Kinesins are molecular motors which utilize ATP hydrolysis energy for unidirectional movement along microtubule (MT). Apart from their function in intracellular cargo transport, kinesins have been implicated in spindle and chromosomal motility and MT-disassembly during mitosis. The motor head of kinesins which binds to both ATP and MT is responsible for the force generation. With every ATP turn-over kinesins move 8 nm forward along the MT lattice. The ATP turn-over that is coupled to force generation in kinesins is allosterically regulated by MTs (tubulin). Although crystal structures of motor head are known in ADP and ATP-like state, due to the lack of structural data for kinesin with its allosteric regulator a complete model for kinesin motility is lacking. Therefore, the proposed project aims to undertake structure-function studies on kinesin-tubulin complex. A major hurdle in structure determination of kinesin-tubulin complex is to inhibit tubulin polymerization. To overcome this, novel strategies will be employed to produce soluble kinesin-tubulin complex suitable for crystallization. The structural findings will be functionally evaluated using biochemical and single molecule studies. Since earlier studies were done by mutating kinesins, the project will also focus on establishing a yeast expression system for the production of recombinant tubulin. Using this purification system extensive mutagenesis in tubulin will be carried out to characterize the kinesin-tubulin interface. The systematic approach of structural and functional studies on kinesin-tubulin complex will enable us to postulate a model and elucidate the mechanism of energy transduction in kinesins.

The Timmermans Lab has recently shown that miRNAs expressed in the epidermis can traffic between cells into subepidermal tissue layers. The knowledge that miRNAs can move from cell-to-cell has changed how we look at miRNA-regulated gene networks. Particularly in the field of developmental biology has miRNA mobility received much excitement, as it presents these riboregulators as possible novel morphogen-like instructive signals. This fellowship would allow me to begin to address many of the unknowns regarding miRNA mobility, how it is mediated and controlled during development. I propose to utilize a miRNA sensor system to establish the developmental contexts in which miRNA mobility can occur. This will also reveal whether miRNA levels affect the range of mobility and whether movement is directional. Mathematical modeling predicts that gradients of small RNAs resulting from mobility can establish discreet domains of target gene expression. The functional relevance of miRNA mobility will be addressed studying the effect of miRNA expression gradients on target gene expression. Finally, miRNAs are likely to move as RNA-protein complexes due to their inherent instability. The mechanism of miRNA mobility is completely unknown. I therefore propose to initiate a forward genetic screen for mutants perturbed in miRNA movement. The outcome of these aims will provide new insights into the mechanism, parameters and breadth of miRNA mobility, and will improve our understanding of the functional relevance of miRNA movement, which will have major implications for the characterization of miRNA controlled networks.

Double-strand breaks (DSBs) occur frequently in the genome by DNA damaging agents or during genome replication. DSBs are hazardous because interaction between DNA ends from different double strand breaks can produce tumorigenic chromosome translocations. The DNA damage response (DDR) has been extensively studied in mammalian cells using UV lasers, γ irradiation or chemical DNA damaging agents. In all of the above systems one cannot visualize the actual DNA break site. The most commonly used "visual" readout that an actual response to DSB has been initiated is the formation of repair foci, which correspond to sites of DSB. To overcome these limitations, I developed a cell system in which a DSB can specifically be induced at a defined genomic site and follow the fate of damaged DNA in living cells in real time. Using this system, I showed that broken ends are positionally stable and unable to roam the cell nucleus and I found that the repair factor Ku80 is required for maintaining the alignment of broken ends. I extended the use of this system to probe how DSBs are recognized in vivo and how DDR pathways are triggered in the context of chromatin. I found that tethering a single component of a DSB repair complex to chromatin is sufficient to elicit the DDR in the absence of DNA damage and that stable association of the mediator of DDR MDC1 induces high local chromatin decondensation. I will use a combination of advanced live cell imaging and biochemistry techniques to further test the importance of nuclear architecture in maintaining genomic integrity and I will identify novel factors involved in DSB repair and alignment of broken chromosomes.

