Expanded malaria interventions, reliant on effective artemisinin-based combination therapies (ACTs), have halved malaria mortality in the past decade. Yet malaria continues to kill over 500,000 children yearly. The situation could rapidly worsen if resistance of P. falciparum (Pf) to ACTs takes hold and spreads. This has begun with the emergence of resistance to artemisinin. Recently, the partner drug piperaquine (PPQ) has encountered resistance, leading to treatment failures in Cambodia, the epicenter of multidrug resistance. Resistance is also emerging in French Guiana. I propose to define the genetic basis of PPQ resistance. Using clinical isolates, I will implement genetic crosses between resistant and sensitive Pf parasites in chimeric FRG-NOD mice that are receptive to Pf infections. I will quantify PPQ susceptibility in the progeny using in vitro dose-response and survival assays. Linkage association studies with whole-genome sequenced progeny will localize candidate determinants, whose role will be ascertained using Cas9-based genome editing. In parallel, I will assess novel mutations in the pleiotropic drug transporter PfCRT that have recently been identified in a subset of PPQ-resistant Cambodian parasites. I hypothesize that resistance is multifactorial and involves altered hemoglobin digestion and availability of reactive heme as a PPQ ligand, along with altered drug transport. I will also define which partner drugs remain effective against PPQ-resistant parasites. Results of this high-risk, high-yield project will provide molecular markers to track PPQ resistance, offer insights into drug action, and inform strategies to combat multidrug-resistant malaria.

Tissue morphogenesis drives several developmental processes and is required for the basic organization of embryos and organs. The three semicircular canals (SC) in the vertebrate inner ear present a fascinating example, whereby the motion-sensing function relies on their complex shape and orientation. Failure of normal development of SC results in vestibular dysfunction. The SC formation requires budding and extension of three projections (anterior, posterior and lateral) into the lumen of a simple epithelial embryonic structure called the otic vesicle. These projections meet and fuse to form pillars, and the spaces around them become the three SC. The patterning and the mechanics underlying the SC formation largely remains unknown. In this research proposal, I aim at studying the cellular dynamics of SC morphogenesis in zebrafish. I will investigate the role of intrinsic and extrinsic forces, and their patterning by morphogens using in toto imaging, molecular-mechanical perturbations, quantitative analysis and theoretical modeling. Specifically, I will test a model for tissue buckling, and the role of local propelling forces by remodeling of the extracellular matrix and intra-cellular forces generated by the actomyosin networks in driving budding of the projections in the otic epithelium. I will also study the signals that pattern the intrinsic and extrinsic forces to result in budding at precise sites, and thus likely to be crucial for the robust topology of the SC. The fundamental, multi-scale, molecular-mechanical principles revealed from this study will be widely applicable to other morphogenetic events involving budding and curvature formation.

The cortical subplate is a transient neuronal circuit that exists only during neocortical development. The activity of subplate neurons is important for connecting thalamic neurons to cortical neurons. Pharmacologically blocking neuronal activity in vivo during cortical development has been shown to interfere with the entry of thalamocortical axons into their normal cortical target area, and silenced axons have been projected to incorrect cortical areas. Here I propose to use the visual cortex as a model system to understand how the activity of subplate neurons contributes to the early wiring of the cortex. I will use single-cell-initiated, monosynaptically restricted retrograde transsynaptic tracing with rabies viruses to label individual subplate neurons and their presynaptic inputs. I will express either membrane-bound GFP or GCaMP6 with rabies viruses and image either the growth or the activity of single subplate neurons and their axonal inputs using in-vivo 3D two-photon imaging. I will then perform post-mortem brain clearing to determine which cell types wire onto individual subplate neurons. Finally, I will use a combination of optogenetic tools expressed within the circuit of a single subplate neuron to show how neuronal communication impacts the wiring of subplate neurons. These experiments will describe the initial wiring of sensory input to the visual cortex, and contribute to the understanding of the basic mechanisms underlying cortex development.

