The first lineage specification in mammals occurs at blastocyst stage, when cells are set aside from pluripotent embryonic cells to make placenta and other supporting tissues for the fetus. In the mouse, trophoblast stem (TS) cells are derived from blastocyst and later give rise to the majority of the placenta. By understanding the genetic regulation of TS cells, we can gain greater insights into the causes behind pregnancy issues resulting from trophoblast deficiencies. Furthermore, as TS cells and embryonic stem (ES) cells are both derived from the blastocyst, understanding the genetic network preventing ES cells from becoming TS cells would allow better control of pluripotency, and improve efficiency during induced pluripotent stem cells production. Building on previous studies of genes necessary for TS formation and maintenance, we will undertake genome-wide approaches to identify the core TS regulatory networks and their interactions with the pluripotency networks. We will mine this dataset for putative novel regulators of TS cell development and differentiation, and test their functions in vitro and in vivo. Mouse TS cells is currently uniquely positioned to provide new insights relevant to human stem cell development and reprogramming, since lineage decisions in different blastocyst cell lines can only be compared and contrasted in the mouse. Furthermore, we can generate transgenic animals to assess in vivo functions of novel regulators to address their importance during trophoblast and placental development. These studies will provide initial data on which to build interaction networks that drive lineage development in both mouse and human development.

In the proposed project, I aim to identify novel determinants for cell polarity and understand the mechanisms regulating polar localization of plasma membrane proteins in plants.
PIN proteins are polar localized transporters for a plant hormone auxin. PIN polarity changes dynamically according to endogenous and external positional cues, and triggers developmental responses. New components regulating polar targeting of PIN proteins will be identified by molecular genetic approach using Arabidopsis mutants with defects in PIN protein endocytosis or exocytosis.
In the aerial tissues, cuticle is specifically deposited on the surface or organs. Recently identified cuticle-precursor transporter PEL1 localizes to the outer side of the plasma membrane in epidermal cells. I aim to identify sequence-based determinants on PEL1 protein of ‘outer’ polarity by expressing series of mutated PEL1 in plants. To identify novel components regulating polar localization of PEL1, mutations and chemicals which inhibit localization of PEL1-GFP will be identified by screening. Identified mutants and compounds will be further used to identify target proteins. In addition, positional cues for cell polarity in shoot epidermis and cell-type specific trafficking regulators will be identified.
By these proposed approaches, I expect to identify new cell polarity determinants that are involved in polar localization along different axis (apical-basal, and outer-inner, respectively), gain insight into their developmental control and mutual regulation.

Over the past decade, noncoding RNA has been recognized as an important regulator in many developmental and essential processes. Preliminary indications suggest that especially long noncoding RNAs (lncRNAs) may have a prominent role in stem cell function and axial organization, two processes involved in regeneration.
In this project we will address the role of lncRNAs in adult regeneration using the emerging model planaria. Planarians are famous for their ability to regenerate missing body parts, and the process of regeneration is readily accessible to experimentation in vivo.
We will use an enriched cDNA library approach to identify planarian lncRNAs, and we will analyze their expression patterns with regard to regeneration. Next, we will use RNAi-mediated knockdown of lncRNAs in combination with several in vivo assays to determine the effect of lncRNA on regeneration potential. Based on their importance in the various regenerative processes, we will select several lncRNAs for detailed analysis of their mode of action using RNA pull-downs and ChIP-based techniques that will be developed for this purpose.
As planarians combine in vivo regeneration -which most other invertebrate models lack- with the availability of RNAi-screening technology to identify lncRNA function, they will be a powerful model to analyze the role of lncRNA in adult stem cell biology, and will provide us with new insights both in the regulation of regeneration and in lncRNA function.