The neural basis of memory is of key scientific and clinical importance, but it is yet unknown how memories are formed and maintained. While it generally agreed that multi-neuronal activity is required for information processing in the brain, the requisite temporal precision of spiking is unknown. We will use the rat spatial memory model system to study this issue. Rats will run in one of two paths after a delay period of wheel-running during which the forthcoming path will have to be memorized for above-chance performance. Using high-resolution silicon probes we will monitor neuronal activity in a region related to short-term spatial memory, the hippocampus CA1 region, and in a main source of input to that region, the entorhinal cortex (EC). We will use photo-stimulation of neurons expressing the archeal cation channel ChR2 in either CA1 or EC to introduce temporal jitter in the spiking activity of the recorded neurons with minimal influence on net discharge. By correlating the temporal jitter during the delay with behavioral performance during the run we will determine the spiking precision required to maintain short-term spatial memory.

Nicotine-sensitive acetylcholine receptors (nAChR) are ligand-gated ion channels that have been
widely conserved throughout evolution. In humans, nAChRs mediate fast synaptic
transmission at neuromuscular junctions and are involved in numerous physiological
and pathological functions in the brain. Despite extensive characterisation of
nAChRs at the molecular level, the cellular mechanisms that regulate AChR
distribution and function within a cell remain largely uncharacterised. In a
genetic screen for mutants resistant to the nicotinic agonist levamisole in C.
elegans, a novel gene required for nAChR function was recently identified. This
gene has not been analyzed up to date in C. elegans, but it has two highly
conserved orthologs in vertebrates: CRELD1 and CRELD2. CRELD2 has been
implicated in cholinergic signaling but its detailed mechanism of action
remains elusive. Here, I propose molecular and genetic approaches to elucidate
CeCRELD functions. Specifically, I will analyze the gene structure and carry out
a detailed analysis of the mutant phenotypes. CeCRELD expression will be
characterized at cellular and sub-cellular levels. A search for physical
interaction partners will be undertaken using genetic and biochemical
approaches. This project should provide testable hypotheses for the function of
CRELD genes in vertebrates and give new insights on the biology of nicotinic
receptors. In combination with my present experience of the zebrafish
system, acquiring a deep knowledge of the C. elegans organism will provide a
means to shuttle efficiently between these two systems in the future building a
solid basis where upon I can further develop my own scientific career.

The process of bone formation is essential to life of all vertebrates. Our understanding of the
molecular mechanisms involved in this process are still limited. Osteoporosis is a common and
often crippling human disease characterized by decreased de novo bone formation by osteoblasts.
The incidence of this disease is growing with the aging of the general population. One condition
favoring an extreme form of osteoprosis is due to loss of function mutations in the LRP5 gene.
This results in a decrease in osteoblast function leading to low bone mass. Recent study in
Gerard Karsenty’s laboratory showed that LRP5 promotes osteoblast function by suppressing
serotonin biosynthesis in enterochromaffin gut cells. Consistently, decrease in serum serotonin
levels results in increased proliferation of osteoblasts and accounts for a protective mechanism
against osteoporosis. This has been verified in both mice and humans in a rare condition called
the high bone mass syndrome. These exiting findings opened up novel research avenues. The
aim of my proposal is to further characterize the interplay between enterochromaffin cells and
osteoblasts and to identify and characterize the molecular pathways involved in regulation of
serotonin by LRP5. To achieve these goals, I plan to use various biochemical, cell biology and
mouse genetics approaches. I expect these studies to contribute to a better understanding of
molecular pathways crucially involved in formation of bone both during normal and disease
development.

