Workshop on "Cell Biology, Fluorescence Microscopy, and Geometry"

Abstracts

Determining optimal thresholds for MELK images

Andrej Borissenko  (Universität Bielefeld, Germany)
Tuesday, June 07, 2005
The new multi-parameter fluorescence-microscopy technique MELK allows to produce a stack of intensity images of the same biological object (for example, liver cells), each image in the stack corresponding to one particular, extra- or intracellular protein of interest.

We present methods of exploring statistical dependence between different protein distributions that are based on the notion of a copula: a function which 'extracts' the dependence structure from the joint distribution of two or more random variables.

To measure the dependence degree between several proteins, we introduce a so-called multi-information function that quantifies, for any two protein distributions, their mutual information -- a measure that estimates how much we can learn about one such distribution from the other. It is shown how this function can be expressed in terms of the copula function.

We use multi-information function also to determine optimal threshold values for individual images in the stack; these threshold values allow us to separate intensities corresponding to `noise' from those corresponding to a fluorescent 'signal'.

A combination of fluorescence microscopy and chemical inhibitors reveals the timing of early T cell receptor-dependent signal transduction

Roland Brock  (Universität Tübingen, Germany)
Tuesday, June 07, 2005
Karsten Köhler#, Annemarie Lellouch§, Susanne Vollmer#, Bernard Malissen§, Roland Brock#
#Group of Cellular Signal Transduction, Interfaculty Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany, Ph.:+49-7071-2977629, Fax: +49-7071-295891, roland.brock@uni-tuebingen.de, §Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranée- Case 906, 13288 Marseille cedex, France
Cellular signal transduction proceeds through a complex network of molecular interactions and enzymatic activities. The timing of these molecular events is critical for the propagation of a signal and the generation of specific cellular responses. Here we introduce the combination of high resolution confocal microscopy with the application of small molecule inhibitors at various stages of signal transduction in T cells to define the timing of signalling events. Inhibitors of Src-family tyrosine kinases and actin reorganization were employed to dissect the role of the lymphocyte-specific tyrosine kinase Lck in the formation and maintenance of T cell receptor/CD3-dependent T cell contacts. Anti-CD3-coated coverslips served as a highly defined stimulus. The kinetics of the recruitment of the yellow fluorescent protein-tagged signalling protein ZAP-70 to T cell receptor complexes was detected by high resolution confocal microscopy. The analyses revealed that at 5 min after receptor engagement Lck activity was required for maintenance of contacts and ZAP-70 clusters. In contrast, after 20 min of receptor engagement, the contacts were Lck-independent. Disruption of the dynamics of the actin cytoskeleton inhibited cell spreading, however, was without effect on cell attachment. In summary, the results indicate that in the early phases of signalling kinases act on the CD3 complex in a way that modulates the avidity of these complexes for a polyvalent ligand similar to the one described for integrin-mediated cell adhesion. The relevance of the timing of inhibitor application provides a pharmacological concept for the maturation of T cell-substrate contacts. References: Köhler et al., ChemBioChem, 6 (2005) 152-161

The human kinetochore: model building and data from fluorescence microscopy

Stephan Diekmann  (Institute of Molecular Biotechnology, Germany)
Wednesday, June 08, 2005
S. Diekmann, J. Sühnel, S. Orthaus, S. Weitkamp-Peters, P. Hemmerich
IMB, Beutenbergstr. 11, 07745 Jena

At each cell division (mitosis), accurate segregation of every DNA chromosome into the two daughter cells is ensured by the assembly of a kinetochore multi-protein complex at each centromere locus. In humans, the centromere is a multifold repeat of α-satellite DNA, each 171 base-pairs in length forming one nucleosome. Centromere nucleosomes are distinct from normal nucleosomes by the exchange of the histone H3 to CENP-A which is very similar in sequence and length. On this repeated structure, several "founding" kinetochore proteins settle. If the α-satellite DNA is deleted from the chromosome, the kinetochore multi-protein complex settles on another stretch of DNA of the chromosome. Thus, there seems to be no direct DNA signal for the complex to form but instead - may be - structural properties.
Our geometrical model building studies of multi-nucleosomal chromatin built of nucleosomes and linker DNA (the DNA between the nucleosomes) indicate that structures without sterical clashes can be formed only for specific linker lengths. Some data suggest that the most compact of these structures is the α-satellite DNA with 171 bp repeat length. By model building, we now place the founding kinetochore proteins onto this structure. By measuring the protein interactions in vitro as well as in the human cell, we experimentally confirm specific details of the model. We measure local distances between GFP-labelled kinetochore proteins by fluorescence microscopy (FRET and FLIM).
From these studies we hope to learn why the kinetochore founding complex settles on α-satellite DNA.

