Clathrin-mediated endocytosis (CME) represents a main pathway that enables cells to take up extracellular and membrane-associated material. Membrane tension represents a major obstacle for vesiculation during CME. In mammalian cells, force provided through actin polymerization is required for formation of deep membrane invaginations and neck constriction for efficient CME under high membrane tension conditions. The goal of the research described in this proposal is to build a structural model for how actin provides force during CME in mammalian cells. By applying state-of-the-art cryo-electron tomography, I will investigate the three-dimensional architecture of the actin network during CME progression in mammalian cells. Using a previously described cryo-electron tomography compatible protein tag, I aim to create a molecular map for actin interactors that mediate CME. Additionally, I propose that the actin network adapts to plasma membrane tension in CME through changes in filament and associated molecule number as well as filament organization. To test this hypothesis, I will manipulate membrane tension prior to sample preparation for examination by cryo-electron tomography. The data obtained will be used to create a comprehensive prediction model for force generation by actin in CME.

The ability to tolerate an evolving definition of ‘self’, is crucial for every living being, however, its origin and complexity is barely understood. Recently, extracellular vesicles (EVs) have been shown to promote tolerance in cancer. Additionally, antigen-presenting cells (APCs) can transport tumor- and self-derived EVs to lymph nodes where immune responses can be generated. As tumors rarely invent pathways but rather use available tools I hypothesize that EV transfer is a yet unrecognized, major mechanism in immune homeostasis. Here, I will assess the impact of self-antigen transfer during immune homeostasis. I will perturb the system with viral infections to discriminate the role of tolerogenic EV-transfer from the transfer during an immune response. I will use mice expressing ZsGreen in alveolar epithelium and infect them with dsRed-labelled influenza to label ‘self’ and ‘foreign’. APCs in draining lymph nodes will be assessed for EV transfer through marker expression. I will visualize transferred EVs with lattice light sheet microscopy, and with large antibodies panels that delineate vesicular pathways, to discriminate handling and fate of EVs associated with influenza from tissue-derived EVs. Both EV types and APCs bearing them will be tested for their potential to elicit an immune response. Furthermore, EVs will be subjected to quantitative proteomics to understand how ‘self’ is encoded. Finally, an in vitro CRISPR screen followed by functional assessment in mice will identify regulators of EV release and uptake. Assessment of EV fate, content and origin in immune homeostasis will lay the foundation for future therapeutics in autoimmunity and cancer treatment.

The evolution of birds from dinosaur ancestors was accompanied by numerous changes in morphology. Of these, the near-complete loss of the tail was arguably the most significant single anatomical modification. While the fossil record has demonstrated the progressive loss of this main appendage, the underlying mechanisms that caused this remain unknown. Our project aims at understanding the developmental pathways that changed over the course of dinosaur to bird evolution giving rise to the modern avian tail. Since this change in morphology occurred quite fast in evolutionary terms, we hypothesize that discrete changes in gene expression potentially had an organ-wide effect on adult morphology, effectively stunting tail formation as a whole. Because non-avian dinosaurs no longer exist, this project will instead use American alligators, as they are the closest living relatives to birds, and will compare their embryonic development and genetics to the domestic chicken. We will focus on differences that exist at an anatomical level, and relate them to tissue and cell dynamics by performing a detailed characterization of posterior axis formation between species, using techniques such as in situ hybridization, immunofluorescence and cell-fate analysis. We will also create for the first time a full transcriptome of tail development in both alligators and chickens. We will use this information to then perform cutting-edge functional studies combining in vivo electroporation, alligator-chicken xenotransplantations and avian germ-line transgenesis, in order to precisely find the genes and cell behaviors that changed in tail development over the course of bird evolution.