Gene expression is controlled by the action of transcription factors which
bind to DNA and influence the rate at which a gene is transcribed.
Previously, by systematically expanding a masterequation for a gene controlled by two or more regulators, a fluctuation-dissipation based method hasbeen identified, which discriminates whether a gene integrates its inputs in asynergistic or additive fashion. This method has turned out to be useful for analyzingexperimental data. In this method it is assumed that the averages are overpopulations of cells in a single biological state and spatial variation of thepopulation is neglected. I seek to develop this physically motivated paradigmfor situations that the regulatory structure does not completely satisfy the idealizedassumptions above.I seek to developthe fluctuation-based methods to treat populations of cells that aretransitioning between states. Thisdeveloped method might be useful forunderstanding regulatory networks for cell fate specification. I will also investigate how these methods canbe modified to treat spatially heterogeneous biological regulatory processes toenable the use of a wider range of experimental data. Successful realization ofthe method will provide deep insights into how noise propagates through regulatorynetworks of higher organisms, and ultimatelymedical diagnostics for diseases resulting from aberrantrecruitment of co-regulators,as in blood cancers.My background and the numerical and analytical tools I havedeveloped as a condensed matter theorist plus the training I will receive fromProf. Dinner’s group at the University of Chicago will assist meto achieve the goals mentioned above.

Wiring specificity in the central nervous system remains among the most complex biological phenomena to date. The Drosophila visual system provides an ideal setting in which to address this due to its genetic tractability and description at the ultra-structural level. Genetic screens have identified several key cell-surface molecules and guidance factors required for the various connections between different photoreceptor and lamina neuron cell types. These molecules somehow control the direction, motility and connectivity of the growth cone, a virtually autonomous structure operating relatively far from the cell body. In many cases questions remain regarding how these guidance molecules are regulated in the growth cone and the signaling cascades they trigger. Emerging evidence from other model systems suggests that posttranscriptional mechanisms including mRNA localization and localized translation in the axon and growth cone play an important part in axonal pathfinding. I, therefore, propose to investigate the regulation of wiring specificity in the visual system through a direct analysis of this posttranscriptional regulation. This will be done through (1) identifying the cis-acting mechanism by which known guidance factors are posttranscriptionally regulated; (2) determining the function of this regulation; and (3) identifying the trans-acting factors that function as regulators of wiring specificity.

An increasing body of evidence indicates that the spatial positioning of genes in the interphase cell nucleus is an important regulatory mechanism in transcription. During the development of the central nervous system, gene expression profiles change dramatically and upon completion of terminal differentiation, neurons express a significant number of genes in response to outside stimuli, which enables the nervous system to adapt its output to the input. While epigenetic modifications such as histone modifications and DNA methylation have been intensively studied in the nervous system, little is known regarding the arrangement of nuclear compartments and nuclear architecture during development and adult function of the nervous system. I have found that the most prominent astrocyte specific gene, glial fibrillary acidic protein (GFAP) is monoallelically expressed in astrocytes, moves toward the center of the nucleus during astrocyte differentiation and that its active allele is located more internally than the inactive one. This indicates that the nuclear spatial positioning of genes is related to differentiation and gene activity in the nervous system. Here I propose to use several complementary approaches to investigate the functional role of gene positioning in the transcriptional regulation of neurons and astrocytes during differentiation and after maturation. The results obtained in the project will provide a new perspective on spatial gene positioning in the nervous system and further insights into the cellular mechanisms of gene expression in the development and function of the brain.

Aneuploidy, a condition where cells harbor an abnormal chromosome numbers, is the leading cause of miscarriages and mental retardation in humans and a hallmark of cancer. However, whether aneuploidy is a cause or a consequence of cancer remains an unsolved question. I will examine the effects of aneuploidy in primary mouse cells by generating a series of cell lines that carry an additional copy of a mouse chromosome. Preliminary data from our earlier studies in four trisomic mouse embryonic fibroblast (MEF) lines have suggested a general proliferative disadvantage. I will extend these analyses by examining more trisomies as well as characterize the effects of trisomy on cell proliferation and metabolism. In addition, as aneuploid cells are delayed in proliferation, cancer cells must develop mechanisms to overcome the proliferative and immortalizing disadvantages caused by aneuploidy. By applying a chemical screening, my aim is to identify genes and pathways that are involved in the suppression of the proliferative disadvantage of aneuploid cells.

