During the resolution (termination) of immune response, macrophages and dendritic cells change their phenotype from activated/pro-inflammatory to reparative/anti-inflammatory. Members of the TAM family of tyrosine kinase receptors (Tyro3, Axl and Mer) play a critical role in this process, and mice lacking these receptors develop broad-spectrum autoimmune disease. However, how TAM receptors orchestrate this switch and how they may crosstalk with other anti-inflammatory pathways remains largely unknown.

In my project, I will investigate the molecular mechanisms of TAM receptor function during the resolution of inflammation. I will use macrophages and dendritic cells derived from the wild-type and TAM knockout mice to determine if the immunosuppression induced by other anti-inflammatory mediators such as glucocorticoids, LXR agonists and PPAR agonists is TAM-dependent, and to dissect the potential crosstalk between these nuclear receptors and TAM signalling pathways. I will also use microarrays and mass spectrometry to study the downstream targets of TAM receptors. These unbiased approaches should reveal a global shift in gene and microRNA expression patterns and enable the identification of novel anti-inflammatory mechanisms. I will further attempt to identify the transcriptional regulators targeted by TAMs, and to dissect the signal transduction pathways involved. Finally, I will verify my findings in vivo using mouse models of autoimmune disease.

Altogether, my studies should increase our understanding of macrophage/dendritic cell reprogramming during the resolution phase of immune response.

In recent years the use of advanced optical techniques has had a strong impact in neurobiology not only for visualizing neuronal structures and signaling processes, but also for controlling neuronal activity. This is possible thanks to a growing list of photosensitive tools that can modify their chemical or conformational structures upon illumination and precisely trigger the propagation of neuronal signals. In addition to the commonly used caged neuroactive compounds, genetically encoded light-sensitive proteins have recently been developed permitting neuronal activation and inactivation. In conjunction with spatiotemporally resolved photo-stimulation techniques, these photoswitchable proteins represent the most promising alternative to electrical stimulation, providing ways to control precisely in space and time the activity of specific types of brain cells.

These approaches require fast, flexible and precise illumination schemes, permitting the selective activation and imaging of sub-cellular regions or multi-cellular ensembles. The Emiliani group has recently developed the first one-photon scan-less holographic microscope where a liquid crystal spatial light modulator (LC-SLM) in the excitation path generates 2D and 3D multiple diffraction limited spots or shaped domains of excitation that can accurately cover a single cell or sub-cellular structures. They showed that shaped holographic illumination for large excitation areas has a significant higher axial resolution than a Gaussian beam, and concentrates all of the laser power on the desired region, thus providing the high intensity needed for fast photostimulation. They have also shown that further optical confinement can be achieved in two photon holographic illumination combined with temporal focusing.

The aim of this proposal is to use this innovative approach to study fundamental properties of central neurons. Holographic photolysis of caged compounds will be used as a novel means for mapping the subcellular distribution, modulation and function in neuronal signaling of ion channels in brain slices. Holographic photoactivation in vivo of the engineered light gated channel LiGluR will be used to elucidate the neural circuit basis of locomotion in zebrafish.

Neuroscientists have measured biophysical properties of individual nerve cells and monitored the activation of large brain areas in intact animals including humans. A coherent understanding of brain functions now requires quantitative insights into the interactions between individual neurons in order to understand how sophisticated computations are performed by neuronal circuits. This question raises a series of conceptual and technical challenges. First, connections between many or even all neurons in a circuit have to be determined by 3-dimensional imaging methods that achieve nanometer resolution throughout very large volumes. Second, activity patterns in the intact brain have to be measured with a resolution of individual nerve cells to quantitatively characterize neuronal computations. Combined measurements of activity and connectivity in the intact brain are then required to understand how the function of a circuit is determined by its structure. Third, activity and connectivity have to be manipulated in order to perturb a circuit and examine the effects onto neuronal computations and behaviors. Fourth, principles underlying neuronal circuit structure and function have to be extracted by mathematical approaches. To address these issues we will focus on the sense of smell in a small vertebrate, the zebrafish, which provides unique advantages. The connectivity among large numbers of neurons will be determined by novel 3-dimensional electron microscopy techniques and analysis methods based on machine learning. Activity patterns will be measured using non-linear optical methods that permit the visualization of activity across thousands of neurons in the intact brain. Advanced genetic methods will be used to manipulate the function of defined neurons in the intact brain. These approaches include optical techniques to precisely control the activity of individual neurons in space and time. Structure-function relationships of circuits will then be analyzed using mathematical methods and computational modelling. This interdisciplinary approach is expected to provide elementary insights into the mechanisms by which neuronal circuits process information and control behaviors, which is essential to understand how higher brain functions emerge from complex communications between large numbers of nerve cells.

