Chemical probes are small molecules that can perturb the activity of a target macromolecule, and thus can be used to understand the biological roles of receptors. Currently, identifying a chemical probe for one specific target requires screening many chemicals to find ones that bind that target. If one screen could be used to find chemical probes for many targets, one could dramatically increase the rate of probe discovery.

Our first goal is to identify novel chemical probes for approximately 40 receptors in S. cerevisiae. This will be done using a novel high throughput metabolomics approach. Recent advances in the Sauer lab allow for the speedy generation of a metabolomic fingerprint of the effect of a compound treatment on the cell. Therefore it should be possible to adopt an innovative approach to chemical screening where compounds are identified that are able to mimic the metabolomic profile of yeast where receptors are genetically perturbed. This will allow us to find chemical probes for those targets. Thus, we will be able to identify many chemical probes for many receptors from a single chemical screen.

Our second goal will be to investigate the roles of these receptors in vivo using our chemical probes. We will investigate what specific metabolomic and transcriptomic changes individual S. cerevisiae nutrient sensors elicit when they activated by a non-metabolizable ligand. This is of interest because it can isolate what the cellular response to receptor activation is, and potentially show why it is adaptive for the cell to respond in that way. Additionally, we will use them to investigate the biological roles of poorly characterized receptors in S. cerevisiae.

The neural circuits underlying vision share a lot of structural and functional similarities between Drosophila and mammals, with motion detection being the best-studied visual modality. The highly stereotyped columnar and layered organization of Drosophila optic lobe neuropils (the visual processing centres) allows a precise study of circuit wiring. Two parallel motion channels have been described in Drosophila, one for detecting moving light edges (ON pathway) and one for detecting moving dark edges (OFF pathway). The main components of these channels have been identified, comprising neurons in the lamina, medulla, lobula and lobula plate neuropils. However, little is known about the specific mechanisms leading to the orderly and stereotypic assembly of these cell types to form functional circuits. In this proposal we offer to study by live imaging the spatiotemporal dynamics of motion detection circuit assembly and the determinants of such formation. It has been proposed that circuits are assembled in the order of neuron generation. To test this hypothesis, we will analyse the assembly order of ON and OFF motion circuit components and study how the developmental specification of cell types is linked to circuit formation. Furthermore, we will investigate whether there are neurons with pioneer roles in circuit assembly, the influence of neuronal soma positioning on axonal and dendritic layer targeting, and the role of spontaneous activity in circuit wiring. Altogether, this project will advance our understanding of the mechanisms governing functional circuitry assembly, which is likely to have broader implications for other neuronal systems.

Autophagy is a ubiquitous process of eukaryotic cell biology, which occurs by the formation and growth of the isolation membrane (IM). The IM engulfs bulk cytosol or targets such as protein aggregates, damaged organelles, and invading pathogens. The IM closes to become the autophagosome, fuses with the lysosome, and its contents are degraded. More than 40 Atg proteins are dedicated to autophagy, thus the “parts list” is known. How the autophagosome initiates, grows, and closes, using these parts is a world-class mystery. We propose to use biochemical reconstitution, cell imaging, and computational biophysics to dissect the uptake of the intracellular pathogen Salmonella (“xenophagy”) and so reveal the physical basis of autophagosome formation. In one major model, derived from yeast data, the IM nucleates from several small vesicles that then fuse into a sheet. The sheet grows by fusion of more small vesicles. In another model, derived from mammalian cell data, the IM is extruded out of a domain of the endoplasmic reticulum (ER). We propose to resolve which of these mechanisms is operative, by reconstituting autophagosome formation, and by directly imaging the process both in vitro and in cells using real-time super-resolution imaging. We will then move beyond distinguishing between these qualitative models, to a quantitative, biophysical paradigm. The arrival, departure, copy numbers, and structures of the relevant protein complexes on and off membranes will be determined in vitro and in vivo. Changes in membrane shape will be correlated with these parameters. These will be input to a computational model accounting for membrane physical properties and protein structures. The complexity of this system, with 10s-100s of copies of more than 40 different proteins, operating on a time scale of minutes and a length scale of two microns, makes this one of the most complex systems to be tackled with such biochemical and biophysical detail.

