Throughout the animal kingdom, social interactions such as parental and mating behavior, territorial defense and predator avoidance are triggered by specific sensory stimuli. In rodents, chemosensory systems provide the main source of information for social interactions. Decades of research has showed that a vast range of animal cues trigger innate social behaviors through the vomeronasal pathway. But how are those cues encoded in the brain to generate specific behavioral responses is still poorly understood. In my proposed work, I firstly plan to dissect the neural coding of critical parameters relevant to social behaviors in awake animals using microendoscopy-based calcium imaging. I will examine the representation of species- and sex- related natural stimuli in the medial amygdala (MeA), a central node of the vomeronasal sensory-motor transformation. Transgenic lines expressing light-activated opsins (ChR2 or C1V1) driven by specific vomeronasal organ receptors (VRs) as well as natural stimuli will be used to uncover the MeA-receptor maps corresponding to discrete vomeronasal inputs. Furthermore, the basic principles of MeA organization will be examined in genetically identified neuronal populations, using markers such as the enzyme aromatase (Ar) and the thyrotropin-releasing hormone (TRH). Finally, taking advantage of chronic imaging by microendoscopy, I seek to find the mechanisms of both instinctive and learned social behavioral changes triggered by past experience. For this purpose, I will observe the representation of both natural pheromones and single receptor population inputs in the MeA during experience-dependent as well as learning-involved social behaviors.

Pharmaceutical drugs represent one of the most powerful methods available for manipulating important aspects of our physiology. As such, their development has been a big driver for the advances in healthcare over the last century. Drugs have been used not only to treat diseases directly, but also as an experimental tool for researchers trying to understand how the body works.
Although the many benefits of pharmaceutical drugs are well known, so are their limitations. Drugs commonly have ‘side effects’ on aspects of our physiology that were previously healthy and, over repeated application of a drug, people often become desensitized, meaning that they require higher and higher doses to achieve the desired effects. One can view both of these limitations as a consequence of the failure of drugs to recapitulate the ‘natural’ modulation of physiology that generally keeps us healthy. Natural checks and balances on cell physiology act by regulating the activity of specific proteins in specific cells in a particular part of the body, while drugs often are much less selective in the nature and location of their activity. Similarly, while the activity of a natural process is modulated in ‘real time’, drug concentrations build up slowly and hang around for a long time. It is, of course, not time to throw the pharmaceutical baby out with the bath water, nevertheless alternative methods of manipulating physiology would be most welcome.
As many as a third of currently prescribed drugs target a specific mode of communication between cells that originates with a large family of proteins called G protein-coupled receptors (GPCRs). We propose developing a group of modified GPCRs that will be light sensitive. These proteins will turn ordinary cells into photoreceptors, allowing us to control their physiology simply by shining light on them. Because only those cells containing these engineered proteins will respond to light, and because its easy to switch light on and off rapidly (or to leave the light on for hours/days), this approach will allow scientists to approach a more ‘natural’ modulation of cell physiology. In the first instance these proteins will be available for researchers to use in cell cultures and/or animals to gain a better understanding of how the body works. Ultimately, we hope that they will be adapted for treating diseases in people.

Myeloid cell infiltration occurs at early stages of melanoma and is correlated with tumor development. Populations of myeloid cells have been proposed to regulate tumor development and maintenance in different manners. In the proposed studies I will identify the mechanisms underlying myeloid cell infiltration into melanoma tumors and elucidate how infiltrating myeloid cells affect tumor cells and vice versa. The zebrafish is unique for high resolution in-vivo imaging of the activity of the myeloid and tumor cells, and transgenic and gene inactivation technology can be used to probe function. A BRAF;p53 model of melanoma has been created by the Zon laboratory, and a specific transgenic using the crestin promoter reports melanomas at the 4 cell stage of tumorigenesis. We plan to watch the myeloid cells migrate to the tumor cells for the first time and describe the populations. Myeloid-expressed GFP transgenic fish will be used to follow the populations. RNA-seq of the infiltrating myeloid and tumor cells, compared to human primary melanoma samples, will define potential chemotactic molecules and genetic alterations in the cells following myeloid and tumor cell contact. I will use a unique plasmid system to express transgenes of the chemotactic factors in the tumors or to inactivate pathways in myeloid cells. This will provide the first genetic evidence for myeloid cell-tumor interactions, and may lead to new therapies directed at these mechanisms.