P-type ATPases are cell-membrane pumps that are of fundamental importance in eukaryotes. The ion gradients created by Ca2+-ATPases or Na+,K+-ATPases are involved in signalling, maintaining the membrane potential as well as secondary active transport. Despite the recent progress in membrane protein crystallography, the lack of detailed structural information currently prevents a better understanding of function and specificity of cation pumps.
One specific aim of this proposed research is to determine the structure of the plasma-membrane Ca2+-ATPase (PMCA) and to understand its regulatory mechanism. The autoinhibited PMCA will be recombinantly expressed in yeast, purified and crystallized. In addition, it is also planned to determine the structure of the activated complex consisting of PMCA and calcium-bound calmodulin (Ca2+-CaM).
In order to deepen our understanding of cation pumps in their native environment, I further propose to investigate Na+,K+-ATPases in native membranes using a multi-technique approach. First, I will analyze complexes of Na+,K+-ATPase isolated from native membranes by mass spectrometry to identify peripheral proteins. In the next step, I plan to assemble those identified multi-protein complexes in vitro and structurally characterize them by cryo-EM. Finally, I plan to use AFM to investigate the oligomeric arrangement of Na+,K+-ATPases in native membranes. This multi-disciplinary project will hopefully expand our knowledge of cation pumps by putting atomic details in the context of the native environment and will concurrently allow me to gain experience in membrane protein crystallography that will complement my expertise in biophysical methods.

Endocytosed cargo transits through endosomal system on its way to a final destination. Transport along the endocytic pathway, specifically between early and late endosome, occurs via multivesicular bodies (MVB) and requires the formation of intraluminal vesicles (ILVs) within the lumen of these organelles. The aim of the proposed study is to elucidate the molecular mechanisms involved in the formation and back-fusion of ILVs and the coordination of these processes with the general membrane trafficking in endosomal pathway. Rab5 and Rab7 GTPases, which are well established in regulation of endosomal transport, seem to be the likely candidates which might influence the formation of ILVs, since they interact with PI 3-kinase hVPS34 and cholesterol, the molecules implicated in intra-endosomal dynamics.
In vivo and in vitro approaches will be used to analyze ILV formation and back-fusion, as well as endosomal trafficking, in response to interfering with Rab proteins. The function of Rab GTPases will be abolished by RNA interference and dominant-negative approaches. The formation of ILVs in vivo will be determined biochemically and by electron microscopy, whereas the analysis of the back-fusion will employ the assay based on the analysis of the release of VSV RNA into the cytosol. Artificial liposomes will be synthesized in vitro and used as a tool to test the effect of Rab GTPases and other candidates on the formation of ILVs. This study will attempt to investigate the role of Rab GTPases in coordination of intra-endosomal trafficking with overall endosomal membrane transport, but also to identify novel candidates involved in the process.

The present project is aimed at development of novel methodological approach to detecting, monitoring and modulating amyloid formation on a basis of in-depth characterization of the membrane-assisted fibrillogenesis. More specifically, during the project execution the following objectives will be pursued: i) to adapt the method of surface plasmon-coupled emission (SPCE) to detection and analysis of protein aggregation on membrane surface, ii) to assess the role of lipid environment in determining the protein aggregation propensity, iii) to examine the influence of different types of amyloid intermediates on molecular organization of lipid bilayer, iv) to define the kinetic parameters of membrane-mediated fibrillogenesis. Short-term Fellowship is of enormous importance in two aspects. First, successful implementation of the project will provide further in-depth insights into molecular basis of conformational diseases and allow elaborating effective strategies for their prevention and treatment. Second, unprecedented opportunity to work in a prominent research group such as CCFT will be highly beneficial for my future professional growth because: i) mastering the novel analytical tool will expand my experimental skills; ii) the proposed work is expected to provide an entry point for my home research group into plasmonics study of lipid-protein interactions; iii) strong world-wide scientific connections of CCFT will be extremely beneficial for my future carrier, because I will have a good practice in collaborative work in a highly dynamic international scientific environment.