Cells that make up the same tissue or organ tend to have a stereotypic physical size, while variability in cell size is a marker of abnormal cell growth. Cell size homeostasis depends on a coordination between growth and cell division cycle. How size homeostasis is achieved in mammalian cells is still debated and contributions remain scarce due to the difficulty in measuring cell size parameters accurately. In this project, I propose to combine two independent and precise methods for cell size measurement: quantitative phase microscopy to measure cell dry mass and fluorescence exclusion, developed in host lab, to measure cell volume. Furthermore, I will introduce well-controlled perturbations in the cell environment, in culture medium and physical environment of cells, such as depletion of nutrient and growth factors, modulation of cell adhesion and spreading, as well as cell confinement. This will provide a general picture of the dynamics of single cell mass and volume as a function of environmental parameters. Specifically, I will:1) record and study fluctuations in mass and volume growth across the cell division cycle and 2) Study mechanisms that couple mass and volume thus modulating cell density. Additionally, physiological relevance will be studied using activation of primary naïve T-cell differentiation in culture, where a drastic increase in cell size precedes asymmetric divisions in size and fate. This work will provide a fundamental understanding of how normal and cancer cells grow and regulate their size. It will also elucidate functional consequences of modifications in mechanisms of mass/volume coupling during the development of specific immune functions.

Though the central task of the brain is to detect important sensory stimuli to shape appropriate behaviors, our mechanistic understanding of the underlying neural processing is scarce. Here I propose a research plan that will explore the neuronal operations that transform visual information into behavior in the superior colliculus (SC). The SC has been assigned a role in extracting visual saliency, processing spatial attention and instructing goal-directed behaviors across species. Technical and methodological limitations, however, have restricted a detailed mechanistic understanding these critical neuronal computations. Here I aim to overcome these barriers by taking advantage of the modern neurogenetic tool-box and adapt the mouse as a model organism to study visual spatial and salience processing in the SC. Head-fixed mice will be trained to report the presence and location of a differing, salient stimulus among a row of identical stimuli in a sensorimotor two-alternative forced choice detection task. Simultaneous optical recording of the activity of thousands of genetically defined SC neurons, in combination with optogenetic manipulations and intracellular electrophysiology, will allow matching neural transformations to the animal’s performance. The proposed design permits testing and comparing neuronal responses to a wide range of stimulus features, the analysis of context dependent neural coding principles (e.g. salience), attention, as well as the interrelationship of neural and psychophysical discrimination capabilities. Together, this multidisciplinary approach will determine how neural circuits encode and transform sensory signals to guide directed behavior.

G protein-coupled receptors (GPCRs) comprise the largest class of human membrane proteins and are targeted by a third of all existing drugs. Remarkably, in a process called biased signaling, different extracellular ligands binding to the same GPCR can trigger distinct intracellular signaling pathways by favoring either G protein or arrestin activation. Despite dramatic progress in the structural biology of GPCRs and their intracellular binding partners, little is understood about arrestin activation and how biased signaling is achieved. In this work, I will use extensive atomistic molecular dynamics simulations to elucidate GPCR-mediated arrestin activation and how it differs from G protein activation. I will closely collaborate with experimental scientists and design complementary experiments that validate and guide the simulations. The research will identify GPCR conformations and phosphorylation patterns that preferentially couple to either G proteins or to arrestins, providing a foundation for the rational design of biased ligands that selectively induce specific signaling profiles. Uncovering how biased signaling occurs at the molecular level is essential not only for a deeper understanding of biological signaling, but also for the development of safer and more efficient drugs, such as superior antipsychotics, medications for heart and pulmonary disease, and powerful pain medications that lack the dangerous side effects of current opiates.

Transparency and enhanced near-infrared (NIR) reflectance are two fascinating and widespread characteristics found in a broad array of animals. However, despite some functional and ecological claims, the biophysical bases for their occurrence in nature are largely unknown. Since the physics of light scattering is well understood, theoretical models have suggested different routes for achieving each phenomenon by modification of variables like refractive index, size, shape and stacking parameters of cellular and extracellular scatterers. The proposed project aims to provide a deep empirical and transdisciplinary analysis of the predicted ultrastructural, physical and chemical properties of transparent and NIR reflecting tissues. To accomplish this objective I propose to use glass frogs (Centrolenidae) as an ideal study system in which both optical phenomena coexist. First, I will design a set up for correct measurements of diffuse and direct transmittance spectra of frogs’ tissues to objectively quantify transparency. Ultrastructure analysis will be performed by TEM, SEM and FIB-SEM to identify main intra and extracellular scatterers and accurately measure their size and distribution. Based on this ultrastructure information I will use Monte Carlo methods, finite element analysis, finite difference time domain, and discrete dipole approximations to model light propagation in order to explain how transparency and NIR reflectance are attained.

