Davide Mazza - mazza.davide@hsr.it
1
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
School of photonics
Cortona, March 30 – April 3, 2014
Protein dynamics in living cells
by fluorescence microscopy
From ensemble average experiments to
single molecule imaging
xVivo – The inner life of cell
http://www.xvivo.net/animation/the-inner-life-of-the-cell/
3
Davide Mazza
mazza.davide@hsr.it
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
28. P. J. Mulholland et al., Nature 452, 202 (2008).
16.
A. Front.
D. Rosemond,
C. 9,M.229
Pringle,
A. Ramírez,
webshence
(from
leaf
litterbasis
toon
detritivores
to fish)activities.
cou1. comA. M. Helton
et al.,
Ecol.
Environ
(2011).
expression and
is the
of
cellular
understanding
of transcription
assembly,
29.transcriptional
M. J. Feio, T. Alves,regulation
M. Boavida, A. Medeiros,
metrics
based
fish,allinvertebrate,
or algalLittle
23 January 2012; accepted 26 April 2
M.
J.
Paul,
J.
L.
Meyer,
Limnol.
Oceanogr. 47, 278
1
2.
C.
J.
Vörösmarty
et
al.,
Nature
467,
555
(2010).
hightheprocess
rates
might
be irreconM. A. S. Graça, Freshw. Biol. 55, 1050 (2010).
informationpled
existswith
about
kinetics
of
this
process
in
live
cells
and
cross-talk
with
transcription-coupled
processes.
,
as
munities) are typically least sensitive. Consequently,
10.1126/science.1219534
3. E. S. Bernhardt (2002).
et al., Science 308, 636 (2005).
30. W. F. Cross, J. B. Wallace, A. D. Rosemond, S. L. Eggert,
cilable
goals
in stream
management.
Second,
Here
we
report
accurate
in
vivo
measurements
of
the
mammalian
understanding
of gene
expression
regulation
comes
from
studies
using
4.
P.
H.
Gleick,
Science
302,
1524
(2003).
litter breakdown—and
potentially
other functionEcology 87, 1556 (2006).
streamFor
managers
currently
rely primarily
struc5. N.
Adv. Ecol.inReseach
44, 2of
(2011).
Pol etIIal.,engaged
the steps of active transcription. We
purified proteins.
instance,
the
subunits
of theonelongating
Pol
II Friberg
al
measures
such
as
whole-ecosystem
metabotural measures to assess stream ecosystem health.
6. D. Hering et al., Sci. Total Environ. 408, 4007
thank
the European Commission and
previously developed a method for the Acknowledgments:
in vivo labelingWe of
mRNA
are well known2 and
thenutrient
crystal structure
of thisprimary
enzymeproduction
explains
lism,
(2010).
In particular,
changes
inspiraling,
biologicalorcommunity
the Swiss State Secretariat for Research and Education for
3,4
transcripts
containing
a series
of repeated
stem-loops (from phage
much of itsstructure
behavior
in vitro . mRNA
transcription
can be decon7. M.reO. Gessner,
E. Chauvet,
Ecol. Appl.
12, 498
(26–28)—can
be used
to complement,
not
funding the RivFunction research project (European Union
(invertebrates, fish,
and algae)
have long
(2002).MS2), which are specifically bound by ancontract
MS2 EVK1-CT-2001-00088),
coat protein fusedwhich
to was supported under
structed intounderpinned
a succession
of
steps:
promoter
assembly,
and
place,stream
established
procedures
to clearance
assess stream
bioassessment
schemes
be8. J. Hilton, M. O’Hare, M. J. Bowes, J. I. Jones,
16. The assay
the Fifth
Framework
Programme.
The constructive comments
green
fluorescent
protein
(GFP)
by
elongation
and
termination.
The
process
of
consists
of
a
human
cell
escape5, followed
ecosystem
highlightsrethe needSci.forTotal Environ.
cause they
provide ahealth.
reliableThis
time-integrated
365,E.66Purvis,
(2006). Kyle W. Karhohs,
Jeremy
Caroline
Mock,
Ericwhich
Batchelor,*
by three
anonymous
reviewers,
substantially improved
transcriptional
initiation
involves
several
structural
changes
line etharboring
gene323
array
into which the
these
stem-loops have been
9. the
C. Perrings
al., Sciencea 330,
(2010).
sponse
to
stressors
such
as
organic
pollution
or
differential diagnoses in environmentalinassesspaper, are greatly appreciated. All basic data are
Alexander
Loewer,†
Galit
Lahav‡
6. Early in initiation,
17. Trends
10. the
M. O. Gessner
et al.,
Ecol.now
Evol.used
25, 372
polymerase as
the nascent
transcript
elongates
integrated
We have
this system
to follow
the synthesismaterials.
of
acidification
(5),
but
biogeographical
constraints
available
in the supplementary
This paper is
ment,
as
is standard
practice
in medicine.
ImporREPORTS
IS
STUDYING
THE
DYNAMICS
OF
YOUR
PROTEIN
IMPORTANT?
(2010).
7,8
dedicated
to the memory of
of our
colleague
Björn Malmqvist,
make
this
approach
difficult
to
standardize
at
.
These
abortive
cycles
polymerase
can
produce
abortive
transcripts
RNA
in
real
time.
Our
method
allows
direct
measurement
Pol
II
breakdown
and
some other
Man, functionally
I. B. Holland,
C. Cole,etK. al.,
Kuchler,
F.
3.tantly,
Materials litter
and methods
are presented as
supporting
phenotype in tissues other than the stem and
11. J. S.C.P.Moore
Ecol.C. Lett.
7, 584 (2004).
who sadly passed away in 2010.
Higgins, Eds. (Academic Press, London, 2003), pp.
material on Science Online.
leaf and accumulation of residual
surface observed
wax
large scales
(10).