A fascinating, unanswered question in biology is how some organisms respond to injury by regenerating the lost body structure. Two fundamental aspects of the problem are first, how does injury trigger a growth and patterning response? Second, how does the regenerating tissue “read” the overall size of the animal to produce an appropriately sized structure? While some molecular pathways have been implicated, a clear mechanism linking physical injury to cellular and molecular response is wholly lacking. Furthermore organ growth control remains a challenging problem in biology. Here we propose to couple the research efforts in three regeneration model organisms, Hydra, Axolotl (salamander) and Drosophila, to quantitative mathematical modeling as a means to address these issues in a novel way.

Drosophila has been a fertile system for identifying and analyzing molecular pathways involved in imaginal disc growth and patterning. We will therefore use the Drosophila imaginal disc as a starting point for identifying and analyzing mechanisms that link tissue injury to a growth and patterning response. Disadvantage: we cannot observe the regeneration in discs as it is happening. Therefore complementarily, we will use two classical regeneration models, Hydra and salamander (Axolotl) where regeneration can be exquisitely controlled and visualized in live animals to build on the physical link between injury and molecular signaling to cellular response. To harmonize the systems, we will all focus on the regeneration of epithelial structures where it is most likely that cellular responses may be similar: the Drosophila wing and leg discs are simple epithelia. In analogy, the Hydra body column consists of a bilayered myoepithelium, and finally, amputation of the Axolotl tail results in regeneration of the spinal cord through a neuroepithelium.

Therefore, we are not looking for molecules responsible for regenerating a particular body structure but for the general mechanisms used to control regeneration and size control of epithelial-based organs. Our specific aim is to come to a deeper mechanistic and integrative understanding of how signaling pathways respond to cell/tissue injury to initiate growth, patterning as well as size control response. For this reason mathematical modeling will help integrate the results obtained in the different phyla to identify shared mechanisms.

This project will investigate the forces between two double-stranded DNA molecules in a protein-free environment. Due to the chiral nature of DNA, these interactions are also chiral and affect DNA conformation and, in turn, are affected by topological constraints imposed on the molecule. Existing (Kornyshev-Leikin) electrostatic theory provides an explanation for the homology-promoted attraction. This theory also predicts a chiral torque that tends to make long DNA molecules wind around each other. The effect will be stronger if the DNA tracts in close juxtaposition are physically identical (i.e., homologous).

The double-stranded helix of DNA in cells is slightly (~30%) underwound relative to relaxed DNAs such as those used to test the homology interaction theory mentioned above. Additionally, cellular forces (in the pN ranges) cause transient extreme under- and overwinding of DNA. These topological states are essential for DNA transactions and without them cells die. The complex interplay between homology and supercoiling is the core subject of this project.

The project will determine: (i) the degree to which homologous tracts attract each other more strongly than non-homologous tracts; (ii) the force keeping two homologous tracts in juxtaposition; (iii) how homology recognition is influenced by supercoiling. These questions will be answered by performing a complementary series of single-molecule experiments and biochemical experiments with circular DNAs of varying degrees of supercoiling. Experiements will then be move to inside live cells. The experiments will be supported and rationalised by a theory, to be developed. This new theory requires the merger of the continuum theory of elastic braids and the theory of chiral electrostatic DNA-DNA interactions. The theory will distinguish juxtapositions of homologous and non-homologous DNA tracts and will allow us to investigate the dependence of supercoiled structures of DNA on structural and environmental parameters, including homology length and type and concentration of counterions.