Some key issues raised by MELK, with audience participation

David Epstein  (University of Warwick, United Kingdom)
Monday, June 06, 2005
The MELK approach to immuno-fluorescence, brilliantly developed by Walter Schubert, produces many aligned images of a biological specimen, each image staining for a different biomolecule. We (that is, the audience and the speaker) will discuss various problems that arise when processing MELK images and approaches to their solution.
a) Noise removal. Many MELK images show a considerable amount of noise which isn't really noise, but rather unwanted signal. This is fluorescence caused by "non-specific binding". What techniques are available for removing, or at least reducing, this unwanted signal? This is related to an attempt to make immuno-fluorescence into a quantitative matter (see b) below). Current practice among biologists is to binarize the signal---either the protein is there or it's not---but that approach discards a lot of the information.
b) Normalization of images. Suppose you take an immuno-fluorescent picture of two different sections related to the same stage of the same underlying biological process. In general, the "scales" on which one sees the two images will differ, probably in a non-linear way. What methods are there of changing scale in each image, so that the two images become more directly comparable?
c) We look at Insulin on the SAME section, first at the 4th MELK cycle, then at the 35th MELK cycle. How do these relate---quantitatively?
d) Segmentation of MELK images into cells. This problem is much more difficult with cell sections where the cells are contiguous, than it is with in vitro material where the cells are usually separate.
e) What kinds of image viewing software is needed for immuno-fluorescence in general, and for MELK in particular. This may be related to b) above.

Dynamic patterns of actin assembly in cell motility and chemotaxis

Günther Gerisch  (Max-Planck-Institut für Biochemie, Germany)
Wednesday, June 08, 2005
Günther Gerisch1, Till Bretschneider1, Annette Müller-Taubenberger1, Kurt Anderson2, and Stefan Diez2
1 Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.
2 Max-Planck-Institut für molekulare Zellbiologie und Genetik, D-01307 Dresden, Germany.
Actin networks are continuously reorganized in cells that rapidly change their shape. Applying total internal reflection fluorescence (TIRF) microscopy at acquisition rates of 10 to 20 Hz, we measured an average growth rate of 3 μm x sec-1 for filamentous actin structures throughout the entire substrate-attached cortex of Dictyostelium cells. New filaments often proceed along pre-existing ones, resulting in bundle formation concurrent with filament growth. In cells that orientate in a gradient of chemoattractant, prominent actin assemblies enriched in the Arp2/3 complex are inserted into the network, primarily at the base of filopods that point into the direction of the gradient. The Arp2/3 complex promotes actin nucleation and branching of the filaments. This complex is inhibited by coronin, a WD40-repeat protein. Accordingly, coronin is recruited to a zone behind the leading edge or to other sites where actin assembly ceases. Arp2/3 and coronin are also involved in the formation of propagating actin waves. These are dynamic assemblies of polymerized actin that are formed on a planar area of the cell membrane remote from a leading edge. Our data reveal two types of dynamic patterns that are generated by the control systems of actin polymerization and depolymerization: a network of bundled actin filaments in the cell cortex and, superimposed on that network, patches and waves formed by the filament branching activity of the Arp2/3 complex. We propose that high turnover rates of actin filaments confer plasticity to the cell cortex that is required for fast changes in cell shape, and that autonomous activation of the Arp2/3 complex is a prerequisite for rapid accommodation to external stimuli.