The PRC1 and PRC2 complexes maintain Hox gene repression during development and also influence tumorigenesis. PRC1/2-mediated gene repression involves PRC2-mediated trimethylation of histone H3 at Lys 27 (H3K27me3) followed by PRC1-mediated chromatin condensation. In contrast, the MLL methyltransferases antagonize gene silencing and promote gene activation by trimethylating H3 at Lys 4 (H3K4me3). Recently, the H3K27-specific demethylase UTX was shown to be an integral subunit of the MLL3/4 complexes, suggesting a possible linkage between H3K4 methylation and H3K27 demethylation. However, the mechanisms underlying the potentially synergistic effects of MLL3/4 and UTX in countering the PRC1/2-mediated chromatin compaction and gene repression are poorly understood. To investigate this problem, this proposal will focus on the RARb2 (and HoxA2) gene locus that is induced by retinoic acid (RA) during cell differentiation. I will use a human EC cell line as a model in conjunction with powerful biochemical assay systems reconstituted with purified factors and recombinant chromatin templates. The specific aims are (i) to analyze dynamic changes in H3K4 methylation and H3K27 demethylation, along with recruitment of related factors, on the RA-induced RARb2 gene, (ii) to assess the function and regulation of factors in RA-induced RARb2 gene activation, (iii) to assemble a highly compacted chromatin template containing the natural RARb2 promoter, and (iv) to use the template in defined cell free systems to analyze the action mechanism of the MLL3/4-UTX complex in countering the H3K27me3- and PRC1/2-mediated RARb2 gene repression and in effecting activation of RARb2 transcription.

Protein synthesis in neurons is spatially and developmentally confined, to control processes as diverse as axon guidance, learning and regeneration. While neuronal RNA localization is extensively studied, little is known about mechanisms regulating the spatial and temporal distribution of ribosomes in neurons. Ribosomes are readily detected in cell bodies, dendrites and growing axons, yet are sparse in mature axons. Upon injury, ribosomes are again detected in axons. Hence, mechanisms should exist to promote or inhibit ribosomal trafficking to axons in response to developmental and external cues. The dynamics of neuronal ribosomes has hardly been explored in-vivo.
I will develop tools to monitor the distribution of ribosomes in the PVD neuron of C.elegans, which grows distinct processes at developmental stages that allow continuous imaging. I will then characterize axonal and dendritic ribosome dynamics in growing and mature processes. Special attention will be paid to the transition from growth to mature stages of the axon, when ribosomes are expected to be eliminated from the process. Ribosomal distribution will also be monitored in a model of axon injury, where localized translation plays a vital role in regeneration.
To understand the molecular mechanisms regulating ribosomal distribution in neurons, I will test mutations in candidate genes for an effect on ribosomal localization, and perform a forward visual genetic screen to identify new mutants. Subsequent mutant analysis will enable to delineate signaling pathways and the underlying cellular machinery that regulate the dynamic distribution of ribosomes in developing or regenerating axons.

Chloroplasts (Plastids) function as the main suppliers of cellular energy in plants. Recent studies have provided significant insights into the evolutionary origin of the plastid-dividing (PD) machinery: 1) The PD machinery is constructed by bacterial-derived and eukaryote-specific proteins, 2) the PD ring, the main structure of the PD machinery, is composed of PDR1-associated poly-glucan. However, the conserved chloroplast division mechanism is not fully understood throughout the plant kingdom. Moreover, some components of chloroplast division in higher plants are different from those in primitive eukaryotes. I would like to investigate which dividing mechanisms have been conserved throughout evolution and how these dividing mechanisms have evolved and changed in different lineages. I will mainly analyze the Arabidopsis PDR1 proteins and conserved unknown proteins identified by proteomic analysis of isolated PD machineries from Cyanidioschyzon merolae. I will analyze these proteins using the chloroplast division analytical methods established in Osteryoung’s lab. In addition, mitochondrial division is similar to plastid division in primitive eukaryotes; therefore, the results generated by my proposal could lead to an understanding of the conserved mitochondrial division mechanism. Further analyses might reveal how eukaryotic host cells engulfed free-living cyanobacteria and a-proteobacteria, which subsequently changed into chloroplasts and mitochondria, respectively. Thus, the results will shed light on the long-standing puzzle of how eukaryotes were established. Successful completion of this study will be indispensable for my career development.

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.