Litter
breakdown
can
help here
Cells
through
molecular
that
often show complex d
have been
with
aTindall,
single
prokaryote
polymerase
initiation
eventsJ. as
well asJ. R.
elongation
in isolation
fromsignals
the other
steps
12. J. B. Wallace,
S. L. transmit
Eggert,
L. information
Meyer,
Webster,
335–355.
4.based
H. Powell,
R.
P. Schultz,
Arch.be
Neurol.
32, 250
methods
can
implemented
at (RNAP)
relatively
on the stem of cer5-2 knockout line suggest
15. Thanks to G. Haughn,
M. Smith,
T.
Hooker,
and
(1975).
9,10
because
biogeography
is
a
minor
issue
(for
examScience
277,
102
(1997).
The dynamic
of the tumor
suppressor
p53 varies depending
releasing
thein order
promoter
of patterns.
transcription.
By usingbehavior
a deterministic
computational
model
. insightful
O.
Rowland
for
their
comments. The
5.transcripts
A. M. cost
Rashotte,
M.resource
A. Jenks, K.escaping
A. Feldmann,
little
orwithout
input
(29)
to13.
assess
that additional wax export
mechanismsseveral
must
Supplementary Materials
J.ofL. the
Tank,
E. J. Sciences
Rosi-Marshall,
N. A. Griffiths,
financial support
Natural
and
Phytochemistry
57,similar
115 (2001).species of the genus
ple,
black
alder
or
exist in plants. Chemical The
analysis
of
the
muin
response
to
double-strand
DNA
breaks,
it
shows
a
series
constrained
by extensive
dataSoc.
sets
tested with transcriptionof repeated pulses.
elongation step
can
beofregulated
byand
pausing
various
times,
Engineering impacts
Research
Council
of Canada,
6.effects
ABC
transporter
motifs were predicted
byother
PROSITE for
pollution
ecosystem
S. A.
Entrekin,
M. L.Canadian
Stephen,
J. N. Am. Benthol.
29, andwww.sciencemag.org/cgi/content/full/336/6087/1438/DC1
tant wax demonstrated that CER5, like many
for11,12
Innovation, BC Knowledge Develas referenced
in (13).
are common
throughout
most of Europe and Foundation
the
computational
we identified
a sequence
of heretofore
precisely timed drug addition
inhibitors,
weare were model,
able to extract
features
of
transcription
.and(2010).
as substrate
demonstrated
using
polymerases
in
Materials
and Methods
118
I –transporters,
CARRIES
INFORMATION
ABOUT
UNDERLYING
MOLECULAR
MECHANISMS.
opmentvitro
Foundation,
the
UBC
Blusson fund
7.that
M.
Jasinski,
Ducos,concern
E. Martinoia,
M.
Boutry,
Plant
areE.prokaryotic
of
to
environmental
managers
ABC
has broad
speciHolarctic),
and
marked
gratefully acknowledged.
We thankE.the
Salk
InPhysiol.
131, 1169
(2003). changes in breakdown
ficity and is capable of transporting
a
variety
Figs.
S1
to
S6
14.
J.
R.
Webster,
F.
Benfield,
Annu.
Rev.
Ecol.
Syst.
17,
pulses
to
instead
produce
a
sustained
p53
response.
This leads
to the expressio
unexplored
and provide a guide for application of the method
to
For eukaryotic cells,
madeJ. O.to D.calculate
endo-Analysis
´nchez-Ferna
´have
stitutethe
for Genomic
Laboratory for pro8.and
R.attempts
Sa
ndez, T.been
G. E. Davies,
stakeholders.
of wax substrates.
We conclude that in plants,
ratetransport
occurred
the
portion
of13(2001).
the pollution
Table S1
567 (1986).
Interactions,
processes,
oligomerization
states…
viding sequence-indexed
Arabidopsis T-DNA insertion
Coleman,in
P. A.
Rea,rising
J. Biol.
Chem.
276, 30231
setThegenes.
ofCER5downstream
genes
and also Reference
alters cell
fate: Cells that experience p53 pu
genous
elongation
run-on
assays
as in other eukaryotes, proteins
of the
WBC/
mutants (project
funded
byother
NSF).
gene Freshw. Biol.
9.speed
C. T. Increasing
Otsuusing
et al., J. Exp.
Bot.
55, 1643
(2004). , reverse-transcription
human
pressure
is accelerating
en(31)
15. V.
Gulis,
K. Suberkropp,
48, 123
gradient,
inanalyses
which
structural
has
been submitted to Genbank, and the accession
10. The
of R.established
Sa´nchez-Ferna´ndez
et al. (8) agreemeasures
ABCG subfamily are key components
of
14
15
DNA
damage,
whereas
cells
exposed
to
sustained
Databases S1 and S2 p53 signaling frequently und
(2003).
(RT)-PCR (such
or fluorescence
in situ
hybridization
(FISH)
specific
no.
ison
AY734542.
with these relationships;
however,
they
erroneously
change
throughout
theand
world,
threatasvironmental
water chemistry,
hydromorphology,
lipid transport systems.
duplicated WBC15/WBC22 in their 2001 work. This
Supporting Online
Material
16.
A.
D.
Rosemond,
C.
M.
Pringle,
A.
Ramírez,
Our results
show that protein dynamics can be an important part of a signal, d
mRNAs, andmetrics
these have
yielded
apparent
elongation
estimates
was corrected
in
(14).
RESULTS
ening
water
security
fororhumans
andranging
aquatic
www.sciencemag.org/cgi/content/full/306/5696/702/
II – CELLULAR
OUTCOMES
DEPEND
ON
PROTEIN
DYNAMICS
based
on
fish,
invertebrate,
algal
comJanuary 2012; accepted 26 April 2012
M. J. Paul, J. L. Meyer, Limnol. Oceanogr. 47, 278
11. G. L. Scheffer et al., Cancer Res. 60, 2589 (2000).
DC1
–1
influencing
fate decisions. 23
J. are
W. Jonker
et al., Proc.
