G.V. Shivashankar
Mechanobiology Institute &
Department of Biological Sciences
National University of Singapore
5A, Engineering Drive
#10-01, T-lab Building
Singapore 117411
E-mail: gvs.shiva@gmail.com
Website: http://mbi.nus.edu.sg/g-v-shivashankar/
Chromatin compaction and dynamics in living cells
1
Thorsten WOHLAND
Departments of Biological Sciences and Chemistry
Centre for Bioimaging Sciences (CBIS)
National University of Singapore
Singapore
E-mail: chmwt@nus.edu.sg
Website: http://staff.science.nus.edu.sg/~chmwt/
Fluorescence Correlation Spectroscopy for the Measurement of Biomolecular Interactions
in live organisms
Abstract:
Biomolecular interactions are strongly influenced by the complex cellular environment in which the
molecules reside. Molecules will not interact in a pairwise fashion but might interact via a third
molecule, might compete for binding or show cooperativity in their interactions. Therefore it is
necessary to establish methods which can quantitatively measure interactions within the
environment of cells and organisms. Fluorescence Correlation Spectroscopy (FCS) and Fluorescence
Cross-Correlation Spectroscopy (FCCS) are widely used tools for this purpose. Both methods are
single molecules sensitive and can determine molecular parameters, including dissociation constants
quantitatively in live tissues and organisms [1-3]. In the first part of this seminar the basics of FCS
and FCCS and their capabilities to extract quantitative information from living specimen will be
discussed.
Conventionally these methods were applied in confocal setups and thus were able to measure not
more than a couple of points per cell due to the risk of inducing photodamage. Therefore new ways
of recording FCS data had to be developed to allow recording of multiple points in live specimen
simultaneously. For that purpose we developed FCS using single plane illumination microscopy, so
called SPIM-FCS [4-5]. In this method a whole plane in an embryo is illuminated by a laser light sheet
and the fluorescence intensity is recorded at a right angle to the illuminated plane by a fast, sensitive
camera. This renders collection of many independent points possible (typically 1000 points but the
record stands above 1,000,000) in each of which the fluorescence fluctuations can be used to
calculate FCS and FCCS functions. Using this approach we have recorded diffusion, flow, and
concentration maps in cells and zebrafish embryos.
References
1. P. Liu., T. Sudhaharan, R.M.L. Koh, L.C. Hwang, S. Ahmed, I.N. Maruyama, and T. Wohland. Investigation of the
dimerization of proteins from the epidermal growth factor receptor family by single wavelength fluorescence crosscorrelation spectroscopy. Biophys. J. (93): 684-698 (2007).
2. X. Shi, Y.H. Foo, T. Sudhaharan, S.-W. Chong, V. Korzh, S. Ahmed, T. Wohland, “Determination of dissociation constants
in living zebrafish embryos with single wavelength fluorescence cross-correlation spectroscopy”, Biophys. J. (97)2:678-686
(2009).
3. X. Shi, L.S. Teo, X. Pan, S.W. Chong, R. Kraut, V. Korzh, T. Wohland, “Probing events with single molecule sensitivity in
zebrafish and Drosophila embryos by fluorescence correlation spectroscopy”, Dev. Dyn., 2009; 238:3156–3167.
4. T. Wohland, X. Shi, J. Sankaran, E. H.K. Stelzer, “Single Plane Illumination Fluorescence Correlation Spectroscopy (SPIMFCS) probes inhomogeneous three-dimensional environments”, Opt. Exp. 2010, 18(10): 10627-41
5. J. Sankaran, X. Shi, L.Y. Ho, E.H.K. Stelzer, and T. Wohland, “ImFCS: A software for Imaging FCS data analysis and
visualization” Opt Expr, (18) 25468-25481, 2010.
2
Alexander GASSEN
German Cancer Research Centre, DKFZ
Division Biophysics of Macromolecules
Im Neuenheimer Feld 580
D-69120 Heidelberg
Germany
E-mail: alexander.gansen@gmail.com
Fluorescence resonance energy transfer as a tool to study biomolecular architecture and
dynamics
Abstract:
The nucleosome is the elemental unit of DNA compaction; its structure and content control the local
morphology of chromatin, which mediates gene regulation by modulating the accessibility of certain
gene regions to nuclear proteins. Many biophysical studies have characterized the shape and size of
single nucleosome particles and nucleosome arrays; yet despite intensive research, little is known
about the dynamic properties of the nucleosome. Over the last decade Fluorescence resonance
energy transfer (FRET) has become a powerful tool to learn more about the dynamic properties of
the nucleosome. FRET is the distance-dependent energy transfer between a donor and an acceptor
fluorophore that are attached to specific sites in a macromolecular complex; changes in architecture
can then be observed via changes in the interfluorophore distance.