We will build the minimal molecular engine required to initiate bacterial division by reconstituting its molecular machinery (the divisome) and reproduce its vital functions in vitro. Cytokinesis is a fundamental process that is known in bacteria in sufficient detail to provide a comprehensive list of its molecular effectors.

Our novel idea is to reconstruct and track the operation of the minimal functional protein set needed to initiate division (the proto-ring) and the two main positioning mechanisms (Min system and nucleoid occlusion) that select the constriction site.

We will use complementary biochemical, biophysical, genetic and imaging methodologies to reconstitute the proto-ring components (FtsZ, FtsA and ZipA) into artificial membrane-bounded compartments that mimic the divisome organization at the cell membrane. The use of biomimetic materials, vesicles and nanodiscs, as substrates on which to reconstitute the machine will be complemented using biovessels to assemble the proto-ring within structures that reproduce the cellular environment with minimal genetic information. Ideal conditions regarding the composition and shape of the membrane compartments will be designed for divisome reconstruction in nanodiscs, biomimetic bilayers, giant vesicles, and membrane tubes pulled to various geometries.

Research will then focus on the reconstruction and stability of proto-rings in biovessels, the interactions and dynamics of proteins within the division ring and how ring constriction is regulated. In vivo top-down strategies will be designed to minimize the components necessary for cytokinesis. The mechanism of proto-ring positioning by Min proteins and the membrane deformation imposed by proteins responsible for its constriction will finally be investigated.

Because of their relative simplicity, bacteria will probably be the first cells to be fully understood at a systems level, and our results will help usher in the new field of synthetic biology of cell division and will open new horizons for pharmacological applications.

One of the main motivations for examining host-pathogen relationships between viruses and cells is for the development of target-specific antimicrobials. Since many aspects of the complex nature of host-pathogen interactions are still enigmatic, even at the most fundamental level, an interdisciplinary and synergistic effort is proposed here to study viral infection and replication in the context of an intact host cell in four dimensions (4D, i.e. in 3D imaging as a function of time) and at a level of resolution never before achieved. Scientists from Geneva (CH), Atlanta (USA) and Norwich (UK) that are still early in their careers with expertise in genetics, live-cell fluorescence imaging, electron cryo-tomography (ECT, 3 dimensional electron microscopy) and mathematical modeling will use a bacterial model system (Caulobacter crescentus) to observe and quantitatively describe how viral particles (bacteriophages) infect and replicate in the live host. The emphasis will be on the mechanisms by which bacteriophages exploit both i) the surface structures on the host for infection (i.e. adsorption, adhesion and genome injection) and ii) the host cell cycle and/or its internal structures (cytoskeleton) for progeny production. C. crescentus is an ideal model system for these studies because it is small enough for whole-cell ECT imaging during bacteriophage infection and replication. Moreover, mutations can easily introduced into C. crescentus and it is easy to enrich for cells that are in a specific cell-cycle phase. These features provide an ideal experimental setting for a marriage of imaging and genetics to define external/internal structures and the cell-cycle parameters of the host that influence bacteriophage infection and replication. These studies, though fundamental in nature and of conceptual relevance for many aspects of biology, could potentially be useful in developing new antiviral treatments.

Adult skeletal muscle stem cells, called “satellite cells”, are mononucleated quiescent residing in a defined anatomical niche adjacent to the myofibre and beneath the basal lamina of the myofibers. In response to muscle injury, muscle stem cells are able to giving rise to both newly differentiated myofibers and self-renewing cells to restore the satellite cell pool. Aged muscle regeneration is impaired due to defective satellite cell function. However, mechanisms involved in their regulation and function during aging remain unknown. The systemic environment is one of the key regulators of muscle stem cell function during aging. In other stem cell compartments, defects in mitotic spindle orientation contribute to the decline of stem cell function. We speculate that, during aging, adult satellite cells have impaired mitotic spindle checkpoint function and through communication with the systemic environment that impact normal satellite cell self-renewal and differentiation, which impairs muscle repair capacity after injury. We intend to study the effects of aging on mitotic spindle/centrosome components of muscle stem cells and their progeny during homeostasis and regeneration and to determine the function and regulation of the mitotic spindle components during muscle stem cell aging. We aim to identify whether spindle components are required for appropriate cell fate decisions and whether disruption of these components is causative to impaired stem cell function during aging. We will also analyze the effects of the systemic environment on mitotic spindle orientation during aging.

The inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. However, during the process of replication, chromatin organization undergoes transient genome wide disruption ahead of replication fork followed by restoration on new DNA. This involves tight coordination of DNA replication and chromatin assembly process in time and space. Here, I propose to dissect molecularly the challenge of chromatin replication. I will isolate chromatin complexes ahead and behind the replication fork and characterize new players involved in chromatin replication. To this end, I will combine standard and novel biochemical techniques with mass spectrometry. This should lead to the identification in an unbiased manner of an array of new players involved in chromatin replication. Most promising ones will be selected for further mechanistic analysis. To this end I will monitor the ability of cells to replicate and divide during replication in the context of chromatin. This work may reveal novel insight to the mechanistic coupling of accurate DNA replication with its organization into chromatin, which is central for development and tumor avoidance.

Ribosomes play a central role in protein biosynthesis, translation, which is a fundamental and essential process in all cells. Mitochondria contain a translational system that is distinct from that of the cell cytoplasm. Mitochondrial ribosomes have remarkable divergence from those of bacteria and the eukaryotic cytoplasm. Most of the mitochondrial ribosomal proteins are unique, having no closely related homologues in cytoplasmic ribosomes. In the proposed project we aim to address several central questions pertaining to the mechanism of mitochondrial ribosome by exploring its three dimensional structure. We take the challenge to crystallize and determine the X-ray structure of the mitochondrial ribosome from bovine heart mitochondria. A crystal structure of the mitoribosome is likely to be a challenging goal. However, we believe the collaboration proposed here that combines the strengths of two groups, V. Ramakrishnan and J. Walker, supplemented by the previous experience of the applicant in determining the structure of large macromolecular complexes, provides a unique opportunity to mount an attack on this problem. Studying the mitochondrial ribosome will have an important medical impact due to the fact that many of the adverse side effects of therapeutically important antibacterial antibiotics are ascribed to inhibitory effects on mitochondrial ribosomes, as well as many genetic diseases. In addition, the structure determination of mitoribosome is expected to require new approaches in structural biology. Therefore, in addressing questions of large complexes protein structural biology, our efforts will yield impact beyond understanding translation mechanisms.

Adult stem cells hold much promise for regenerative medicine. Typical therapies employ their direct injection into tissues, but this does not promote robust regeneration as few cells remain in the tissue post-injection. Supporting scaffolds similar to natural extracellular matrix (ECM) are likely necessary, but current scaffolds do not sufficiently mimic key properties provided by natural ECM: stiffness, porosity, and adhesivity. As a result, the adult stem cells do not receive the appropriate environmental stimuli required for them to change into mature tissue, such as muscle and bone, and thus become stuck in an intermediate state that is not useful for regenerative medicine. In this research project, we will generate a new class of porous foam materials that are better at mimicking these three key environmental cues than current classes of biomaterials to more effectively drive adult stem cells to change into mature tissue cells.

These porous foams we will create will use a new building material for biology: diblock copolymers, which is a polymer chain containing separate repeating units of water loving (hydrophilic) and water avoiding (hydrophobic) polymer. To make the foam, copolymers will be placed in a solution of water droplets (hydrophilic) mixed in oil (hydrophobic), i.e. a solution called an emulsion. Given each side of the chain’s desire to be in or out of water, the copolymer will align at the water-oil interface and be crosslinked together to form the foam structure. Stiffness and porosity can be varied in these materials by changing the density of crosslinking or the size of the water droplets in the emulsion, respective. To change adhesivity, we will have the polymer form adhesive “nano-domains” forcing the cells to attach to the foam at discrete points, just as with natural ECM for muscle and bone. By displaying these three key properties within the foam at values typical of muscle and bone, it will better mimic the typical environment seen in the body. Thus the adult stem cells can then receive a more complete set of signals allowing them to become a mature cell rather than being stuck at an intermediate state.