Natl.
Acad.
U.S.A.
99,no Materials
References and Notes from 1.1 tomunities)
2.5 12.
kilobases
(kb)
min
Kinetics of Pol cellular
II transcription
. ToSci.stretches
date,
assay
been
biodiversity
(2).
Large
of
theandhas
landscape
typically
least
sensitive.
Consequently,
Methods
10.1126/science.1219534
(2002).
15649 (2002).
1. L. Kunst, A. L. Eg.
Samuels, Prog.
Lipid Res. 42, 51 (2003).
Figs.
S1 toinflammation,
S4
Circadian
rhythms,
Response
to
toline
DNA
13.in
L. Falquet
al., Nucleic
Acids
Res. 30,
235
(2002).
2. M. Koornneef, C. J. Hanhart,
F. Thiel, J. Hered.
80,measure
breakdown—and
other
Europe
andpotentially
other
parts
ofII functionthe
world
areincharwith adamage…
stable integration of approximately 200
developed
tolitter
theet various
steps
of
Pol
transcription
a We used a cell
5 July 2004; accepted 3 September 2004
14. P. A. Rea et al., in ABC Transporters from Bacteria to
118 (1989).
17, eachto
al measures
such today
as whole-ecosystem
metaboacterized
by highly
industrialized,
inten- repeats of ells
some instances,
dynamical pro
use cassette
molecular
networks
a gene
at asignaling
single locus
containing
256
living cell. For
instance,
although
abortive
initiation
is widely believed
18
lism,
nutrient
spiraling,
or
primary
production
sively
managed
agriculture
the large-scale
sense,repeats
interpret,andanda respond
stimuli. cillation
to occur, the dynamics
of this
event are
unknown, and
including
whether upstream lacO
minimal to
cytomegalovirus
(CMV)frequency or signal d
(26–28)—can
used
to complement,
not
reapplication
of fertilizers.
This, in
combination
with
shown
toaalter gene expression (1
Recent
advances
in
time-lapse
microscopy
initiating polymerases
are be
committed
to entering
processive
elongapromoter
coupled
to
a
tetracycline-operator
cassette
controlling
sized free I0B" binds to nuclear NF-0B,
place,
established
procedures
to
assess
stream
leading
to
export
of
the
complex
to
the
other
sources
suchDNA,
as atmospheric
de- gene
or intoitscontrol
cellular different
have
many mRNA
signaling
Oscillations
in
NF-.B
Signaling
tion or whether
they
maynutrient
dissociate
from the
and the(10).
probthatrevealed
encodes athat
functional
with molecules
24 MS2 repeats
3¢
cytoplasm
This complex, but not free
ecosystem
health.has
This
highlights
the need
fornutrient
16,19
Jeremy
Purvis,
Kyle
W. Karhohs,
Mock, detect
Eric Batchelor,*
position,
resulted
in widespread
point to a ric
show
complex
dynamical
(1–13).
ability of each event.
Furthermore,
no assay
exists that
can
measure
untranslated
region
(Fig.Caroline
1).behaviors
We could
theInlocusThese
using examples
the
I0B",
is the
targetpolfor E.
I0B"
phosphorylation
Control
the
Dynamics
differential
diagnoses of
in environmental assessby IKK (11, Alexander
12).
Loewer,†
Galit
Lahav‡
elongation speed
template
within a live
cell. Accurate lactose repressor fused to red (Fig. 1b,e,h,k) or cyan (Fig. 1n,r,v)
Oscillations in the temporal response of
ment, on
as ais chromatin
standard practice
in medicine.
Impor-
p53 Dynamics Control Cell Fate
WHY
C
A R T Ip53
C L EDynamics
S
Control Cell Fate
Gene tantly,
Expression
NF-0B activity
litter breakdown and some other functionally
have been observed by
15shift
JUNE
2012 onlyVOL
336 SCIENCE www.sciencemag.org
electromobility
assay (EMSA)
in
Cells
through molecular signals that often show complex dynamical
D. E. Nelson, A. E. C. Ihekwaba,
M. Elliott,
R. Johnson,
based methods
canJ. be
implemented at relatively
studies of I0B$
andtransmit
( knockoutinformation
mouse em1 1Department of1 Anatomy and
1 Structural
1
1 Einstein College of Medicine, Bronx, New York 10461, USA. 2Laboratoire de Ge
´ne´tique
´culaire,
Biology,
Albert
Centre on the stimulus;
C. A. Gibney, B. E. Foreman,little
G. Nelson,
V. See, input
C. A. Horton,
bryonic fibroblast
cell populations
and have
patterns.
The dynamic
behavior of the tumor suppressor
p53Mole
varies
depending
cost or
resource
(29)5inEcole
order
to assess
3The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan
1
1
4 UMR-8541,
´
National
de
la
Recherche
Scientifique,
Normale
Supe
rieure,
75005
Paris,
France.
been
simulated
in
a
computational
model
D. G. Spiller, S. W. Edwards, H. P. McDowell, J. F. Unitt,
in
response
to double-strand
DNA breaks, it shows a series of repeated pulses. Using a
effects
pollution
and
other3 ecosystem
impacts
4Integrative
6
7 Ganof
7 Israel.
(13).Inc.,
In theLos
absence
time-lapse 94024,
single-cellUSA. Correspondence should be addressed to R.H.S.
University,
Ramat
52900,
Bioinformatics,
Altos,ofCalifornia
E. Sullivan,
R. Grimley,
N. Benson,
D. Broomhead,
computational
model,
we
identified
a sequence of precisely timed drug additions that alter p53
2
1
analysis,
it
has
remained
unclear
whether
that
are
of
concern
to
environmental
managers
(rhsinger@aecom.yu.edu).
