Biophysics by the sea 2016 program and abstract book
International conference on fluorescence super-resolution microscopy, spectroscopy, molecular cell mechanics and theoretical neurophysics
26th. -30th. september 2016
Pollentia resort, Alcudia, Spain
Event organizer:
Georg August University
Third Institute of Physics
Dirk Hähnel
37077 Göttingen
Formation of low mass protostars and their circumstellar disks
Biophysics by the sea 2016 program and abstract book
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Scientific program
Monday 26.09.2016
15:45 - 16:00 WELCOME MESSAGE
Session: Imaging technics and applications I Chair: Jörg Enderlein
16:00 - 16:30 Markus Sauer (Julius-Maximilians-University Würzburg, Germany)
Super-resolution fluorescence imaging by dSTORM: Where next?
16:30 - 16:50 Fabian Zwettler (Julius-Maximilians-University Würzburg, Germany)
Expansion Microscopy meets dSTORM
16:50 - 17:10 Andrea Schulze (Julius-Maximilians-University Würzburg, Germany)
Local motions within the Hsp90 molecular chaperone machinery observed by fluorescence
quenching
17:10 - 17:30 Wim Vandenburg (Katholieke Universiteit Leuven, Belgium)
Enhancing the performance and applicability of SOFI using new probes and analysis strategies
17:30 - 18:00 COFFEE BREAK
Session: Imaging technics and applications II Chair: Christoph Schmidt
18:00 - 18:30 Theo Lasser (École polytechnique fédérale de Lausanne, Switzerland)
Super-resolution optical fluctuation imaging
18:30 - 18:50 Sebastian Letschert (Julius-Maximilians-University Würzburg, Germany)
Quantification of immune receptors on primary tumor cells
18:50 - 19:10 Jan Thiart (Georg-August-University Göttingen, Germany)
TrackNTrace: A simple and extendable open-source framework for developing single-molecule
localization and tracking algorithms
19:10 - 19:40 Thomas Jovin (Max Planck Institute for Biophysical Chemistry Göttingen, Germany)
Extended Excitation FLIM (eeFLIM)
19:40 – 21:00 DINNER
21:00 - … POSTER SESSION
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Tuesday 27.09.2016
Session: Biophysics of the cell I Chair: Theo Lasser
9:00 - 9:30 Christoph Schmidt (Georg-August-University Göttingen, Germany)
Broken detailed balance at mesoscopic scales in active biological systems
9:30 - 9:50 Samaneh Rezvani (Georg-August-University Göttingen, Germany)
Osmosis and force fluctuation of non-adhering cells
9:50 - 10:10 Tim Meyer (UMG Georg-August-University Göttingen, Germany)
Engineered Myocardium for heart repair and Drug Screening
10:10 - 10:30 Florian Rehfeld (Georg-August-University Göttingen, Germany)
Mechanics Matters for Cells: Forces, Elasticity, and Cytoskeleton
10:30 - 11:00 COFFEE BREAK
Session: Biophysics of the cell II Chair: Markus Sauer
11:00 - 11:30 Stefan Klumpp (Georg-August-University Göttingen, Germany)
Surface motility and colony growth in bacteria
11:30 - 11:50 Dieter Klopfenstein (Georg-August-University Göttingen, Germany)
May the force be with you: how actin filaments are stabilized during muscle contraction
11:50 - 12:10 Galina Kudryasheva (Georg-August-University Göttingen, Germany)
Mechano-Sensitivity is Cell Type Specific
12:10 - 12:30 Donna Arndt - Jovin (Max Planck Institute for Biophysical Chemistry Göttingen, Germany)
Generation 3 Programmable Array Microscope (PAM) for Adaptive, high speed, large format
optical sectioning
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Thursday 29.09.2016
Session: Single molecule spectroscopy and applications in biophysics I Chair: Thomas Jovin
9:00 - 9:30 Ben Schuler (University Zürich, Switzerland)
Single-molecule spectroscopy of unfolded and intrinsically disordered proteins
9:30 - 9:50 Jan Sykora (J. Heyrovsky Institute of Physical Chemistry of the CAS Prague, Czech Republic)
Are protein hydration and dynamics important factors in the enzyme kinetics? – Fluorescence
study on Haloalkane-dehalogenases
9:50 - 10:10 Roman Tsukanow (Georg-August-University Göttingen, Germany)
Investigating conformational dynamics of DNA hairpin and Holliday junction using single-
molecule fluorescence techniques
10:10 - 10:30 Erik Holmstrom (University Zürich, Switzerland)
Probing the biophysics of nucleic acids chaperones using single-photon single-molecule FRET
10:30 - 11:00 COFFEE BREAK
Session: Single molecule spectroscopy and applications in biophysics II Chair: Fred Wouters
11:10 - 11:30 Sebastian Isbaner (Georg-August-University Göttingen, Germany)
Dead-time correction of fluorescence lifetime measurements and fluorescence lifetime
imaging
11:30 - 11:50 Alexey Chizhik (Georg-August-University Göttingen, Germany)
The fluorophore out of anything
11:50 - 12:10 Ingo Gregor (Georg-August-University Göttingen, Germany)
Non-linear image scanning microscopy
12:10 - 12:30 Daja Ruhland (Georg-August-University Göttingen, Germany)
Determining absolute values of fluorescence quantum yield using a nanocavity
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Friday 30.09.2016
Session: Neuroscience Chair: Dieter Klopfenstein
9:00 - 9:30 Elisha Moses (Weizmann Institute of Science, Israel)
Dynamics in Networks of Cultured Neurons
9:30 – 9:50 Andreas Neef (Max Planck Institute for Dynamics and Self-Organization Göttingen, Germany)
A slow receptor speeds up cortical processing
9:50 - 10:10 Christian Tetzlaff (Georg-August-University Göttingen, Germany)
Self-organization of computation in neural systems by interaction between homeostatic and
synaptic plasticity
10:10 - 10:40 Fred Wouters (UMG Georg-August-University Göttingen, Germany)
Light Sheet Microscopy for Clinical Histopathology
10:40 - 11:10 COFFEE BREAK
Session: Biophysics of the cell III Chair: Ingo Gregor
11:10 - 11:30 Kengo Nishi (Georg-August-University Göttingen, Germany)
New analysis method for passive microrheology
11:30 - 11:50 Moritz Kalhöfer-Köchling (Georg-August-University Göttingen, Germany)
Generic Three Dimensional Modelling of Beating Flagella and Cilia
11:50 - 12:10 Kareem Elsayad (Vienna Biocenter, Austria)
Unravelling and understanding the mechanical properties of plants using
Brillouin Light Scattering Microspectroscopy
12:10 - 12:25 CLOSING REMARKS
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Abstracts oral presentations
(Listed alphabetically by last name)
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
The fluorophore out of anything
Alexey Chizhik
III. Institute of Physics – Biophysics, Georg-August-University Göttingen
We all, those who work in the field of fluorescence microscopy, got used to buying
fluorophores from manufacturers, which are believed to produce any kind of dye our
experiments may ever require. A couple of mouse clicks – and a vial of colorful solution is
standing on our table. The reverse of the medal is high price, often impossibility of any
chemical modification of the dye or even unknown chemical structure, and finally, sad but
true, improper characterization of the fluorophore’s physico-chemical properties.
A decade ago, in 2004, Scrivens and co-workers accidentally found a way around it, probably
even haven’t been realizing it first1. What they reported was fluorescent carbon-based
impurities, which they observed as a result of purification of carbon nanotubes. Because
“impurities” is what one normally gets for free, or even against one’s will, the publication was
followed by a tsunami of works, where researchers reported on cheap and simple synthesis
of various fluorophores that consisted mostly of carbon nanoparticles and numerous types of
surface chemical groups. It turned out that thermal treatment, or simply put, combustion of
basically any organic substance leads to generation of fluorescent carbon nanoparticles, which
have been often called “carbon dots” or “carbon nanodots”. The ways of synthesis reported
strike imagination: “carbon dots from orange juice”, ”carbon dots from milk”, “carbon dots
from waste paper”2. In recent years, a lot of efforts have been made to understand the
mechanism of their fluorescence as well as to develop more advanced ways of synthesis in
order to achieve high monodispersity of particles and homogeneity of their photophysical and
structural properties3. In this talk I am going to provide you with an overview of the most
prominent works in this field and to present you our own recent results.
1. X. Xu et al. Journal of the American Chemical Society 2004, 126, 12736-12737.
2. C. J. Reckmeier et al. Opt. Express 2016, 24, A312-A340.
3. S. Ghosh et al. Nano Letters 2014, 14, 5656-5661.
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Unravelling and understanding the mechanical properties of plants using
Brillouin Light Scattering Microspectroscopy
Kareem Elsayad
Advanced Microscopy, VBCF, Vienna Biocenter, Vienna
Brillouin Light Scattering (BLS) spectroscopy is an all-optical label-free technique which allows
for the determination of the viscoelastic properties of a sample. BLS is generally a very weak
process, based on the interaction of light with thermal density fluctuations, and thus
challenging to implement for life-science/biomedical applications. Recent advances in
spectrometer and camera designs have however made it possible to perform BLS
measurements on live cells, opening the door to a new means of studing the mechanical
properties of biological systems. Here I will discuss the use of BLS Microspectroscopy and
correlative Fluorescence – BLS Microspectrocopy to map the viscoelastic properties of cells
and tissue in 3 dimensions, focusing on its use to understand the mechanical properties of
plant cells. 3 dimensional mapping of the mechanical properties of plant cells is particularly
interesting given the delicate balance between extracellular matrix (cell wall) mechanical
properties and turgor pressure involved in defining cell shape, assuring “correct”
development, for maintaining the structural integrity of the organism as a whole, and
ultimately determining their survival subject to all types of environmental perturbations.
Firstly I will give an introduction to BLS including experimental setups and the physical
principles it is based on. I will then discuss some details of the quantities that are and can be
extracted from a BLS measurement and how and to what extent they may be compared to or
compliment results obtained from alternative measurements of the mechanical properties of
and within cells - such as those obtained using microrheology and perturbation-deformation
techniques such as Atomic Force Microscopy (AFM). I then will present a series of studies on
different live plant cells and tissue we have performed focusing on the physical and biological
significance of the obtained results. Finally I will summarize the strengths of the technique, its
limitations and some of the current challenges, along with an outlook of what we are working
on, and some planned and potential future applications in biophysics research as well as
medical diagnostics.