The function of human body to a large extent depends on direct cell-to-cell communication mediated by gap junctions composed of intercellular channels. They permit the exchange of ions and small molecules between cells. Calcium homeostasis modulator 1 (CALHM1) is a recently characterized gap junction-like ion channel involved in the Alzheimer’s disease (AD) and in taste-stimuli-promoted neurotransmitter ATP release from taste sensing cells. CALHMs 2–3 are associated with neurodegenerative Creutzfeldt-Jakob’s disease.

The molecular architecture of CALHMs remains poorly understood. No three-dimensional structures of CALHMs are available and only one structure of gap junction protein is known. This limits the mechanistic understanding of ion and ATP recognition, translocation across the membrane, and release by CALHMs (as well as by most large pore channels). Thus the signal transduction underlying the CALHM-associated neurodegenerative diseases is poorly understood.

To decipher permeation and regulatory mechanism of CALHM, I will integrate biophysical, electrophysiological, cell-based analyses to investigate the structure and function of (1) human CALHM cytoplasmic domains, (2) the full length CALHMs, and (3) CALHMs in complex with ligands such as ATP, calcium and cobalt (blocker of the channel) ions and antibodies. These studies will reveal the overall architecture of at least one CALHM alone and in complex with its ligands, provide atomic level understanding of how CALHM may function in healthy individuals and patients. Structure of CALHM-ligand complexes may also provide a platform for the rational design of therapeutically valuable molecules for clinical interventions.

Over the past tens of thousands of years, humans have expanded into a hugely diverse range of environments. This expansion has been accompanied by dramatic phenotypic changes. As a result of this, many traits vary substantially across modern human populations, often with important medical implications. Understanding which traits have been under selection, and which genomic loci have responded to this selection is important both for our understanding of human history, and for our knowledge of the genetic architecture underlying phenotypic variation in modern humans. One exciting new source of information about this evolution comes from recently developed technology to extract DNA sequence data from ancient samples. Though this data is relatively easy to analyze, it cannot be generated in large quantities, which limits it usefulness. Conversely, data from modern humans is easy to obtain, but statistically difficult to analyze. This project aims to tackle the challenging problem of developing methods that can combine both of these sources of information in order to take advantage of both of their strengths. This will allow us to answer questions both about the role of selection in the evolution of specific genomic loci, and about the relative importance of different types of selection. In particular, we will be able to test the hypothesis that polygenic selection, or weak selection on large numbers of loci, is responsible for much of the recent phenotypic evolution of humans.

To determine the neurons and mechanisms a fly employs to integrate blends of male pheromone and food odor, I will proceed in four steps. 1)I will build a reference library of flies’ individual behavioral responses to blends of male pheromone and food odors. 2)I will identify the activated receptor neurons using loss of function mutants, and resolve their downstream neurons based on literature and recent mapping data of the lateral horn neurons from the Jefferis group. The lateral horn is the most promising site of integration. 3) I will identify the integrator neurons by recording from the downstream neurons using electrophysiology and calcium imaging. If the level of neuronal activity depends on the presence of both food odor and male pheromone, then the targeted neurons are likely the integrators. I will backtrace signals to confirm that the identified, and not other upstream neurons, are the integrators. I will also verify that stimulating their presynamptic inputs is sufficient to trigger behavioral integration. 4)I will reversibly inactivate the neurons to eliminate the reponses to blends, which tests whether the neurons are necessary. I will stimulate the neurons to recapitulate behavioral integration, which tests whether the neurons are sufficient. Last, I will modulate the excitability of the integrator(s) and measure input levels. If required input levels change coordinately with changes in excitability, then activity within the integrators is critical for behavioral integration. Together the results will advance our understanding on how behavioral choices are elicited at the neuronal level.