D. B. Kell, M. R. H. White *
asynchronouspulses
single-cell
oscillations
occur a sustained p53 response. This leads to the expression of a different
to
instead
produce
and stakeholders.
Impossibile visualizzare l'immagine. La memoria del computer potrebbe essere insufficiente per aprire l'immagine oppure l'immagine
in 2007;
single cells
following NF-0B stimulation
Received
18
April;
accepted
June;
published
doi:10.1038/nsmb1280
potrebbeby
essere
danneggiata.
Riavviare il computer
e aprire
di nuovo
il file.
Se viene 28
visualizzata
di nuovo
la x rossa,involves
potrebbeonline
essere
Signaling
the
transcription
factor
nuclear
factor
kappa
B
(NF-.B)
its 5 August
1,2, Yaron
1,3, Valeria
1, Yehuda
3, Shailesh
1,
necessario eliminare l'immagine e inserirla di nuovo.
of downstream
genes de
and
also
alters cell
Cells Mthat
experience
p53 pulses recover from
Xavier
Shav-Tal(15),
Turris
Brodyfate:
Shenoy
is accelerating
en(8, Darzacq
14). Likeset
calcium
signaling
NF-0B
release from inhibitor kappa B (I.B) inIncreasing
the cytosol, human
followed pressure
by translocation
4
1
&
Robert
H
Singer
Robert
D
Phair
could
be
a
complex
dynamic
oscillator
using
DNA
damage,
whereas
cells
exposed
to
sustained
p53
signaling
frequently
undergo senescence.
into the nucleus. NF-.B regulation
of I.B! transcription
a delayed
vironmental
changerepresents
throughout
the world, threatperiod and/or amplitude to regulate transcripnegative feedback loop that drives oscillations in NF-.B translocation. SingleOur results show that protein dynamics can be an important part of a signal, directly
ening water security for humans andWeaquatic
tion of transcription
target genes. in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic
cell time-lapse imaging and computational modeling of NF-.B (RelA)
imaged
796
VOLUME
14 influencing
NUMBER
9cellular
SEPTEMBER
2007 NATURE STRUCTURAL & MOLECULAR BIOLOGY
fate
decisions.
We
have
used
fluorescence
imaging
biodiversity
(2).
Large
stretches
of
the
landscape
localization showed asynchronous oscillations following cell stimulation that
measurements of promoter
binding,
initiation
andofelongation.
Photobleaching of fluorescent RNA polymerase II revealed several
NF-0B
(RelA)
and
I0B"
fluorescent
fusion
decreased in frequency with increased
I.B! transcription.
Transcription
of
kinetically
distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent
in Europe
and other parts
of the world
areproteins
char(11, 16) to study oscillations in
target genes depended on oscillation persistence, involving cycles of RelA
MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model
RelA
N-C localizationells
(N-Cuse
oscillations)
in signaling networks to
acterized
today byconsequences
highly industrialized,
intensome min
instances,
dynamical
properties such as osmolecular
–1, much faster
phosphorylation and dephosphorylation.
The functional
of NFthat fits
our data was validated
using specific
inhibitors.
Polymerases elongated at 4.3 kilobases
than
HeLa
(human cervical
carcinoma) cells and
Davide
Mazza
Raffaele
Institute
.B signaling may thus depend on number,
period,
and amplitude
of oscillations.
sively
managed
agriculture
and
the
large-scale
cillation
frequency
or
signal
duration, have been
respond
to
previously
documented,
andsense,
entered ainterpret,
paused stateand
for unexpectedly
longstimuli.
times.San
Transcription
onsetScientific
was inefficient,
with
onlyCentro
4
SK-N-AS cells Ehuman S-type neuroblasto1%
of with
polymerase-gene interactions
leading
to completion
of an mRNA.
Our systems approach,
quantifying
both expression
polymerase
and
Di
imaging
Sperimentale
mazzad@mail.nih.gov
application of fertilizers. This, in combination
shown
to
alter
gene
(1,
3, 6, 8, 11, 13–16)
Recent
advances
in
time-lapse
microscopy
ma cells that have been associated with
mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of
cytoplasm
of
unstimulated
cells
by
binding
NF-0B is a family of dimeric transcription
deregulated
NF-0B
signaling
(17)^.
In
SKother nutrient sources such as atmospheric
de- processes
have inrevealed
that many signaling molecules or to control cellular differentiation (7, 12, 17).
vivo.
N-AS cells
expressing
RelA fused at the C
to I0B proteins. NF-0B–activating stimulitranscriptional
factors (usually RelA/p65:p50) that regulates
1
14402
1
1
In vivo dynamics of RNA polymerase II transcription
C
position,
hasthe
resulted
widespread
nutrientterminus
pol- toshow
complex
dynamical
activate
inhibitorin
kappa
B kinase (IKK)
cell division, apoptosis, and inflammation
the red
fluorescent
protein
behaviors (1–13). In
These examples point to a rich mode of regula-
WHAT
INSTRUMENT TO USE?
OPTICAL MICROSCOPY looks like a good candidate.
Cells are transparent to visible light.
Relatively low phototoxicity (but beware of near UV radiation).
With sufficient resolution (~200nm) to visualize intracellular compartments
What kind of microscopy?
WIDEFIELD
ILLUMINATION
Sensitive
Fast
Out of focus blur
5
TIRF
ILLUMINATION
Sensitive
No out of focus blur
Limited to surface
Davide Mazza
mazza.davide@mail.hsr.it
LASER SCANNING
(e.g. CONFOCAL)
No out of focus blur
Not that fast
Not that sensitive
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
The missing link.
Light microscopy
Viability
Electron microscopy
© T. Hatano, NIG, Japan
Immunofluorescence
© CBIM, Imperial College, UK
© M.Neguembor, HSR.