Insects were the first animals to evolve flight capabilities and can perform a wide array of extreme aerial maneuvers. This complex animal behavior arises from the interplay between the intricate biological structures and functions of an insect, and the aerodynamics they must master to fly. Over the past 60 years, the general underlying principles governing unsteady flight have become significantly better understood. Nevertheless, we are far from having a complete understanding of this phenomenon. Little is known about how insects perform and control maneuvers. Moreover, we have little insight into the structure function relationship between insect morphology and flight.

Recently, the Cohen group developed a new technique capable of fast and accurate extraction of wing and body kinematic data from high speed 3D videos of free flying insects. I propose to use these techniques to conduct a quantitative, comparative study of flight maneuvers and control mechanisms in different species of Drosophila and their mutants. I will focus on two maneuvers, forward flight and ascending flight, in Drosophila melanogaster and grimshawi. These two species are distant relatives with a significant size disparity, and qualitative differences in the gross features of their flight patterns have already been observed. These comparative studies will elucidate the origin of these differences. Finally, I plan to test the validity of the principles that I discover using minimal robotic models. Ultimately, these experiments will form the basis for a broader systematic study on the evolution of insect flight strategies and control mechanisms.

DNA double strand breaks (DSBs) are dangerous lesions that, if improperly repaired, may lead to chromosomal rearrangements and other genetic alterations, and eventually to cancer. Previous studies have suggested a role for the Ubiquitin-Proteasome System (UPS) in DSB repair, and have speculated that its proteolytic activity may be required to degrade components of the DSB repair machinery. We have recently demonstrated this idea for the first time, showing that the repair protein Mms22 is a UPS target, and that failure to degrade Mms22 results in impaired DSB repair. Our results suggest that the impaired DSB repair caused by UPS dysfunction can, in part, be attributed to the specific accumulation of additional UPS targets that still await discovery.
In this proposal we devised a systematic screening approach for identifying yeast and human proteins involved in DSB repair via their proteasome-mediated degradation. Such a list will facilitate the understanding of the regulated removal of repair proteins from DNA, a broad problem that currently is not well understood. It will also serve as the basis for detailed mechanistic follow-up studies as well as for novel therapeutic approaches. The second aim of this proposal is to gain further mechanistic insight into the role of the UPS mediated degradation of Mms22 in the repair of DNA DSB. Chromatin immunoprecipitation (ChIP) analysis will be used to draw a global view of the effect of Mms22 degradation on the cellular response to DNA DSBs. This may serve as a paradigm to understand how additional repair proteins are regulated by the UPS.

One function of the eyes is the detection of color information. In the Drosophila retina, color detection is achieved by the inner photoreceptors R7 and R8, which project to the medulla part of the optic lobes. The medulla is a highly complex structure composed of ~40,000 neurons organized in 10 layers perpendicular to R7 and R8 projections. There are more than 60 subtypes of medulla neurons, and the projections of each pair of R7 and R8 are surrounded by columns composed of more than 35 of these neurons.
My main objective is to understand how the medulla neuropil forms. My postdoctoral project has 3 specific aims: (1) to define the developmental origin of the neuroblasts forming a given column, (2) to determine the exact neuronal content and the developmental mechanisms leading to the formation of medulla columns, (3) to identify genes controlling the formation of specific medulla neuronal subtypes. To achieve this, I will use a combination of approaches based on Flip-out clone analysis, Chromophore Assisted Light Inactivation (CALI), Brainbow, live imaging, brain culture and a piggyBac-based genetic screen. This work will be critical for understanding the neural circuitry processing Drosophila color vision.