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Non-linear image scanning microscopy
1Ingo Gregor, 2Robert Ros, 1Jörg Enderlein
1III. Institute of Physics – Biophysics, Georg-August-University Göttingen
2Department of Physics and Center for Biological Physics, Arizona State University, Tempe AZ
ingo.gregor@phys.uni-goettingen.de, www.joerg-enderlein.de
Nowadays, multiphoton microscopy can be considered as a routine method for the
observation of living cells, organs, up to whole organisms. Second-harmonics generation (SHG)
imaging has evolved to a powerful qualitative and label-free method for studying fibrillar
structures, like collagen networks. However, examples of super-resolution non-linear
microscopy are rare. So far, such approaches require complex setups and advanced
synchronization of scanning elements limiting the image acquisition rates. We describe theory
and realization of a super-resolution image scanning microscope [1, 2] using two-photon
excited fluorescence as well as second-harmonic generation. It require only minor
modifications compared to a classical two-photon laser-scanning microscope and allows
image acquisition at the high frame rates of a resonant galvo-scanner. We achieve excellent
sensitivity and high frame-rate in combination with two-times improved lateral resolution. We
applied this method to fixed cells, collagen hydrogels, as well as living fly embryos. Further,
we verified the excellent image quality of our setup for deep tissue imaging.
[1] Müller C.B. and Enderlein J. (2010) Image scanning microscopy. Phys. Rev. Lett. 104(19), 198101.
[2] Sheppard C.J.R. (1988) Super-resolution in confocal imaging. Optik (Stuttg) 80 53–54.
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Dead-time correction of fluorescence lifetime measurements
and fluorescence lifetime imaging
Sebastian Isbaner, Narain Karedla, Daja Ruhlandt, Simon Christoph Stein, Anna Chizhik,
Ingo Gregor, Jörg Enderlein
III. Institute of Physics – Biophysics, Georg August University, Göttingen
Dead-time artifacts can dramatically influence the shape of Time-Correlated Single Photon
Counting (TCSPC) histograms such as fluorescence lifetime curves [1]. These artifacts occur at
high count rates, which limit the acquisition speed in Fluorescence Lifetime Imaging
Microscopy (FLIM). We present an algorithm that corrects the distortions of TCSPC histograms
which are caused by constant electronics and/or detector dead-times [2]. We verified the
algorithm with Monte-Carlo simulations and fluorescence lifetime measurements.
Furthermore, we performed FLIM measurements on densely labeled cells at various excitation
powers and corrected the lifetime and intensity values for each pixel. Our correction method
is not restricted to TCSPC measurements only, but can be applied to any periodic single-event
counting or timing measurement. Since it corrects dead-time artifacts for both lifetime and
intensity, the algorithm could be beneficial for example for lidar or time-resolved fluorescence
anisotropy measurements.
[1] W. Becker, Advanced Time-Correlated Single Photon Counting Techniques (Springer, 2005).
[2] S. Isbaner, N. Karedla, D. Ruhlandt, S.C. Stein, A. Chizhik, I. Gregor, and J. Enderlein, Opt. Express 24, 9429-9445 (2016)
11. 11
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Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Generation 3 Programmable Array Microscope (PAM) for
adaptive, high speed, large format optical sectioning
Donna J. Arndt-Jovin, Anthony H. B. de Vries, Thomas M. Jovin
Laboratory of Cellular Dynamics, Max-Planck-Institute for Biophysical Chemistry, Göttingen
djovin@mpibpc.mpg.de
We report on the current version of the optical sectioning programmable array
microscope(PAM) implemented with a digital micro-mirror device (DMD) as a spatial light
modulatorutilized for both fluorescence excitation and emission detection. The PAM is based
on structured illumination [1]. A sequence of HD (1920×1080) binary patterns of excitation
light is projected into the focal plane of the microscope at the 18 kHz binary frame rate of the
TI1080p DMD. The resulting sequence of patterned emissions is captured in a single
acquisition as two distinct images: conjugate (ca. “on-focus”) consisting of signals impinging
on and deviated from the “on” elements of the DMD, and the non-conjugate (ca. “out-of-
focus”) of those falling on and deviated from the “off” elements. The sectioned image is gained
from a weighted subtraction of the conjugate and non-conjugate images. This procedure
allows for a high duty cycle (typically 30 to 50%) of on-elements in the excitation patterns and
thus functions well with low light intensities, preventing saturation of the fluorophores. The
corresponding acquisition speed is also very high, limited only by the bandwidth of the
camera(s) (100 fps full frame with the current sCMOS camera) and the optical power of the
light source (lasers, LEDS). In contrast to the static patterns typical of SIM systems, the
programmable array allows optimization of the patterns to the sample (duty cycle and feature
size), as well as enabling a wide range of microscopy applications, ranging from patterned
photobleaching, (FRAP, FLIP) and photoactivation, spatial superresolution (SIM, etc.),
automated adaptive minimized light exposure (MLE) [2], and photolithography. This work is
supported by BMBF VIP Grant 03V0441 (iPAM: "Intelligentes" Programmierbares Array
Mikroskop).
[1] de Vries, A., N. Cook, S. Kramer, D. Arndt-Jovin and T. Jovin (2015). "Generation 3 programmable array microscope (PAM) for high
speed, large format optical sectioning in fluorescence." Proc. SPIE 9376(93760C): 1-15
[2] W. Caarls; B. Rieger, A.H.B. de Vries, D.J. Arndt-Jovin, T.M. Jovin (2010). “Minimizing light exposure with the programmable array
microscope”, J. MICROSCOPY, 241, 101-110
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Extended Excitation FLIM (eeFLIM)
Thomas M. Jovin, Nathan P. Cook, and Donna Arndt-Jovin
Laboratory of Cellular Dynamics, Max-Planck-Institute for Biophysical Chemistry, Göttingen
tjovin@mpibpc.mpg.de
The usual dogma in the field of time-domain fluorescence lifetime determination is that “the
shorter the excitation pulse the better”. We overcome this requirement by recording the
integrated emission of an emitting species excited with a rectangular light pulse with a
duration substantially longer than the anticipated lifetimes. Sensitive and accurate
determinations of the mean intensity-weighed lifetime are feasible. A series of successive
determinations (≥2) are taken in the region corresponding to constant excitation intensity and
at integration times > 6·the longest lifetime in the sample population. These points correspond
to a straight line, the slope and position of which are referenced to a companion measurement
of a sample with 0 lifetime (e.g. scattered excitation light) or known lifetime so as to yield the
absolute mean lifetime. That is, the displacement on the integration time (gate width) axis is
given by the lifetime (Fig. 1). The mixtures can be of arbitrary heterogeneity. For a
twocomponent system (e.g. a binding reaction), the mean lifetime can be expressed
analytically as a function of the fraction of species engaged in FRET. The mean lifetime is very
useful in numerous other applications, including single molecule determinations. We have
implemented eeFLIM in an imaging system based on the gated intensified camera PI-MAX4-
1024EMB of Princeton Instruments using laser diodes for excitation. This camera features
excellent spatial resolution and linearity (emCCD detector), and powerful software +
electronics for control of multimode acquisition and external synchronization. The system is
very sensitive and allows real-time full-field (1K×1K) FLIM at rates that can exceed 1 Hz.
Some important advantages of eeFLIM can be emphasized: (1) the rectangular excitation
pulses (e.g. 10-50 ns) are easy to generate and provide very high pulse energies and thus
intense response signals; it is anticipated that light sources based on pulsed LEDs will be more
versatile (wide spectral range, no speckle) and cost effective. (2) virtually all the light emitted
per pulse (discounting detection efficiencies) is utilized; (3) the temporal resolution is tens of
ps; long-lived emissions (delayed fluorescence, phosphorescence) can also be measured; (4)
lifetime image calculations are very fast, involving only simple, linear, noniterative
calculations. (5) eeFLIM is also applicable to single or array detectors and TCSPC detection.
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Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Figure 1. Validation of eeFLIM. Mean normalized integrated signals from images
of IRF (scattering from focal plane) and 3 fluorescence dye solutions. The inset
highlights the horizontal (temporal) displacements (equal to the lifetimes) of the
4 measured dyes: Rhodamine B (1.6 ns), Coumarin 6 (2.5 ns), Rhodamine 110 (3.8
ns), and dianionic Fluorescein (4.1 ns).
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Generic Three Dimensional Modelling of Beating Flagella and Cilia
Moritz Kalhöfer-Köchling, Steffen Mühle
III. Institute of Physics – Biophysics, Georg-August-University Göttingen
m.ka-koe@gmx.net
Flagella and cilia are motile, hairlike cell appendages, providing forces, capable of self
propulsion and transport for example of waste in the trachea. Strong effort has been put into
the elucidation of the underlying mechanisms driving the dynamics of axonemal beating, and
although the structure of flagella and cilia is well understood, they still wait to be revealed.
Most research in this field has been focused on two dimensional models, yielding an accurate
description of the typical, whip-like, beating motion of spermatozoa. Yet, also helical and
other three-dimensional movement patterns have been observed, demanding new,
augmented models. Using the natural frame as an advanced description of three-dimensional
filaments and incorporating modern operator splitting techniques for the numerical tasks, we
could model helical beating patterns on the basis of a generic and simple physical model. The
model takes anisotropic drag, internal active elements and a propelled cell body into account,
providing a formidable springboard for the implementation of further physiological concepts.
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
May the force be with you: how an actin binding protein stabilizes filaments
during muscle contraction
1Eugenia Butkevich, 1Kai Bodensiek, 1,4Nikta Fakhri, 1Kerstin von Roden, 1,2Iwan A. T. Schaap,
3Irina Majoul, 1Christoph F. Schmidt, 1Dieter R. Klopfenstein
1III. Institute of Physics – Biophysics, Georg-August- University Göttingen
2Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB)
Göttingen
3Institute of Biology, Center for Structural and Cell Biology in Medicine, University of Lübeck,
4Department of Physics, Massachusetts Institute of Technology, Cambridge, MA
Dieter.Klopfenstein@phys.uni-goettingen.de
Actin filament organization and stability in the sarcomeres of muscle cells are critical for force
generation. We have identified and functionally characterized a C. elegans drebrin-like protein
DBN-1 as a novel constituent of the muscle-contraction machinery. In vitro, DBN-1 exhibits
actin-filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles
of C. elegans. During muscle contraction cycle, DBN-1 alternates location between myosin-
and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-
bands where it likely regulates proper spacing of a-actinin and tropomyosin and protects actin
filaments from the interaction with ADF/cofilin. DBN-1 loss-of- function results in the partial
depolymerization of F-actin upon muscle contraction. Taken together, our data show that
DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship
between actin-binding proteins such as a-actinin, tropomyosin and ADF/cofilin and possibly
strengthening actin filaments by bundling.