Cell geometry, hormones and mechanical stress have all been involved in Cell division plane orientation (CDPO), yet the corresponding molecular mechanisms remain unknown notably because 1- CDPOs are more variable than initially anticipated, 2- the relation between microtubule dynamics and CDPO robustness has not been analyzed, 3- the mechanotransduction pathways controlling CDPO are unknown. Using 4D live imaging, quantitative image analysis and biomechanics, I will analyze how microtubule dynamics is used, or filtered out, to generate robust cell division planes, and in turn how tissue tension feed back on microtubule dynamics to control CDPO. Using the Arabidopsis shoot apical meristem as an experimental system, I will draw a correlation map between microtubule dynamics, cell shape and growth, cell cycle, and CDPO and identify the spatio-temporal scales at which these correlations exist. I will then test the corresponding causalities using mutants affected in microtubule functions and CDPO robustness (TTP complex), mechanosensing (e.g. FER, DEK1), and micromechanics (compression, ablation, inducible lines and mosaics with cell wall defects). I will thus challenge the robustness of cell divisions and characterize the mechanotransduction pathways controlling CDPO.

Autophagy is a vital cellular process in all eukaryotes in which cytoplasmic organelles, proteins, and pathogens are inexplicably engulfed in a double membrane and transported to the lysosome for degradation and recycling. While the biochemistry is still poorly understood, defects in autophagy are known to result in different pathologies including inflammatory, neurodegenerative, and neoplastic diseases. More than 30 proteins orchestrate autophagy, including the single membrane protein Atg9 which participates in different complexes that regulate the initiation and trafficking of the autophagosome. Although protein partners and molecular mechanisms are relatively known for Atg9 in yeast, not much is known for the mammalian counterpart. Elucidation of the atomic structures of Atg9 and its complexes from yeast and human would provide important mechanistic insights, and may guide the detection of additional partners for the mammalian homolog, and the development of new pharmaceuticals. I propose to use leading methods in cryogenic electron-microscopy (CryoEM) to determine the structures of both the yeast and human Atg9 proteins alone, and in various functionally important complexes. Specifically, I would focus on electron micro-diffraction (MicroED), electron crystallography, and single particle reconstructions (SP). Overall I aim to join the Gonen laboratory to gain new expertise in membrane protein structural biology, as well as in the aforementioned cryoEM methods, and to encounter new biological systems by exploring the otherwise inaccessible membrane protein complexes involved in autophagy, a crucial yet poorly understood process.

Pediatric high-grade gliomas (pHGG) are the number one cause of cancer-related death in children. Intra-tumor heterogeneity is widely accepted as a major bottleneck for successful cancer treatments and has not been characterized in pHGG. Heterogeneity in clinical tumor samples is governed by cancer cells genetics, their differentiation lineages, cancer stem cell programs and the tumor micro-environment (TME). We recently established the application of single cell RNA sequencing (scRNA-seq) to adult gliomas, demonstrating the ability to identify diverse tumor subclones, redefine cancer stem cell programs and characterize their TME. Here, we propose to extend these approaches to pHGG in order to: (i) Characterize the cellular states within pHGG patient samples, and to identify putative cancer stem cells and differentiation programs; (ii) Evaluate the capacity to model pHGG through analysis of patient-derived cellular and mice models; (iii) Compare the cellular states of distinct pediatric and adult glioma subclasses, leveraging datasets from multiple projects towards an integrated view of glioma classification and biology; (iv) Use the emerging collection of cellular states in adult and pediatric gliomas to deconvolute the compendiums of bulk glioma samples, and model each tumor as a combination of distinct cellular states. This will extend our work to hundreds of glioma samples measured in bulk, and would facilitate the association between tumor composition and clinical features. Through these studies we expect to fundamentally change the understanding of pediatric glioma and provide guidance for new therapeutic approaches.