Feeling and Influencing Active Intracellular Polymer Networks with Light

Josef Käs  (Universität Leipzig, Germany)
Tuesday, June 07, 2005
The cytoskeleton, a compound of highly dynamic polymers and active nano-elements inside biological cells, mechanically senses a cell's environment and generates cellular forces sufficiently strong to push rigid AFM-cantilevers out of the way. These forces are generated by molecular motor-based nano-muscles, and by polymerization through mechanisms similar to Feynman's hypothetical thermal ratchet. Light has been used to observe cells since Leeuwenhoek's times; however, we use the forces caused by light described by Maxwell's surface tensor to feel cells. The optical stretcher exploits the nonlinear, thus amplified response of a cell's mechanical strength to small changes between different cytoskeletal proteomic compositions as a high precision cell marker that uniquely characterizes different cell types. Consequentially, the optical stretcher detects tumors and their stages with accuracy unparalleled by molecular biology approaches. This precision allows us to isolate adult stem cells for regenerative medicine without contamination through molecular markers. In addition to probing cytoskeletal structure, optical gradient forces can also influence cytoskeletal activity allowing us to manipulate neuronal growth. The specific opto-molecular interactions are complex since cells, which cannot modulate diffusion by the parameters found in the Einstein equation (temperature, viscosity, molecular size), exhibit rich multifaceted behavior including ballistic transport and anomalous diffusion.

Brownian dynamics with memory

Gerald Kneller  (CNRS d'Orléans, France)
Wednesday, June 08, 2005
The classical theory of Brownian motion is based on the Markovian hypothesis of the underlying stochastic process, implying short time memory effects. Diffusion processes in complex systems, such as biological macromolecules, are, in contrast, characterised by long-time memory effects which lead to an algebraic decay of the respective correlation functions. Recent simulation results show the meaning of "complexity" in this context and demonstrate that fractional Brownian dynamics is a good model for the internal dynamics of proteins. The model can be applied on the short pico- to nanosecond time scale seen by quasielastic neutron scattering, as well as on the millisecond to second time scale seen by single molecule fluorescence spectroscopy. The results are discussed from astatistical physics point of view and possibilities for applications to the analysis of macroscopic transport processes are outlined.

Single cell studies of bacterial two component systems (work in progress)

Eduardo Mendoza  (Ludwig Maximilians Universität München, Germany)
Tuesday, June 07, 2005
*Pamela David1, Orland Gonzalez1, Kirsten Jung2, Judith Leierseder3, Eduardo Mendoza1, 3 and Joachim Rädler 3
* Authors in alphabetical order
1 University of the Philippines Diliman 2 Biology Department, Ludwig-Maximilians-University 3 Physics Department, Ludwig-Maximilians-University
The response of biological systems to external stimuli is generally measured from an entire population. However the individual response of the cells is often widely distributed. Many effects that could elucidate the biological network are averaged out. In order to address the variance and underlying stochastic processes of signal transduction systems single cell analysis techniques shall be developed. The dynamics of gene expression in bacteria will be monitored by quantitative and time resolved image processing of GFP-hybrid proteins. This allows acquiring time traces of the protein content of individual cells, which are used as input for mathematical models. The analysis of many traces in an automated procedure measures the distribution of gene expression in a population. Single cell studies are likely to access stochastic events in the signal transduction pathway, which are the key to identify the functionality of molecular modules. The talk will report on the progress of a recently initiated joint experimenter-modeller study of the EnvZ/OmpR system in E.coli. On the experimental side, techniques developed from the study of artificial virus transport in eukaryotic cells are being adapted by J. Leierseder, K. Jung and J. Rädler. On the computational side, approaches from modelling other two component systems (such as KdpD/KdpE in E.coli) with S-Systems will be adjusted by P. David, O. Gonzalez and E. Mendoza, to integrate data from the current experiments and the literature. In particular, simulated annealing will be used to estimate parameters directly from the time-course data generated.