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Probing the biophysics of nucleic acids chaperones using
single-photon single-molecule FRET
Erik D. Holmstrom, Daniel Nettels, Benjamin Schuler.
University of Zurich
Much like proteins, nucleic acids can fold into intricate 3D structures with specific biological
functions. However, in order to do so they must avoid any potential non-functional
conformational traps that often complicate the folding process. Nucleic acid chaperones are
an emergent class of proteins that function to alleviate this notorious folding problem,
enabling efficient formation of natively folded RNAs and DNAs. These chaperones facilitate
many nucleic acid-dependent processes, including critical steps in the life cycles of many
viruses. However, a detailed mechanistic understanding of the chaperoning process has
remained elusive, especially for viral proteins that are often intrinsically disordered.
Recently, we have started to uncover some of the structural and dynamical aspects of nucleic
acid chaperone activity using a variety of single-photon single-molecule FRET techniques.
Specifically, we chose to study the interaction between a model DNA hairpin and the
nucleocapsid domain of the Hepatitis C virus core protein (HCVncd), which is a non-specific,
intrinsically-disordered nucleic acid chaperone that facilitates viral genome dimerization. By
independently observing both components of this nucleoprotein interaction with smFRET, we
have been able use single-photon analysis methods to characterize multiple structural and
dynamical changes in both the chaperone (i.e., HCVncd) and its model substrate (i.e., DNA
hairpin). These findings have been used to construct a structurally and kinetically motivated
molecular mechanism that explains this interesting biophysical process.
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Surface motility and colony growth in bacteria
Stefan Klumpp
Institute for Nonlinear Dynamics, Georg-August- University Göttingen
Max-Planck-Institute of Colloids and Interfaces, Potsdam
Motile bacteria move through a variety of mechanisms, which employ different molecular
machines. Often, physical forces play a key role. I will discuss this using the role of mechanical
interactions in twitching motility as an example. Twitching motility is a mode of motion on
surfaces that is driven by the retraction of type IV pili, filamentous appendages that pull the
cell forward through cycles of growth, attachment to the surface and retraction into the cell,
driven by APTases at the base of the pili. In some bacterial species multiple pili pull the cell in
different directions simultaneously. Thus, the pili perform a two-dimensional tug-of-war.
Tugof- war-like interactions, where molecular motors exert forces on each other, were
previously studied for bidirectional cytoskeletal transport. I will review this case, which is one-
dimensional and show that the tug-of-war provides a mechanism for persistent directionality.
In the two-dimensional case, the tug-of-war is less efficient at doing so than in one dimension,
as will be shown for the case of the twitching motility of N. gonorrhoeae, where an additional
mechanisms for directional memory was predicted theoretically and confirmed
experimentally [1]. N. gonorrhoeae bacteria use twitching to find each other in order to
initiate the formation of colonies. As a second topic, I will discuss the growth of planar colonies
and its interplay with the adhesion between cells that is also mediated by the type IV pili. To
that end, a minimal model for mixed colonies of cells of different adhesion is presented [2].
The model effectively combines differential adheision with rangeexpansion-like growth.
[1] R. Marathe, C. Meel, …, B. Meier, S. Klumpp, Nature Comm. 5, 3759 (2014)
[2] J.J. Dong and S. Klumpp, unpublished
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Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Mechano-Sensitivity is Cell Type Specific
Galina Kudryasheva, Florian Rehfeldt
III. Institute of Physics – Biophysics, Georg-August- University Göttingen
galina.kudryasheva@phys.uni-goettingen.de
Nowadays it is widely acknowledged that cellular fate is dependent on the mechanical
properties of their micro-environment. Cells sense the stiffness of their surrounding with
contractile acto-myosin stress fibers through focal adhesions and react to such physical stimuli
by altering their bio-chemical pathways. Human mesenchymal stem cells (hMSCs) are an
especially striking as their differentiation towards various cell types can be guided not only by
chemical induction, but also by tuning the extracellular matrix stiffness. While the entire
differentiation process can take several days up to weeks, the structure and dynamics of stress
fibers can be used as an early morphological marker and theoretically modelled using classical
mechanics with an active spring model [1]. We use this approach to analyze the mechanical
cell-matrix interactions of hMSCs and several types of differentiated cells.
We plated cells on elastic poly-acrylamide hydrogels covering the whole physiological range
of stiffness given by Young’s moduli E from 1 to 130 kPa. Using immunofluorescence we
visualized stress fibers and analyzed the cytoskeletal morphology [2]. Analyzing cell area and
cytoskeletal order parameter we could assign an effective cellular stiffness that shows
distinct differences during the differentiation process and for different cell types. Our
experiments show that cellular susceptibility to the substrate elasticity is highly cell type
specific and dependent on acto-myosin contractility.
[1] A. Zemel et al. Nat.Phys. 6, 468–473 (2010)
[2] B. Eltzner et al. PLoS One 10 (2015)
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Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Super-resolution optical fluctuation imaging
1Theo Lasser, 1,5Hendrik Deschout, 1,4,5Tomas Lukes, 1Azat Sharipov, 1Daniel Szlag,
1Lely Feletti, 2Wim Vandenberg, 2Peter Dedecker, 2Johan Hofkens, 3Marcel Leutenegger,
1Arno Bouwens, 1Jochem Deen, 1Adrien Descloux, 1Aleksandra Radenovic
1Laboratory of Nanoscale Biology & Laboratoire d’Optique Biomédicale,
Ecole Polytechnique Fédérale de Lausanne
2Department of Chemistry, University of Leuven, Heverlee
3Abteilung NanoBiophotonik, Max-Planck-Institut für biophysikalische Chemie, Göttingen
4Department of Radioelectronics, FEE, Czech Technical University, Prague
theo.lasser@epfl.ch, http://lob.epfl.ch, www.voirestsavoir.ch
Super-resolution optical fluctuation imaging (SOFI) allows 3D sub-diffraction fluorescence
microscopy of living cells. When analyzing the acquired image sequence with an advanced
correlation method, i.e. high-order cross-cumulant analysis, super-resolution in all three
spatial dimensions can be achieved.
In this talk we will introduce the underlying principles of SOFI and point to its differences and
shared characteristics with prominent SMLM methods.
Novel SOFI 3D imaging for life cell imaging, a combined PALM-SOFI framework used for
imaging the dynamics of focal adhesion with additional insights into molecular parameters
will be shown to demonstrate the unique potential of SOFI.
20. 20
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Quantification of immune receptors on primary tumor cells
1Sebastian Letschert, 2Thomas Nerreter, 2Michael Hudecek,
2Hermann Einsele, 1Markus Sauer
1Department of Biotechnology & Biophysics, Julius-Maximilians-University Würzburg
2Department of Hematology and Medical Oncology, Medical Clinic and Policlinic II,
University Hospital Würzburg
sebastian.letschert@uni-wuerzburg.de
T-cells as an important component of everyone’s immune system are able to detect antigens
specifically on the surface of their target cells. Only few antigen molecules per cell are enough
to activate T-cells and to initiate an immune response which leads to the elimination of the
target cell. During the last 20 years this ability was utilized in cancer therapy to develop gene-
modified T-cells which specifically detect and destroy cancer cells. This was realized by
encoding and expressing a synthetic membrane receptor called CAR (chimeric antigen
receptor) in patient T-cells. CARs have the ability to bind tumor specific antigens and activate
the CAR T-cell. (1, 2)
To analyze these target molecules, fluorescence flow cytometry systems as for example FACS
are the methods of choice. However, despite its brilliant sensitivity common flow cytometry
instruments are not able to significantly distinguish between positive cells with only a few
surface molecules and the negative control. Localization based super-resolution microscopy
methods share the potential to extract single-molecule information from fluorescently labeled
cells.
In this study we present a live-cell labeling strategy for screening of antigen-positive (CD19)
cancer cells from multiple myeloma patients. Furthermore, we performed direct stochastic
optical reconstruction microscopy (dSTORM)(3, 4) of these cells in order to analyze and
quantify CD19 molecules as a possible target for a myeloma specific CAR T-cell
immunotherapy. The aim is to combine and compare the benefits of flow cytometry (high-
throughput) and dSTORM (high sensitivity, single-molecule information) to analyze and
quantify low-abundance immune receptors on cancer cells.
(1) Jensen and Riddell, Immunol Rev, 257 (2014), 127–144.
(2) Sommermeyer et al., Leukemia, 30 (2016), 492–500.
(3) Heilemann et al., Angew Chem Int Ed, 47 (2008), 6172–6176.
(4) van de Linde et al., Nat Protoc, 6 (2011), 991–1009.
21. 21
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Engineered Myocardium for heart repair and Drug Screening
Tim Meyer, Malte Tiburcy, Susanne Schlick, Wolfram-H. Zimmermann
Institute of Pharmacology and Toxicology, University Medical Center Göttingen
Tissue engineered organ surrogates evolve rapidly as advanced tools for safety and efficacy
screens. Human pluripotent stem cells are today available to engineer human organoids under
controlled and highly reproducible conditions. Human cardiomyocytes from embryonic and
induced pluripotent stem cells can be reconstituted in collagen-hydrogels to facilitate self-
assembly into engineered human myocardium (EHM) for applications tissue replacement
therapy and screens for cardio-active drugs (Figure 1).
Here we present advances in automated tissue generation and analysis focusing on the newly
developed 48 well format for high throughput screening
Figure 1: Concentration response curves of 12 well established reference compounds tested with Engineered Heart Tissue in
an organ bath setup. The Screen identified 5 compounds as positive inotropes, 3 as negative inotropes, and 3 showed a
concentration dependent biphasic inotropic behavior. Our screening platform identified all test compounds according to
their known pharmacologic profiles.
.
22. 22
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Dynamics in Networks of Cultured Neurons
Elisha Moses
Department of Physics of Complex Systems, The Weizmann Institute of Science, Rehovot
elisha.moses@weizmann.ac.il
Cultured networks of neurons from hippocampus constitute a fascinating reductionist model
for biological computation. While individual neurons retain the physiological characteristics
as in the intact brain, the structure and connectivity in the network are considerably simpler
to measure and analyze, and therefore to engineer and design. We show that disconnected
single neurons oscillate independently of each other, and that when the network is connected
they synchronize into periodic network bursts in which all neurons fire together. This behavior
is attributed to Kuramoto-Strogatz like behavior for the synchronization of pulse-coupled
oscillators. We investigate how initiation of this burst is brought about, and find that the
recruitment of a minimal cohort of firing units plays a crucial role in the process. Activation of
the whole network is well described by a theoretical model of percolation invoking the need
for ‘quorum’ decision making.