Cell transitions are characterized by a wide range of cell physiological modifications. As cells progress towards a disease or developmental fate, they change their gene expression, shape, size, motility, polarity, metabolism and signaling, in addition to interactions with their niche. While we know a considerable amount about the molecular processes regulating many cell behaviours, how changes in these dynamic behaviours are coordinated over time is unclear. Superimposed upon many developmental transitions is cell heterogeneity. Cells in clonal populations show considerable heterogeneity in just about any property one can measure. Does this heterogeneity reflect different cell paths towards a common fate, or differences in developmental timing between cells? This generic problem in developmental and disease biology is formalized by two alternative hypotheses: either cells follow a stereotypical linear path from state A to state B; or cells move between A and B via a variety of paths, with different behaviours shown by different cells. Using live imaging of transcriptional bursts in single cells, in combination with measurement of multiple dynamic cell properties, we will generate a multi-parameter description of the dynamic sequence(s) of events occurring during a developmental transition. This will illuminate the implications of heterogeneity on cell behaviour.

Endocytosis plays key roles in various cellular events. However, it remains largely unknown when, where and how endocytosis occurs in each cellular activity, mainly because it is impossible by conventional imaging techniques to visualize the structural dynamics of endocytosis in living cells. Here, I aim to unravel the nature of endocytosis by taking advantage of both two-photon microscopy and high-speed atomic force microscopy (AFM). I will optically identify endocytosis events using two-photon microscopy and visualize the events with nano-meter scale using high-speed AFM. Since endocytosis is involved in a variety of cellular activities and mediated by various molecules, I hypothesize that endocytosis consists of various structural forms. First, I will investigate the location, time-course and shape of endocytosis taking place in cell lines to classify the dynamic structures of endocytosis. Second, I will identify the molecules responsible for each type of endocytosis by gain- and loss-of-function analysis of candidate genes in cell lines. Third, I will reveal the functional relevance of each type of endocytosis by observing when, where and how endocytosis occurs in neurons during synaptic plasticity, in which endocytosis is known to play an essential role. Furthermore, I will clarify the spatio-temporal relationship between molecular activity, endocytosis events and structural changes of dendritic spines during synaptic plasticity by combining two-photon uncaging of caged glutamate, two-photon fluorescent life time imaging microscopy and high-speed AFM. This proposed study may provide new insights into the nature of endocytosis overlooked by conventional techniques.

Movement precision is ultimately determined by the resolution of the motor circuit at the level of single neurons and synapses. However, other factors such as muscle quality/quantity and joint flexibility also affect locomotion. Hence, it is not clear to what extent the fineness of the motor circuit determines physical ability. I propose to tackle this question using Caenorhabditis elegans as a model system. C. elegans moves with a sinusoidal pattern and has no skeletal structures or joints, enabling us to directly test how the synaptic pattern of the motor neurons affects locomotion. In worms locomotion is regulated by 6 types of motor neurons. Each neuron in the same class innervates a non-overlapping segment of the muscle field by restricting its synapses to a distinct sub-axonal region. This type of innervation generates a phenomenon we term “synaptic tiling”. Recently we developed a genetic marker to visualize synaptic tiling between two motor neurons, and showed that Plexin-dependent axon-axon interaction is critical for establishing synaptic tiling. There are, however, many questions that remain unanswered, for example: 1. What are the other components in the Plexin signaling pathway? 2. How is tiling regulated in other neurons? 3. What effects do synaptic tiling defects have on locomotion? To answer these questions, I will attempt to identify the additional molecules involved in synaptic tiling; develop tools to stably distinguish neurons within the same class; and utilize high-sensitivity markers to monitor the effect of synaptic tiling on locomotion. With these studies I hope to use the power of genetics to determine how synaptic patterns affect locomotion.