New tools for automatic and interactive exploration of multivariate image data in biomedicine

Tim Nattkemper  (Universität Bielefeld, Germany)
Monday, June 06, 2005
Tim W. Nattkemper, Thorsten Twellmann, Axel Saalbach
Applied Neuroinformatics Faculty of Technology Bielefeld University
The analysis of multivariate image data is a field of research becoming increasingly important in a broad range of applications from remote sensing to biomedical microscopy imaging. While traditional scientific image processing and visualisation techniques are often not suitable for the analysis of this kind of data, applications of machine learning methods have shown promissing results regarding image segmentation or image fusion. In this talk we will present systems for the automatic detection and classification of cells in micrographs and a new approach for image fusion based visualization of multivariate image data.

Vibration and Cell Activity - Cell Activity and Vibration

Ulrich Randoll  (Matrix-Center-München, Germany)
Wednesday, June 08, 2005
Vibrations an rhythms, as a result of organs and single cells, are well known in the field of clinical medicine. Also the meaning of their changes until a total stop (death) have been described for decades.
In cardiology and sports medicine for example it is well established to use time patterns (cell vibrations as a result of cell activity) for diagnostics.
On the other hand, to use time patterns (electrical, magnetical, mechanical) specifically for therapy is new and still strange for some medical doctors, but opens the new field of vibrational medicine.
Biophysics ( Synergetics, Cybernetics, non linear thermodynamic of irreversible processes, Chaos-Theory) of today gives the idea how biological structures are the result of physico-chemical processes, that are driven by body intrinsic and / or body external rhythms. Such bio-informative fields interact the whole span of life and stabilize dynamically.
In the early 90th researchers in the department of traumatology at the University of Erlangen-Nuremberg showed in high resolutiuon videomicroscops cellular oscillations depending from the biophysical environment. More and more it was recognized, that geometry is the informational link between time- and space-pattern.
They started to find out how far "basic evolutionary time patterns", as natural conductor-frequencies are disturbed in diseases and how far they can be systematically and continuously restored and brought back to a synchronous cooperation up to the macroscopic level again.
Following this dynamic approach it was necessary to develop the Matrix-Rhythm-Therapy, a "Rhythmic Micro-Extension-Technique", which uses the so far neglected time-structure (time-pattern) of the organism.
This innovation got the PCT and US patent and is ready for use in present day modern medicine. Clinically evaluated studies have been done in rehabilitation-clinics of the LVA and also at DaimlerChrysler, Stuttgart.

Dissecting Gene Expression by Hybridisation in situ (FISH) and Indirect Immuno Fluorescence