(1) Penn Y., Segal M. and Moses E. “Network synchronization in hippocampal neurons”, Proceedings of the National Academy of Sciences
USA 113 (12), 3341–3346 (2016).
(2) J. Soriano, M. Martínez-Rodríguez, T. Tlusty, E. Moses. "Development of Input Connections in Neural Cultures", The Proceedings of
the National Academy of Sciences USA 105, 13758-13763 (2008). doi: 10.1073/pnas.0707492105
(3) J.-P. Eckmann, E. Moses, O. Stetter, T. Tlusty, C. Zbinden, “Leaders of neuronal cultures in a quorum percolation model” Frontiers in
Computational Neuroscience, 4 Article 132, doi:10.3389/fncom.2010.00132 (2010).
23. 23
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
A slow receptor speeds up cortical processing
Andreas Neef
Bernstein Center for Computational Neuroscience
Biophysics of neural information encoding
Max-Planck-Institute for Dynamics and Self-Organization, Göttingen
A population of cortical neurons encodes common input in the population firing rate. The
transfer function, input --> firing rate, is shaped by the properties of several ion channels.
However, the cut-off at high input frequencies, this is, the temporal precision encoding, is
restricted by one property of one channel type: the voltage dependence of the sodium
channels in the axon initial segment (Focault-Trocme 2005).
Several experimental studies observed that a more slowly fluctuating background input
promotes a larger bandwidth of encoding but physiological relevance and mechanistic
explanations for this observation could not be found. We studied the main gateway of sensory
information into the brain and present both, physiological relevance and mechanistic
explanation for the increased bandwidth through slow input fluctuations.
The relay cells of the cortical gateway cells utilize a very unusual NMDA receptor that is not
blocked at rest (Fleidervish 1998), this results in an unusually slowly fluctuating background
input. As a consequence the relay cells are able to transmit the sensory input without
attenuation until 200 Hz which doubles the reliability of thalamocortical spike transmission.
We found that two different potassium channel types, KCNQ and Kv1, are required to couple
the correlation time of input fluctuations to the bandwidth of information encoding and
present a mechanistic model of this coupling.
24. 24
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
New analysis method for passive microrheology
1Kengo Nishi, 2Maria L. Kilfoil, 1Christoph F. Schmidt, 3Fred C. MacKintosh
1III. Institute of Physics - Biophysics, Georg August University Göttingen
2Univ. of Massachusetts, Amherst, MA
3Department of Physics & Astronomy, Faculty of Sciences, Vrije Universiteit Amsterdam
kengo.nishi@phys.uni-goettingen.de
Passive microrheology is an experimental technique used to measure the mechanical
response of materials from the fluctuations of micron-sized beads embedded in the medium.
Microrheology is well suited to study rheological properties of materials that are difficult to
obtain in larger amounts and also of materials inside of single cells. In one common approach,
one uses the fluctuation-dissipation theorem to obtain the imaginary part of the material
response function from the power spectral density of bead displacement fluctuations, while
the real part of the response function is calculated using a Kramers-Kronig integral. The high-
frequency cut-off of this integral strongly affects the real part of the response function in the
high frequency region. Here, we discuss how to obtain more accurate values of the real part
of the response function by an alternative method using autocorrelation functions.
[1] B. Schnurr, F. Gittes, F. C. MacKintosh, and C. F. Schmidt, Macromolecules, 1997, 70, 7781-7792.
25. 25
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Mechanics Matters for Cells: Forces, Elasticity, and Cytoskeleton
Florian Rehfeldt
III. Institute of Physics – Biophysics, Georg-August-University Göttingen
rehfeldt@physik3.gwdg.de, www.florian-rehfeldt.de
The mechanical properties of microenvironments in our body vary over a broad range and are
as important to cells as traditional biochemical cues. An especially striking experiment of this
mechano-sensitivity demonstrated that systematic variation of the Young’s elastic modulus E
of the substrate can direct the lineage differentiation of human mesenchymal stem cells
(hMSCs) (1).
To elucidate the complex interplay of physical and biochemical mechanisms of cellular
mechano-sensing, well-defined extracellular matrix (ECM) models are essential. While elastic
substrates made of poly-acrylamide (PA) are widely in use, they have the potential drawback
that the precursors are cytotoxic and therefore do not allow for 3D culture systems. Here, a
novel biomimetic ECM model based on hyaluronic acid (HA) was successfully established that
exhibits a widely tuneable and well-defined elasticity E, enables 2D and 3D cell culture and
enables us to mimic a variety of distinct in vivo microenvironments (2). Quantitative analysis
of the structure of acto-myosin fibers of hMSCs on elastic substrates with an order
parameter S, reveals that the stress fiber morphology is an early morphological marker of
mechano-guided differentiation and can be understood using a classical mechanics model (3-
5). Furthermore, the cytoskeleton also dictates the shape of the nucleus and lends support to
a direct mechanical matrix-myosin-nucleus pathway (6).
[1] Engler, A. J., S. Sen, H. L. Sweeney, and D. E. Discher. 2006. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 126:677-
689.
[2] Rehfeldt, F., A. E. X. Brown, M. Raab, S. Cai, A. L. Zajac, A. Zemel, and D. E. Discher. 2012. Hyaluronic acid matrices show matrix
stiffness in 2D and 3D dictates cytoskeletal order and myosin-II phosphorylation within stem cells. Integrative Biology 4:422-430.
[3] Zemel, A., F. Rehfeldt, A. E. X. Brown, D. E. Discher, and S. A. Safran. 2010. Optimal matrix rigidity for stress-fibre polarization in
stem cells. Nature Physics 6:468-473.
[4] Zemel, A., F. Rehfeldt, A. E. X. Brown, D. E. Discher, and S. A. Safran. 2010. Cell shape, spreading symmetry, and the polarization
of stress-fibers in cells. J Phys-Condens Mat 22.
[5] Paluch, E. K., C. M. Nelson, N. Biais, B. Fabry, J. Moeller, B. L. Pruitt, C. Wollnik, G. Kudryasheva, F. Rehfeldt, and W. Federle. 2015.
Mechanotransduction: use the force(s). BMC Biology 13:1-14.
[6] Swift, J., I. L. Ivanovska, A. Buxboim, T. Harada, P. C. D. P. Dingal, J. Pinter, J. D. Pajerowski, K. R. Spinler, J.-W. Shin, and M. Tewari.
2013. Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation. Science 341.
26. 26
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Osmosis and force fluctuation of non-adhering cells
1Christoph F. Schmidt, 2Todd M. Squire, 1Samaneh Rezvani
1 III. Institute of Physics – Biophysics, Georg-August-University Göttingen
2Department of Chemical Engineering, University of California. Santa Barbara, CA
srezvani@physik3.gwdg.de
Cells sense their micro-environment through biochemical and mechanical interactions. They
can respond to stimuli by undergoing shape- and possibly volume changes. Key components
in determining the mechanical response of a cell are the viscoelastic properties of the
actomyosin cortex, effective surface tension, and the osmotic pressure. We use custom-
designed microfluidic chambers with integrated hydrogel micro windows to be able to rapidly
change solution conditions for cells without any hydrodynamic flow. We use biochemical
inhibitors and different osmolytes and investigate the immediate response of individual cells.
Using a dual optical trap makes it possible to probe suspended rounded-up cells by active and
passive microrheology to quantify the response to the various stimuli.
[1] F. Schlosser, F. Rehfeldt and C. F. Schmidt, Phil. Trans. R. Soc. B 370, 0028 (2014)
[2] Joel S. Paustian and Todd M. Squires, Phys Rev 3, 041010 (2013)
27. 27
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Determining absolute values of fluorescence quantum yield using a nanocavity
Daja Ruhlandt, Alexey Chizhik, Jörg Enderlein
III. Institute of Physics – Biophysics, Georg-August-University Göttingen
daja.ruhlandt@phys.uni-goettingen.de
The fluorescence quantum yield (QY), which is the ratio of the number of photons emitted by
a fluoreophore to the number of photons absorbed by it, is one of the key photophysical
properties of fluorescent species. It determines the suitability of an emitter for applications
such as labeling of biological samples, but its value is also needed for data evaluation in
techniques such as metal-induced energy transfer (MIET). There exist several methods for
determining the QY experimentally, for example by comparing the fluorescence to a
fluorescent standard of known QY, by doing a thermal lensing measurement or by using an
integrating sphere. All of these methods are either technically challenging or can suffer from
inaccuracies typically occurring in referential measurements.
We have developed a reference- and calibration-free technique for determining absolute
values of fluorescence QY using a tunable metallic nanocavity. It requires only very small
amounts of low-concentration chromophore solution and can even be used for multicolor
samples. Furthermore, we have employed the method on dyes in lipid bilayers, enabling us to
monitor changes in QY that are induced by the local chemical environment.
28. 28
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Super-resolution fluorescence imaging by dSTORM: Where next?
Markus Sauer
Department of Biotechnology & Biophysics, Julius-Maximilians-University Würzburg
Super-resolution microscopy by single-molecule photoactivation or photoswitching and
position determination (localization microscopy) has the potential to fundamentally
revolutionize our understanding of how cellular function is encoded at the molecular level.
Among all powerful high-resolution imaging techniques introduced in recent years localization
microscopy excels at it delivers single-molecule information about the distribution and,
adequate controls presupposed, even absolute numbers of proteins present in subcellular
compartments. This provides insights into biological systems at a level we are used to think
about and model biological interactions. We briefly introduce basic requirements of
localization microscopy, its potential use for quantitative molecular imaging, and discuss
present obstacles and ways to bypass them. We demonstrate the advantageous use of
dSTORM for quantitative imaging of synaptic proteins, the study of plasma membrane
organization, and the molecular architecture of multiprotein complexes. Finally, we outline
how dSTORM can be used advantageously to improve next generation medical therapies.
29. 29
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Broken detailed balance at mesoscopic scales in active biological systems
Christoph F. Schmidt
III. Institute of Physics – Biophysics, Georg-August-University Göttingen
Systems in thermodynamic equilibrium are not only characterized by time-independent
macroscopic properties, but also satisfy the principle of detailed balance in the transitions
between microscopic configurations. Living systems function out of equilibrium and are
characterized by directed fluxes through chemical states, which violate detailed balance at the
molecular scale.
I will report on a method to probe for broken detailed balance and demonstrate how such
non-equilibrium dynamics is manifest at the mesosopic scale. The periodic beating of an
isolated flagellum from Chlamydomonas reinhardtii exhibits probability flux in the phase
space of shapes. With a model, we show how the breaking of detailed balance can also be
quantified in stationary, non-equilibrium stochastic systems in the absence of periodic motion.