Specificity
6
Resolution
Davide Mazza
mazza.davide@mail.hsr.it
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
GFP: bridging viability and specificity
Viability
Specificity
Resolution
Von Dassow, 2009
GFP-tagged Histone H2B in live zebrafish embryo
7
Davide Mazza
mazza.davide@mail.hsr.it
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Some dynamics can be measured directly
Example: massive translocations between different compartments
NF-kB translocation
(Sung and Agresti, 2009)
8
Davide Mazza
mazza.davide@mail.hsr.it
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Mobility of dispersed particles
Tracked particles
one image every 5 sec!
whole movie ~ 500s!
Linear fit of mean square displacement
D = 0.0096 µm2/s
A0 =0.239
4.5
4
3.5
MSD [µm]
3
2.5
2
1.5
1
0.5
Aggregates of Ab heavy chain.
(w/ M. Mossuto and R. Sitia, HSR)
9
0
0
Davide Mazza
mazza.davide@mail.hsr.it
20
40
60
Time[s]
80
100
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Equilibrium:
a molecular Where’s Waldo
It can be difficult to understand what individual molecules do,
even when selectively labelling your protein of interest.
Tumor suppressor p53-GFP
in live fibroblasts (30 min after dex stimuli)
10
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Binding to ~immobile scaffolds is a
widespread event in the cells
chromatin
cytoskeleton
In the nucleus, these interactions regulate
transcription, translation and DNA repair.
B
2 J/m2 UV
p53-Venus (AU)
800
Interfering with protein 400
dynamics
cellular
fate.
0
mulus-dependent dynamics of p53 in single cells
allows interfering with
Batchelor et al
0 5 10 15 20
5 10 15 20
Time (h)
p53-Venus (AU)
400 ng/ml NCS
800
400
C
p53-Venus (AU)
0
5 10 15 200 50 10
5 15
10 20
15 200 5 10 15 20
TimeTime
(h) (h)
Time (h)
Time (h)
F
First pulse
800
400
400 ng/ml NCS
100 ng/ml NCS
200 ng/ml NCS
400 ng/ml NCS
0
0 2009)
5 10
(Batchelor
0 5et al.,Nat.
10 15Cancer
20 Rev,
Time (h)
15 20
Time (h)
0 5 10 15 20
Time (h)
0 5 10
Tim
B
8 J/m2 UV
p53-Venus (AU)
D
100 ng/ml NCS
p53-Venus (AU)
A
800
400
0
0 5 010 515102015 20
Time (h)
hours Time
after irradiation
(h)
800
400
0
0 5 10 15 200 50 10
5 15
10
(h)
TimeTim
(h
hours Time
after irradiation
D
2
4
800
6
CELL DEATH
400
8
(Batchelor et al., Mol Sys Biol, 2011)
10
02012)
(Purvis
et
al.,
Science,
0 5 10 15
0 5 10 15 20
1.4
Time (h
Time (h)
f p53-Venus (AU)
Time (h)
0 5 10 15 20
Time (h)
2.2
DNA DAMAGE
1.8 REPAIR
p53-Venus (AU)
100 ng/ml NCS
The toolbox of
cellular dynamicists*
Photoperturbation microscopy
Correlation microscopy
Single Molecule Imaging
* http://en.wiktionary.org/wiki/dynamicist
13
Davide Mazza
mazza.davide@hsr.it
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Photo-perturbation techniques
Modify the fluorescence properties of a subpopulation of molecules
by using a pulse of intense light.
Fluorescence Perturbation techniques (FPT)
Photobleaching
Techniques
Photoactivation
Techniques
i-FRAP
Inverse FRAP
FRAP
Fluorescence
Recovery
after Photobleaching
FLIP
Fluorescence Loss
Into Photobleaching
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Instrumentation for FRAP
A Widefield microscope
+
CCD
+
High intensity source
(laser)
A Confocal Laser
Scanning Microscope
(CLSM)
OR
with
Acusto-optic or Electro-optic
modulator devices
15
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Instrumentation for FRAP
A Widefield microscope
+
Fast image collection system
+
High intensity source
(laser)
A Confocal Laser
Scanning Microscope
(CLSM)
OR
with
Acusto-optic or Electro-optic
modulator devices
16
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Fluorescence recovery after
photobleaching
Fluorescence Recovery
Normalized Intensity
After Photobleaching (FRAP)
p53 dynamics in living cells
(whole movie 30 s)
Time
17
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Qualitative analysis of FRAP data
F(t) Immobile
Fraction
FASTER PROTEIN
ER
W
O
SL
EIN
T
O
PR
t 50 > t 50 > t 50
18
Mobile
Fraction
t Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Can we be more quantitative?
YES BUT WE NEED SOME MATH
Free diffusion
Axelrod, D et al.(1976), Biophys J 16, 1055--1069.
Soumpasis, D. M. (1983), Biophys J 41, 95--97.
Anomalous sub-diffusion
Saxton, M. J. (2001), Biophys J 81, 2226--2240.
Diffusion in heterogeneous systems
Siggia, E. D. et al. (2000), Biophys J 79(4), 1761--1770.
Diffusion and binding problems (hit and run)
Sprague, B. L.; et al. (2004) Biophys J, 86, 3473--3495.
Mueller et al. , B. L. et al. (2008), Biophys J.
19
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Selecting a model for the FRAP experiments
Equations deduced from
the choices made
!!
!
∗
= !!! !−!!"!+!!""!
!"
!
!!
= +!!∗!"!−!!""!!! ! ! ! ! !
!"
With proper initial and
boundary conditions lead
to model for experimental
data
20
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
What
model
to
be
chosen?