In this lecture I will review the basic concepts of FRET and its potential and limitations for exploring
nucleosome structure and dynamics. I will discuss strategies to efficiently perform FRET experiments
on the ensemble level and in a single molecule format. Examples will illustrate how FRET can.be used
to follow changes in nucleosome structure induced by spontaneous linker DNA dynamics, changes in
DNA sequence, histone and DNA modifications and nucleosome disassembly and reassembly. The
final part of my talk will discuss advanced data analysis strategies, which can provide high resolution
data on single molecule dynamics on the sub-ms timescale.
3
Bai LU
206B Life Science Building
Penn State University
University Park PA 16801
USA
Email: lub15@psu.edu
Website: http://www.personal.psu.edu/lub15/blogs/bai_lab_at_penn_state_university/
Imaging Methods: Timelapse Fluorescence Microscopy
Abstract:
Using timelapse fluorescence microscopy and custom-made image analysis tools, single live cells
with fluorescent markers can be tracked over multiple rounds of growth and division. This technique
allows us measure the cell-to-cell variability and dynamics of gene expression. In the mean time, it
provides information on the cell morphology, cell cycle stage, age and mobility. In addition, by
following cell growth and division, this method generates a cell pedigree that is essential for study of
inheritance. Combined with a flow-cell device, we can change the growth condition of cells while
carry out these single cell measurements. In this workshop, I will talk about the technical aspect of
this method, and its application to different biological problems.
4
Jie YAN
Mechanobiology Institute
Department of Physics
National University of Singapore
Singapore
Email: phyyj@nus.edu.sg
Webpage: http://www.physics.nus.edu.sg/~biosmm/research.html
Study micromechanics and interactions of DNA and protein using the magnetic tweezers
single-molecule manipulation techniques
Abstract:
Single-molecule manipulation techniques can apply force to single bio-molecules and
examine their force responses. Such techniques have been extensively applied to the
studies of the micromechanics of DNA, proteins and their interactions at a nanometer
spatial resolution and a sub millisecond temporal resolution in physiological buffer
solutions. Atomic Force Microscopy, Optical Tweezers, and magnetic tweezers are three
commonly used single-molecule manipulation techniques. Among which, the magnetic
tweezers technique has several unique advantages, including the low cost to build the
instrument and the capabilities of twisting molecules, applying constant force without
feedback control, easy implementations of constant loading rate control, and easy
combination with fluorescence imaging. This talk will first introduce the three commonly
used single-molecule manipulation techniques and their basic operating principles. Then it
will focus on the magnetic tweezers techniques with deeper technical details and different
ways of designing the magnetic tweezers setups. Finally, it will introduce the applications of
the magnetic tweezers technique to studies of micromechanics of bio-molecules and their
interactions.
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Laura FINZI
Emory University
Department of Physics
Atlanta GA 30322-2430
USA
Email: lfinzi@physics.emory.edu
Website: http://www.physics.emory.edu/faculty/finzi/
Tethered Particle Microscopy (TPM), technique, analysis and applications
Abstract:
In this lecture I will describe a very simple single molecule technique called Tethered Particle
Microscopy (TPM). TPM is a technique that can be used to detect conformational changes in
any polymer molecule. It has most often been applied to study DNA, DNA-protein
interactions, and the activity of DNA motor enzymes. TPM permits estimation of the
extension of a DNA molecule attached at one end to a glass surface, and at the other end to
a microscopic bead exhibiting Brownian motion. The DNA tether restricts range of motion of
the bead and the average amplitude of excursions of the bead from the anchor point
reflects the DNA tether length. Excursions of the bead depend on the physical properties of
the DNA-bead system, and the physical and chemical properties of the surrounding solution.
I will discuss the TPM experimental setup, data analysis methods and a few applications.