This project will investigate locomotion control in undulatory animals and transitions between different environmental media. It will test the idea that a single control principle could explain different modes of locomotion in vertebrate and invertebrate animals for different morphologies in different environmental media (water and ground). The control principle is based on “sensory synchronization” of local neural oscillators. It merges ideas from central pattern generators and from sensory-driven locomotor models. We postulate that flexible motor patterns for body-limb coordination during swimming, crawling and walking can be obtained in a distributed network of neural oscillators that rely to a large extent on (multimodal) sensory feedback signals for synchronization. In other words, we postulate that the synchronization between oscillations in body and limbs, and hence the generation of gaits, is due to mechanical interactions of the body with the environment and the resulting sensory signals more than to direct neuronal (or central) coupling between the involved neuronal oscillators. We also postulate that many observed features of various gaits are due to changes in the environment rather than to changes in the descending commands.
This idea and related predictions will be tested by investigating Polypterus (a walking fish), salamander, and centipede locomotion. These three species represent a diversity of life that has overcome the problems associated with neuro-control in amphibious environments. Our research tools combine animal locomotion studies, neuromechanical simulations, and robotic experiments. The neuromechanical simulations and the robots will play an essential role in decoding the complex interplay between central pattern generators, sensory feedback, the musculoskeletal system, and the different environments. The interdisciplinary approach will allow us to investigate whether the same control principle could not only facilitate adaptations to growth, lesions, perturbations, and different environments during the lifetime of the animal, but also to evolutionary adaptations (e.g. changes of ecological niches and of morphologies) in particular the transition from aquatic to terrestrial environments in vertebrates and invertebrates, a key step in evolution.

In photosynthesis, light-harvesting complexes (LHCs) capture solar energy and feed it to the downstream molecular machinery. However, when light absorption exceeds the capacity for utilization, the excess energy can cause damage. Thus, LHCs have evolved a feedback loop that triggers photoprotective energy dissipation. The critical importance of photoprotection for plant fitness has been demonstrated, as well as its impact on crop yields. However, the mechanisms of photoprotection ? from fast chemical reactions of molecules to slow conformational changes of proteins ? have not yet been resolved. Despite extensive studies of the native photosynthetic apparatus, previous experiments have not been able to control architecture in order to distinguish between hypotheses of the mechanisms of photoprotection. To overcome this barrier, we take a novel synthetic structural biology approach. We build minimal regulatory units using model membranes that we measure and model to gain a multi-timescale understanding of photoprotection, which is core to natural light harvesting. Taking this synthetic structural biology approach is a significant experimental challenge in three distinct fields; biochemical and molecular biology development is required to construct a well-defined and well-characterized sample, new spectroscopic analyses and collection strategies are required to adapt optical experiments to probe these samples, and theoretical advances are required to model these length scales.
Uniquely, photoprotection regulates a photophysical process, providing an intrinsic spectroscopic handle to uncover how information flows within the multi-timescale phenomena of biological regulation. LHCs and other biological systems perform meso- and macroscopic functions efficiently and robustly even when exposed to the changing and fluctuating environment that is a hallmark of natural systems. Furthermore, biological systems adapt and even reprogram their function in response to the environment. Unveiling the design and working principles of such responsive and highly autonomous behaviors exhibited by molecular systems is one of today’s grand challenge areas from the standpoint of both theoretical and experimental research; we incisively explore such behaviors in the context of photosynthetic light harvesting through the work proposed here.