We further demonstrate such broken detailed balance in the non-periodic fluctuations of
primary cilia of epithelial cells. This analysis provides a general tool to identify non-equilibrium
dynamics in cells and tissues.
30. 30
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Single-molecule spectroscopy of unfolded and intrinsically disordered proteins
Ben Schuler
University of Zurich
Single-molecule spectroscopy provides a versatile way of probing distance distributions and
dynamics in biomolecules. We have been using these techniques extensively for probing the
physical properties of unfolded and intrinsically disordered proteins over a wide range of
conditions. For a complete picture of structure and dynamics, however, the integration with
other methods, including theory and simulations, can be essential. I will illustrate this point
with a recent example where we address a long-standing controversy regarding the
denaturant-dependent collapse of unfolded proteins
31. 31
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Local motions within the Hsp90 molecular chaperone machinery
observed by fluorescence quenching
1Andrea Schulze, 1Gerti Beliu, Dominic A. Helmerich, Jonathan Schubert, 2Laurence H. Pearl,
2Chrisostomos Prodromou, 1Hannes Neuweiler
1Department of Biotechnology and Biophysics, Julius-Maximilians-University Würzburg
2Genome Damage and Stability Centre, University of Sussex, Brighton
andrea.schulze@uni-wuerzburg.de
The 90-kDa heat shock protein Hsp90 is a molecular chaperone that facilitates the folding and
activation of a wide array of cellular “client” proteins essential for signal transduction. Hsp90
is frequently implicated in the formation of cancer because it stabilizes some key
oncoproteins. But the mechanism by which the chaperone works is elusive (Taipale et al.,
2010). Hsp90 undergoes large conformational rearrangements during its ATP-dependent
chaperone cycle, resembling a molecular clamp that opens and closes. Structural studies
provide snapshots of a network of distinct local conformational changes at a scale of ~1 nm
that may limit the rate constant of ATP-hydrolysis (Ali et al., 2006). For the first time, we
detected local motions site-specifically by a contact induced quenching mechanism that is
based on a photoinduced electron transfer (PET) reaction between fluorophore and
engineered tryptophan side chains (Doose et al., 2009). We could show that several specific
structural rearrangements, which are crucial for the functionality of the chaperone machinery,
appeared to cooperate. The ATPase activity of Hsp90 was reflected in the kinetics of these
local motions. We found some elements that undergo structural rearrangement to be highly
dynamic on a sub-millisecond scale already in the nucleotide-free state. The restructuring of
the ATP-lid, which folds over the bound ATP in the nucleotide-binding pocket, is a crucial step
of the ATPase cycle. We observed that this structure rearranged in a two-step process.
Furthermore the activating co-chaperone Aha1 mobilized the lid already in the nucleotide-
free state of Hsp90 (Schulze et al., 2016).
Ali, M.M.U., Roe, S.M., Vaughan, C.K., Meyer, P., Panaretou, B., Piper, P.W., Prodromou, C., and Pearl, L.H. (2006) Nature 440(7087): 1013-
1017.
Doose, S., Neuweiler, H., and Sauer, M. (2009) ChemPhysChem 10(9-10): 1389-1398.
Schulze, A., Beliu, G., Helmerich, D.A., Schubert, J., Pearl, L.H., Prodromou, C., and Neuweiler, H. (2016) Nature chemical biology 12(8): 628-
635
Taipale, M., Jarosz, D.F., and Lindquist, S. (2010) Nature Reviews Molecular Cell Biology 11(7): 515-528.
32. 32
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Are protein hydration and dynamics important factors in the enzyme kinetics? –
fluorescence study on Haloalkane-dehalogenases
1Jan Sýkora, 2Jan Brezovský, 1Mariana Amaro, 3Silvia Kováčová, 1Avisek Ghose,
2Zbyněk Prokop, 2Koen Beerens, 2Šárka Bidmanová, 2Radka Chaloupková, 3Kamil Paruch,
2Jiří Damborský, 1Martin Hof
1Department of Biophysical Chemistry, J. Heyrovsky Institute of Physical Chemistry,
Czech Academy of Sciences, Prague
2Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic
Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Brno
3Department of Chemistry, Faculty of Science, Masaryk University, Brno
jan.sykora@jh-inst.cas.cz
The hydration and mobility of proteins are believed to profoundly affect their function1.
However, only a few approaches for monitoring these characteristics within the relevant
protein regions are available. Here we describe two general methods for site-specific analysis
of the extent of hydration and degree of the mobility in enzyme Haloalkane Dehalogenase.
The first approach is based on recording „time dependent fluorescence shift“ (TDFS)2 placing
the dye in the tunnel mouth of this enzyme3,4. In the latter approach, environment sensitive
coumarin dye is inserted in the selected region employing the technology of the “unnatural
aminoacid”5. By means of the steady state spectroscopy the degree of hydration can be
determined including the presence of ‘structured water’6. Finally, the „gating“ dynamics of
the enzymes can be traced by following the photoinduced electron transfer (PET) between
the selected tryprophan and properly positioned fluorescence dye7. Both the hydration and
dynamics monitored within the biologically relevant regions of the dehalogenase enzymes is
then compared with their enzyme kinetics of various mutants, which can bring the deeper
insight into the functioning of these enzymes.
[1] Levy, Y.; Onuchic, J. N. Annu. Rev. Biophys. Biomolec. Struct. 2006, 35, 389.
[2] Horng, M. L. et al. J. Phys. Chem. 1995, 99, 17311.
[3] Amaro, M. et al. J. Phys. Chem. B 2013, 117, 7898.
[4] Sykora, J. et al. J. Nat. Chem. Biol. 2014, 10, 428.
[5] Summerer, D. et al. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 9785.
[6] Amaro, M. et al. J. Am. Chem. Soc. 2015, 137, 4988.
[7] Sauer, M.; Neuweiler, H. In Fluorescence Spectroscopy and Microscopy; Engelborghs, Y., Visser, A. J. W. G., Eds.; Humana Press: 2014;
Vol. 1076, p 597.
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Self-organization of computation in neural systems by interaction between
homeostatic and synaptic plasticity
Christian Tetzlaff
III. Institute of Physics - Biophysics, Georg-August-University Göttingen
Bernstein Center for Computational Neuroscience, Georg-August-University Göttingen
Max-Planck Institute for Dynamics and Self-Organization, Göttingen
tetzlaff@phys.uni-goettingen.de
The ability to perform complex motor control tasks is essentially enabled by the nervous
system via the self-organization of large groups of neurons into coherent dynamic activity
patterns. During learning, this is brought about by synaptic plasticity, resulting in the
formation of multiple functional networks – commonly termed as ‘cell-assemblies’. A
multitude of such cell assemblies provide the requisite machinery for non-linear computations
needed for the mastery of a large number of motor skills. However, given the fact that there
exists considerable overlap between the usage of the same neurons within such assemblies,
for a wide range of motor tasks, creation and sustenance of such computationally powerful
networks posses a challenging problem. How such interwoven assembly networks self-
organize and how powerful assemblies can coexist therein, without catastrophically
interfering with each other remains largely unknown. On the one side, it is already known that
networks can be trained to perform complex nonlinear calculations [1], such that, if the
network possesses a reservoir of rich, transient dynamics, desired outputs can be extracted
from these reservoirs in order to enable motor control. On the other side, cell assemblies are
created by hebbian learning rules that strengthen a synapse if pre- and post-synaptic neurons
are co-active within a small enough time window [2]. Therefore, it appears relatively
straightforward to combine these mechanisms in order to construct powerful assembly
networks. However, given that the self-organization of neurons into cell assemblies by the
processes of synaptic plasticity induces ordered or synchronized neuronal dynamics, which
can destroy the required complexity of a reservoir network, such a combination remains a
very challenging problem [3]. Furthermore, simultaneous creation of multiple cell assemblies
can also lead to catastrophic interference if one cannot prevent them from growing into each
other. In this study, we exploit for the first time the interaction between neuronal and synaptic
processes acting on different time scales to enable, on a slow timescale, the self-organized
formation of assembly networks (Fig. 1), while on a faster timescale, to conjointly perform
several non-linear calculations needed for motor fine-control. Specifically, by the combination
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
of synaptic plasticity and synaptic scaling [4], as a homeostatic mechanism, we demonstrate
that such self-organization allows executing a difficult, six degrees of freedom, manipulation
task with a robot where assemblies need to learn computing complex non-linear transforms
and - for execution - must cooperate with each other without interference. This mechanism,
thus, permits for the first time, the guided self-organization of computationally powerful sub-
structures in dynamic networks for behavior control. Furthermore, comparing our assembly
network to networks with unchanging synapses ("static" networks) shows that it is indeed the
embedding of a strongly connected assembly that creates the necessary computational
power.
[1] Buonomano DV, Maass W. Nat. Rev. Neurosci 2009, 10:113-125.
[2] Palm, G. et al. Biol. Cybern., 108:559 -572, 2014.
[3] Klamp, S. and Maass, W. J. Neurosci., 33(28):11515 11529, 2013.
[4] Tetzlaff, C. et al. PLoS Comput. Biol., 9(10):e10003307, 2013.
Figure 1: Cell assembly size and
computational performance are correlated.
(A) Input driven formation of cell assemblies
brought about by the interaction long-term
potentiation (LTP) and synaptic scaling (Syn.
Sca.). (B) With more learning trials the
assembly grows and integrates more
neurons. We measure this by arbitrarily
defining assembly size by that set of neurons
connected with efficacies larger than half
the maximum weights. (C) Parallel to the
outgrowth of the cell assembly the error of the
system to perform several linear and non-linear
calculations decreases.
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
TrackNTrace: A simple and extendable open-source framework for
developing single-molecule localization and tracking algorithms
Jan Thiart, Simon Christoph Stein
III. Institute of Physics – Biophysics, Georg-August-University Göttingen
jthiart@phys.uni-goettingen.de, www.joerg-enderlein.de
Super-resolution localization microscopy and single particle tracking are important tools for
fluorescence microscopy. Both rely on detecting, and tracking, a large number of fluorescent
markers using increasingly sophisticated computer algorithms. However, this rise in
complexity makes it difficult to fine-tune parameters and detect inconsistencies, improve
existing routines, or develop new approaches founded on established principles. We present
an open-source MATLAB framework for single molecule localization, tracking and super-
resolution applications.