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Ching-Hwa KIANG
Office: 226 Brockman Hall
Department of Physics & Astronomy
6100 Main Street - MS 61
Rice University
Houston, TX 77005-1892
Email: chkiang@rice.edu
Website: http://www.chkiang.rice.edu/
Single Molecule Manipulation Methods - Atomic Force Microscope and the Applications of
the Jarzynski Equality
Abstract:
Single-molecule manipulation technique provides a unique tool for a close-up investigation of the
complex biological properties and interactions. During the force measurement, a single molecule is
being pulled while its force response is monitored. However, quantifying these nonequilibrium data
and using them to understand the structure-function relationship of biosystems has been
challenging.
I will describe free energy reconstruction from these nonequilibrium measurements from AFM, using
the recently derived nonequilibrium work theorem, i.e., Jarzynski's equality and compare the results
with those from other phenomenological approaches. I will also discuss the implication of
mechanical versus chemical unfolding of proteins, using titin I27 as a model system. Finally, I will
present single-molecule studies of other systems such as overstretching transitions of DNA and
mechanical activation of protein, and discuss implications and challenges of single-molecule force
studies.
References
1. Phys. Rev. Lett. 105 (2010) 218104.
2. J. Phys. Chem. B 113 (2009) 10549.
3. Phys. Rev. E 79 (2009) 041912.
4. Phys. Rev. Lett. 99 (2007) 068101.
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Mark C. WILLIAMS
Department of Physics
Northeastern University
111 Dana Research Center
Boston, MA 02115
USA
Email: ma.williams@neu.edu
Website: http://nuweb.neu.edu/mark/
Quantifying DNA interactions with optical tweezers
Abstract:
I will discuss the design use of optical tweezers for quantitative study of DNA and DNA interactions.
As DNA is stretched with optical tweezers, the force-extension curve is strongly altered in the
presence of ligands that bind to double- and single-stranded forms of DNA. These ligands can alter
the length of the molecule, its elasticity, and the relative stability of the two forms of DNA. For
example, small molecules that intercalate between the DNA base pairs increase the length of DNA,
and this length increase can be used to precisely quantify the ligand-DNA binding energy. For more
complex small molecules such as Actinomycin D, slow binding kinetics can be directly measured,
allowing complete characterization of the energy landscape for DNA-ligand binding. DNA bending
proteins such as eukaryotic HMGB proteins alter the flexibility of the molecule, and the change in
DNA persistence length can also be used to quantify DNA-protein binding affinity. In addition, these
measurements allow protein-DNA interaction kinetics to be measured. In the case of HMGB
proteins, I will show how we can independently measure microscopic and macroscopic dissociation
events. We find that these proteins dissociate rapidly from local DNA binding sites, while remain
associated overall to the DNA molecule for a much longer time. Optical tweezers are therefore an
excellent tool for quantifying the thermodynamics and kinetics of a variety of important DNA
interactions.
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Kunio TAKEYASU1
Y.Suzuki1, A.Yoshida1, N.Sakai2, A. Yagi2, Y.Uekusa2 and K.Karasi2
1
Laboratory of Plasma Membrane and Nuclear Signaling
2
Corporate R&D Center, Olympus Co. Tokyo 192-8512, Japan
Graduate School of Bio studies
Kyoto University, Kyoto 606-8501
Japan
Email: takeyasu@lif.kyoto-u.ac.jp
Website: http://www.lif.kyoto-u.ac.jp/labs/chrom/main.html
Biological application of fast-scanning atomic force microscopy
Abstract:
Molecular imaging at nano-meter scale in millisecond time region and force measurement at picoNewton level are indispensable for understanding of the action mechanisms of biological
macromolecules. Techniques and devices of atomic force microscopy (AFM) have been developed
over the last two decades and now fulfill such requirements. We have been using these techniques
to investigate genome folding mechanisms and membrane protein dynamics. Here we summarize
our previous studies (1-11) and report most recent unpublished experiments. Topics include: (i)
biophysical properties of DNA and DNA-binding proteins, (ii) nucleosome and beyond in eukaryotes,
(iii) visual dissection of action mechanism of DNA-targeting proteins (Fig. 1), (iv) capturing the
motion of membrane proteins at work. We also describe the development of a high-speed AFM
combined with inverted fluorescent microscopy (FM), aiming at the elucidation of dynamic
structure-function relationships of biological macromolecules on live cell membrane.