Mitochondria are organelles that perform fundamental functions in eukaryotic cells. They are responsible of cellular respiration, energy generation and an array of biosynthetic processes. Additionally, they play a key regulatory role in programmed cell death. In medicine they are widely studied, as mitochondrial dysfunction/disease have enormous health consequences, due to their involvement in aging and in a number of pathologies including, cancer, metabolic disorders and heart disease.
Despite their importance, and the effort of numerous researches, multiple aspects of mitochondrial biology are still unclear. In this project we propose to exploit a unique system to develop novel tools to study and to interact with mitochondria, with potential groundbreaking applications. Midichloria mitochondrii is an intracellular bacterium that presents a characteristic that makes it unique: it is able to enter the mitochondria of its host cells. This unique interaction has been discovered recently, and has still widely uncharacterized. Our multidisciplinary team envisioned an integrated experimental approach, using state of the art technologies in the fields of bioinformatics, -omics, imaging, biochemistry, and cellular biology to investigate this novel biological system.
This research project will allow us to understand the evolution and the functional details of the relationship between M. mitochondrii and its host at the organismal, cellular and molecular level. We will use this novel information to develop an in vitro model system that will allow us to use this unique bacterium to interact with mitochondria in novel ways. This approach will ultimately lead to the development of new experimental tools to characterize mitochondrial mechanisms, including mitochondrial fusion/fission, inner and outer membrane repair, metabolism and apoptotic signaling.

Demography (i.e. a population’s history of size changes, gene flow, and divergence events) affects patterns of genetic diversity, and has implications for studying evolution, adaptations, and disease. However, demographic inference is complicated by “ghost populations,” unobserved populations in the past that admixed with the ancestors of sampled populations. Inferring this hidden structure is challenging both because the number of ghost populations is unknown, and also because signals of cryptic structure may be distorted by other evolutionary forces. I will address this problem by developing a flexible method capable of estimating unknown population structure jointly with other sources of genetic diversity. Our method will combine models from Coalescent theory with machine learning techniques for inferring graph structure, and will estimate the number of ghost populations, their demographic history, and the distribution of fitness effects for mutations arising in these ghost populations. Furthermore, our approach will allow us to estimate allele frequencies in ancestral populations, which will be useful for identifying adaptive introgressed regions from ghost populations, and for addressing questions such as the adaptive consequences of gene flow from ghost populations. We will apply our new method to diverse human whole-genomes to shed light on open questions in recent human evolutionary history, such as the relationship between modern African populations and the out-of-Africa population that gave rise to modern Europeans and Asians, or the extent to which variants selected in particular populations have spread more broadly by admixture and introgression.

Propagation of a-synuclein in the brain is the hallmark feature in the pathogenesis of Parkinson’s disease (PD). Patients with PD exhibit marked differences in the rate of a-synuclein propagation, but the reasons for this variability are unknown. I hypothesize that the rate of a-synuclein propagation varies because it is affected by various genetic modifiers. In addition, I hypothesize that individuals with PD are mosaics for a-synuclein mutations in the brain, a type of genetic variation that is missed by standard screening methods, which further impacts on a-synuclein propagation. In order to investigate these hypotheses, I am going to generate human iPSc-derived neuronal cultures expressing a construct in which mutant a-synuclein that is fused with mCherry on its C-terminus is also fused to a GFP tag on its N-terminus through a 2A self-cleaving peptide. Neuronal cultures expressing wild type (WT) a-synuclein fused with BFP on its C-terminus will also be generated. I am then going to mix these neuronal cultures to generate cultures of various mosaic percentages for the a-synuclein mutation. This system will enable the identification of single neurons that have uptaken WT or mutant a-synuclein through propagation based on their positivity to GFP, mCherry and/or BFP. I am then going to use a CRISPR-Cas9 high throughput screening system combined with high content imaging techniques developed at the host institution to assess the effect of thousands of genetic factors on the rate of propagation in the mosaic neuronal cultures, followed by RNA sequencing to determine molecular signatures that underlie the propagation process and that are specific for each genetic modifier.