Klaus Scherrer  (Institut Jacques Monod, CNRS, France)
Wednesday, June 08, 2005
The expression of the α-globin gene domain was studied in the chicken erythroleucemic AEV cells by systematic Northern blotting and in situ hybridisation, using riboprobes of 15 fragments. A 33 kb-long Full Domain Transcript (FDT) runs through the entire globin domain including, from the 5' to 3' side, a putative LCR, an upstream silencer or terminator element, a CpG island acting as transcriptional modulator, the π-, αD- and αA-globin gene cluster, and the 3'-side enhancer and silencer elements. The precise function of the globin FDT is not clear as yet; the possibility is not excluded, however, that it may be the physical precursor of the individual globin pre-mRNAs. Most interestingly, we were able recently to give evidence for the first time, that the main function of high Mr RNAs of globin FDT-type may be at the level of the organisation of the nuclear matrix where they act as a structural backbone, assembling RNA-binding proteins.
Among the latter are the Prosomes (PS) which constitute a population of particles (Mr 720 kD) built of variable combinatories of 2 x 14 protein subunits. They were first observed as sub-complexes of cytoplasmic untranslated mRNPs, often associated to the cytoskeleton, as well as of nuclear pre-mRNPs. Some of their subunits have RNase activity. However, they constitute also the proteolytic core of the 26 S proteasomes. Acting on the biosynthetic as well as catabolic pathways they are involved, thus, in protein homeostasis. Studying the nuclear phase of the Prosomes on Rat Myoblasts we had identified them as components of the nuclear matrix, attached to chromatin, DNA and RNA; they where found to form specific but variable distribution patterns, lining up in particular around the nucleoli. On AEV cells, immune-fluorescence using monoclonal Abs specific to PS and the 19S Proteasome Regulator (ATPase) subunits Rpt3 (S6b, TBP7) and Rpt5 (S6a, TBP1), was combined with in situ hybridisation of globin riboprobes.
In transformed AEV cells, globin transcripts appear in the nuclear lumen and concentrate around the nucleoli. After induction of hemoglobin synthesis in these thermo-sensitive cells, transcripts move to two RNA processing centres (PCs) where partially processed transcripts accumulate. Interestingly, within the PCs, globin RNA co-localises with nuclear PS patches were, specifically, the p23K-type PS accumulate. Surprisingly, sites of coincidence of p23K-type PS and globin mRNA appear in the cytoplasm as well, at sites where the untranslated globin mRNPs concentrate. This indicates that globin transcripts might be transported on the nuclear matrix from the PCs to the nuclear periphery and into the cytoplasm along "Prosome trails".
Preparing nuclear matrices from AEV cells we found once more the 23K-type prosomes as constituents of the matrix networks lining up, as in myoblasts, around the nucleoli. Simultaneous hybridisation in situ with riboprobes for the globin genes and extra-genic FDT segments showed the presence of these transcripts in the matrix network as well. Treatment with RNase suppressed the globin signals completely and removed up to 90 % of the 23K prosomes. In contrast and most interestingly, the 19S regulator complex of the proteasome was not sensitive to RNase treatment indicating, that the 20S prosomes but not the 26S proteasomes are on the RNA matrix.
Carrying over the prosomes and other RNP proteins from the chromatin to the nuclear periphery during processing, pre-mRNA may preserve protein alignments assembled originally on the DNA-derived nuclear matrix. The integration of primary transcripts of the globin FDT-type allows suggesting, that they may represent the organisational principle of an RNA-dependent part of the nuclear matrix within which processing and mRNA transport proceed. Acting as a 3D-backbone for a dynamic part of the nuclear matrix, at instars of the precursors of ribosomal RNA (pre-rRNA) and ribosomes which form the nucleoli, the apparently useless length of primary transcripts and their introns finds a logical interpretation. Fragments of coding information would thus be inserted into supporting RNA forming 3D structures, as are in case of proteins the sites of catalytic or other activities, allowing specific processes to be controlled in space and time during gene expression.

Topological proteomics: Exploring molecular networks directly in the cell

Walter Schubert  (Universität Magdeburg, Germany)
Monday, June 06, 2005
Understanding how proteins are temporally and spatially arranged to generate function is a major challenge of the post-genomic era, as existing methods for analysing protein-protein interactions seldom reveal when and where interactions occur in vivo. Here this challenge is addressed by combining three advances: a technique capable of mapping hundreds of different molecules at light microscopic resolution in a single sample; a method for selecting the most significant protein groupings by representing the resulting data as multidimensional binary vectors; and a system for imaging the distribution of these groupings in a 'toponome map'. This approach reveals new hierarchical properties of protein network organisation, in which the frequency distribution of different protein groupings obeys Zipf's law. It also provides a rapid route to distinguish new diagnostic features and therapeutic targets in human diseases, and offers a powerful way to identify the hub proteins around which networks are organized.

Perspectives for confocal single molecule analysis in cells and membrane systems

Petra Schwille  (Technische Universität Dresden, Germany)
Monday, June 06, 2005
Single molecule flourescence studies have come of age. While the detection and analysis of single biomolecules was still per se a difficult task to accomplish some years ago, and while some challenges still remain for in vitro applications, one of the greatest goals of modern biophysics is to follow single molecules in their native environment, i.e. the living cell or organism. Here, additional problems arise, such as the increased optical background, difficulties of labeling or controlling concentrations and environmental conditions, and, last but not least, the huge complexity of biological systems in general. To study protein interactions in situ, many precautions have to be taken to account for the increased complexity, and the employed methods such as Fluorescence Correlation Spectroscopy may have to be accommodated, e.g. to integrate complex stoichiometry or ternary interactions. Here we discuss the perspectives of FCS and related techniques as potential tools for single molecule based proteomics. Protein-protein interaction measurements with complex stoichiometry are introduced, as well as the use of controllable model systems for analyzing the dynamics of membrane bound proteins.