References:
[1] K. Takeyasu et al., Cytogenet Genomes Res.,
107: 38-48 (2004).
[2] K. Hizume, S.H. Yoshimura and K. Takeyasu
Biochemistry, 44: 12978-12989 (2005).
[3] M. Yokokawa et al. EMBO J., 25: 4567-4576
(2006).
[4] R.L. Ohniwa et al., EMBO J., 25: 5591-5602
(2006).
[5] M.
Yokokawa
et
al.,
IEE
Proc.
Nanobiotechnology, 153: 60-66 (2006).
[6] J.L. Gilmore et al., Biochemistry. 48: 1049210498 (2009).
[7] N. Crampton et al., Proc. Natl. Acad. Sci. USA
104: 12755-12760 (2007).
[8] S. Otsuka et al., Proc. Natl. Acad. Sci. USA, 105:
16101-16106 (2008).
[9] M. Yokokawa, K. Takeyasu & S.H. Yoshimura J.
Microscopy 232: 82-90 (2008).
Fig.1 Analysis of elongation process of E. coli RNAP. (A) Time-lapse
[10] H. Takahashi et al., Biophysical J., 99: 2550-2558
images of a RNAP–DNA complex obtained at 2 frames per second.
(2010).
Images every 2 s are selected and sorted (the elapsed time is shown
[11] Y. Suzuki et al., Ultramicroscopy, 110: 682-688
in each image). Branched structures emerging from the polymerases
(2010).
were indicated by allows in inset. Scale bar, 100 nm. (B) AFM images
[12] Y. Suzuki et al., Biophys. J., 101: 2992-2998
(2011).
of atranscription complex in air condition. (C) The contour lengths of
[13] M. Yokokawa & K. Takeyasu, FEBS J.,
the shorter DNA arms were measured at every 0.5 s and plotted.
doi:10.1111/j.1742-4658.2011.08222.x (2011).
[14] Y. Suzuki et al., Interdisciplinary Review, Nanomedicine and Nanobiotechnology, 3: 574–588 (2011).
[15] Y. Suzuki et al., FEBS Lett., dx.doi.org/10.1016/j.febslet.2012.06.033 (2012)
9
Pakorn(Tony) KANCHANAWONG
Mechanobiology Institute
Department of Bioengineering
National University of Singapore
Singapore
Email: biekp@nus.edu.sg
Website: https://sites.google.com/site/kanchanawonglab/home
Nanoscale Architecture of Integrin-Mediated Cell-Matrix Adhesions
Abstract:
Cells depend on integrin-based adhesions—commonly called Focal Adhesions (FAs)—to
carry out several biological functions such as migration, mechanosensing, and remodeling of
the extracellular matrix (ECM). FAs form the main mechanical linkages between the ECM
and the actin cytoskeletal machinery and are complex molecular assemblies containing >100
different types of proteins. Despite central roles of FAs in many cellular processes, the
molecular architecture underlying FAs function has remained largely unknown. Here we
applied a 3-dimensional superresolution imaging technique, interferometric PhotoActivated
Localization Microscopy (iPALM), to map protein localizations within FAs. iPALM provides 3dimensional superresolution via the combination of single molecule localization (x,y) and
simultaneous multiphase interferometry (z), allowing spatial resolution better than 20 nm
with photoswitchable fluorescent proteins. To image FAs, we create fusion between
important FA proteins and tdEos or mEos2 photoswitchable fluorescent proteins, which
were then individually expressed in human osteosarcoma cells (U2OS) grown on fibronectincoated substrate. Within FAs, we observed distinct and characteristic localizations of each
protein along the vertical dimension, revealing that FA proteins are organized at the
nanoscale level into a multilayer architecture. These results suggest how FA proteins are
assembled into nanoscale structure to mediate interdependent processes of adhesion,
signaling, force transduction, and actin cytoskeletal regulation.