Monitoring membrane potential is a necessary step in understanding neuronal activity. Invasive methods cannot provide the spatial resolution and coverage required for such information. On the other hand, optical methods that provide high spatio-temporal information demand optical sensors with large signal amplitude, photostability, live-cell compatibility, and fast response to voltage variations. Existing optical voltage sensors do not match all these requirements. Recently, semiconductor nanorods (NRs) with asymmetric structures have been shown to exhibit prominent voltage sensing capabilities. Their high luminescence and photostability will also allow for nanoscale resolution and reduction in photodamage of live cells. Therefore, here I aim to explore ways for functionalizing these particles with peptides of DNA origami for membrane insertion. I will evaluate the insertion mechanisms by measuring their orientations and accurate positions with respect to the lipid bilayer using single-molecule Metal-Induced Energy Transfer (smMIET) imaging. After stable insertion, I aim at characterizing their voltage sensing properties and performance with fast optical imaging techniques. In the end, I will explore their application in neuronal cultures by measuring action potentials and subthreshold events in individual cells and across the network. Membrane potentials in subcellular sections such as dendrites and spines will be monitored with high spatial resolution as a function of their morphology and structural dynamics, which has not been achieved to this date.

My project aims to identify neuronal circuits that are spared with severe spinal cord injury and function as motor command relay pathways during reactivation of the lumbar spinal cord. I will use a neuroprosthetic platform that supports bipedal stepping and implant an electro-chemotrode over the lumbar spinal cord of mice. First, I will optogenetically activate motor cortex projections in severely contused mice and assess whether immediate will-powered motor control is regained over completely paralysed hindlimbs during spinal stimulations (Aim 1). Next, I will use a combination of conventional tracers, such as Fast blue, and viral tracers that are regulated by a Cre promoter in vGluT2 Cre or 5-HT Cre transgenic mice to label and identify brainstem projections to the lumbar spinal cord. Moreover, I will prove these brainstem projections are required to rely motor cortex commands by chemogenetically silencing them with the DREADD technology, which should abolish any leg motor function (Aim 2). Mice will undergo 8 weeks of actively engaging neurorehabilitation with spinal electrochemical stimulation. I will assess recovery of will-powered voluntary motor function and remodeling of spared pathways (Aim 3). I will laser-microdissect cell bodies of motor command relay neurons in the brainstem and assess the transcriptional profile with RNA sequencing to identify which molecular cues are involved in circuit reorganization (Aim 4). Taken together, these results will establish the causal relationship between circuit reorganisation of residual descending projections and voluntary motor control with electrochemical neurorehabilitation in severe spinal contusion injuries.

Cell division and differentiation are strictly coordinated during development. Although it is known that differences in S-phase length are essential for embryogenesis our understanding of its causes and consequences is extremely poor. The second cell division of C. elegans is stringently asynchronous and is well-suited to study developmental control of replication regulation in a multi-cellular organism.
Studying replication initiation is limited by the availability of specific assays. I will develop a novel live assay to visualise the recruitment of replication factors towards origins in single cells within the C. elegans embryo. With the LacO/LacI system I will generate functional origins and measure the binding of fluorescently tagged replication factors by confocal imaging dissecting consecutive steps of DNA replication. This assay will permit to follow the dynamics of replication factors in real-time and to reveal changes in S-phase control during development. On this basis, I will perform an RNAi-based high-throughput screen for novel regulators of genome duplication in the C. elegans embryo. This unique screen implemented in a whole organism will reveal novel interactions between DNA replication and developmental signalling pathways. In a complementary approach I will generate mutants bypassing the normal cell cycle regulation in the embryo. I will examine potential changes in cell fate adoption through transcriptional analysis and will measure differences in their sites of replication. This approach will clarify if replication control plays a direct role in differentiation and illustrate a physiological role for S-phase differences in development.