Somitogenesis, the process by which the body axis is periodically segmented into epithelial structures known as somites, occurs in a bilaterally symmetric fashion. Preliminary observations indicate that some somite pairs exhibit transient asymmetries before attaining their final symmetric alignment. The mechanisms that facilitate this recovery and ensure symmetric somite formation between the left (L) and right (R) sides, are however unknown. Using real time SPIM, I propose to investigate this question in zebrafish embryos by performing a spatiotemporal quantification of processes that mediate symmetric somitogenesis. I will first systematically quantify the extent to which the dynamics of somite formation is correlated between the L and R sides under non-perturbed conditions. Following this, I plan to investigate the role of canonical signaling pathways such as Nodal, Notch and retinoic acid in facilitating somite symmetry. Here, I intend to perform a quantitative comparison of somite formation dynamics between the two sides upon local activation or disruption of these signals. Finally, I will test a novel hypothesis where mechanical cues originating from actomyosin contractility and cell adhesion facilitate somite symmetry through an active mechanical interaction across the notochord. I will test this by performing local mechanical perturbations on one side followed by a quantification of somite dynamics on the contralateral side. The proposed work here thus provides a live study of the emergence of somite symmetry for the first time and aims to reveal contributions of biochemical and mechanical processes that facilitate symmetric somite formation.

The gastrointestinal tract is essential for the absorption of water and nutrients, the induction of mucosal immune responses and the maintenance of a healthy gut microbiota. Virtually all aspects of gastrointestinal physiology are controlled by the enteric nervous system (ENS), an extensive network of neurons and glial cells that is intrinsic to the gut wall. ENS interacts with components of its ‘outer’ microenvironment (gut microbiota, metabolites and nutrients) and ‘inner’ microenvironment (immune cells and stromal cells). A number of reports have demonstrated that the gut microbiota and immune system contribute to the development and homeostasis of the central and peripheral nervous system, but the mechanisms by which these cellular systems regulate the assembly and maturation of neural circuits are currently unknown. Recent studies have raised the possibility that maternal and/or early postnatal microbiota influence the organization and function of the ENS. In this proposal, we will take advantage of established expertise and reagents (including transgenic and germ-free mice) to analyse the role of the microbiota and associated products (including microbial metabolites) and the adaptive immune system on the development and functional maturation of the ENS. Our aim is to identify molecular and cellular cascades that are regulated by gut microflora and directly or indirectly control the assembly and function of intestinal neuroglial networks. This proposal will provide insight into the molecular pathways that underpin the effects of environmental factors (such as microbes) on the development and functional maturation of the central and peripheral nervous systems.

CRINKLY-type receptor kinases are unique plant membrane receptors with key roles in plant growth and development, in different tissues and throughout the plant's life cycle. Their cellular functions are poorly characterized and it is unknown what ligands they respond to.

I propose to combine protein biochemistry and X-ray crystallography with experiments in the model plant Arabidopsis to define what ligands CRINKLY receptors sense and how these ligands act in concert with the receptors to control very different developmental processes in different parts of the plant.

Specifically, my project aims to employ in vitro and in vivo biochemical screens to identify ligand candidates for CRINKLY receptors and to validate these interactions in genetic and structural detail. Next, I want to combine structural biology and kinetic kinase assays in vitro with reverse genetic experiments in Arabidopsis to understand ligand-induced receptor activation of CRINKLY receptors. Finally, I would like to define down-stream signaling targets of activate CRINKLY complexes in different plant tissues, by performing in vivo interaction screens.

This project sets the stage to define in molecular terms the functions of a new class of membrane receptors in plants. Identification of bona fide CRINKLY ligands will enable us to better understand plant membrane receptor activation mechanisms, the connection to downstream signaling events, and the cellular roles of CRINKLY receptors in plant development.