How does the brain process the complex, dynamic signals that we encounter in the natural environment? And are there different central circuits active for processing the different aspects of sensory stimuli, or are shared neural substrates involved? In the case of vision, we have a handle on these questions, with an understanding of elementary features and their representation in the brain. In the auditory system, especially above the thalamus, the issues are complex and the organizing principles much less clear, with no overall framework yet to guide our understanding of auditory processing. With respect to these questions, songbirds provide a very useful model. They learn to sing using auditory feedback, and show lifelong perceptual learning of other birds’ songs. They possess a hierarchical network of central auditory areas, analogous to those in mammals, which likely subserves these auditory behaviors. The combination of a rich set of behaviorally relevant auditory stimuli and tasks and of an identified neural circuit opens up the possibility of fundamental understanding of how auditory networks function in perception and learning. To this end, we propose an international collaboration to combine neurophysiological recordings of multiple neurons in awake songbirds; new theory-based approaches to extracting complex feature selectivity and analyzing population coding in these neurons; and state-of-the art anatomical techniques to map the connectivity of neurons at electrophysiologically identified sites and to create a functional wiring diagram. Our work will be guided by, and test the following hypotheses: a) there is a systematic and hierarchical organization of feature response properties across the avian forebrain, including specialized pathways for different aspects of complex sounds, such as frequency and time, b) the collective activity of neuronal populations across these areas reveals a further organization of sound encoding not evident in the properties of neurons considered individually, and c) this network can be dynamically reconfigured by bottom-up influences (such as stimulus statistics), top-down influences (behavioral task), and learning (of adult song discriminations). Our goal is a principled picture of how this circuit is organized to analyze and learn complex natural sounds, with potential relevance to all vertebrates.

Dynamic actin networks serve to generate, transmit and resist forces on the cellular scale by providing mechanical strength to the cytoplasm. Despite advances in our biochemical knowledge of how actin networks are assembled in vivo, very little is known of how dynamic actin networks with close-to-physiological architecture respond to mechanical stimuli at the molecular level. By combining total internal reflection microscopy (TIRFM) and atomic force microscopy (AFM), we will set up an experimental system that allows for the visualization of the active, force generating region of in vitro reconstituted actin networks in a biochemically and mechanically defined environment.

As a starting point we will investigate the biochemical response of reconstituted, Arp2/3-generated, dendritic actin networks to mechanical load. We will perturb network architecture and dynamicity in a controlled manner by systematically changing key physical and biochemical parameters, like the bulk concentration or local surface densities of the various key organizing proteins to determine how the force response and organization of the network is altered. This approach will uncover details of the molecular mechanisms by which the microscopic interactions of actin filaments and actin binding proteins lead to the emergence of specific global mechanical behavior.

Sedentary animals attach to substrates with glues that work underwater, a feat we do not master in our technology. A well-known example is the blue mussel who uses its beard, called the byssus, to attach the soft mussel tissue to the hard substrates it lives on. The blue mussel byssus is made of protein. In contrast to the multi-thread pure protein byssus of the blue mussel, its cousins called the Anomiidae attach via a byssus plug made from a single thread that is calcified, i.e. contains calcium carbonate crystals in addition to proteins. Almost nothing is known about this strange attachment system and in particular the adhesive. We will investigate this intriguing solution to sticking in place using an interdisciplinary approach where we will understand both the mechanical function and the biomolecules involved in the adhesion. This is done by joining the forces of materials chemists and biochemists.

In the project we will use state of the art tools to investigate the structure on the 1 nm to 10 µm length scale of the byssus with focus on the adhesive. In this range of distances molecules interact with each other and develop structures that have macroscopic functions. We will measure the strength of the byssus attachment system. By prodding the surface with a sharp needle, a technique called nanoindentation, we will measure the local mechanical performance on the µm scale. Thereby we will unravel the mechanical performance of the different structural elements. In particular, we can measure the mechanical properties of the adhesive layer itself. Through these experiments we will understand the function of the byssus but also how this function comes about. To do this we will investigate which molecules are involved and how they are built up from amino acids. We will also determine if the animal uses specially modified amino acids, as is sometimes the case in such systems in particular in the blue mussel. To fully understand how these biological glues function, we will measure the structure and self assembly capabilities of selected molecular moieties. We will also measure how they interact with specific surfaces to unravel their role in the adhesive process.

Through this interdisciplinary project we will develop a deep understanding of how these animals attach. This may provide inspiration for novel adhesives for use in e.g. biomedical applications.