It has been postulated that cancer stem cells (CSC), an identifiable subpopulation of cancer cell that can differentiate and repopulate cancer, exist. In this context, cancer cells follow strict hierarchy with CSC being at the apex of it. However, depending on the subtype and malignancy of cancer, cell fate plasticity had been observed where non-CSC dedifferentiates to acquire CSC function. However, the nature and regulation of CSC formation and plasticity has been difficult to understand in vivo. For example, what kind of niche signaling is responsible for cancer cell plasticity? What is the epigenetic or transcriptomic changes in cancer cell plasticity? In this project, I aim to address these questions in vivo using highly novel technique involving transgenic mice carrying recombinase-based state machine (RSM), which enables each individual cell within cancer to keep a memory of its history and sequence of changes in cell fate. The proposal will involve generation and validation of mammalian RSM that can label each cell with different cell fate history (e.g. original CSC and non-CSC that had dedifferentiated to CSC) with different fluorescent proteins. I will then cross the transgenic mice harboring RSM with mouse models of breast cancer and colon cancer can accurately identify heterogeneity in individual cancer cell fate in vivo and in situ. By examining the relative locations of cancer cell fate plasticity in intact tumor mass, and FACS sorting of cells with different fate history for transcriptomic or epigenetic analysis, I aim to dissect the extracellular factors and intracellular signaling events that govern cancer cell plasticity in vivo.

Spatial organization of chromatin and related genes is of key importance for genome expression and maintenance. Unlike stem cell differentiation, for T-cell activation gene regulation happens on a much faster timescale as the immune response is critical for the bodies defense mechanism. The chromatins’ three-dimensional organization as well as its interaction with nuclear lamina and the consequential impact on gene regulation is poorly understood. This proposal will in its first step focus on how mechanical forces can directly influence cellular functions such as the T-cell activation and furthermore elucidate how they ultimately change gene expression. We hypothesize that mechanical force triggers structural/conformational changes of the nuclear lamina network; hence genes encoded in chromatin proximal to lamina can be up/downregulated. Here, we propose to develop 3D and 4D Super Resolution Microscopy (SRM) technologies to enable us to determine the 3D structure of lamina associated chromatin domains, resolve how such domains are organized in 3D during the fast process of T-cell activation. We specifically aim to:
1. Establish imaging methods to measure tension and the dynamic structural re-organization of cytoskeleton, nuclear envelope and chromatin during T-cell activation.
2. Study the effect of perturbations of the mechanobiological pathway that links external cues to activation.

3. Link these perturbations to gene expression and spatial location of genetic loci and transcripts.
This project requires synergistic interplay between the two team members, which brings together early career researchers in T-cell biology, single molecule biophysics, structural biology and SRM.

Cytoskeleton networks are the main vehicles of cell mechanics. Coordinated network dynamics is responsible to processes as diverse as cell motility and cytokinesis. Understanding the principles governing motion in these networks requires studying in vitro assemblies of purified components which can be subjected to finely tuned conditions. Recent works in purified systems have shown them to be unstable: either extremely contractile to the point of network collapse; or extensile and turbulent. This proposal delineates a research plan for studying active gels of microtubule filaments and kinesin motors as a prototypical cytoskeleton. Its main goal is to understand how asymmetric contractile and extensile modes of deformation emerge from inherently symmetrical network elements. My experimental approach will be to develop tools for modifying and controlling network dynamics on three length scales. At the filament scale, I will use the SNAP-BG crosslink to decorate filaments with motors. I expect that motors crosslinked at the minus end of a microtubule will encourage contractile motion and that plus-end crosslinked motors will encourage extensile motion. I will elongate the minus-end crosslink with photosensitive short DNA. Irradiating a mixed sample of minus-end and plus-end crosslinked motors with UV, I will be able to spatiotemporally tune contractility on the micron scale. Macroscopic stresses in the network will be measured and applied through a patterned soft substrate. The multiscale approach combined in a single set-up will allow us to uncover mechanisms responsible for contractility in cells and provide us with novel tools for designing biomimetic systems.