Understanding of what controls plant growth has been a focal point for plant scientists since the dawn of botanical research, yet basic concepts of growth physiology remain poorly understood. Plant growth dynamics involves complex molecular networks integrating the effects of many factors; here we focus on its dependence on photosynthetic and metabolic performance. Recently, novel ground-breaking technologies paved the way for complex analyses of plant growth and metabolism with the implementation of 13CO2-based metabolite flux-profiling. In addition, a new desert green alga, Chlorella ohadii, was isolated, exhibiting unparalleled growth and photosynthetic rates, potentially providing us with essential, missing information on the photosynthetic machinery and what sets the upper growth limits. We propose a system-oriented approach dissecting the specific impact of photosynthetic & metabolic performance on plant growth by focusing on photosynthetic cell models - green algae of varying growth rates. Comparative metabolic flux analysis based on microfluidics platform is expected to yield important insights on potential factors and pathways controlling growth-rate of photosynthetic cells, to be further characterized for the underlying mechanisms at the cellular level and relevance for the whole plant scale. Identification of patterns, shunts or ratios exhibiting significant kinetic alterations and playing a potential role in increased algal growth rate may serve as a source for potential targets for plant growth improvement. In an era when many are seeking for ways to increase biomass for food and energy production, the importance of these questions can't be overestimated.

Background: The visual system is organized in a hierarchical manner, with the complexity of neuronal computations increasing from the retina to higher brain regions. How complex feature detectors, such as face cells – which selectively respond to images of faces – are built from simpler feature detectors remains unclear. Developments in virus-mediated transsynaptic circuit tracing techniques allow for unprecedented access to presynaptic inputs converging onto individual postsynaptic neurons. By combining such strategies with in vivo 2-photon calcium imaging, neuronal response properties can be studied across synaptically-connected networks, providing insights into how the feature selectivity of presynaptic neurons drives tuning of a postsynaptic cell.

Specific Aim 1: Using vivo 2-photon calcium imaging, we will measure visually driven responses in inferotemporal cortex during presentation of sets of images including faces and non-faces, and identify face cells.

Specific Aim 2: We will perform single cell-initiated circuit tracing to identify the location of the presynaptic neurons providing input to individual face cells.

Specific Aim 3: We will perform multi-site in vivo 2-photon calcium imaging to characterize the feature selectivity of the presynaptic neurons that provide input to individual face cells and make a model describing how the specific inputs combine to drive face selective responses.

Outcomes: These experiments will provide deep insights into the circuit level organization that creates a complex and behaviorally relevant feature selectivity.

The effector function of IgG antibodies critically depends on its N-glycosylation. Thus, the glycan represents a point of potential immunomodulation. The N-glycan is a bi-antennary complex type oligosaccharide with micro-heterogeneities implying variable levels of terminal sialic acid, galactose, fucose, and bisecting N-acetyl glucosamine residues. Different glycoforms of the IgG-Fc significantly influence the magnitude of effector functions by affecting the binding affinity of the IgG receptor-binding region (Fc) to diverse Fc receptor family members. It has been recently demonstrated that core fucosylation dramatically reduce FcgRIIIA binding, in vitro cytotoxicity, and in vivo IgG-mediated cellular depletion. Conversely, the effect of sialylation was dependent on the status of the core fucosylation. These studies however, are limited to a small number of symmetric N-glycans. The effect of arm-selective glycan modifications, as well as bisecting asymmetrical N-glycan, on the antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC) pathways remains to be further addressed. We propose to remodel IgGs by synthetizing an unprecedented panel of glycoforms, which will allow for the biological assay and for the structural studies. Herein, we will employ a novel library of recombinant human glycosyl hydrolases and glycosyl transferases that exhibit high branch selectivity in order to access to a large number of asymmetrical structures. We suggest that the access to a range of well-defined glycoforms may provide an exhaustive description of the role that carbohydrates play in improving the efficacy of mAb effector functions.