More than a decade of research on angiogenesis and VEGF has provided evidence that VEGF-A plays multiples roles in endothelial cells, controlling both physiological and pathological angiogenesis. Recent studies illustrate how endothelial Dll4/Notch signaling, stimulated by VEGF-A, regulates the sprouting response by limiting tip cell formation in the stalk. In turn, the spatial distribution of VEGF-A regulates the shape of the ensuing sprout and determining stalk cell proliferation. Computational modeling of the interaction between VEGF-A gradients and Dll4/Notch predicts that synchronous Notch oscillations occur under pathologically high VEGF-A levels found in tumours environment, providing a potential mechanistic explanation for defective vascular patterning in tumours. I will therefore explore (a) the existence of such Notch-signalling oscillations as the basis for defective patterning in tumour angiogenesis and in experimental models of high VEGF-A levels, (b) I will test a new ES cell sprouting model complemented with tumour cells to allow dynamic observations of endothelial tip and stalk cells with tumour cells, and finally (c) I will explore predictions derived from the computational model how to normalize the sprouting pattern in pathologically high VEGF concentrations in the experimental models and in tumours.

Since their discovery over 100 years ago, enzymes are a topic of intensive research. However, only in recent years a more profound understanding of the catalytic mechanisms of different enzymes is starting to emerge based on the combination of biochemical and structural data. Despite this progress, still little is known about how protein flexibility and dynamics contribute to the catalytic reaction. Single enzyme studies have the unique capability of resolving temporal and spatial heterogeneities. They allow for the observation of enzymatic reactions at the level of individual enzymatic turnover events thereby providing dynamic information which is not accessible at the ensemble level.

Current single molecule approaches are either based on single molecule force or fluorescence spectroscopy. We intend to combine the area of single molecule fluorescence spectroscopy with the newly emerging field of nanobioelectronics. Electronic sensing of biological reaction events with nanoscale field-effect transistors (nanoFETs) is extremely powerful and has recently been shown to provide single molecule sensitivity. We intend to establish a novel charge-based nanoFET single enzyme assay to study the action of proteases on synthetic peptide substrates. The assay will be designed such that it utilizes peptides carrying fluorescence and charge labels allowing us to monitor the same enzymatic reaction with both a fluorescence and a charge-based detection scheme. This will allow us to validate the signals from both detection schemes and to compare both assays with respect to their sensitivity and time resolution, which are crucial determinants for the level of detail which can be obtained from a single molecule experiment. Our ultimate goal is the integration of nanoFET and fluorescence detection giving access to both independent parameters (charge and fluorescence) from the same protease molecule simultaneously. We expect that our combined approach will yield unrivalled details about the catalytic reaction cycle of proteases such as the order of product release and lay the foundations for a broadly applicable single molecule detection scheme for enzymes acting on charged substrates.

Filopodia are small cell protrusions playing important mechanical and sensory roles for a variety of different cell processes. They have recently been observed pulling invasive bacteria towards the host cell before infection occurred. However the exact mechanics and the functionality behind the filopodial retraction mechanism are far from being understood.

We want to shed light onto the molecular principles behind the filopodial retraction mechanism when it is triggered by pathogens such as Shigella. To measure the mechanics we will use an optical trap combined with confocal microscopy. We will quantify the time resolved force that a single filopodium exerts onto beads that are coated with different bacterial ligands or other effectors inducing retraction. By simultaneously observing the movement of fiduciary marks placed on the filopodial actin core and by locally perfusing drugs that interfere with cytoskeletal components inside the filopodium we intend to decipher the underlying molecular principles. The local perfusion of drugs that affect the early stage of filopodial adhesion to beads or to the Shigella bacterium will help to define the signaling pathway leading to retraction.

These experiments together with theoretical modeling will allow to better understand the mechanisms behind filopodial retraction. For example this might help to understand how filopodia sense the stiffness of the substratum in order to guide cell migration. Also these measurements will likely have impact on our understanding how pathogens approach to the cell body and may therefore facilitate the search for new therapeutics targeting early events of pathogen recognition.