Complex, multicellular organisms are made up of many cell types, each performing different functions. Understanding the evolution of cells' social interactions is hard: multicellularity evolved long ago and dissecting individual interactions is difficult. We therefore evolve social behavior in two microbes, E. coli and budding yeast. Comparing a prokaryote and a eukaryote will suggest hypotheses for how social interactions between cells evolved in plants and animals.
We will use sucrose utilization as a model to study social evolution. Sucrose hydrolysis releases one molecule of glucose and one of fructose, highly preferred carbon sources for many microbes. In yeast, the enzymes that hydrolyze sucrose are extracellular public goods, whereas in E. coli they are plasmid-encoded, private goods.
We will use genetic engineering, experimental evolution, theory, and simulation to investigate three aspects of social evolution: cooperation, the origin of multicellularity, and the division of labor. We have four aims:
1) Engineer and evolve differentiation within multicellular yeast aggregates to test the idea that undifferentiated, multicellular aggregates evolved before the division of labor.
2) Study the role of plasmid transfer in bacterial cooperation. Does the production of secreted public goods by plasmids select for the faster transfer of plasmids to neighboring cells?
3) Ask how prior history and initial conditions determine the trajectory of evolution. We will engineer yeast and bacteria so they can evolve from similar starting points. We will vary whether sucrose-harvesting systems are public or private, whether they are transmissible or chromosomal, and whether cells can differentiate.
4) We will compare our experiments with three different methods to model social evolution: analytical theory, numerical simulation, and evolving digital organisms within computers.
The proposed work will benefit from a) combining engineering and evolution as experimental approaches, b) comparing different approaches to model these problems, c) strong interaction between theory and experiment, d) the interplay between expertise in yeast and bacterial cell biology, and e) regular meetings to exchange results, critiques and ideas that will influence future research.

Toxoplasma gondii is an obligate intracellular parasite infecting an estimated 30% of the human population worldwide. The infection it produces can pose serious health risks in immunocompromised patients or during pregnancy. Because it can infect almost any warm-blooded animal Toxoplasma is considered one of the most successful parasites in nature. My goal is to understand how Toxoplasma has evolved to be such a generalist, what mechanisms confer this ability and what cost this has on transmission in the most relevant hosts. I will do this in light of recent information that the interaction between Toxoplasma and a given host is highly dependent upon allelic differences (both sequence polymorphisms and copy number variants) between effectors secreted by the parasite into the host cell. My working hypothesis is that this genetic diversity is a result of selection for the ability to infect multiple hosts and I will test this by repeatedly passing a recent, natural isolate of Toxoplasma, with and without mutagenesis, through macrophages from different host species and assessing the transcriptomic, genomic and epigenomic changes that occur within the parasite as a result of selection in each host. I expect to see changes in sequence and/or expression of the effectors that reflect the optimal interaction for each host species.

Heart cell differentiation is driven by the sequential action of dedicated transcription factors. These factors orchestrate the activity of thousands of genes at each step of cardiac development by binding to a constellation of regulatory elements. We aim at understanding how the interplay between these transcription factors and cis-regulatory elements underlie proper execution of the mammalian cardiac gene expression programs.
In order to isolate precisely staged cell types we will use a well-controlled ex-vivo system based on the directed differentiation of mouse embryonic stem cells into beating cardiomyocytes, through step-wise commitment to mesoderm and cardiac precursor lineages. DNAseI-sequencing experiments will be performed to create genome-wide maps of cis-regulatory elements and identify transcription factors that bind them. Stage-specific transcription factor regulatory networks will be reconstructed using established computational methods. Overlaying the information from detailed maps of chromatin topology we will obtain from high-throughput Chromosome Conformation Capture experiments will reveal targets of these cis-regulatory elements. Integration with other available datasets, such as RNA-seq, will allow precise understanding of the temporal changes in the structure of the (epi)genome that underlie transcriptional dynamics during cardiac cell differentiation. Finally, analysis of mutant ES cells as well as mouse embryos will provide mechanistic insights as to how disruption of key transcription factors or regulatory elements impairs the execution of cardiac gene expression programs, heart morphogenesis and heart function.