Different models can fit the same FRAP curve equally well
Normaized Intensity 1.0 0.9 Different in-vivo FRAP studies result
in different binding estimates
(and different biological interpretations)
One binding state + diffusion
0.8 0.7 0.6 0.5 residence time = 3.7 s
0.4 0 Normaized Intensity 1.0 2 4 6 Time [s] 8 10 12 Two binding states
(no diffusion)
0.9 TF
# of binding
states
Residence
time
Bound
fraction
GR1
1 (non-specific)
13 ms
85 %
Max2
2 (non-specific /
specific)
6 s / 14 s
95 %
p533
1 (non specific)
2.5 s
43 %
AR4
1 (specific)
90 s
20 %
0.8 Many of these differences are not biological but
due to different protocols/models used to
analyze FRAP data
0.7 0.6 1st residence time = 0.26 s
2nd residence time = 4.6 s
0.5 1 Sprague et al., Biophys J, 2004 3 Hinow et al., Biophys J, 2006
0.4 0 2 4 6 Time [s] 8 10 12 2 Phair et al., Mol Cell Biol 2004 4 Farla et al., J Cell Sci, 2005
“With four parameters I can fit an elephant, and
with five I can make him wiggle his trunk.”
J. von Neumann
O. Levenspiel, Chemical Innovation, 2000
22
Davide Mazza
mazza.davide@hsr.it
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
And this is only one of the problems of
photo-perturbation techniques
1. Bulk Experiments.
Only information on the average behavior
Rare events are lost
Needs modelling
2. Requires “high” levels of tagged protein (µM concentration range)
Potential problems with overexpression.
3. Photoperturbation.
Potential photodamage of the cells.
23
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Also FRAP is a local measurement
Can answer “how fast do protein move” at position x,y,z.
Won’t tell you how fast protein moves from A to B.
24
Davide Mazza
mazza.davide@hsr.it
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Expanding the scope of FRAP
Fluorescence loss into
photobleaching (FLIP)
Fluorescence Perturbation techniques (FPT)
Photobleaching
Techniques
Photoactivation
Techniques
i-FRAP
Inverse FRAP
FRAP
Fluorescence
Recovery
after Photobleaching
25
FLIP
Fluorescence Loss
Into Photobleaching
Normalized intensity
time
Davide Mazza
mazzad@mail.nih.gov
Time
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
FLIP Example: Search of p53 for its targets
FLIP detects a slow-down in the p53 search
upon DNA damage
1
10 µ
m
Normalized Intensity
0.9
0.8
0.7
0.6
0.5
0.4
Ctrl
10 Gy IR
0.3
0.2
(P. Rainone)
0
20
40
Time [s]
60
80
And this is only one of the problems of
photo-perturbation techniques
1. Bulk Experiments.
Only information on the average behavior
Rare events are lost
Needs modelling
2. Requires “high” levels of tagged protein (µM concentration range)
Potential problems with overexpression.
3. High doses of irradiation during photobleaching.
Potential photodamage of the cells.
4. Local measurement.
27
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Photoactivation/Photoconversion/
Photoswitching
Different mechanisms for “turning on”
FPs fluorophores
28
The first engineered photoactivatable
protein (PA-GFP, Patterson, 2002)
shows change in abs spectrum upon
activation
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Little summary
Dendra2 photoactivation
in HEPg2 cells
Fluorescence Perturbation techniques (FPT)
Photobleaching
Techniques
Photoactivation
Techniques
i-FRAP
Inverse FRAP
with Dr. M. Crippa
29
FRAP
Fluorescence
Recovery
after Photobleaching
Davide Mazza
mazzad@mail.nih.gov
FLIP
Fluorescence Loss
Into Photobleaching
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
And this is only one of the problems of
photo-perturbation techniques
1. Bulk Experiments.
Only information on the average behavior
Rare events are lost
Needs modelling
2. Requires “high” levels of tagged protein (µM concentration range)
Potential problems with overexpression.
30
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
SOMETIMES LESS IT’S BETTER
DECREASING CONCENTRATION
OF FLUORESCENT PROBE
FRAP
FLIP
Fluorescence
fluctuation techniques
Probe concentration
µM
µM
nM
pM to nM
Photobleaching
Yes
Yes
No
No
1 µm
>1 µm
0.2 µm
0.02 µm
Local measurement
Yes
No
Yes/no
No
Bulk technique
Yes
Yes
Yes
No
Resolution
31
Davide Mazza
mazzad@mail.nih.gov
Single
molecule
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Fluorescence fluctuation
spectroscopy (FCS)
Slower
protein
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Getting non-local information from
fluctuation measurements
Correlating fluorescence fluctuations between
different pixels in time.
STICS: Spatiotemporal image
correlation spectroscopy
(Hebert et al., Biophys J, 2005)
RICS: Raster Image Correlation
Spectroscopy
(Digman et al., Biophys J, 2005)
Pair correlation spectroscopy
(Digman and Gratton, Biophys J, 2009)
(credit: M. Digman)
33
Davide Mazza
mazza.davide@hsr.it
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Modelling of FCS faces the same
problems of FRAP
Temporal Image correlation spectroscopy
of p53 in living cells
34
Davide Mazza
mazza.davide@hsr.it
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Modelling of FCS faces the same
problems of FRAP
35
Davide Mazza
mazza.davide@hsr.it
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
SOMETIMES LESS IT’S BETTER
DECREASING CONCENTRATION
OF FLUORESCENT PROBE
FRAP
FLIP
Fluorescence
fluctuation techniques
Probe concentration
µM
µM
nM
pM to nM
Photobleaching
Yes
Yes
No
No
1 µm
>1 µm
0.2 µm
0.02 µm
Local measurement
Yes
No
Yes
No
Bulk technique
Yes
Yes
Yes
No
Resolution
36
Davide Mazza
mazzad@mail.nih.gov
Single
molecule
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
A nice add-on
Advantage: if sample sparse – we can localize single molecules with precision
higher than the resolution limit.
