Research aims/Tasks of the project

 Task 1: FLIN Development and application {partner 2 (coordinator)}.

(i) Set-up of a world-first FLIN prototype in Berlin following two different strategies : (a) 4Pi microscopy and (b) two-colour PSF (partners 2 and 6). By applying ultra-low background time-gated surface-selective total internal reflection fluorescence (TIRF) imaging (partner 3), a minimal-invasive system results for long-period observation (LPO) of live cells and single molecules, thus preserving the native living state (partners 2 and 6). (ii) Improvements of several parameters of the current generation of fluorescence imaging TSCSPC (time- and space-correlated single photon counting) detectors, such as temporal and spatial resolution, throughput, photocathode sensitivity, and background noise (partners 2 and 12). Partner 12 will here provide crucial expertise in detector technology. (iii) Application of dual-colour and PSF-modelling to approach 5 nm precision and 10 nm spatial resolution (partners 2 and 6).
(iv) Utilization of novel picosecond (ps) FLIN/FLIM and several of its applications (such as ps anisotropy imaging) to study the behavior of artificial and biological motors, and intracellular protein interaction and dynamics (all partners).
(v) Application of FLIN/TSCSPC detectors for ultra-parallel fluorescence screening (partners 2 and 13)
(vi) Transferring TSCSPC detector technology within the network to Partner 4 (Partner 2,4)

Task 2: Super-background free TIRF {partner 3 (coordinator)}.

(i)Development of a super background-free microscope for intravital long-term observation of single molecules, motors and nanocrystals with enhanced signal-background ratio, by applying (ai) time-gated TIRF and lock-in detection, (b) surface-selective detection of near-interface dipoles by directional emission collection, (b) locally enhanced non-linear excitation by a surface-plasmon resonance enhanced evanescent field (partners 2 and 3).
(ii) Software to control this novel microscope technique will be tested and improved (partner 11) and the performance of background-free TIRF will be compared with the newly developed Pulse Evanescent Field Fluorescence Lifetime Sampling (PEFFLS) technique (partner 1).
(iii) Super-background free TIRF will be used to study molecular engines and cellular protein dynamics (all partners).

Task 3: Design and implementation of a controlling software prototype for the FLIN prototype in Berlin{partner 11 (coordinator)}.

(i) This software system will synchronize (a) an x-y Scanner by PiezoJena GmbH (b) a Leica z-Focus (c) a CCD camera (d) different devices. As a result the software system will produce a number of image stacks.
(ii) Development of a software system for controlling and integrating the Quadrant-Anode (QA) detector and super background free TIRF (partners 2, 3 and 6).
(iii) Implementation of several analysis modules for TSCSPC (time and space correlated single photon counting).
(iv) Controlling software and analysis modules in the arivis platform software system.

Task 4: The rotary engine ATP synthase {partner 1 coordinator}.

Purified thermostable ATP synthase will be re-incorporated into liposomes and immobilized onto functional surfaces (partner 1). By introduction of FRET labels and utilizing FLIN structural changes of the enzyme involved in intermediate elastic energy storage will be determined (partners 1, 2 and 6). Obtained results will be modeled (partner 5), the results of the modeling may conversely predict additional elastic elements stimulate further FRET/FLIN experiments, finally resulting in detailed mapping of the major structural changes involved in energy conversion. Engineered forms of the enzyme will be produced that respond to chemical signals, testing the usability of the experimental system as a nano-sensor. Insights into regulation/switching of ATP synthase will be screened for possible utilization for bio-mimetic nano-machines (partner 9). Observation of many individual rotary motors at the same time will place the action of ATP synthase into a cellular context and provide detailed insight into the distribution of active ATP synthase molecules within the cell membrane as well as into the response of energy metabolism to external agents such as antibiotics. We anticipate for the last step contributions of partner 6 (cellular context of motor action) and also partners from the Cell Imaging Cluster.

Task 5: Theoretical modelling of molecular motors and rotors {partner 5 (coordinator)}.

