Cognet, Laurent

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Live-Cell Super-Resolution Imaging of Endogenous Ligand-Activated Protein Dimers by Combining Upaint and Single Molecule FRET 

Sunday, February 16, 2014 201aof free NADH indicated by a distribution shifted more toward the shorter life-
time as compared to the phasor distribution of the cytoplasm. Also within the
nucleus, two distinct lifetime distributions were observed with the perinuclear
region having more bound fraction of NADH and an abundance of free NADH
in the central nucleolus region. A similar observation was reported in myoblasts
[Microscopy research and technique,75(12),1717-22,(2012)]. Furthermore,
within the cytoplasm there are some regions with more bound NADH. This
could be due to presence of more protein bound NADH within the mitochon-
dria. The phasor analysis also revealed a unique long lifetime distribution
which arose from small granular like structures within the iPS cells. These areas
also had higher intensities compared to rest of the cells. This could be liquid
droplet-associated granules (LDAGs) which were previously observed by
Stringari et al [Journal of biomedical optics 17(4),046012,(2012)] in embryonic
stem cell. Hence, we show mapping of metabolic activity of iPS cells at subcel-
lular resolution employing a non-invasive, label-free, optical microscopy
technique.
Work supported in part by NIH grants P50GM076516, P41GM103540 and
UH2TR000481-0.
1014-Pos Board B769
Pulsed Interleaved Excitation Fluctuation Imaging: Method and
Application to HIV-1 Assembly
Jelle Hendrix, Waldemar Schrimpf, Matthias Ho¨ller, Don C. Lamb.
Ludwig-Maximilians-Universita¨t, Mu¨nchen, Germany.
Pulsed interleaved excitation (PIE) is the method of nanosecond alternating
excitation with time-resolved detection and allows accurate, independent
and quasi-simultaneous determination of fluorescence intensities and lifetimes
of different fluorophores. We have combined PIE with fluctuation imaging
methods (PIE-FI) such as raster image correlation spectroscopy (RICS) and
number and brightness analysis (N&B) on a home-built multicolor laser/sam-
ple scanning microscope. PIE-FI is a novel method that allows robust and ac-
curate measurements of concentrations (down to pM), diffusion and
stoichiometry, crosstalk-free dual-color measurements to probe protein-
protein interactions, dual-color fluorescence lifetime measurements to quan-
tify Fo¨rster resonance energy transfer and even allows resolving different spe-
cies in images on the basis of their fluorescence lifetime rather than their color.
We have applied PIE-FI to study the HIV-1 Gag protein inside live cells. Dur-
ing de novo synthesis of HIV particles in infected cells, this protein assembles
at the plasma membrane and creates the shell that ultimately defines the size
and shape of budded virions. Little is known about interactions of HIV Gag
with cellular and/or viral components inside the cytosol of infected cells.
We investigated the wild-type HIV Gag as well as some critical site-specific
mutants that influence membrane and/or RNA and/or auto-interactions of
Gag to gain more insight into these early stages of HIV assembly.
1015-Pos Board B770
Live-Cell Super-Resolution Imaging of Endogenous Ligand-Activated
Protein Dimers by Combining Upaint and Single Molecule FRET
Laurent Cognet.
University of Bordeaux, Bordeaux, France.
Molecular interactions are key to many chemical and biological processes
like protein function. In many signaling processes they occur in sub-
cellular areas displaying nanoscale organizations and involving molecular
assemblies. The nanometric dimensions and the dynamic nature of the inter-
actions make their investigations complex in live cells. While super-
resolution fluorescence microscopies offer live-cell molecular imaging with
sub-wavelength resolutions, they lack specificity for distinguishing interact-
ing molecule populations. Here we combine the uPAINT super-resolution mi-
croscopy method (1) and single-molecule Fo¨rster Resonance Energy Transfer
(FRET) to identify dimers of endogenous receptors induced by ligand binding
and provide super-resolved images of their membrane distribution in live
cells (2). By developing a two-color universal-Point-Accumulation-In-the-
Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor
receptors (EGFR) activated by EGF are studied at ultra-high densities,
revealing preferential cell-edge sub-localization (3). We will also discuss
the versatility of uPAINT when using different fluorescent ligand systems
(antibodies, activating ligands, synthetic ligands etc...) for targeting and im-
aging different types of membrane proteins with superresolution, including
endogenous receptors (1-3).
References:
(1) Giannone et al, Biophys. J., 99 , 4 (2010) 1303.