Recently recognized to occur by the hundreds or thousands in the genomes of nearly all organisms, regulatory RNAs control complex biological functions ranging from embryonic development to bacterial virulence. Most regulatory RNAs function by base-pairing with target mRNAs causing altered protein synthesis. In bacteria, small RNAs (sRNAs) constitute the best-studied class of non-coding regulators estimated to control up to 20% of all genes in a given organism. Base-pairing of an sRNA with a target mRNA can occur by several mechanisms with different outcomes for the regulator: the sRNA can be degraded, sequestered, or stabilized. The factors dictating which mechanism will occur for a given sRNA-mRNA pair are currently only poorly understood, and it is not known how these mechanisms globally affect sRNA function at the systems level. To address both questions, we will study the sRNAs controlling group behavior and virulence in the major human pathogen, Vibrio cholerae. This system relies on four homologous sRNAs, Qrr1-4, and one additional sRNA regulator, VqmR. For both types of sRNAs we have defined distinct sets of target mRNAs allowing us to probe base-pairing regimes to classify mechanisms of regulation. We will study the molecular principles underlying target selection by the sRNAs, and examine their impact on crosstalk among target mRNAs. We will test the role of sRNA-controlled regulatory mechanisms on stochastic gene expression and screen for network motifs operating at the post-transcriptional level. We expect that our work will provide insight into some of the most general features of RNA-based regulation applicable beyond the limits of a single RNA or organism.

Contrary to the traditional view of sensory cortex as a passive filter for sensory input, recent research indicates that even computations in primary sensory cortex are strongly modulated by the current behavioral demands of the animal. However, the afferent pathways that mediate this type of modulation and the consequences for processing in the cortical circuit are only beginning to emerge. While several recent studies have focused on the role of neuromodulation in this process, here I propose to dissect how non-specific thalamic afferents from MGm/PIN modulate the function of auditory cortex for learning and memory expression. Using in vivo 2-photon calcium imaging and patch-clamp recordings in combination with viral and transgenic targeting strategies, and optogenetic activity manipulations, I will investigate the role of these afferents in auditory fear conditioning as a robust and highly controlled learning paradigm. Previous converging evidence from lesions and recordings suggests that MGm/PIN is a critical area for fear learning and memory expression, but how it communicates with auditory cortex during this process is completely unknown. Interestingly, these axons prominently target neocortical layer 1 (L1), which controls dendritic integration in pyramidal neurons and disinhibitory circuits, and has been suggested to play a critical role in top-down modulation and association. In addition to dissecting how brain-wide interactions between specific neuron types contribute to learning and memory, I expect my investigation will reveal important general principles of how cortical circuits are modulated for processing of salient stimuli.

Testosterone (T), estrogen (E), and progesterone are vital for the development and function of diverse organs, including reproductive tissues, bone and the brain. Recent studies have also uncovered the fundamental principles whereby sex hormones control neural circuits underlying complex social behaviours. Dysregulated sex hormone signaling is correspondingly important in various diseases, including tumors of reproductive tissues, osteoporosis, neurological and psychiatric illnesses. Sex hormone signaling is classically believed to be initiated by the passive diffusion of the lipophilic hormone across the cell membrane followed by binding to cytosolic ligand activated transcription factors. The published literature however provides tantalising (and compelling) hints of diverse transport mechanisms for the mediated uptake of sex hormones across the membrane, thus questioning the passive diffusion theory, which also fails to explain why a lipohilic molecule would passively move from the hydrophobic membrane to the hydrophilic cytosol. Additionally, sex hormones also initiate rapid non-transcriptional effects, thus demanding the identification of non-canonical sex hormone receptors. Given the vital role of sex hormones in health and disease, I propose to use powerful unbiased approaches to identify these transport and signaling mechanisms, with a focus on T and E. I propose to use a CRISPR library based genetic screen and CLICK chemistry based proteomic screen to identify novel T and E transport mechanisms and receptors. I also plan to study the functions of candidate genes, in the establishment of sexual dimorphism in the mouse brain and in governing social behaviour.