Although the hippocampus is critical to episodic memory, rules governing neuronal sequences content remain elusive. The present project aims to increase our understanding of cellular and network basis of this cognitive function. In order to pursue this goal, we will use neuron-size light emitting diodes (µLED) silicon probes to allow independent optogenetic control of 4 to 8 small fractions of CA3 pyramidal cells, combined with high-density silicon probes recordings. By this method, I will excite different groups of neurons at specific location and at specific times while mice are running in a circular maze, to imprint an artificial place field pattern. This artificial pattern should be subsequently be replayed during theta sequences and SPW-Rs. Further, fast pulse light protocols combined with real-time SPW-R detection will allow to implement specific manipulation of these sequences during offline replay periods. Finally, I will empirically contrast the ability of CA3 recurrent system to complete patterns upon artificial partial sequence activations, a hallmark property of the network that plays a key role in learning and memory. I believe that the present project will advance significantly our understanding of the neural sequences, a basic process underlying cognition.

Tumour masses comprise of heterogeneous cell populations which shape the tumour microenvironment and drive response to treatment. As such, dormant and senescent (D/S) cancer cells significantly differ from proliferative cancer cells and interact in an intricate manner with neighbouring cells; however the processes regulating such interactions have not been precisely described. In particular, while D/S cancer cells express genes related to immune response and secrete pro-inflammatory factors, immune cells can induce and maintain cell dormancy and senescence during tumour immunoeditting. The current burst of interest for cancer immunotherapy requires researchers to exactly understand the crosstalk between tumour cells and the immune system, therefore in this proposal the influence of D/S cancer cells on the response of breast cancer primary tumours and brain metastases (BCBM) to T-cell mediated apoptosis will be interrogated in the context of treatment with anti-PD1 agents. A multidisciplinary platform, integrating intravital (in vivo) imaging tools, unique reporters of cell behaviours, proteomics and transcriptomics analyses will be developed. The host lab has invented cutting edge intravital imaging technologies to monitor cell behaviour in live tissues and in complex in vivo environments, and this platform will allow us to unravel under-studied fundamental mechanisms driving the crosstalk between D/S cancer cells and neighbouring cells. Overall, this proposal will provide much needed information on the biology of tumour cell dormancy and senescence and might open new avenues for the development of novel therapeutics in breast cancer.

In addition to transcribing protein-coding messenger RNAs (mRNAs), RNA polymerase II (RNAPII) synthesizes numerous non-coding RNA (ncRNA) species. Many of these ncRNAs are transcribed from regulatory regions such as enhancers or divergent mRNA promoters. There is great interest in evaluating roles for such ncRNAs in gene regulation; however, their activities and the mechanisms underlying biogenesis are currently unclear. Recent work indicates that RNAPII initiates transcription of mRNAs and ncRNAs using the same DNA sequence information and protein machinery. Nonetheless, fundamental differences arise during RNAPII elongation: ncRNAs are typically short, unspliced and unstable. Probing the mechanisms that dictate the efficiency of transcription elongation and coupled RNA processing/stabilization at non-coding vs. coding loci will thus uncover fundamental regulatory principles and illuminate ncRNA formation and decay. I will study these processes in mouse ES cells, with a focus on the ncRNA originating upstream and antisense of the Oct4 gene. Despite high-level initiation of this ncRNA, RNAPII typically terminates within 500nt, generating an unstable transcript. Thus, a fluorescent reporter integrated at this region exhibits moderate expression. Accordingly, sequences inserted upstream of the reporter will impact fluorescence if they alter RNA formation or stability, enabling sequence identification through a FACS-based strategy. With this system, I will test specific models for elongation control and 3’-end formation. Additionally, I will perform live-cell transcript visualization to assess the dynamics and impact of the Oct4 ncRNA transcripts on mRNA synthesis.

The capability to develop functional teeth has been independently lost several times in the evolutionary history of vertebrates. Tooth loss can occur either during ontogeny, known as ontogenetic odontogenic truncation and reported in many extant and extinct phylogenetically disparate vertebrate lineages, or during the process of tooth replacement, present in some mammals. Recent studies have shown that truncation of odontogenesis is associated with the evolution of horny beaks, but detailed developmental mechanisms involved in regulating these processes remain unclear. This research proposal aims to understand the mechanisms mediating both ontogenetic odontogenic truncation and the epithelial keratinization during beak formation. Using an integrated approach including immunohistochemistry, whole mount in situ hybridization and omics analyses, and various model animals including chicken, ducks and crocodiles, the proposed project aims to investigate the factors that initiate and maintain the species-specific temporo-spatial pattern of the ontogenetic odontogenic truncation, and what factors have determined the developmental fates of tooth and beak. Results from these experiments and simulations will provide new insights into tooth development, renewal and beak formation processes, and crucial information will be acquired in understanding the molecular mechanisms of the ontogenetic odontogenic truncation.