Traditionally, bone has been viewed as a relatively static tissue only fulfilling mechanical and scaffolding functions. In the past decade however, this classical view has considerably evolved towards a more complex picture. Through the secretion of an osteoblast-derived molecule, osteocalcin (Ocn), the skeleton regulates functions as important and diverse as glucose metabolism, energy expenditure and male fertility. The diversity of Ocn’s known functions, together with the powerful inhibition of bone mass accrual exerted by the brain, was a logical incentive to test if the skeleton, through a feedback loop, would signal back to the brain. Consistent with this hypothesis, we uncovered a dialogue of functional relevance between the bone and the brain occurring at several stages during life, and relying on the bone derived hormone Ocn. Post-natally, Ocn crosses the blood-brain barrier and binds to neurons of the brainstem, midbrain and hippocampus. Ocn enhances the synthesis of several neurotransmitters, inhibits GABA synthesis and favors adult neurogenesis. All together, these functions prevent anxiety and depression and favor spatial learning and memory. During embryonic development, maternal Ocn crosses the placenta and mediates the maternal influence on brain development and functions of the offspring. Although we showed that Ocn has an important effect on brain, the mode of action by which this hormone is acting on fetal brain development and adult neurogenesis remain elusive. Indeed, we propose here to identify these biological and molecular mechanism(s) and to enter in a broader search for the Ocn receptor(s) and the downstream cascade triggered by Ocn in the brain. Because of this unexpected bone-brain crosstalk, we believe that this application is novel and has the potential to be truly transformative for the field of bone biology.

Influenza A causes around 300.000 deaths worldwide each year. Because of a high degree of recombination of its surface epitopes long lasting immunity cannot be achieved. Influenza like many pathogens has developed immune evasion strategies. An important mechanism of antigen delivery for presentation to T cells, autophagy, is targeted by Influenza. The consequence for viral immunogenicity is unknown.
I aim at unraveling the molecular mechanisms underlying the fusion of autophagosomes with antigen loading compartments. To elucidate the cross-talk between viral proteins and the autophagy machinery I will investigate matrix protein 2 (M2) of Influenza A. M2 was shown to cause a block of autophagosome/lysosome fusion, which in turn leads to an accumulation of viral antigens in autophagosomes. I will identify the cellular players interacting with M2 and the mechanism of regulation. Furthermore, the role of autophagy in antigen presentation after Influenza A infection will be addressed applying an inducible mouse model of autophagy deficiency in pneumocytes, the main target of Influenza A infection in mice.
By integrating all findings (molecular mechanism of antigen delivery at steady-state and in viral infection, manipulation of the host machinery by M2, role of autophagy in viral immunogenicity) I will gain a broad overview of the regulation of autophagosome fusion in health and disease. This will help the field to advance in finding new ways to target Influenza infections. However, the implications of these findings are not necessarily limited to infection, as autophagy also plays a major role in many other diseases, such as neurodegenerative diseases.

