mitotic
chromosome compaction

Walther et al., 2018, J Cell Biol,

doi: 10.1038/jcb.201801048
Data mining can estimate stoichiometry, temporal order and
rates of chromosome proteins dis/assembly

100 000

200 000

50 000
100 000

Cai, Hossain et al., 2018, Nature, doi: 10.1038/s41586-018-0518-z

Labeling co-replicating domains by incorporation of
fluorescent nucleotides to unravel chromatin organisation

N rounds of cell division

Correlative confocal and STORM imaging

Confocal STORM Overlay Zoom-in Clustering Zoom-in

Xiang and Roberti et al., JCB, 2018,

doi: 10.1083/JCB.201709074.
Can we resolve DNA loops in cells?
Super-resolution: 2 kb instead of 2 Mb

HeLa cell HeLa genome

• ~3 sets of 23 chromosomes

• ~9 billion basepairs

• >2 m DNA folded into ~700 µm3

Optically resolvable genomic distance

• Diffraction limited (Airy Scan confocal; ~140x140x400 nm)
2 000 000 bp (~10 000 nucleosomes)

• Super-resolution (4Pi STORM; ~20x20x20 nm)

2 000 bp (~10 nucleosomes)
 ChromoTrace algorithm

• Compute physical adjacency graph

• Reversible suffix tree finds unique

genome sequence anchors

• Extends linear genome sequence

onto 3D FISH coordinates

Ø 30 nm 3D resolution and
10 differently “colored” FISH probes
can resolve 50% of the genome

Collaboration with
Carl Barton and Ewan Birney, EMBL-EBI Barton, Morganella et al., 2018, PloS Comp Biol 14:e1006002
Mapping the Protein Network of Cell Division
in Space and Time
The gene network of human cell division

Silence each gene The human genome on

one by one 150 siRNA microarrays

High throughput 200 000

time-lapse imaging time-lapse movies

Computational analysis of the 20 000 000

movies phenotypically-annotated cell divisions

~600
Functional genome annotation
human cell division genes

Neumann, Walter et al, 2010, Nature 464:721

What is the dynamic network of 600 proteins required to
drive human cell division?
A homozygous knock-in cell line resource:
60 mitotic genes and counting…

M20
M17
H20
Clone:

H5
paired CRISPR/Cas9 nickases

+
modified from Ran et al. 2013 AUKRKB-GFP
+
double-strand break
homology arm
& homologous recombination endogenous
AUKRKB

Donor Fluorescent
Gene
plasmid

homology arm anti-AURKB

AURKB-mEGFP
Homozygous (three alleles) clonal HeLa cell line (M17)
Koch et al., 2018, Nature Protocols, doi: 10.1038/nprot.2018.042.
FCS-calibrated 4D imaging of mitotic proteins

Concentration
calibration
High resolution 4D
Low resolution and
cell detection
Automated
Confocal
Microscope

H2B-mCer3, NES-mCer3
molecules
per voxel

Politi et al., 2018, Nat Protoc, doi: 10.1038/nprot.2018040

Building the canonical human dividing cell

Imaging time
20 cells
Protein 1

20 cells
Protein 2

20 cells
Protein 3
…

Protein 572 …

Computational integration of 4D localisation data from

10 000 cells allows to measure protein similarity in time and space
Parametrising protein localisation:
SURF interest point clusters

Cai, Hossain et al., 2018, Nature, doi: 10.1038/s41586-018-0518-z

Unsupervised 4D clustering maps proteins to six dynamic
mitotic localisation patterns

Cai, Hossain et al., 2018, Nature, doi: 10.1038/s41586-018-0518-z

Reconstructing protein networks from dynamic mitotic
localisation patterns

Cai, Hossain et al., 2018, Nature, doi: 10.1038/s41586-018-0518-z

From genes to proteins – modeling human cell division
www.mitocheck.org
Mitotic Cell Atlas - a VR experience

David López Tabernero

Understanding cell division at the beginning of
mammalian life
Understanding the causes of infertility