Direction selectivity is a feature of selected neurons in the visual cortex that is conserved in evolution. The emergence of cortical direction selectivity has been extensively studied but its circuit mechanism is still debated. My goal is to understand the circuit mechanism of cortical direction selectivity that is independent of retinal direction selectivity. I plan to approach this by using two animal models in which retinal direction selectivity has been disrupted. I will investigate the neuronal circuits of those cortical direction-selective neurons which retain their specificity in these animal models by performing single-cell-initiated functionalized rabies tracing and single-cell electrophysiological recordings. I will ask the three following questions: Do axon terminals of the principal cells of the lateral geniculate nucleus (LGN) fully lose direction selectivity after the abolishment of retinal direction selectivity, or could cortical feedback from layer 6 entrain direction selectivity in a subset of LGN cell axon terminals? What is the synaptic input profile underlying retina-independent cortical direction selectivity in pyramidal neurons? What is the contribution of the local presynaptic networks to the direction-selective tuning of pyramidal neurons with retina-independent cortical direction selectivity? This work will provide insights into how cortical selectively for environmental features arises.

Eukaryotic genomes are organized into a highly-compacted nucleoprotein complex known as chromatin. Hi-C and high-resolution microscopy studies reveal organization of chromosomes at multiple levels, from chromosome territories, to MB-scale functional domains that are spatially separated from each other, to shorter contact domains often called topological associated domains (TADs). While genetic studies in cell culture coupled with Hi-C or high-resolution microscopy reveal key roles for a variety of factors in higher-order chromatin folding, our understanding of chromatin fiber folding largely derives from biochemical studies in vitro. Mostly, these in vitro studies rely on artificial, homogenous chromatin templates that do not reflect the heterogeneous local folding properties of in vivo chromatin.
The aim of this project is to approach higher-order chromatin folding and chromosome organization biochemically. Micro-C detects internucleosomal interactions, which refer to local chromosome folding, by identifying nucleosomal DNA sequences and is therefore equally suitable for in vivo and in vitro studies. I will use chromatin prepared from cells (ex vivo) and in vivo-like in vitro reconstituted chromatin for chromatin compaction studies. With Micro-C I will be able to measure autonomous salt-dependent chromatin folding driven by all endogenous factors (ex vivo chromatin) or solely by histone-DNA interactions. Biochemical manipulation will allow to remove or test individual candidate factors.
This biochemical approach will help to decipher the regulatory role of higher-order chromatin organization on DNA templated processes, such as gene regulation.

Synthetic approaches toward redesigning cellular circuits and signaling networks have accelerated our understanding of sophisticated cellular behaviors. In particular, exploiting optogenetic approaches on neurons to manipulate neuronal activities have significantly expanded our comprehension on the nature of neuronal circuitry, whereas those approaches were applied much less in immune cells. Indeed, repurposed and/or engineered immune cells have been successfully used as cancer treatments, but they are only sometimes successful and can have harmful side effects. We hypothesize that these attempts have been limited because they have been restricted to a limited number of wild-type like signaling inputs. Here, through evolution-based approaches we intend to engineer chimeric antigen receptors (CARs) in T cells as a model system to answer a simple, yet, fundamental question regarding finding the maximal potency and unique phenotypes of T cell-based immune responses. Also, we offer a generalizable design scheme for safely controlling CARs through an optogenetic module. Newly engineered CARs could be of great use in eradicating cancer, and unique combinations and sequences of T cell signaling components could provide unexplained mechanisms and further understanding of the evolutionary nature of designed immune system. In order to achieve the goal, we build CAR intracellular domain libraries, and with a properly designed activity-based selection strategy we anticipate to find novel classes of CARs with extraordinary potency of immune responses.