Localization precision depends
on the number of photons N detected
over background
σ ~ 1/√N
Reduction of out of focus signal
WIDEFIELD
ILLUMINATION
INCLINED (HILO)
ILLUMINATION
TIRF
ILLUMINATION
Images of 100nm fluorescent beads in aqueous solution - Frame rate 100fps
Tokunaga et al., Nat. Methods 2007
Single plane illumination for SMT
Supplementary
1
Gebhardt etFigure
al. Nat.
a
Meth 2013
b
C1
M3
2=;3%/>329;
;0%808
D3
@A310$;3<A1
3;;?&3%9134%$
456021370
$&3>>4>
89&:;0$
819<0
/0102134%$
456021370
D1
!"#$%&
!''$(
#)!$%&
#*"$%&
L1
D2
D4
L2
T1
T2
T3
c
M5
M1
+,--.
M2
!
Setup of the reflected light sheet microscope. (a) Scheme of the setup. Lasers are collimated (telescope
T1 – T3) and aligned (mirrors M1 and M2 and dichroic beamsplitters D1 and D2). A cylindrical lens
telescope and cylindrical lens C1 create an expanded and collimated line that overfills the back aperture
of the illumination objective. After the illumination objective, laser beams are reflected off an AFM
cantilever and focused to a diffraction limited sheet. Alternatively, laser beams are focused by L1 and
reflected by dichroic D4 for wide field illumination. Fluorescent
light is collected by the imaging objective
Davide Mazza
San Raffaele Scientific Institute
40
and focused by L2 onto an electron-multiplying CCD
(EMCCD)
chip. (b) Photograph of the illumination
Centro Di imaging Sperimentale
mazza.davide@hsr.it
objective. A home built xyz-device is clamped to the objective and holds the AFM cantilever. (c) Close
up of the illumination objective and the metal device that holds the AFM cantilever. (d) bright field image
Intranuclear single molecule tracking.
I - Bright and photostablefluorescent label.
II - Possibility of tuning concentration of label without changing the actual
concentration of the TF à FRAP, FCS and SMT at the same expression levels.
Post-translatonal labeling system (HaloTag)
SMT of tumor suppressor p53
p53 show a saltatory motion, alternating between a free and a
bound state
p53-wt in living H1299 cell nucleus
(25fps – shown15fps– FOV 12x12 mm)
Automatic tracking (Grier and Crocker algorithm) – hand checking of detected tracks
Kymograph analysis of binding events
Bright field + fluo
time
SMT
Movie 25 fps –
displayed RT
Kymograph analysis of binding events
Bright field + fluo
Kymograph (bkg subtracted)
time
SMT
Movie 25 fps –
displayed RT
Binding properties of p53 to chromatin
Distribution of residence
times for bound p53
•  Distributed exponentially (for > 95%).
•  Bound fraction ~ 20% à Mostly free.
•  Average is ~2s à Transient binding.
Bound fraction 18% ± 3%
Average residence time 1.7 ± 0.2 s
if you really like modelling…
Time [s]
Cross validation with
ensemble techniques
Displacements [µm]
Bound fraction 22% ± 5%
Average residence time 1.8 ± 0.2 s
(Gebhardt J.C.M. et al., Nat. Meth., 2013)
(Mazza D. et al., Nat Meth., 2013)
p53 binding dynamics are modulated
over time upon IR
Average residence time on chromatin [s]
10 Gy IR
0hrs
1.5hrs
2.5hrs
6!
5!
4!
3!
10 Gy
IR!
2!
1!
0!
1!
2!
3!
4!
Time after irradiation [hrs]!
5!
4.5hrs
time
Upon IR p53 binding shows
“oscillatory” dynamics
UV result in modulation of p53 binding
with a distinct temporal profile
Average residence time on chromatin [s]
8 J/m2 UV
0hrs
1.5hrs
2.5hrs
6!
5!
4!
3!
10 Gy IR!
2!
8J UV!
1!
0!
1!
2!
3!
4!
Time after irradiation [hrs]!
5!
4.5hrs
time
Upon UV p53 binding shows
a sustained increase
formed in a custom-made cuvette [19] (Hellma) by a plan apochromat illumination objective
(10x, NA 0.28, Plan Apo, Mitutoyo) as depicted in Fig. 1.
Work in progress
PolII-GFP
p53-HaloTag
With H. Mueller and J.G. McNally (submitted)
acquisition rate 5 fps – displayed 15 fps
Fig. 1. (a) Schematic representation of the instrument. Illumination light was focused into the
sample chamber by a 10x apochromat objective to form a thin light sheet in the focal plane (z0).
The sample could be positioned coarsely by a 3-axes translation stage (X, Y, Z) and more
accurately by an additional z piezo stage (Zp). Fluorescence was collected by a high NA 40x
water-immersion objective, cleared up by notch and bandpass filters (NF, BF) and detected
with a camera (EMCCD). A cylindrical lens (C) was used to shape the PSF for 3D localization.
Fast image analysis was used to determine this information in real-time and feed it back to the
z piezo stage in an active feedback loop to keep a particle near the focal plane. (b) Detailed
view of the specimen within the sample chamber. Fluorescent particles located below (1) or
above (3) the focal plane resulted in an elliptical PSF as observed on the camera chip (z0’).
Focusing the excitation light with an air objective through the 2 mm glass wall (BK7) of
the sample cuvette into aqueous solutions introduced spherical aberration, which resulted in
focal shifts when moving the sample chamber in x-direction. These shifts could automatically
be compensated by moving the objective with a motorized translation stage coupled to the xaxis of the sample stage. Chromatic aberrations in the illumination path caused by the
refractive index mismatch between air, glass wall and aqueous medium were well below the
Rayleigh limit for all light sheet configurations. The sample cuvette was placed in a custommade holder making it accessible for e.g. a micro-injection device. It was magnetically
attached to a motorized 3-axes stage (3x M-112.12S, Physik Instrumente) equipped with an
additional closed loop piezo z-stage for fast and accurate positioning perpendicular to the
image plane (P-611.ZS, Physik Instrumente).