The methylation of arginine residues is a common post-translational protein modification that plays poorly understood roles in the nervous system. Our work will test the hypothesis that methylation of synaptic proteins is an important regulator nervous system function. Our preliminary findings indicate that numerous synaptic proteins are methylated in vivo, and that at least two members of the PRMT (protein arginine methyltransferase) family, PRMT1 and PRMT8, localize to synaptic sites. We also find that PRMT8 and the postsynaptic scaffolding protein Shank3 (SH3 and multiple ankyrin repeat domains 3) physically interact in vivo, and that PRMT8 can methylate Shank3 in vitro. Further, we find that pharmacological inhibition of arginine methylation or manipulation of specific PRMTs in cultured neurons can alter dendritic spine density and synaptic function. We are proposing to more thoroughly characterize the effects of pharmacological PRMT inhibition in cultured neurons, and then examine the contribution of individual PRMTs to the observed phenotypes. Next, we will characterize the effects of methylation on Shank3 function as well as performing a proteomic screen to identify additional arginine methylated synaptic proteins. Finally, we will explore the importance of arginine methylation in vivo by characterizing the structural, electrophysiological and behavioural phenotypes of PRMT1 and PRMT8 conditional knockout mice. Together, these experiments will allow us to test the importance of this previously un-described modification of synaptic proteins, and if successful will establish arginine methylation as a novel regulator of brain function and plasticity.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) of eukaryotic cells leads to ER stress eliciting the unfolded protein response (UPR). The UPR is a network of intracellular signaling pathways that adjusts the protein folding capacity of the ER according to the demands of the cell. The transmembrane kinase/RNase Ire1 functions as a signal transducer and represents the most conserved branch. Ire1 oligomerizes in the ER membrane upon stress-induced activation. However, the relationship between different oligomeric states and the activity of Ire1 remains elusive. The goal of the proposed project is to mechanistically dissect how different assembly steps of Ire1 trigger its activation. Using state-of-the-art biochemical and biophysical methods, I will study the assembly steps and structure of higher-order oligomers of Ire1. To this end, I will implement two nanobiological approaches. I will study the structure of full-length Ire1 embedded in artificial lipid bilayers (nanodiscs). In addition, I will exploit the DNA origami nanotechnology to scrutinize the activity of defined oligomeric states assembled on DNA scaffolds. Both approaches will also help to decipher the interaction of Ire1 with RNA substrates. I will further integrate these in vitro results into Ire1 signaling in vivo: By using novel genetically encoded tags, I will determine the localization and ultrastructure of Ire1 signaling platforms in the ER membrane by correlated light and electron microscopy. These analyses hold the promise to significantly enhance our understanding of the molecular activation mechanism of Ire1 and the cellular organization of ER stress signaling.

In humans, nucleotide excision repair (NER) is the exclusive mechanism for the removal of DNA damage induced by ultraviolet light and thus protects against the primary cause of skin cancer development. NER must detect minuscule DNA modifications obscured in chromatin, initiate chromatin decondensation and then assemble the NER repairosome. NER employs two damage detection complexes with remarkably contrasting properties. XPE recognizes chromatinized DNA damage and induces ubiquitin dependent chromatin decondensation, but is dispensable for the recruitment of the NER machinery. XPC recognizes major DNA helix distortions but has no affinity for lesions in chromatin, yet it is essential for NER in living cells. Consequently, damage handover must occur in order to initate NER, presumably by the formation of an as yet unidentified bipartite damage detection supercomplex. Here I propose to study the damage handover mechanism and aim to provide experimental and structural evidence in support of the existence of an XPE-XPC-DNA damage handover complex. I will determine the structure of this 0.5MDa nucleoprotein complex by X-ray crystallography and chemical cross-linking followed by mass spectrometry will guide the crystallization of representative subcomplexes. In vitro reconstitution of damage handover and structure-guided mutational analysis will help to dissect the damage handover mechanism in a stepwise manner. Insight into the regulation of damage handover will be validated in living cells. This study will provide a detailed understanding of a process whose malfunction is the cause of a severe genetic disease that condemns its sufferers to life without sunlight.