It has been a long-standing question what mechanisms counter antibiotic resistance in nature. Are there any widespread multidrug strategies in soil bacteria for avoiding, inhibiting, or bypassing resistance mechanisms against antibiotics, whose biosynthetic pathways evolved a hundred million years ago, but still exist across natural microbial communities? One of the well-known examples of naturally occurring combination strategies is the co-production of beta-lactam antibiotics and beta-lactamase inhibitor in Streptomyces spp. which has led to the development of a potent drug combination for clinical use known as Augmentin. However, our knowledge on this issue is extremely limited due to the shortage of systematic investigations. It is important to emphasize that the extraordinary capacity of soil bacteria to produce multiple natural products may reflect an ecological advantage for them. Therefore, I hypothesize that a systematic investigation of the combinatorial biosynthesis of secondary metabolites, including antibiotics and non-ribosomal antimicrobial peptides, could reveal novel drug combinations. First, using state-of-the-art computational approaches I will explore the co-occurrence of biosynthetic genes of known secondary metabolites and antibiotics in the genome sequence of soil microorganisms which presumably co-evolved under strong selection pressure. Second, I will explore the capacity of novel candidate combinations i) to restore susceptibility of the resistant bacteria, ii) to synergistically inhibit bacterial growth or iii) to invert the selective advantage of resistance mutations.

Plants have evolved sophisticated mechanisms to recognize non-self molecules, allowing them to deploy effective immune reactions against a myriad of pathogens. Sometimes, however, plants mistakenly identify their own molecules as foreign and induce autoimmunity, causing severe growth defects including plant death. Autoimmunity is a hallmark of a syndrome that is seen in certain intra- and interspecific hybrids and that is known as hybrid necrosis. A particularly interesting case in Arabidopsis thaliana is that of the RPP7 and RPW8 loci, where three different pairs of alleles from different wild strains interact in F1 hybrids to cause hybrid necrosis. RPP7, which confers resistance against oomycetes, encodes members of the well-studied family of NLR immune receptors. The biochemical function of the RPW8 coiled-coil domain proteins that primarily confer resistance to fungi is less well understood than that of NLRs. I propose to reveal the mechanisms by which interactions between RPP7 and RPW8 proteins activate immune responses, taking advantage of the allele-specificity of these interactions. By interpreting these findings in the context of normal immunity, my work will not only advance our understanding of genetic incompatibility and autoimmunity, but will also contribute to optimizing combinations of favorable immune alleles in crop breeding.

Cells are constantly making millions of proteins that need to be synthesized correctly, folded, trafficked, and assembled to their functional state. All of these steps are monitored by the cells for mistakes or failures, and these failed products are degraded to avoid their accumulation. These quality control (QC) pathways are emerging as major contributors to neurodegeneration. The newly discovered ribosome-associated quality control (RQC) pathway represents one of the most cleverly imposed QC pathways to allow cotranslational surveillance of erroneous translation products. Recent studies have identified the factors involved in RQC pathway and defined its primary steps: recognition and splitting of stalled ribosomes into subunits, signaling of the heat shock response, ubiquitination of nascent chains, and extraction of polyubiquitinated nascent chain from the 60S subunit for degradation. I propose to dissect the mechanism of two unresolved steps of the RQC pathway and define its endogenous clients. Aim1 will investigate an essential step in signaling the heat shock response: the addition of C-terminal Alanine and Threonine (CAT) tails to stalled nascent chains. I will reconstitute this unusual mRNA-independent polypeptide elongation reaction in vitro to identify the minimum required components, define the energy source, and order the primary steps of this process. Aim2 will reconstitute nascent chain extraction in vitro to determine how a stalled polypeptide is delivered from the ribosome to the proteasome. Aim3 will identify the physiological clients of the RQC by trapping in vivo intermediates and identifying associated mRNAs or partially synthesized nascent chains.