Understanding how activity propagates through the brain is a major focus of neuroscience. The ability to eavesdrop on the activity of single neurons has been utilized for decades, but only recently have we begun to appreciate the complexity of neural networks. Through the use of new techniques, this topic can now be addressed at a mesoscopic level, which has the powerful advantage of being able to examine brain-wide interactions. Importantly, these methods can be used in awake and behaving animals, which is critical for understanding normal brain function. I will utilize these novel tools to investigate the interactions between the cerebral cortex and basal ganglia, which represents a ubiquitous architecture for supporting sensory, motor, and associative functions. The cortex is commonly described as a serial arrangement of sensory to motor regions, yet a multitude of pathways allow for an omnidirectional flow of information. In the first set of experiments, I will use widefield imaging to characterize how activity propagates throughout the cortex during a visual discrimination task. In the second set of experiments, I will combine this imaging with electrophysiological recordings of the striatum to investigate bidirectional interactions between the cortex and basal ganglia. In the third set of experiments, I will selectively inactivate corticostriatal projections to test the causality of this pathway in the interactions observed in the first two experiments. Together, these results will provide a step towards understanding how multiple areas work in parallel to drive sensory-guided movements.

Signalling pathways allow cells to quickly respond to changes in their environment. Defects in pathways such as MAPK have been directly implicated in many diseases such as cancer, Alzheimer’s, and diabetes. Our ability to understand and engineer these pathways has been hindered by their complexity, with pathways involving thousands of protein interactions, and technological limitations preventing the study of pathways in the context of the whole cellular system.

I propose to develop and leverage highly-multiplexed gene synthesis and single-cell genome-wide expression profiling techniques in order to engineer novel signalling pathways in human cells. I will develop microfluidic based gene synthesis methods to create libraries of protein kinase variants with altered interaction specificities and introduce these pathway components into human cells in a step-wise approach. At each stage, cells will be exposed to different stimuli and the interactions of each kinase variant with endogenous pathways will be mapped out by determining differences in their transcript expression profiles using a multiplex approach based on drop-seq. These interactions will be used to design subsequent stages in the pathway, with the aim of developing a fully orthogonal pathway at the final stage.

I will use this approach to: (i) elucidate a set of fundamental design rules governing the protein interactions in these pathways. (ii) design and test pathways capable of perturbing native pathways for use as research tools and in therapeutic applications. (iii) implement fully orthogonal signalling pathways for use in mammalian synthetic biology applications.

The objectives of the planned research are to characterize the dynamic structure of a G-protein coupled receptor in micelle and phospholipid nanodisc environments in solution. The research will rely on the directed evolution technologies developed in the laboratory of Dr. Andreas Plückthun at the University of Zurich for expressing functional GPCRs at high yield in E.coli. This approach has enabled bacterial expression of isotope labeled neurotensin receptor NTR1 suitable for NMR studies in solution. The Plückthun group also contributes numerous functional assays and important insights into GPCR biology. The Wagner laboratory will pursue a characterization of the dynamic structure of NTR1 using NMR. We will use new 15N-detected and other advanced experiments to assign backbone and methyl-bearing side chains of the receptor. We will use relaxation dispersion and other methods to characterize internal motions of the receptor in the presence of agonists and antagonists in order to elucidate allosteric mechanisms by which the GPCR transmits signals. We have furthermore developed methods to place receptors in phospholipid nanodiscs where the surrounding membrane scaffolding protein is covalently circularized to produce circular nanodiscs (cNDs) of exact size with tunable diameters from 11 to 80 nm. Thus, the diameter of the cND nanodisc can be tuned to include a single copy of the receptor. The cND-enbedded NTR1 can be complexed with heterotrimeric G protein and/or arrestin, expressed in insect cells, which has also already been worked out by the team. Dynamic changes of the GPCR upon such interactions and upon agonist antagonist interactions will be followed by observing changes of methyl group chemical shifts and dynamics. Our preliminary results provide for the first time a complete map of NMR backbone and methyl signals of a GPCR suitable to characterize dynamics of signal transduction. The technology of monitoring dynamic events upon ligand binding in the near-native membrane environment of cNDs offers unique tools for elucidating molecular mechanisms of dynamic GPCR signaling. This project will significantly complement and extend our knowledge about GPCR mechanisms beyond recent advances in GPCR x-ray crystallography, as it is not constrained by the necessity to grow crystals of particular states, and adds information on dynamics previously inaccessible.