A novel molecular dynamics (MD) program is developed for simulations of nanostructured materials and molecular motors. The program will be used to study the synthetic molecular motor recently proposed and designed in our group, as well as synthetic structures of partner 9. The results will be used to develop simple phenomenological models of motor behavior that could be generalized for more complex synthetic and biological motors. These simulations will also be used to advise partner 9 about possible more efficient structures. MD of ATP synthase subunits will be performed in collaboration with Partners 1, 2 and 6. Coarse grain simplified Langevin Dynamics (LD) will be implemented into our program and simple simulations of the rotary and linear biomotors will be attempted. The development of these new models will be based on the recently updated AMBER united atom force field and implicit solvents. Input from the Cell Imaging Cluster will be essential for successful setup of the model.

Task 6: Photosynthesis in individual chloroplasts in vitro and in vivo{partner 4 (coordinator)}

Photosynthesis in green plants takes place in chloroplasts, which have dimensions of several micrometers in all directions. The chloroplasts contain so-called stacked grana (hundreds of micrometers in diameter) containing the multi-protein complexes, called photosystem II where oxygen is produced after absorption of sunlight and the subsequent transportation of excitation energy of hundreds of picoseconds. The latter process can be followed via time-resolved fluorescence spectroscopy. The unstacked regions contain besides ATP-synthase also photosystem I with different fluorescence lifetimes and spectral composition. The overall process is highly regulated and depends strongly on light conditions in vivo. These processes are crucial for plant survival but their physical nature is only poorly characterized and understood. It will now be possible to study these processes by applying FLIN to chloroplasts in vitro and in vivo. Light conditions will be varied in a controlled way and the response of PSI and PSII will be measured for the first time (partners 1, 2, 3, 6 and 11) and modeled (partner 5).

Task 7: Synthetic robust chemical nanomachines {partner 9 (coordinator)}.

Structures with mechanically interlocked components are potentially useful for molecular machine-type applications because they permit controlled, large amplitude, movement and positioning of mechanically interlocked components with respect to another. The aims of the project are two-fold.
(i) Attach fluorescent probes to synthetic switchable molecular machines to produce catalysts and sensors, smart molecules (metal-based molecular devices), and molecular machines (artificial muscles, pistons and motors). The new, labeled molecular machine will be characterized using standard chemical procedures and fluorescence spectroscopy (partners 4 and 7).
(ii)Analyse such nanoscale molecular motions using fluorescence and the capabilities of the proposed FLIN instruments to image individual switchable molecular machines on surfaces and in polymer films during their operation (partners 1-4).

Task 8: Probe development {Partner 7 (coordinator)} .

Traditionally, fluorescent probes have been made by organic synthetic chemistry, but the advent of green fluorescent proteins (GFPs) has allowed the construction of recombinant indicators. Earlier work on indicators for calcium ion (cameleons) is currently being extended to generate probes sensitive to ligands of the steroid receptor superfamily that directly regulate gene expression. We will make protein fusions of such receptors (e.g. the estrogen receptor) with ECFP and EYFP (and other variants) that upon conformational change caused by ligand binding to the receptor exhibit an increase of energy transfer efficiency between the two fluorescent protein tags. A robust fluorescent readout will allow high throughput screening of agonists and antagonists of nuclear receptors. High throughput testing using laser scanning cytometry (LSC) will be implemented (partner 10). Chemiluminescence resonance energy transfer-based sensors for calcium will be developed with the aim of simultaneous imaging calcium dynamics in two or more subcellular compartments of live cells. Fluorescent proteins will be fused to aequorin as the calcium sensor in order to minimize interference with physiological signal transduction elements such as calmodulin. Protein fusions with recently described fluorescent proteins will also be created: several pairs amenable for FRET have been described: MiCy/mKO, mOrange/mCherry and T-sapphire/mOrange(mKO). These will be tested to replace the commonly used pair CFP/YFP by partners 1, 6, 8 and 10. We will also use the photochromic protein Dronpa, able to withstand many cycles of bleaching (503 nm) and photoactivation (390 nm), to study protein translocation to the nucleus, and interactions of receptor tyrosine kinases and motor proteins in various subcellular compartments (partners 8 and 10). Genetically engineered cell lines, either fluorescently tagged with visible fluorescent proteins or modifiable with FlAsH/ReAsH tags, as well as quantum dot-based applications will be developed for the in vivo experiments.

