Project
1: Profiling Extracellular RNA Comm
unication in Action
Proj
ect 2
: B
iolog
ical Re
cognition of Nanoparticles
Our
Imag
ing
Stra
tegi
es
j
p
j
Zebrafish as a Model for Nano-BioimagingYuya Hayashi
(formal supervisor: Claus Oxvig) e-mail:
[email protected]: mbg.au.dk/yuya-hayashi/
MOLECULAR BIOLOGY AND GENETICS DEPARTMENT OF
AARHUS UNIVERSITY AU1. "Differential Nanoparticle Sequestration
by Macrophages and Scavenger Endothelial Cells Visualized in Vivo
in Real-Time
and at Ultrastructural Resolution" in ACS Nano (2020) doi:
10.1021/acsnano.9b072332. "Tracing the In Vivo Fate of
Nanoparticles with a Non-Self Biological Identity" in ACS Nano
(2020) doi: 0.1021/acsnano.0c05178
Recent publications from the group
dpf, days-post-fertilization. mpi/hpi,
minutes-/hours-post-injection.CLSM, confocal laser scanning
microscopy. CLEM, correlative light-electron microscopy. TEM,
transmission electron microscopy.
Intravenous microinjection of nanoparticles
500 μm 3 dp
f em
bryo
Brig
ht-F
ield
x
x
y y
z
20 μm
Bloo
d ve
ssel
sM
acro
phag
esN
anop
artic
les
100 μm
Real-Time Bioimaging and Screening Electron microscopy (TEM)
20 μm
Bloo
d ve
ssel
sM
acro
phag
esN
anop
artic
les
20 μm
3 mpiM
acro
phag
esN
anop
artic
les
tnf-a
lpha 20 μm
1 hpi 6 hpi30 mpi
Chemical fixation
20 μm TEM
(pse
udo-
colo
ured
)Bl
ood
vess
els
Mac
roph
ages
Nan
opar
ticle
s ca
ptur
edby
mac
roph
ages
Nanoparticle Sequestration
Nanoparticle Clearance
Macrophage Activation
4D Live-cell Imaging
3 μm 500 nm
3 μm
1 μm
50 μm
Tolu
idin
e Bl
ue
Correlative Light-Electron
Microscopy (CLEM)
Mac
roph
ages
Nan
opar
ticle
sO
verla
y(C
LSM
+ T
EM)
TEM
Cryo-fixation (high-pressure freezing)
Control (unmodified) + FBS protein corona
20 μm
Bloo
d ve
ssel
s70
nm
SiO
2 nan
opar
ticle
s
3 mpi 30 mpi 3 mpi 30 mpi
100 nm
Protein coronas visualized by TEM (right).SiO2 nanoparticles (70
nm) with a corona
pre-formed of fetal bovine serum (FBS)were negatively stained
for TEM imaging.
Inset shows a pristine (unmodified) nanoparticle.
20 μm
Bloo
d ve
ssel
sEx
trace
llula
r ves
icle
s(p
Hlu
orin
-labe
lled)
GFP-TrapMagnetic beadsY Y
YY
Fluorescent tag fusedto an extracellular loop
of tetraspanins
Affinity purification
2. RNAseq
1. LC-MS/MS
Whole-embryo homogenization
Endogenously-labelled fluorescentextracellular vesicles
circulating in the blood
Live
imag
ing
plasmidTol2 transgenesis vector Cell type-specific promoter
Tetraspanin-fluorescent tag
Cargo profiling1. Proteins2. Small RNAs
What we doeeing is believing. Starring zebrafish as a model
organism, we image life at the
nanoscale “visualizing” how innate immu-nity works at the
interface with blood.
Of particular interest are macrophages:the cells that eat/clean
up foreignmaterials, pathogens and dead cells to maintain
homeostasis of our body.
How do they recognize nanoparticles cir-culating in the blood?
What are their role in inflammation? With zebrafish, we seek
an-swers to these questions in a manner not possible using cell
cultures or rodent models.
This year, we offer two exciting projectpossibilities in which
you will play a role applying genetics/molecular biology techniques
andlearning complex image analysis methods.
S
ransgenic lines with a cell type-specific fluorescent protein
re-porter allow us to study the dynamic behaviour of
macrophages
and how injected nanomaterials are cleared from the
bloodstream.Electron microscopy (TEM) can then complement the
real-time
observations by visualizing those processes at the
nanoscale.
A marriage of the two imaging approaches is CLEM, by which we
can link the fluorescent reporters
(i.e. cell-type identity) to ultrastructure observed by TEM.
T
hat lies at the interface of biological receptors
andnanoparticles? Today, it is widely accepted that cells
“see” the proteins adsorbed to nanoparticles (a.k.a. “protein
corona”) rather than the bare particle surface.
Using proteomics and imaging/screening approach-es, we study how
protein coronas determine the
fate of nanoparticles injected into the blood.
Internal collaboration partner:Prof. Duncan Sutherland
(iNANO)
W
xtracelluler vesicles (EVs) were once believed to be just waste
released from cells. Over the past decades we have
started to appreciate the many faces of EVs – they are indeed
bags filled with secrets. EVs have a size range from nano- to
micrometres, and thus the imaging approaches we use for
bionanoscience have technical overlaps.Using zebrafish as an in
vivo model, we are also develop-ing a new method to profile the EV
cargo "in transit" from donor cells to understand what messages are
conveyed for cell-to-cell communication.
Internal collaboration partner:Prof. Jørgen Kjems
(MBG/iNANO)
E