Compartmentalized localization of mRNAs has been recognized as an important regulator of fundamental processes that govern cell polarity, cell fate, cell movement and differentiation including early embryonic patterning and asymmetric cell division. Several mechanisms are known to enable non-random distribution of transcripts, such as localized protection from degradation, local anchoring and directed movement along elements of polarized cytoskeleton. These mechanisms are mediated by a number of RNA binding proteins that can influence nuclear export, cytoplasmic movement and accessibility of mRNA to the ribosomes.
Although initially described in specialized cells and for a few genes, mRNA compartmentalization is now recognized as a mechanism used by all cell types including fibroblasts and epithelial cells. The extent by which regulated subcellular patterning exists across the transcriptome, and in which forms, is however not clear. Recent developments in technologies to quantify and visualize single transcripts in thousands of single cells in an automated manner at high-throughput, now make it possible to approach this unexplored but fundamental aspect of the transcriptome with large-scale approaches. This allows systems-level analyses of the types of transcript patterning and their correlation with other cellular properties at an unprecedented scale, as well as mapping the complexity of the molecular regulatory interactions involved. In this proposal, I will undertake such an approach, whereby I will focus on a set of gene transcripts encoding for proteins involved in endocytosis and ubiquitination of intracellular cargo.

Why are scars created? In lieu of regeneration, adult mammals patch skin injuries by scarring, the deposition of a fibrotic extracellular matrix that impedes organ function. In contrast to scarring setups in adults, the injured fetal back skin regenerates a complete replica of the original tissue, without scarring. Adopting a cellular genealogy view of scarring, we identified the coexistences of two unique embryonic fibroblast cell lineages in the back skin, one of which lacks scar forming capacity (Engrailed-1 negative fibroblasts; ENFs), whereas the second serves as the primary contributor to all back skin fibrotic developments (Engrailed-1 positive fibroblasts; EPFs). Cell lineages analyses demonstrate that early fetal dermis is predominantly ENF-derived, while mature dermis is primarily EPF-derived. I propose that distinct ontogenic programs in fibroblast lineages dictate the succession of EPFs over ENFs, culminating in the alternating dermal responses to injury, from scarless regeneration to skin fibrosis. The research objectives: (i) characterizing EPFs/ENFs cellular dynamics during fetal/adult transition phase (ii) characterizing EPFs/ENFs molecular changes during fetal/adult transition phase (iii) testing candidate genes to inhibit scar development. My overall goal is to identify the mechanisms giving birth to mammalian scar, and develop the knowledge basis for future therapeutic strategies that ameliorate fibrotic programs. Further, the lack of scarless/scar transitions in some mammalian species, where adult skin injuries heal without scars, adds evolutionary perspectives to this proposal, with biological & clinical inputs that extend beyond this immediate field.

Myopathies are extremely debilitating diseases. Muscle wasting, which is a systemic response to cancer-induced cachexia, is generally caused by excessive proteolysis. The main proteolytic pathways involved in general muscle atrophy are the ubiquitin-proteasome system (UPS) and the autophagic-lysosome pathway. However, the role of these pathways in muscle wasting induced by tumors remain poorly understood. Recently, Drosophila has been described as a model of muscle wasting, showing that tumors cause muscle wasting through systemic decrease of insulin signaling. I propose to study the role of UPS activity and autophagy in muscle degeneration by using a Drosophila model of muscle wasting. In particular, I will determine whether the manipulation of these proteolytic pathways contributes to pathogenesis, or can serve a protective role in muscle degeneration. In aim 1, I will characterize the muscle wasting phenotype by measuring rates of protein degradation, and also analyze the transcriptional profile of muscles during recovery after restoring systemic insulin signaling. In aim 2, I will study in detail the UPS activity in muscle wasting. And in aim 3, I will investigate how insulin signaling and other regulators control UPS and autophagy mediated proteolysis in this model of muscle degeneration.