Synapse formation and maturation are essential for the normal establishment and remodeling of neuronal circuits. Impairments in these functions contribute in the pathogenesis of brain disorders such as autism and mental retardation. Neuroligins are postsynaptic proteins that are considered key players in these processes. Incorporation of an autism-associated mutation in neuroligin 1 was shown to have dominant-negative properties and interfere with normal neuroligin function in vitro. I propose to generate a transgenic mouse with the ability to overexpress this mutant neuroligin form in a tissue-specific, inducible and reversible manner. Perturbations in the synaptic properties of these mice will be analyzed by immunohistochemistry,electrophysiology and behavioral assays. Special attention will be given in the ability of this mouse to model autism-spectrum disorders (ASDs).By taking advantage of the specificity and the inducible properties of mutant neuroligin 1 expression, these animals will also be used to transiently interfere with the synaptic strength of forebrain circuits during the process of learning and memory. This approach promises to shed light in the role of neuroligins in the development of ASDs as well as dissect neuronal circuits involved in animal cognitive processes. This project will provide me the opportunity to learn new techniques in order to study synaptic transmission (e.g: histochemical and electrophosiological) as well as to apply my currect knowledge in mouse behavior to develop new tools for understanding neuronal circuitry. This marks a significant change from my previous research which was focused in the development of anxiety disorders.

Axonal pruning and synapse elimination reorganize neural circuits in many parts of the developing mammalian nervous system. Much of what is known on this process comes from the study of the neuromuscular junction where axons compete for the occupation of the same synaptic sites on muscle fibers. Muscle fibers, however, are end-organs, so the result of this competition cannot have any downstream effects. The objective of this project is to understand the temporal and spatial relationships between circuit rearrangements that occur during development at different levels of the synaptic relay between brain and salivary end-organ. In order to do accomplish these aims I will use advanced physiological and imaging techniques with de-novo transgenic mice expressing different mixtures of multiple fluorescent proteins in each neuron ("Brainbow" mice) in order to follow these developmental reorganizations over time. This research will provide novel information on the postnatal development of a two level pathway allowing me to test, for the first time, the particular hypothesis that the purpose of synapse elimination is to allow a circuit-wide rearrangement in a neural pathway such that each cell upstream in a polysynaptic pathway drives the output of a limited synchronously active territory in the downstream synaptic target.

The dentate gyrus of the hippocampus is one of the brain regions in which neurons are generated during adulthood. Increasing evidence supports the view that adult newborn granule neurons play an important role in hippocampus-dependent learning and memory. In recent years, it has been reported that immature new neurons approximately one month after their birth may make particularly significant contributions to memory formation. However, at what exact stage the young new neurons become important for memory formation, and the memory processes they mediate, have not been yet determined. Therefore it is necessary to identify the specific functions of young new neurons in memory formation in order to understand the mechanisms of newborn-neuron-based memory processing in the hippocampus. Identifying a specific function of young new neurons in learning and memory requires the establishment of new temporally and spatially controlled ablation techniques. Here we propose to develop a virus-based ablation system capable of specifically ablating young newborn neurons at different times during memory processing. Furthermore, we will evaluate the direct effect on memory formation resulting from virus-based specific elimination of young newborn neurons. Through these analyses, we aim to determine the specific function of young newborn neurons as a component of learning and memory. The achievement of the goals of this project will provide us with new insight into the issue of what kind of memory young newborn neurons are involved in, and give us an idea as to how newborn neurons compute memory information.

White fat in different depots is heterogeneous with respect to its metabolic functions and impact on overall glucose and lipid homeostasis. Accumulation of excess visceral fat (central or “apple-shaped” obesity) is associated with insulin resistance and increased risk of type 2 diabetes and metabolic syndrome, whereas excess of subcutaneous adipose (peripheral or “pear-shaped” obesity) does not have such a risk.
Identifying the pathways that distinguish visceral from subcutaneous fat is essential for finding solutions for the metabolic complications of obesity. Recent work has identified several differentially expressed genes. Among those Glypican-4 (Gpc4) is very interesting in that it is one of the few that is highly correlated with the level of obesity, as measured by body mass index, and the fat distribution as measured by waist-hip ratio.
Gpc4 is a poorly characterised cell surface heparan sulphate proteoglycan, suggested to facilitate or enhance the interaction of growth factors with their receptors. The overall goal of this proposal is to address the role of the differential expression of Gpc4 in subcutaneous vs. visceral fat and its relationship to obesity.
The functional consequences of down- and upregulation of Gpc4 expression will be addressed using a battery of cell biological and biochemical methods. Adipocytes and preadipocytes will be phenotypically and functionally analysed, and Gpc4 interaction partners will be identified using mass spectrometry and characterised to relate them with the observed phenotypes. This project will not only provide insights into Gpc4 function in different fat depots but also unravel its role during weight loss and gain.