"Lasagne": a tool for interactive exploration of multivariate image data

Peter Serocka  (Universität Bielefeld, Germany)
Monday, June 06, 2005
As new technologies for gathing multivariate image data arise in many areas, the number of obtainable dimensions or layers per data set has increased dramatically. To quickly explore such data we visualize the similarity of all pixels in the data set with a given reference pixel. Two aspects are essential: first, providing a good similarity measure or metric which helps detecting relevant features that are not a-priori known. Second, computing this metric extremely fast for all pixels in the data set.

We present our tool Lasagne, which implements different metrics and delivers high interaction rates: The similarity image is generated in virtually the same instance as the user points to a pixel on the screen. This allows for visually scanning the data set in a contiguous way - a new experience in looking at multivariate images, revealing smallest details as well unexpected features.

Micro-Rotation Imaging- a novel system and new mathematical methodologies for three-dimensional fluorescence imaging in individual, non-adherent living cells.

Spencer L. Shorte  (Institut Pasteur, France)
Tuesday, June 07, 2005
Quantitative three-dimensional (3D) imaging of single living cells comprises an important tool for studying cell biology, cellular signalling and processes of infection and virulence therein. To date, conventional 3D imaging is limited mainly to methods using z-stack axial sampling whereby a sample volume is imaged repeatedly while mechanically shifting the microscope objective focal plane in small (100-500nm) axial steps. The resulting planar axial ¿z-stack¿ image series can then be processed and rendered in 3D using computer-software. However, among the main disadvantages of ¿through-stack¿ 3D imaging methods is the prerequisite that targeted samples must be stabilised by attachment to an optically transparent surface, precluding 3D visualization of many important non-adherent cell types, for example lymphocytes. Driven by the need to overcome this limitation we present a new micro-manipulation device (the Micro-Rotator*) that uses dielectric field control, enabling individual, non-adherent cells to be immobilised in suspension, and rotated around a fixed focal plane, through any chosen vector including the z-axis. We show that image series acquired during these z-axis rotation protocols effectively comprise fixed viewpoint 3D ¿movies¿ of compartmentalized fluorescence localised to single, living cells. Inasmuch as this method does not require axial movement of microscope or image detector components, and uses x,y plane sampling in one focal plane only, it offers a two- to three-fold superior 3D spatial resolution, and greatly diminishes (or removes completely) artefacts due to axial optical defects. As such, micro-rotational 3D imaging presents a distinctly new technological advance over existing axial through-stack methods, and opens new avenues requiring development of novel mathematical approaches to solve the reconstruction problem.
Work funded by EU-FP6-NEST programme in a grant to SLS, in consortium AUTOMATION (www.pfid.org/AUTOMATION)
*Patent: Shorte, S.L., Müller, T., Schnell, T. ¿Method and device for 3 dimensional imaging of suspended micro-objects providing high-resolution microscopy¿ Patent Number; EP 02 292 658.8; 25 October 2002

Signal evaluation and cell motion

Angela Stevens  (MPI MIS, Germany)
Tuesday, June 07, 2005
Mathematical models for cell motion due to diffusive and cell-surface bound signals are discussed. It is shown that different types of signal evaluation by the cells can lead to different macroscopic structures on the cell population level. Thus the analysis of pattern forming processes may be able to give hints on possible underlying mechanisms of signal detection during chemosensitive motion.

Digital light microscopy techniques for the study of living cytoplasm

Dieter G. Weiss  (Universität Rostock , Germany)
Tuesday, June 07, 2005
See this  PDF abstract.

Date and Location

June 05 - 08, 2005
Max Planck Institute for Mathematics in the Sciences
Inselstraße 22
04103 Leipzig
Germany
see travel instructions

Scientific Organizers

Andreas Dress
Max Planck Institute for Mathematics in the Sciences
Leipzig
Contact by Email

Jean-Pierre Bourguignon
Institut des Hautes Études Scientifiques
Bures-sur-Yvette

Administrative Contact

Antje Vandenberg
Max Planck Institute for Mathematics in the Sciences
Leipzig
Contact by Email

05.04.2017, 12:42