 Task 9: Motor proteins in cell polarity: {Partner 8 (coordinator)} .

Coordination with kinesin I and dynein motors on single microtubules. Involvement in polarity establishment. mRNA (tau, bicoid, oskar, gurken) will be fluorescently labeled in three different ways: fluorescent nucleotide incorporation during in vitro transcription from plasmids containing cDNA of interest and microinjected in living cell; microinjection of fluorescent beacon molecules specific for the mRNA of interest and GFP-tagged bacteriophage protein MS2 interacting with 6 MS2 binding sites inserted in the 3'UTR. Kinesin, dynein and proteins involved in vesicular trafficking will be tagged with GFP spectral variants. By utilizing co-single particle tracking and FLIN, coordinated transport of mRNA and motors and co-factor proteins will be analysed (partners 2 and 6) in microtubules labeled (tau-GFP) living sample (neuronal cells; oocyte of drosophila). Data will be analysed and modelled (partner 5) to determine the mechanisms of the coordination of motors and cofactors in polarity. Mechanical tension will be applied on the cellular cortex utilizing either multiple optical trap and fibronectin coated beads or stretched collagen gel. Dynamics of mRNA and motors will be analysed in real time under stress made possible by to the coupling of 2-photon excitation FLIN and nanomanipulation devices. Motor structural changes will be determined in response to load (applied with optical tweezers) in in vitro experiments applying appropriate FRET labels, including those developed in Task 8 (partners 1, 2, 3, 7 and 9).

Task 10: Linear biological motors in postsynaptic signaling: {Partner 6 (coordinator)}.

Using the novel high resolution FLIN technique we will study the recruitment and transport of membrane associated guanylate kinase homologues as for instance Discs large (Dlg), SAP97/hDlg or SAP90/PSD95. Particularly, the energy dependent interactions of these molecules with the submembraneous cytoskeleton and related motor-proteins of the kinesin superfamily will be investigated by double-transfection and stimulation and/or controlling and varying the local energy supply (eg ATP concentration) (partners 1, 2, 3, 8 and 9). Results of long term dual-colour FLIM / FLIN observations of linear motor activity (kinesin) and intracellular protein interaction will be modeled (partner 5). Obtained computer simulations will conversely lead to a better understanding of the dynamics within the postsynaptic signaling machinery during normal and pathological function. Insights into the regulation of the linear motor-cargo activity and comparison of this motor with the distribution and the regulation of rotary motors / molecular engines in the membrane (partners 1 and 4) may stimulate the development of artificial molecular machines at synaptic membranes.

Task 11: Exploring molecular interactions in protein kinase signaling networks: {partner 10 (coordinator)} .

The association states (homo- and heteorodimers, oligomers, clusters) of the receptor tyrosine kinase proteins, the stability and dynamics in space and time of their supramolecular receptor assemblies are will be one main area of investigations. The effect of important molecular modulators with potential diagnostic and therapeutic consequences (eg. integrins, IGFR, mucosialoprotein complexes, heat shock proteins) and the lipid membrane environment will be assessed as well. As a sign of activation or inhibition by modulators and relevant ligands, we shall endeavor to explore how the signal spreads on all the way to the nucleus through transient molecular interactions. These latter investigations will include space- and time-dependent correlated monitoring of second messengers (e.g. calcium), activated and deactivated effectors (phospho- and dephospho- forms of enzymes), as well as the dynamics of molecular interactions, localization, and translocation between cellular compartments of MAP kinases, activated Stat transcription factors, protein kinases B and C (partners 2, 3, 6 and 8). Genetically engineered cell lines either fluorescently tagged or modifiable with FlAsH tags, visible fluorescent proteins and quantum dots will be used for the experiments (partner 7). We will study the interaction of the inhibitor of Fibroblast Growth Factor signalling, Sprouty, with the adaptor protein Grb2 by FRET (partner 7). If this interaction can be established in live cells, the effect of several point mutations in Sprouty tested to be critical for its function will be tested.