Our cells are faced with more than 10.000 DNA lesions per day. Failure to repair such lesions can lead to cell death or the accumulation of genetic aberrations, which could contribute to cancer development. Efficient repair of DNA damage, however, is complicated by the fact that genomic DNA is packaged, through histone and non-histone proteins, into a condensed structure called chromatin. The repair machinery has to circumvent this natural barrier to gain access to damaged DNA and repair DNA lesions properly. We found that the ATP-dependent INO80 chromatin remodeling complex helps to overcome this barrier at sites of DNA damage. INO80 is recruited to chromosomal double-strand breaks (DSBs), where it removes histones to facilitate the accessibility and processing of damaged DNA. Along with INO80 more than 50 different proteins involved in diverse aspects of the DNA damage response are recruited to a DSB. Some of these proteins may interact with the INO80 complex to regulate its activity through post-translational modification of distinct subunits. However, the interplay between INO80 and any of these proteins remains an enigma. Therefore, we propose genetic and biochemical approaches to identify and characterize (I) proteins that interact with INO80 during the DNA damage response and (II) possible post-translational modifications of INO80 subunits. Their role in the DNA damage response will be examined in order to decipher the mechanism that regulates INO80’s activity at chromosomal DSBs. These studies will extend our basic knowledge on the pivotal role that chromatin remodeling complexes play in the signaling, processing and repair of DNA damage.

In eukaryotes, most intracellular membrane fusion reactions are mediated by the formation of a tight core-complex, consisting of complementary SNARE-proteins that are present in both fusing membranes (trans-complex). Of all SNARE-mediated fusion reactions, neuronal exocytosis is best characterized. This is a heterotypic fusion reaction, meaning that the synaptic vesicles and the plasma membrane each contain a different and complementary set of SNAREs. Other intracellular fusion reactions, such as the less well characterized endosomal fusion, are homotypic and both membranes contain a full set of SNAREs. It is unknown what prevents instant formation of a core complex within the same membrane (cis-complex), which would deplete all free SNARE-proteins. Furthermore, it is unclear in which combination active SNAREs need to be distributed between the membranes for effective fusion of endosomes and which regulatory factors may be involved. In this research proposal, it is planned to address these questions using an in vitro approach involving SNAREs reconstituted in liposomes. Using a fluorescence resonance energy transfer (FRET)-based approach, fusion mediated by endosomal SNAREs will be characterized, and a search for regulatory factors will be carried out.

Understanding how cells respond to internal and external forces is important for many biological processes, ranging from cell division to movement and differentiation. A principal determinant of the mechanical properties of living cells is the cell cortex, a shell of cross-linked actin filaments attached to the plasma membrane. This cortical actin network not only provides mechanical strength to the cell; myosin motor proteins create internal stresses within it that can lead to mechanical instabilities, active contractions, and deformations. The aim of this project is to understand the properties of such active, thin actin networks. To this end, we will develop a reconstituted model system that mimics the cell cortex, consisting of a thin actin network linked to a phospholipid monolayer adsorbed to an oil/water interface. By combining advanced microscopy and interfacial rheology, we will systematically study the relation between the structure and composition of this reconstituted cell skin and its mechanical properties. By incorporating myosin motors in the gel layers, we will be able to study, for the first time, active flows and contractions under well-controlled conditions. It is anticipated that these in-vitro studies of mechanics, (in)stability, contraction, and flow in active actin/myosin gel layers will reveal important details on the dynamic behavior of living cells.