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Gan LU
Centre for BioImaging Sciences
Department of Biological Sciences
National University of Singapore
Singapore
Email: dbsganl@nus.edu.sg
Website: http://staff.science.nus.edu.sg/~lugan/
Cryo-EM basics, with an emphasis on tomography
Abstract:
Electron cryomicroscopy (Cryo-EM) is a technique to study the structure of proteins,
macromolecular complexes and cells in a life-like state using an electron
cryomicroscope.The sample is prepared in way such that all the biological molecules are
trapped in a frozen-hydrated state, without any fixation, dehydration, or staining that could
damage or destroy the structures of interest. Furthermore, the image features come from
the atoms in the proteins, lipids, and nucleic acids, and therefore represent the “true”
biological structure. Finally, using image-processing techniques, one can obtain a 3dimensional structure from a set of 2-dimensional images.
In this lecture, I will give an overview of cryo-EM including sample preparation,
image-processing concepts, and image interpretation. I will then go into more detail about a
specialized form of cryo-EM called electron cryotomography (cryo-ET), a method to obtain
3-D reconstructions of “unique” objects like cells, at moderate resolutions. To conclude, I
will show some recent applications of cryo-EM to a wide-range of biological questions.
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Bo HUANG
University of California at San Francisco
1700 4th St MC 2532
UCSF Byers Hall 303A
San Francisco, CA 94158
Email: bo.huang@ucsf.edu
Website: http://huanglab.ucsf.edu/index.html
Stochastic Optical Reconstruction Microscopy (STORM)
Abstract:
As one of the super-resolution microscopy techniques invented in the past few years, Stochastic
Optical Reconstruction Microscopy (STORM) overcomes the diffraction limit of optical microscopy
through single-molecule detection of photoswitchable fluorescent proteins or dyes. By stochastically
activate sparse subsets of fluorophores in a sample and subsequently determining their positions,
STORM has achieved a lateral spatial resolution of 20-30 nm, which is more than an order of
magnitude better than that in conventional fluorescence microscopy. The incorporation of threedimensional (3D) single-molecule localization, e.g. with a cylindrical lens or by multi-focal plane
imaging, further enabled 3D STORM of a whole cell with 50-60 nm axial resolution. Multicolor
imaging using differently colored photoswitchable fluorophores or dye pairs has provided the
opportunity to examine the interactions between biomolecules and cellular structures. Live cell
STORM imaging have been implemented by fast recording or combination with single-molecule
tracking. STORM has now already showed its power in structural biology, cell biology and
neurobiology applications.
12
Renjith PADINHATEERI
Department of Biosciences and Bioengineering
Indian Institute of Technology
Mumbai 400076
India
Email: ranjithp@iitb.ac.in
Website: http://www.btc.iitb.ac.in/~ranjith/
Understanding nucleosome kinetics
Abstract:
Nuclesomes in a chromatin are dynamic -- they constantly bind, dissociate and slide along DNA with
the help of ATP-dependent remodelling machines. In this lecture, we will discuss fundamental
principles that are necessary to understand nucleosome kinetics.
We will discuss how simple physical models of nucleosome assembly can explain many of the
experimentally observed nucleosome positioning. Then, we will examine how one can use stochastic
simulations to predict kinetics of nucleosome organisation.
13
Roy BAR-ZIV
Department of Materials and Interfaces
The Weizmann Institute of Science
Rehovot
Israel, 76100
Email: roy.bar-ziv@weizmann.ac.il
Website: http://www.weizmann.ac.il/materials/barziv/
Synthetic cellular functions on a biochip: Gene expression and protein assembly
Abstract:
The ability to compartmentalize functions is essential for the emergence of complexity, as in a
eukaryotic cell and in electronic devices. One way to obtain compartmentalization in man-made
biological systems is to spatially localize the expression of genes and their products. We developed a
photolithographic biochip for localizing DNA and proteins with sub-micron precision and tunable
density. We assemble micron-size DNA brushes as localized units of expression with high density
that is comparable to DNA in a bacterium. In vitro transcription-translation in a DNA brush differs
from solution reactions, with rates controlled by DNA density and gene orientation; we suggest that
DNA brushes form boundary-free compartments. Gene expression on the chip can be cascaded from
one location to another, thus forming a basis for regulatory devices. We show that proteins
synthesized on the chip diffuse to predetermined traps, where they can assemble into structural
complexes. The gene expression biochip opens possibilities for engineering synthetic systems, such
as protein factories and template-based assembly lines, as well as environmental sensors.
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