The purpose of this software is to facilitate the development, distribution, and comparison of
methods in the community by providing a unique, easily extendable plugin-based system and
combining it with a novel visualization system. This graphical interface incorporates
possibilities for quick inspection of localization and tracking results, giving direct feedback of
the quality achieved with the chosen algorithms and parameter values, as well as possible
sources for errors. This is of great importance in practical applications and even more so when
developing new techniques. The plugin system greatly simplifies the development of new
methods as well as adapting and tailoring routines towards any research problem's individual
requirements.
We demonstrate its high speed and accuracy with plugins implementing state-of-the-art
algorithms and show two biological applications.
36. 36
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Investigating conformational dynamics of DNA hairpin and Holliday junction using
single-molecule fluorescence techniques
1,2Roman Tsukanov, 3Menahem Pirchi, 2Toma E. Tomov, 2Yaron Berger, 2Miran Liber,
2Dinesh Khara, 2Eyal Nir, 3Gilad Haran
1III. Institute of Physics – Biophysics, Georg August University Göttingen
2Department of Chemistry and the Ilse Katz Institute for Nanoscale Science and Technology,
Ben-Gurion University of the Negev, Beer Sheva
3Chemical Physics Department, The Weizmann Institute of Science, Rehovot
DNA is a highly-designable, easily modified and cost-efficient biological molecule. DNA hairpin
and Holliday junction are responsible for genetic recombination and other important
biological processes. The interconversion rates of synthetic DNA hairpin and Holliday junction
molecules can be programmed by designing its sequence and changing the environment of
the molecule (ionic strength, temperature, viscosity, pH and etc). These properties make DNA
hairpin and Holliday junction perfect dynamic model molecules for development and
validation of single-molecule fluorescence techniques and approaches. I will discuss the
implementations of Probability Distribution Analysis and photon-by-photon Hidden Markov
Model in DNA hairpin and Holliday junction conformational dynamics study on a broad time-
scale.
(1) Benedict E. K. Snodin et al (2015), Introducing Improved Structural Properties and Salt Dependence into a Coarse-Grained Model of
DNA
(2) Tsukanov R. et al (2014) Acc. Chem. Res., 47 (6), 1789–1798.
(3) Tsukanov R. et al (2013) J. Phys. Chem. B, 117(50), 16105-09.
(4) Tsukanov R. (2013) J. Phys. Chem. B, 117(40), 11932-42
37. 37
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Enhancing the performance and applicability of SOFI using
new probes and analysis strategies
Wim Vandenberg, Sam Duwé, Peter Dedecker
Department of Chemistry, Katholieke Universiteit Leuven
wim.vandenberg@chem.kuleuven.be, www.chem.kuleuven.be/pd
In the past decade, one after the other, new ways of achieving super-resolution have been
thought up and implemented, targeting different niche parts of the imaging field. One of these
techniques, superresolution optical fluctuation imaging or SOFI (1) is targeting an audience
concerned with the robustness of the analysis (2). As such it’s truly in high background low-
signal situations (such as living systems) that SOFI comes in to its own. The technique is based
on a statistical analysis of several hundred images taken of a sample in which the label shows
fluorescence dynamics (blinking), the precise nature of this blinking is often irrelevant making
many different labels suitable (3,4). In the last couple of years SOFI has matured to deliver
multi-color (4) as well as 3D (5) imaging in living cells. In this contribution we will describe a
continuing focus on our part to quantify and enhance the robustness of SOFI in live cells. On
the one hand this work has focused on the development of fluorescent proteins with
increased bio-compatibility and good performance in SOFI microscopy (6). On the other hand
this work has focused on the development of a statistical framework which allows for the
model free quantification of the quality of SOFI datasets as well as an enhancement of the
SOFI analysis, allowing for the doubling of temporal resolution by using all available
information (7).
[1] Dertinger et al., “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)”
[2] Geissbuehler at al., "Comparison between SOFI and STORM"
[3] Dertinger et al., “Superresolution Optical Fluctuation Imaging with Organic Dyes”
[4] Dedecker et al., “Widely accessible method for superresolution fluorescence imaging of living systems”
[5] Geissbuehler at al. Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging”
[6] Duwé et al., “Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution
Microscopy”
[7] Vandenberg et al., “Model-free uncertainty estimation in stochastical optical fluctuation imaging (SOFI) leads to a doubled temporal
resolution”
38. 38
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Light Sheet Microscopy for Clinical Histopathology
1,3,5Robert Ventzki, 1,3Gertrude Bunt, 2Peter Herrmann, 4Philipp Ströbel, 2Michael Quintel,
1Wolfgang Brück, 1,5Fred Wouters
1Institute for Neuropathology, University Medical Center, Göttingen
2Clinic for Anesthesiology, University Medical Center, Göttingen
3Technology Platform Clinical Optical Microscopy (CLINOMIC),
University Medical Center, Göttingen
4 Institute for Pathology, University Medical Center, Göttingen
5Laboratory for Molecular and Cellular Systems, University Medical Center, Göttingen
fred.wouters@gwdg.de
Light sheet microscopy opens a new window on pathological tissue that aids in its spatial
description and understanding in the same way radiology has transitioned from 2D X-ray
images to 3D visualization tools like CT and MRT. It offers large field-of-view tomographic
imaging possibilities at the mesoscopic scale that adds valuable consiliary information to
histological assessments of human biopsy material. Optical clinical imaging is rapidly catching
up with recent developments in microscopy.
Pathological evaluation is typically performed on thin tissue slices. Volumetric information on
pathological material widens the pathologist’s view and aids statistical conclusions. However,
pathological practice imposes real-world constraints: biopsy material is collected directly in
formalin (without washing or perfusion), tissue clearing should be quick (within a day) and
compatible with routine pathological lab workflows.
Our clinical light sheet microscope platform is based on a function-maintaining modification
of a small animal imaging system (Olympus OV100). A custom-designed breadboard holds all
components for light sheet formation and imaging. The system contains a computer-
controlled objective and filter turret, allowing imaging at different magnifications. The
microscope is used in conjunction with a new rapid and efficient clearing protocol for formalin-
stored tissue that can also be used with archival paraffin-embedded tissue blocks as starting
material.
This system is the starting point for the design of a new, simple light sheet microscope for use
in a clinical setting. We will show the utility of the light sheet microscope with representative
examples from the neuro/pathological practice.
39. 39
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Expansion Microscopy meets dSTORM
1Fabian Zwettler, 1Felix Rüdinger, 1Markus Sauer
1Department of Biotechnology & Biophysics, Julius-Maximilians-University Würzburg
fabian.zwettler@uni-wuerzburg.de
Single molecule localization microscopy (SMLM) and the recently developed technique
Expansion Microscopy (ExM) 1 are two different approaches that achieve the visualization and
investigation of proteins and other biological molecules with nanoscale precision. SMLM
techniques such as direct stochastic optical reconstruction microscopy (dSTORM) bypasses the
diffraction limit of light microscopy by photoswitching or –activation of a sparse subset of all
fluorophores, localization of single molecules by fitting a two dimensional Gaussian function
to the photon distribution (PSF) of single fluorophores, and reconstruction of a super-resolved
image. In contrast to this technique, ExM increases the effective resolution through physically
magnifying the specimen. Therefore the specimen is embedded in a dense swellable polymer
in which a modified fluorescent tag is targeted to a biomolecule of interest. Additionally this
label is anchored into the polymer mesh. By adding water the polymer expands isotropically
in all dimensions and enables a 4.5x magnification of the specimen. This process improves the
spatial resolution down to roughly 60-70 nm in lateral direction on a diffraction-limited
microscope. By combining dSTORM with Expansion Microscopy we are able to further improve
the spatial resolution to molecular dimensions. Our new approach is a highly promising tool
that can be used advantageously to investigate the 3D molecular architecture of biomolecular
complexes and machines.
[1] Chen, F. Tillberg, P. W. & Boyden, E. S. Expansion microscopy. SCIENCE 347, 543–548 (2015).
40. 40
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Abstracts poster presentations
(Listed alphabetically by last name)
41. 41
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Self-Organized Memory Allocation by Hebbian Cell Assemblies
Johannes M. Auth, Timo Nachstedt, Christian Tetzlaff
III. Institute of Physics – Biophysics, Georg August University Göttingen
Bernstein Center for Computational Neuroscience, Göttingen, 37077, Germany
Max Planck Institute for Dynamics and Self-Organization, Göttingen, 37077, Germany
jauth@phys.uni-goettingen.de
Declarative memory denotes the storage of facts and concepts from perceived stimuli. The
formation of such memories, in particular their allocation in neural circuits is still an
unresolved problem. In general, different stimuli to be learned have to trigger the formation
of different memory representations. In addition, each learned stimulus has to maintain its
assignment or allocation to its specifically formed memory representation. Experimental
findings imply that variations in neural excitability due to a complex cascade of proteins that
make individual neurons more susceptible form a memory representation of a new stimulus
[1]. Furthermore, the concept of synaptic tagging, which assumes cascades of plasticity-
related proteins, is assumed to locally determine the synapses involved in the memorization
process [2]. However, both ideas require complex, highly specialized cascades of several
proteins to allocate memories. Here, we show in a theoretical model that the allocation of
memory can already be solved by the self-organized dynamics of synaptic plasticity. The
system consists of three neuronal populations: an input population projects activity patterns
(stimuli) through random excitatory connections on a second, recurrently interconnected
memory population. All feed-forward as well as the recurrent synapses are adapted by a
combination of Hebbian synaptic plasticity and synaptic scaling [3]. An inhibitory population
is mutually connected to the recurrent layer to provide global competition. Interestingly, first
of all, our model successfully forms stable memory representations: presenting a given
stimulus to the recurrent layer causes a locally clustered group of neurons to become strongly
interconnected with each other (Hebbian cell assembly [4]). Furthermore, presenting another
stimulus of sufficient dissimilarity to the first one causes the formation of another memory
representation. Remarkably, if the stimuli are quite similar to each other, both are allocated
to the same memory representation. In addition, the system shows the dynamics of
competitive memory recall, i.e. differentiating recognition [5], as the activation of one
memory representation fully suppresses others. In summary, the here-presented simple but
biologically plausible concepts of stimulus-dependent self-organization of plasticity provide a
promising approach to the question of how memory allocation is coordinated in the brain.
42. 42
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
[1] Yiu A. P. et al. Neuron 2014, 83(3): 722-735.
[2] Rogerson T. et al. Nat Rev Neuroscience 2014, 15(3): 157-169.
[3] Tetzlaff C. et al. PLoS Comput Biol 2013, 9(10):e1003307.
[4] Hebb, D. O.: The organization of behavior: A neuropsychological approach. John Wiley & Sons 1949
[5] Wills T. J. et al. Science 2005, 308(5723): 873-876.