Recent epidemics of Ebola and Zika are stark reminders of the enormous impact of viruses on public health. Despite many recent technological advances in related fields, systematic methods to investigate virology have lagged behind, so that many of the global principles underlying viral infection remain unknown. One critical area of study is how viruses evade the host immune response. During infection viral peptides are uploaded on the major histocompatibility class I molecule (MHC-I) and presented to cytotoxic T-cells. Some viruses respond by inhibiting the MHC-I pathway globally, but many infected host cells maintain an active presentation pathway, suggesting the existence of additional viral response mechanisms. Since traditional studies investigate only a few peptides at a time, we have not achieved a comprehensive view of the collection of immunogenic peptides in each virus and the causal effect of mutations on immunogenicity. During my postdoctoral research, I will perform a systematic investigation of immunogenic peptides in hundreds of viruses and characterize the regulatory mechanisms underlying their syntheses. To this end, I will combine computational and high-throughput molecular approaches including synthetic library design, mass-spectrometry and ribosome profiling. In addition to investigation of wild-type antigens, I will design sequences to match the mutations acquired by viruses during transmission and evolution to infer their causal effect on immunogenicity. Analysis of this data should reveal basic principles of viral immune evasion, the regulatory mechanisms involved and the forces driving viral evolution during large outbreaks.

Without a simultaneous tactile stimulus, the spatial acuity of thermal stimuli is extremely poor leading to gross misjudgments of stimulus location on the skin surface. However, the spatial receptive fields of thermoreceptors are estimated to be only a few millimeters. This suggests that the poor thermal spatial acuity observed in human perception must be occurring centrally, but how these thermal inputs are processed remains largely unknown. Here, using a combination of behavior, electrophysiology and optical techniques in the hind paw system of mice, I aim to address the spatial integration properties of tactile and thermal stimuli to uncover neural mechanisms of somatosensory perception. I hypothesize that the tactile acuity of the mouse hind paw will be significantly better than the thermal acuity as quantified by the spatial discrimination performance of the animal and the spatial receptive fields in the thalamocortical circuit. Spatial acuity of the mouse will be quantified using a head-fixed, paw-tethered behavioral paradigm (Aim 1). Neural integration of tactile and thermal inputs will be measured using thalamic and cortical recordings in mice performing this spatial acuity task (Aim 2). Finally, spatial perception of the mouse will be manipulated using a combination of optogenetic interventions (Aim 3). While there is significant perceptual evidence of the differences of thermotactile integration, we have not yet been able to link the behavior to the neurophysiological underpinnings. Gaining fundamental knowledge of how thermotactile stimuli are integrated would provide a critical framework to interpret both natural and atypical patterns of neural activity.

Phase transitions are emerging as a new paradigm for establishing membraneless organelles in cells. These phase separated compartments are complex; they contain many biomolecular species, each of which may prefer one phase “state” over another. However, for most intracellular phase transitions, little is known about composition, especially in different phase states, hindering comprehensive dissections of their physiological role. My goal is to develop methods for assaying the biochemical composition of intracellular phase transitions as a function of phase state. I will employ chemical proteomics, in which photo-reactive amino acids are integrated into phase separating bait proteins in cultured cells via genome expansion techniques. Combined with recently-developed approaches for control of intracellular phase transitions, photo-crosslinked complexes in different phase states will be isolated from cells and analyzed using quantitative mass spectrometry. Dynamic biochemical profiles describing which components are “locals” and which are “tourists” in the phase diagram will then be used to generate targeted hypotheses regarding cellular function. I will apply this approach to the stress granule component and ALS-associated protein FUS, whose biochemical profiles I will use to inform reconstitution of functional stress granules in vitro. This general methodology will provide a launchpad for advancing our understanding of the cellular stress response, intracellular organization by phase transitions, and the human diseases associated with aberrant biomolecular aggregation.