(2) Winckler et al, Sci. Rep., 3 (2013) 2387,
(3) Giannone et al Methods Biol Mol 950 (2013) 95,1016-Pos Board B771
Development of Pump-Probe Nanoscopy Architecture
Kseniya Korobchevskaya, Paolo Bianchini, Silvia Galiani,
Marco Scotto d’Abbusco, Colin Sheppard, Alberto Diaspro.
Istituto Italiano di Tecnologia, Genoa, Italy.
Although modern optical microscopy allows the achievement of sub-
diffraction resolution, most of the current techniques rely on a fluorescence
contrast mechanism. Moreover, deep tissue imaging remains a challenging
task especially for thick and highly scattering biological objects. The
infrared absorption/saturation microscopy method is designed to overcome
these issues [1, 2], having InfraRed Nanoscopy (IRN) as an instrumental
perspective. The main idea behind IRN is an absorption/saturation effect
similar to conventional pump-probe in which the first pump beam modifies
the carrier density inside the sample, followed by intensity changes in the
transmitted probe beam. By introducing an additional doughnut shaped
pump beam one can transiently saturate the induced transition in the periph-
ery of the focal spot, and collect the signal generated from the central sub-
diffraction area. This is useful mainly for infrared absorption microscopy.
The IR range operates in the transparency window of biological tissues
and by selecting specific wavelengths one can observe selected vibrational
modes. Therefore, such a technique could be applied for the imaging
of non-fluorescent species, which is important for various biological
applications.
In this work we outline the architecture of a saturated transient absorption
microscope, directed towards the development of a microscopy platform
comprising a pump-probe setup and STED nanoscope [3]. Pump and
probe wavelengths can be tuned from the visible (400-500 nm) to the near
IR (800-1000 nm) spectral ranges, according to the sample properties.
This permits accessing explicit spatial and dynamical information about
the sample for applications in cellular biophysics and nanochemistry.
We also indicate the possibility of implementing 3D super-resolution
capability.
Supporting grants: LANIR, FP7/20012-2015, 280804 and PRIN, MIUR
2010JFYFY2_002.
1. C. Silien, et al., Opt. Express 20(28), 29694-29704 (2012).
2. W. Pu et al., Nat. Photonics 6, 449-453 (2013).
3. S. Galiani, et al., Opt. Express 20(7), 7362-7374 (2012).1017-Pos Board B772
Super-Resolution Imaging and Single Molecule Tracking of the Nuclear
Protein Emerin
Anthony M. Fernandez, Fabien F. Pinaud.
University of Southern California, Los Angeles, CA, USA.
Emerin is an integral protein of the nuclear membrane expressed in
most somatic cells. When under-expressed or mutated, it leads to Emery-
Dreifuss Muscular Dystrophy. This disease is characterized by cardiac ab-
normalities and progressive muscle wasting. The molecular bases of these
phenotypes are not well defined. Previous attempts at characterizing emerin
localization and diffusional mobility have relied on ensemble confocal fluo-
rescence microscopy. This approach did not reveal differences between
normal and mutated versions of emerin, perhaps due to the limited optical
resolution of confocal and FRAP techniques. Here we employ single mole-
cule tracking and super-resolution optical imaging of emerin to further
characterize emerin diffusion behaviors and localization at the nanometer
scale.
We performed single protein tracking of photoactivable-TagRFP-emerin
fusions in live cells and study their dynamics in the nuclear membrane.
Diffusion analyses indicate the co-existence of four diffusing sub-
populations. One emerin sub-population is associated with the endoplasmic
reticulum diffusion, two are located exclusively to the nuclear membrane, and
a nearly immobile sub-population also locates to the nuclear membrane.
Single molecule pulse-chase experiments reveal that the two nuclear-
exclusive sub-populations accumulate at different rates, providing further
evidence of their mutual uniqueness and their differential distribution
between the inner and outer nuclear membrane. Further studies with
disease-associated emerin mutants revealed that a single point emerin mutant
has a 250% increase in diffusion coefficient for one of the nuclear membrane
sub-populations when compared to the wild type.
We also map the 3D nanoscale organization of emerin at the nuclear
membrane using super-resolution imaging of SNAP-tag-emerin fusions.
Analysis of emerin clusters was performed with 3D Ripley’s K-function
to compare the distribution of wild-type and mutated versions of emerin.


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Live-Cell Super-Resolution Imaging of Endogenous Ligand-Activated Protein Dimers by Combining Upaint and Single Molecule FRET

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