43. 43
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Physarum polycephalum utilizes specialized function regions when exposed to light
Felix Bäuerle, Karen Alim
Max Planck Institute for Dynamics and Self-Organization, Göttingen
felix.baeuerle@ds.mpg.de, http://bpm.ds.mpg.de
The slime mold Physarum polycephalum is a bright spark among the amoebas. It has received
the honor of being called ‘intelligent’ multiple times: i.e. for finding the shortest path in a
maze, anticipating events in time, choosing a balanced diet, or providing a transport network
closely resembling the Tokyo subway system in efficiency. Yet, strikingly the organism - while
growing tube-like structures on centimeter sizes - is still one single cell with lack of any
centralized control system or specialized organs. How is Physarum able to adapt its entire
morphology to a complex environment while lacking any kind of nervous system? I want to
answer this question by studying its yielding reaction to light stimuli. Experiments show that
during blue illumination – a known repellent - the organism divides behaviorwise: illuminated
and non-illuminated parts change differently to facilitate mass transportation away from
illuminated regions. This happens in two distinct phases. The first acts as a rearrangement
period whereas the second constitutes the efflux peak until complete pruning. Asymmetric
contractions of the illuminated tubes may act as a driving factor to cause efflux. Meanwhile
the non-illuminated region rearranges to deposit the incoming mass in the organism’s
periphery. This in turn means that Physarum can utilize transient specialized function regions
to compensate for missing predetermined organs.
44. 44
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Click-PAINT with quenched tetrazine-dyes
Gerti Beliu, Andreas Kurz, Markus Sauer
Department of Biotechnology and Biophysics, Julius-Maximilians-University Würzburg
www.super-resolution.de
We observed that some fluorescent dyes are quenched efficiently when functionalized as
tetrazine derivative for covalent labeling of proteins using modified amino acids and click
chemistry. We identified electron transfer from the electron donor tetrazine to the dye’s
excited singlet state as underlying fluorescence quenching mechanism. Using steady-state and
time-resolved fluorescence spectroscopy as well as fluorescence correlation spectroscopy
(FCS) we demonstrate that tetrazine forms ground- and non-fluorescent excited state
complexes with an association constants with fluorescent dyes belonging to the class of
rhodamine and oxazine dyes such as ATTO 488, and ATTO 655, respectively, besides dynamic
collisional quenching. Upon covalent coupling to click chemistry partner amino acids the
electron donating properties of the tetrazine moiety are reduced and fluorescence is released
reflected in a 7-13 fold increase in fluorescence intensity. Here, we use this coupling-induced
de-quenching advantageously for super-resolution imaging of membrane receptors by PAINT.
Since the fluorescence increases upon binding higher probe concentrations and even epi-
illumination schemes can be used for PAINT super-resolution microscopy we termed the
method “Click-PAINT”. We validate the potential of Click-PAINT by super-resolution imaging
of three different receptors on live and fixed cells and compare the data with classical
dSTORM. The new fluorescently quenched tetrazine dyes might also be useful for intracellular
labeling of modified amino acids without washing steps.
45. 45
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Modelling growth-induced wrinkling of elastic biofilms
Horst-Holger Boltz, Stefan Klumpp
Institut für nichtlineare Dynamik, Georg-August-University Göttingen
Microbial biofilms, macroscopic aggregates of microorganisms, have been an important
subject of study in the recent years due to their biological, medical and technological
relevance. Planar biofilms are large multicellular structures of microorganisms adherent to a
substrate providing mechanical support as well as supply of nutrients leading to a
predominantly flat growth. The formation of these structures is usually accompanied by the
production of an extracellular matrix formed by so-called extra-cellular polymeric substances
(EPS). Thus, an elastic film is created that is growing due to the ongoing cell growth and
division as well as the continued production of EPS. This growth leads (provided sufficient
anisotropy or inhomogeneity) to residual and dynamic stresses that are relieved by a non-
planar pattern-formation (wrinkling). Apart from its high practical relevance this interplay of
growth and elasticity (morphoelasticity) poses an interesting challenge to any form of
analytical or numerical treatment. We present a coarse-grained discrete elements model with
well-defined elastic properties (suitable for any quasi-two dimensional elastic medium) as well
as locally adjustable, possibly anisotropic growth and use this to study the mechanics of this
problem numerically. We find that usually found morphotypes within rdarbiofilms (red, dry
and rough) can be explained by a somewhat minimal model of differential growth. Also, we
argue that the mechanisms leading to this pattern formation are so fundamental that they can
give a glimpse into the physical interactions of cells and extracellular matrix. Additionally, we
present molecular dynamics simulations of (inevitably small) early stage bacterial colonies
where we are particularly interested in the emergence of macroscopic an orientational (quasi-
nematic) order that could break the underlying rotational symmetry within the biofilm giving
a possible mechanism for anisotropic growth.
46. 46
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
FRET‐based structural analysis of ion channel regulation at the nanoscale
René Ebrecht, Gertrude Bunt
Clinical Optical Microscopy, Institute of Neuropathology, University Medical Center
Göttingen
gbunt@gwdg.de
A precise and tight regulation of ion channel activity is a prerequisite for proper cellular
functioning. Regulatory mechanisms use the binding of several regulatory and modulatory
proteins to the channel, along with structural arrangements within the channel subunits.
Inhibition by Ca2+/CaM binding is one of the most important regulation mechanisms for the
voltage-dependent potassium channel eag [1]. The channel contains multiple intracellular
domains that mediate Ca2+-mediated calmodulin binding [2], but the structural mechanism
behind CaM-mediated channel inhibition is not yet fully understood. Here we show, using
FRET imaging for the binding of CaM to heag1, that the two C-terminal binding domains, BD-
C2 and BD-C1, are the predominant binding sites in the native channel. Both sites can bind
CaM independently. Deletion of the N-termini results in reduced CaM binding, however the
binding domain in the N-terminus is not involved. Here we show that the N- and C-termini of
the channel subunits, by their direct intermolecular interaction, cooperate in CaM Binding to
the C-terminal binding domains. A 'transverse' Interaction between the N- and C-terminal tails
of the channel subunits support the binding of calmoldulin to the binding sites at the C-
terminus, likely forming a structural pocket that is required for efficient binding.
(1) Schönherr et al. EMBO J. (2000), 19 (13):3263-71
(2) Ziechner et al. FEBS J. (2006), 273(5):1074-86
47. 47
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Single-molecule Brightness Analysis by Stroboscopic Imaging
in Nanofluidic-Channels
Hao Cheng, Simon Stein, Jan Thiart, Ingo Gregor, Jörg Enderlein
III. Institute of Physics – Biophysics, Georg-August-University Göttingen
hcheng@gwdg.de, www.joerg-enderlein.de
Molecular brightness is an essential parameter for single-molecule studies. Accurately and
quantitatively determining the brightness in solution helps to disentangle complex mixtures
of molecular species and facilitates many biomedical studies, e.g. determining molecular
stoichiometry or detecting single binding events. Previous technologies employing
fluorescence-fluctuation-spectroscopy to deduce single-molecule brightness distribution are
restricted to rigorous experimental conditions and complicated statistical model, which
achieve limited success. Here we present our progress on observing and interrogating
individual molecules during their diffusion or transportation in nanofluidic-device. Utilizing the
full-glass-chip with channels height less than 200nm, molecular movement is physically
confined to the focus plane. It enables the investigation and manipulation of fast diffusing
molecules by directly imaging, therefore we can obtain the single-molecule brightness
distribution with superb accuracy. A high-speed stroboscopic imaging method is combined
with active flow control system to produce images that can be instantly analyzed by well-
established single-molecule localization techniques. With its high-throughput, thousands of
individual molecules are investigated within only several minutes. We prove its single-
molecule sensitivity for determining molecular stoichiometry through the measurement of
multiple Atto647N-labelled short DNA fragments. Enjoying in the convergent development of
lab-on-a-chip and single-molecule approaches, our platform opens up tremendous
opportunities for further biomedical application.
48. 48
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Localization of cell adhesion points using dual color MIET
Anna Chizhik, Carina Wollnik, Daja Ruhlandt, Alexey Chizhik , Narain Karedla, Dirk Haehnel,
Ingo Gregor, Florian Rehfeldt, Jörg Enderlein
III. Institute of Physics – Biophysics, Georg-August-University Göttingen
anna.chizhik@uni-goettingen.de
We present the result on axial localization measurements of cell adhesion points with nm
accuracy. We used the recently developed metal-induced energy transfer (MIET) imaging,
which allows us to measure the axial localization of a fluorophore with 2-3 nm accuracy [1].
The principle of MIET imaging is based on the energy transfer between a fluorescent molecule
and a metal surface, which results in the molecules de-excitation rate acceleration and can be
observed as a shortening of the molecule’s fluorescence lifetime [1,2]. Because energy
transfer rate is monotonically dependent on the distance of a molecule from the metal layer
within near first 200 nm, the fluorescence lifetime can be directly converted into a distance
between the emitter and metal surface within this range of distances. Here, for the first time
we present the results of the dual-color MIET measurements correlated with FRET imaging
[3]. This allows us to simultaneously measure the axial localization of actin filaments and
vinculin and to monitor the areas where the distance between actin and vinculin is within
FRET-range, that is does not exceed 10 nm. By combining the realms of MIET and FRET
microscopy we achieve unprecedented axial resolution based on absolute and relative values
obtained by these methods.
[1] Chizhik, A. I. et al. Nature Photon. 8, 124-127 (2014).
[2] Karedla N. et al. ChemPhysChem, 15, 705–711 (2014).
[3] Förster, Th. Ann. Physik 437, 55-75 (1948).
49. 49
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Filling the usability gap: Bioinformatics solutions for Image-Scanning Microscopy,
Stochastic Optical Fluctuation Imaging, and Surface Single Molecule Experiments
Dirk Hähnel, Narain Karedla, Anna Chizhik, Alexey Chizhik, Simon Christoph Stein, Anja Huss,
Sebastian Isbaner, Qui Van, Ingo Gregor, Jörg Enderlein
III. Institute of Physics – Biophysics, Georg-August-University Göttingen
dirk.haehnel@phys.uni-goettingen.de, www.joerg-enderlein.de
Recent years have seen a tremendous increase of new and novel methods in the field of
superresolution fluorescence microscopy. Furthermore even better methods for increasing
axial resolution of fluorescence imaging have been introduced by our group very recently. Our
group has developed powerful methods: Confocal Spinning Disc Image-Scanning Microscopy
(CSDISM)1,2, Superresolution Optical Fluctuation Imaging (SOFI)3,4,5,6, and Metal Induced
Energy Transfer (MIET)7,8. However, new microscopy techniques that provide not only
enhanced image quality and resolution, but they are also simple enough for finding broad
application. To bridge the ultimate usability gap for end-users, we present simple soft- and
hardware solutions for CSDISM and SOFI which enable potential users to implement them in
an easy and straightforward way into their existing microscopy systems. In the case of CSDISM,
we have integrated the method into the environment of the widely used and popular
MicroManager Open Source Imaging platform. This allows any researcher who already has a
commercial Confocal Spinning Disk microscope to easily implement the image-scanning
option and thus to double the spatial resolution. For SOFI, we have developed a dedicated
hardware based on a Freely Programmable Gate Array (FPGA) which converts, in real time,
image movies taken by high-speed CCD systems into SOFI cumulant images. Thus, all
algorithmic complexities and numerical workload of SOFI calculations are taken care of.