Nuclear organization and epigenetic modifications of chromatin are crucial determinants of gene expression, closely linked to physiological variation and disease. Current methods are not adequate to answer how the chromatin is modified and organized for particular gene loci at the single cell level. I propose to develop a framework for highly-multiplexed in situ visualization of chromatin and its modifications, in the architectural context of the whole cell nucleus. Epigenetic modification markers will be immunolabeled together with the protein targets important for architectural organization, activity, cellular identity and physiology. These targets will be visualized using the principle of the super-resolution DNA-PAINT/Exchange-PAINT approach, developed by the host laboratory of Prof. Peng Yin. Following, defined genomic sequences will be specifically marked on the same sample by Oligopaints, dual-functionality FISH probes recently designed in the laboratory of the co-mentor Prof. Ting Wu.

I will first advance both the Exchange-PAINT and the Oligopaint approaches to achieve high-levels of multiplexing (up to 100 different markers) at <50 nm resolution. Next, I will validate my methodology on isolated chromatin, and in cultured cells by targeting epigenetically well-characterized gene loci. I will then apply it to investigate how the chromatin is changed during neuronal differentiation of induced pluripotent stem cells (iPSCs), specifically for highly-regulated genes. Finally, I will implement it on mouse brain sections, and probe different types of cells, to contrast the neurons differentiated from iPSCs in culture with terminally differentiated neurons in the tissue.

A biosignature is a feature in the rock record of unequivocal biological origin. Biosignatures are used as proxies to investigate the extent of life on Earth in the absence of preserved biological material. Despite the requisite for a biosignature to be derived from a process unique to biological activity, very few biosignatures have been empirically tied to a biological process. Microbial Fe reduction is one of the most primitive and geochemically significant biological processes. Dissimilatory metal reducing bacteria (DMRB) use soluble or insoluble Fe(III) as a terminal electron acceptor during Fe respiration. Microbially mediated reduction of Fe(III)-bearing minerals will lead to patterns of Fe speciation in the growth substrate, however, this putative biosignature has not been empirically linked to Fe respiration. Here we propose to empirically identify geochemical patterns of microbial dissimilatory Fe reduction using a combination of spectromicroscopy and genomic approaches. Using controlled mono-cultures, we will identify gene expression profiles consistent with active dissimilatory Fe reduction. Substrate-level Fe speciation patterns will be characterized by near-edge X-ray absorption fine structure spectroscopy (NEXAFS). Comparing the mono-culture gene profiles of dissimilatory Fe-reduction to metatranscriptomes from community cultures enriched for DIRBs we will be able to identify active dissimilatory Fe reduction and verify Fe speciation patterns in the substrate using NEXAFS. Genomic insight into mechanisms of interaction between microbial communities and their abiotic substrates will lead to geochemical proxies for metabolic activity.