Nuclear constriction is an important requirement for all cells migrating through narrow pores inside extracellular matrix or between other cells. Upon encountering pathogenic bacteria or viruses, dendritic cells (DCs) internalize antigens and are triggered to then infiltrate lymphatic vessels. Here, the applicant first aims to visualize nuclear constriction of DCs migrating through confined, microfabricated matrices of collagen as well as during their process of entering lymphatic vessels. Nuclear squeezing during DC migration will be observed in mouse ear explants and in the footpad of living mice. The applicant hypothesizes that the layer of lamina just under the nuclear envelope protects the against DNA damage during nuclear squeezing. Intermediate filament polymer Lamin A/C is speculated to contribute to mechanical rigidity of the nucleus, and thus its function during nuclear deformation will be evaluated in this proposal. Lamin A/C will be either mutated at a site found in Muscular Dystrophy, depleted with KO cells, or increased with transient transfection, and the effect this has on DC survival through constrictive matrix will be assessed. The extent of DNA damage caused by nuclear constriction will be assayed for and whether Lamin A/C alleviates the damage will be determined. The final part of the proposal will highlight the importance Lamin A/C has for allowing DCs to reach and enter lymphatic vessels in a mouse model. The proposal will combine the efforts from multiple disciplines by applying what is learned about nuclear squeezing from microfabricated devices to an in vivo context in mice.

Nonmembrane-bound compartments in eukaryotic cells are dynamic intracellular organelles performing multifarious cellular functions. Recent studies suggest that phase separation of proteins with Low Complexity Domains (LCDs) may be a unifying principle of assembly of such compartments. However, regulatory mechanisms and molecular regulators of reversible condensation/dissolution are unknown. Recently, kinase activity of dual-specificity kinase DYRK3 was shown to couple stress-induced RNA granule dynamics with mTOR signalling in a manner dependent on its cycling between the granule and cytosol phase. I propose to investigate the mechanism, based on a working hypothesis, of DYRK3 cycling between liquid-unmixed and –mixed phases via N-terminal LCD auto-phosphorylation. I will also perform an RNA interference (RNAi) and chemical compound screen to identify the phosphatase, which counteracts the phosphorylation event in DYRK3, thereby completing the DYRK cycle of partitioning between two phases. This is followed by an analysis of stress-induced changes in spatio-temporal dynamics of DYRK3 activity by performing FRET/FLIM assays inside cells. Finally, I will test how generalizable is the mechanism of DYRK3 cycling among DYRK family members (DYRKs, HIPKs and PRP4s) and whether there is a single mechanism on how the reversible condensation/dissolution of proteins through LCDs works. This research would reveal a novel mechanistic principle shared by a class of kinases to regulate nonmembrane-bound compartmentalization and the signaling driven by it and will thereby be a major advance for the field of cellular compartmentalization.

Intrinsically disordered proteins (IDPs) are widespread in prokaryotes and eukaryotes, and fulfill important functions mainly associated with signaling and regulation. This proposal addresses fundamental questions in a recently discovered subgroup of IDPs, intrinsically disordered chaperones that use the plasticity of intrinsic disorder for their action. My aim in this proposal is to portrait experimentally driven stress response chaperone networks in prokaryotes and eukaryotes, focusing on intrinsically disordered (ID) chaperones. The long-term goal is to map relationships between key proteins maintaining protein homeostasis under diverse stress conditions, and being able to manipulate this network by design of novel chaperones. In this study we will focus on ID chaperones, which undergo conditional unfolding by specific stress conditions (e.g., heat, oxidation, acid and others). The involvement of intrinsic plasticity in molecular recognition is usually explained by such terms as promiscuity and flexibility. Beyond these notions it still remains unclear why specific intrinsically disordered protein were selected to recognize signaling proteins, DNA, small ligands or aggregation-prone proteins. In this proposal, we aim to explore the putative links between intrinsically disordered regions (IDRs) and chaperone activity. In order to establish such links we plan to increase the number of members of this novel protein family. We plan to derive a database of functionally defined IDRs, which will serve to develop a computational toolbox for identification of novel intrinsically disordered chaperones within bacteria and yeast proteomes.