Fluorescence was collected through the coverslip bottom of the sample cuvette (0.17 mm)
by a long working distance water immersion apochromat objective (CFI Apo LWD Lambda S
40x NA 1.15, wd 0.60 mm, Nikon). Scattered excitation light entering the detection pupil was
rejected by placing appropriate notch filters (NF01-[488, 532 and 633]U-23.7-D, Semrock)
and, if necessary, additional band pass emission filters into one of the filter turrets. For
(Spille et al. Opt Expr. 2012)
fies the constant focus step $z between planes. Scale bars, 1 Mm.
BRIEF
(~67%) of this type of grat- shift is introduced
by aCOMMUNICATIONS
carefully calculated geometri
tically to ~93%) by using a tion of the MFG pattern (Fig. 1d) and is dependent on
us
e image in the N × N arr
Objective Tube lens
MFG CCG prism
f
an MFM amicroscope
with f order
so that each
duplicate
ifferent MFG to image the a focus shift $z × (mx + N × my) (Fig. 1c). The ma
y
Primary
Fourier
Sample
image
plane MFG distortion determines the step size $z. To
obtaining
5 plane
× 5 = 25 planes,
the
lay
Final
ens,
Excitation lasers
image
.
(488
and
560
nm)
mm)
Glane)is
14
14
sat it
d
13
CCG Prism f
b
12
ne
13
11
cona
b
c
f109
12
r
8
deep
f
238 nm
7 PSF
MFG
–4∆z –3∆z –2∆z
11
parate
bjece focus
–∆z z = 0 +∆z
–2 –1 0 1 2
ch it
x (m)
Time (s)
T
+2∆z +3∆z +4∆z
7
plane
I
2.5
rise
6
MFG.
5
660 nm
2.0
Primary
4
ion,
1.5
image
3
Fourier
orrect
1.0
2
plane
–2 –1 0 1 2
void
Final image
1
z (m)
0.5
ays of
g
h Meth, 2013) 0
d
(Abrahamsson
et al., Nat
sine
he MFG.
z
2
e
hase
T
1Time (s)
1
2
0
0
40
0
40
0
20
0
1, 0
20
0
00
0
1,
80
60
0
Distance (µm)
20
0
y (µm)
0
1,
00
1, 0
20
0
0
Amplitude (a.u.)
00
0
20
0
max
ion (m)
4
z (µm)
min
80
0
40
20
0
min
.u.)
1
60
x (µm)
max
0
Amplitude
(a.u.)
Camera
2
Viability
Specificity
Resolution
Experimental Imaging Center
San Raffaele Research Institute
NOWMilan
HIRING.
- Italy
Marco E. Bianchi
Alessandra Agresti
Samuel Zambrano
Giovanni Pietrogrande
Advance Microscopy
and Nanoscopy Unit
Carlo Tacchetti
Teresa Leva
Paolo Rainone
How does a single molecule look like
How do we recognize a single molecule?
Single molecules bleach suddenly.
Individual GFP molecules
spattered on a coverslip
time
The payoff is high! (2)
tumor suppressor p53
Histone H2B
Single cell analysis of:
- Dynamic behavior of molecules
- Interactions.
- Oligomerization.
- Number of molecules.
Mazza et al, Nucl Ac Res, 2012 w/ Bianchi ME, Chromatin Dynamics unit
Acquisition speed up to 100 images/s (3x faster than video rate).
Epi, TIRF and inclined illumination.
Localize individual molecules with high precision: 20 nm (10x higher than resolution
limit)
SMT of a stably bound protein
Histone H2B is tightly bound to
chromatin
H2B displacements as function of time
Movie collected 25 fps (displayed 15 fps)
Field of view 13 x 13 mm2
Bound Fraction: 98%
SMT of tumor suppressor p53
p53 show a saltatory motion, alternating between a free and a
bound state
p53-wt in living H1299 cell nucleus
(25fps – shown15fps– FOV 12x12 mm)
Automatic tracking (Grier and Crocker algorithm) – hand checking of detected tracks
Binding properties of p53 to chromatin
Distribution of residence
times for bound p53
•  Distributed exponentially (for > 95%).
•  Bound fraction ~ 20% à Mostly free.
•  Average is ~2s à Transient binding.
Bound fraction 18% ± 3%
Average residence time 1.7 ± 0.2 s
Example: transcription factor dynamics
during cellular reprogramming
Scientific Report
2011
Overexpression of three different
transcription factors (MASH1, LMX1a,
NURR1) induces direct reprogramming
of Fibroblasts into Dopaminergic
neurons.
62
ffaele Scientific Institute
What does it happen to the transcription
factors during the reprogramming?
Davide Mazza
mazzad@mail.nih.gov
Scientific Report
2011
(Vania Broccoli)
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
Example: transcription factor dynamics
during cellular reprogramming
When all three factors are expressed, the dynamics of each of them are slower
than when only one of them is expressed
Normalized Intensity
1.0
0.8
0.6
0.4
1_day_solo
1_day_all
0.2
50
Mash - Only
40
Mash - All
30
20
10
0
Lmx1a dynamics
Time to reach 80%
recovery [s]
Time to reach 80%
recovery [s]
Mash1 dynamics
14
12
10
8
6
4
2
0
Lmx1a - Only
Lmx 1a - All
0.0
0
10
20
30
40
50
Time [s]
(T. Leva and M. Perino)
63
Davide Mazza
mazzad@mail.nih.gov
San Raffaele Scientific Institute
Centro Di imaging Sperimentale
GFP: bridging viability and
specificity
Isolated from Aqueorea Victoria
(Shimamura, 1960)
64
Applied as genetically
encoded marker
(Prasher and Chalfie, early 90’s)
Davide Mazza
mazza.davide@mail.hsr.it
Improved and modified
(Tsien et al., ongoing)
San Raffaele Scientific Institute
Centro Di imaging Sperimentale