Only 30% of all conceptions progress to live birth

[Santos et al. (2010) Reproduction and Larson et al. (2013) BMC Medicine]

Conception Loss pre-/post-implantation Miscarriage

60% 10%
Principle of Light-sheet microscopy

Mouse Embryo Confocal Light Sheet

Illumination
(lower NA)
80 μm

transparent sphere Illumination, fluorescence collection Fluorescence collection

80 µm diamter (high NA) (high NA)

Key advantages of light sheet compared to confocal microscopy:

• Lower overall light dose
• Higher imaging speed and therefore throughput
An inverted light-sheet microscope for mouse embryology

Key advances of mouse light sheet microscope

• Lower overall light dose
• Higher imaging speed and throughput
• Sample isolation from environment, no embedding Strnad et al., 2016, Nature Methods 13:139
Commercially available from Bruker/Luxendo
• Standard microdrop oocyte/embryo culture
Disclaimer: J.E. is scientific co-founder of Luxendo
with Lars Hufnagel
The first in toto imaging of the beginning of mammalian life
Watching the beginning of mammalian development in real time

Inner cell mass

100 %

% ICM in branch
50 %

0%

trophectoderm

Strnad et al., 2016, Nat Methods,

doi: 10.1038/nmeth.3690
 Parental genomes remain separate during the first division

Mouse zygote

H2B-mCherry
Maternal Kinetochores
Reichmann et al., 2018, Science, Paternal Kinetochores
doi: 10.1126/science.aar7462
Dual zygotic spindle formation requires spindle alignment

mid-prometaphase late-prometaphase metaphase

Mouse
zygote

Reichmann et al., 2018, Science, doi: 10.1126/science.aar7462

Defects in dual spindle alignment lead to bi-nucleated
blastomeres
mid-prometaphase late-prometaphase metaphase
Mouse
zygote

Human 2-Cell embryo

Reichmann et al., 2018, Science, doi: 10.1126/science.aar7462

Defects in dual spindle alignment lead to bi-nucleated
blastomeres
mid-prometaphase late-prometaphase metaphase
Mouse
zygote

Human 2-Cell embryo First division mouse zygote

(Experimentally increased pro-nuclei distance)

Reichmann et al., 2018, Science, doi: 10.1126/science.aar7462

Summary

• Correlative light and electron microscopy

resolves the structural dynamics of membranes and protein complexes

• 3D Super-Resolution Microscopy
resolves the dynamic structure of protein complexes and chromosomes

• High-throughput “proteomic” imaging

maps dynamic protein networks in space and time

• Light-Sheet Microscopy
visualizes physiological processes in embryos and organoids in real time
 External Collaborators

Stefan Hell Ralf Jungmann

EMBL Collaborating Daniel Gerlich Jan-Michael Peters

Ellenberg Group
Groups and Facilities
Adele Rickerby
Recent Alumni: Martin Beck
Stephanie Alexander Ewan Birney
Bianca Nijmejier Takashi Hiiragi
Andrea Callegari
Sandra Correia
Birgit Koch Wolfgang Huber External funding
Yin Cai Lars Hufnagel
Nathalie Daigle
Shotaro Otsuka Rainer Pepperkok
Manuel Eguren
Antonio Politi Jonas Ries
Merle Hantsche
Maria Julia Roberti Yannick Schwab
Jean-Karim Heriché
Petr Strnad
Julius Hossain
Vilma Jimenez Advanced Light Microscopy
Moritz Küblbeck Facility (ALMF)
Euro-BioImaging:
Franziska Kundel
Yu Lin Electron Microscopy Core
Antje Keppler Facility (EMCF)
Øyvind Ødegård
Frauke Leitner
Judith Reichmann
Federica Paina Mechanical workshop
Arina Rybina
Kelly Sheehan-Rooney Electronic workshop
Isabell Schneider
Nike Walther
Laboratory of animal
resources (LAR) Industrial partners

J.E. is co-founder and

advisor to Luxendo
 Ellenberg Lab
2019

Want to join the lab?

Contact Jan: jan.ellenberg@embl.de