We propose to apply a unique combination of molecular biology, physical chemistry and nuclear magnetic resonance to addressing a major unsolved problem in the study of biological systems: how to observe the dynamics of cellular function deep inside living organisms with high sensitivity and spatial resolution. Existing optical technologies such as fluorescent proteins are difficult to detect in opaque tissues, while high-resolution non-invasive techniques such as magnetic resonance imaging (MRI) lack sensitive genetically encoded reporters. To bridge this gap, we will develop genetically encoded imaging agents for hyperpolarized MRI – an advanced physical technique capable of increasing the sensitivity of MRI by up to five orders of magnitude. These imaging agents are based on a special class of proteins that interact with xenon, a biocompatible noble gas that can be hyperpolarized and introduced into the body by inhalation. Xenon dissolves in blood and is distributed throughout the body, enabling xenon-based MRI (Xe-MRI) of tissues such as the brain. We recently discovered that gas vesicles (GVs), a unique class of genetically encoded protein nanostructures, can bind hyperpolarized xenon and produce Xe-MRI contrast at picomolar concentrations (an improvement of ~10,000-fold over the state of the art). Based on this discovery, we propose to develop GVs as ultra-sensitive genetic reporters of mammalian cell migration, viability and function in vivo. To achieve this goal, we will characterize the physico-chemical properties of GVs and optimize them via their DNA sequence for optimal Xe-MRI detection. We will then transplant the genes encoding GVs into mammalian cells of interest, and develop methods to use Xe-MRI to image GV-labeled cells in vivo. To demonstrate utility, we will use this technology to image the trafficking of immune monocytes to sites of neuroinflammation. This research is enabled by the unique and complementary expertise of the co-investigators. Schröder introduced the NMR methodology enabling sensitive imaging of Xe-MRI reporters and is an expert in physical chemistry and NMR. Shapiro discovered the use of GVs as genetically encoded imaging agents and is an expert in biomolecular and cellular engineering and in vivo MRI. Successful implementation of this proposal will transform multiple areas of pre-clinical biomedical research.

Over the last few years, significant advances in identifying genetic loci underlying phenotypic variation have been made. However, our mechanistic understanding of how these genetic variants exert their effects and manifest in a phenotype remains sparse. Proteins are arguably the most relevant biomolecules defining the phenotype of a cell, unraveling the genetic factors determining protein abundance is therefore crucial to link genotype to phenotype. Here, I propose to systematically characterize the genetic architecture of protein abundance variation at high resolution using a novel, powerful approach. Single-cell protein abundances will be quantified via GFP tags in very large populations of mutagenized yeast cells. Mutants exhibiting extreme protein levels will be analyzed by next-generation sequencing and causal mutations will be directly identified using state-of-the-art statistical methods from rare variant association studies. From the resulting data, I will extract general principles of the genetic architecture underlying protein abundance variation, including the number of loci involved, their effect sizes, and whether they are enriched in non-coding DNA, non-coding RNAs, or coding regions of specific classes of genes. For selected proteins with orthologs involved in human disease, I will use engineered yeast strains to dissect the mechanism by which the specific genetic polymorphisms exert their effects on protein levels. I expect these analyses to provide refined knowledge of the genetic network and cellular processes involved in the corresponding human diseases and to reveal potential candidates for medical intervention.

Transmission electron microscopy of frozen hydrated specimens (cryo-EM) has recently enabled reconstruction of many macromolecular complexes, offering insight into their function. However, high resolution cryo-EM structure determination so far has been restricted to relatively large, rigid assemblies due to the low visibility of biological matter in conventional TEM. Similarly to Zernike phase contrast in optical microscopy, the signal from transparent samples can be amplified by interference of the scattered wave with a retarded transmitted wave. We are developing such a phase contrast (PC) TEM based on laser control of the electron wave function. Laser-induced potential allows for stable, lossless electron retardation, immune to electron beam damage. Improved image contrast will enable extending cryo-EM methods to smaller protein complexes or to those with greater structural variability. We propose to use PC TEM to investigate the mechanisms of chromatid segregation in mitosis by studying the structure of the budding yeast protein complex Dam1. Dam1 is a microtubule-binding part of the yeast kinetochore, an organelle coupling spindle microtubules to chromatids. Dam1 oligomerizes to form rings that hold on to the receding microtubule end, transducing the force pulling sister chromatids apart. Unassembled Dam1 presents a challenge for cryo-TEM reconstruction due to its small size and partially disordered structure, making it a perfect test target for PC TEM. The details of Dam1 structure are expected to elucidate the mechanisms of its coupling both to the microtubule and to proteins tethering it to the kinetochore, making an inroad for unraveling kinetochore complexity.