Furthermore we will present our recently developed software tool for smart automated single
molecule on surface experiments termed (SIMA). This is an effective tool to save time and
enables the researcher to conduct complex measurements. SIMA increases the comparability
of single molecule measurements, and reduces bleaching to the absolute possible minimum.
[1] Müller and Enderlein, “Image Scanning Microscopy”;
[2] Schulz, Pieper, and Clever, “Resolution Doubling in Fluorescence Microscopy with Confocal Spinning-Disk Image Scanning Microscopy”;
[3] Dertinger et al., “Achieving Increased Resolution and More Pixels with Superresolution Optical Fluctuation Imaging (SOFI)”;
[4] Dertinger et al., “SOFI-Based 3D Superresolution Sectioning with a Widefield Microscope”;
[5] Dertinger et al., “Advances in Superresolution Optical Fluctuation Imaging (SOFI).”; Dertinger et al., “Fluctuation Imaging ( SOFI )”
[6] Geissbuehler et.al., “Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging”;
[7] Chizhik et.al. “Metal-induced energy transfer for live cell nanoscopy”;
[8] Karedla et.al. “Single-Molecule Metal-Induced Energy Transfer (smMIET): Resolving Nanometer Distances at the Single-Molecule Level”
50. 50
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Long-term information storage by the collective dynamics of multi-synaptic
connections
Michael Fauth, Florentin Wörgötter, Christian Tetzlaff
1Bernstein Center for Computational Neuroscience, Göttingen
Excitatory synapses in cortex typically reside on dendritic spines. Although cortical synapses
play an important role in long-term memory, these spines undergo a remarkably high turnover
[1,2]. This poses the question how information can be stored on a variable substrate as
synapses. As a possible solution, we propose that information is stored and retained by the
collective dynamics of multiple synapses. Such a collective dynamics can already be found on
the connection between two neurons, which can consist of multiple synapses. More precisely,
the experimentally obtained distribution of the number of synapses on these connections,
which are bimodal with peaks at zero and multiple synapses, can only emerge from a collective
dynamics of the involved synapses [3]. Modelling studies showed that this collective dynamics
can emerge from the interaction of synaptic and structural plasticity [4,5] and that it can be
influenced by external stimulation such that the neurons become either unconnected or
connected with multiple synapses [5].
Here, we investigate the information storage and retention of these collective dynamics with
a simple stochastic model of structural plasticity, where synapses are created with a constant
probability and removed with a probability depending on the number of existing synapses and
the external stimulation. Using information theoretic measures, we show that the collective
dynamics yielding the bimodal distributions of the number of synapses enables information
retention on time scales orders of magnitudes longer than the typical lifetime of a synapse.
Thus, the conflict of spine turnover and long- term memory can be resolved by storing
information in the collective dynamics of multiple synapses. Yet, at different external
stimulation levels where the collective dynamics yield distributions with a single peak either
at zero or at multiple synapses, information about the initial conditions decays quickly. This,
however, implies that these stimulations can be used to learn new information orders of
magnitude faster than it is forgotten. We confirm this by using these stimulations to store an
image in a population of multi-synaptic connections. Indeed, this image can be retained orders
of magnitude longer than it took to store it. Thus, learning can be faster than forgetting, which
is also a necessary prerequisite to solve the plasticity-stability dilemma in learning and
memory on the time scale of structural changes.
51. 51
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
[1] Yang G, Pan F, Gan WB (2009) Stably maintained dendritic spines are associated with lifelong memories. Nature 462: 920-924.
[2] Xu T, Yu X, Perlik AJ, Tobin WF, Zweig JA, et al. (2009) Rapid formation and selective stabilization of synapses for enduring motor memories.
Nature 462: 915-919
[3] Fares T, Stepanyants A (2009) Cooperative synapse formation in the neocortex. Proceedings of the National Academy of Sciences.
106:16463–16468.
[4] Deger M, Helias M, Rotter S, Diesmann M.(2012) Spike-timing dependence of structural plasticity explains cooperative synapse formation
in the neocortex. PLoS Comput Biol. 8:e1002689.
[5] Fauth M, Wörgötter F, Tetzlaff C (2015) The formation of multi-synaptic connections by the interaction of synaptic and structural plasticity
and their functional consequences, PLOS Comput Biol. 11(1):e1004031
52. 52
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Gating mechanosensitive channels in bacteria with an atomic force microscope.
1Renata Garces, 2Samantha Miller, 1Christoph F. Schmidt
1III. Institute of Physics – Biophysics, Georg-August-University, Göttingen
2The Institute of Medical Sciences, University of Aberdeen
The regulation of growth and integrity of bacteria is critically linked tomechanical stress.
Bacteria typically maintain a high difference of osmotic pressure (turgor pressure) with
respect to the environment. This pressure difference (on the order of 1 atm) is supported by
the cell envelope, acomposite of lipid membranes and a rigid cell wall. Turgor pressure is
controlled by the ratio of osmolytes inside and outside bacteria and thus, can abruptly
increase upon osmotic downshock. For structural integrity bacteria rely on the mechanical
stability of the cell wall and on the action of mechanosensitive (MS) channels: membrane
proteins that release solutes in response to stress in the cell envelope. We here present
experimental data on MS channels gating. We activate channels by indenting living bacteria
with the cantilever of an atomic force microscope (AFM). We compare responses of wild-type
and mutant bacteria in which some or all MS channels have been eliminated.
53. 53
BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
Functional and genetic dissection of mechanosensory organs of Drosophila
1, 2C. Guan, 1N. Scholz, 1R. J. Kittel, 1T. Langenhan
1Institute of Physiology – Neurophysiology, Julius-Maximilians-University Würzburg
2III. Institute of Physics – Biophysics, Georg-August-University Göttingen
chonglin.guan@phys.uni-goettingen.de
Larval chordotonal neurons provide fundamental sensory information as they convert
mechanical stimuli into biological responses (stretch, touch and sound). They are
monociliated, bipolar nerve cells that reveal genetic and functional parallels with inner hair
cells of the mammalian ear [1, 2]. Here we have developed a preparation to directly record
from sensory neurons of the lateral chordotonal organ (lch5) during mechanical stimulation.
This method enables to correlate the neuronal electrical output with defined mechanical
input. We have used this setup to characterize basal functional lch5 parameters including time
course of response during continuous mechanical stimulation and the recovery time between
successive bouts of stimulation.
Previously, we identified the calcium-independent receptor of α-latrotoxin
(dCIRL/Latrophilin), a member of the Adhesion class of G protein-coupled receptors (aGPCR),
as a mechanoreceptor [3]. We found that dCIRL modulates lch5 neuron activity by adjusting
the mechanogating properties of ionotropic receptors known to produce receptor potentials
that subsequently lead to the generation of nerve impulses. Furthermore, our results indicate
that the extent of the extracellular NTF of dCIRL shapes mechanosensitivity of the lch5. These
experiments provide new insights into the mechanobiology of dCIRL and establish
chordotonal organs as interesting sites to study the molecular machinery involved in the
perception of mechanical challenges.
[1] Eberl, D. F., Hardy, R. W. & Kernan, M. J. Genetically similar transduction mechanisms for touch and hearing in Drosophila. J Neurosci
20, 5981-5988 (2000)
[2] Nadrowski, B., Albert, J. T. & Gopfert, M. C. Transducer-based force generation explains active process in Drosophila hearing. Curr
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BBTS 2016 - BIOPHYSICS BY THE SEA 2016
Fluorescence Spectroscopy, Microscopy and Molecular Cell Mechanics and Theoretical Neurophysics
The role of competition in memory organization
1Juliane Herpich, 1, 2Florentin Wörgöttier, 2Christian Tetzlaff
1
III. Institute of Physics – Biophysics, Georg August University Göttingen
2Bernstein Center for Computational Neuroscience, Göttingen
Humans are able to perform cognitive strategies to solve problems they are faced with. Thus,
they generate a huge variety of strategies which cannot all be hard wired in their neuronal
networks which consist of a finite number of neurons. One hypothesis is that neural entities
are reorganized to participate in different cognitive purposes. Therefore, different entities are
exploited, recycled, and redeployed and, thus, put to different uses without losing their
original function [1]. Given this idea, to enable an accurate reaction according to a given
situation, humans adaptively organize the learned memories of previous experienced
environmental stimuli. However, the neuronal principles for the functional reorganization of
the brain, thus, for rewiring the links between memories are still unknown.
Here, we use an adaptive neuronal network model depending on the interactions of synaptic
plasticity [2, 3] and synaptic scaling [4]. Hebbian synaptic plasticity adapts the efficacies of
synapses dependent on the corresponding neuronal activities [5]. With the intertwined
mechanism of synaptic scaling, thereby, synaptic plasticity yields the formation of strongly
interconnected subgroups of neurons (cell assemblies; CAs) [6]. These CAs serve as neuronal
representations or memories of specific environmental stimuli [5]. As we are interested in the
functional organization of the brain, we started to investigate the interaction between two
memories. We describe the dynamics for the representation of each memory by
homogeneous populations and drive the CAs with different external stimuli. It is shown that
neuronal competition (synaptic plasticity combined with synaptic scaling) is mandatory for the
formation of CAs [4, 7]. Here, we investigate the role of competition between memories for
their functional rewiring. Therefore, we combine synaptic plasticity with different generic
synaptic scaling mechanisms. Thus, we gradually increase the influence of synaptic scaling
from a constant to a more complex and activity-dependent condition. Interestingly, increased
competition between both CAs leads to the formation of different functional interactions
between them. Dependent on the external drive and the internal competition the two-
memory system is capable to build up different functional links between these memories such
as association, discrimination, and sequence [8].
This work describes different forms of functional organization of memories in the brain.