Although potentially of fundamental importance for many phototrophs on Earth, the physiological mechanisms and molecular underpinnings that allow survival over long periods of dark remain a mystery. Diatoms, the dominant oceanic eukaryotic photosynthetic organisms, specifically Fragilariopsis cylindrus (polar pennate diatom with a sequenced genome) and Thalassiosira gravida (representative Arctic centric diatom), will serve as models to characterize the physiological, cellular, genomic, epigenomic, and metabolic state of cells during prolonged darkness and the return of light. Complementary expertise in our team (culturing of polar species, optics, photochemistry, genomics) will allow us to identify key adaptive mechanisms used by diatoms.
Realistic light transitions from fall to early spring will then be simulated to grow F. cylindrus and T. gravida in specially designed bioreactors under stable nutrient and cold temperature conditions. Cellular energy flow and allocation will be monitored before, during and after a simulated 6-month polar night: light absorption coefficients and pigment composition, absorption cross-sections and concentrations of PS1 and PS2, electron production by PS2, redox states of the plastoquinone pool, linear and alternative electron flows, cellular content of electron acceptors, concentrations and activity of RUBISCO, rates of C-fixation, respiration, heterotrophy, and reduction level of storage compounds. F. cylindrus cells will also be examined for their transcriptional and translational activity, and will be subject to transcriptome and metabolome analyses. Mass spectrometry and immunoblotting performed on chromatin will identify major changes in DNA methylation and histone tail modifications. Features of interest will be subject to chromatin immunoprecipitation and DNA sequencing. DNA methylation and histone marks will be aligned to the reference genomes. Chromatin and gene expression states arising in response to prolonged darkness and the return of light will be defined, informing metabolic maps and physiologies. In parallel, organellar structures will be assessed by electron microscopy. Candidate genes encoding key processes for dark survival and recovery will be studied by producing transgenic strains of F. cylindrus, in which these specific genes will be knocked-out.

Animals have to constantly make decisions about what to do next. Especially, when navigating in the natural world, they have to cope with an abundance of potentially conflicting cues. Hence, in order to interact with the environment in a meaningful way, animals have to extract the currently relevant sensory features and select an appropriate, experience- or internal state-based, behavioral action. How this is achieved by the nervous system is poorly understood in any sensory modality and organism.

Landmark studies in primates have already revealed some principles of decision making but, due to technical limitations, could only focus on confined brain regions. However, understanding the complex process of how an animal selects the right behavior requires complete explorations of the hierarchical and parallel processing steps within neuronal assemblies in different parts of the entire brain.

The larval zebrafish is becoming the model of choice for studying brain-wide sensory processing as it offers a unique combination of tools: Larval zebrafish possess a variety of innate and robust behaviors in response to sensory stimulation, while giving access to state-of-the-art rapid whole-brain imaging techniques with cellular resolution.

Adapting successful concepts used in classical primate literature, I will use larval zebrafish as a modern model to investigate general principles of sensory integration, stimulus competition, decision making, and action selection. This will permit unprecedented detailed explorations of how neuronal circuits in an entire vertebrate brain extract features from a complex sensory world in order to decide what to do next.

In living cells, molecules organize into complex assemblies on the micrometer scale. Among these structures is the bacterial cell wall - a rigid exoskeleton composed of peptidoglycan chains cross-linked by short peptides. This two-dimensional polymer surrounds the cell and defines its shape during growth and division. Although its synthesis is the target of many antibiotics, how bacteria build the cell wall is largely unknown, even for a relatively simple cell like E. coli. To shed light on the mechanism of cell wall assembly I will develop an experimental assay to reconstitute peptidoglycan synthesis in vitro. Starting from the enzyme PBP1b and its substrate lipid II I will build a PG layer on a biomimetic membrane and quantitatively characterize the interactions, which can lead to a continuous, two-dimensional polymer. Using this novel reconstituted system, I will not only access the molecular mechanism of cell wall assembly, but also identify how local interactions contribute to the large-scale structure of cell wall, which ultimately determines the shape of the bacterial cell.

Filopodia are thin, actin-rich plasma membrane protrusions, which function as sensory organelles of cells. Despite a wealth of information on the physiological functions, the molecular mechanisms underlying their assembly and dynamics are incompletely understood. Filopodia are characterized by high negative membrane curvature and high density of phosphoinositides. Their formation and dynamics are controlled by an array of actin-binding and signaling proteins, many of which interact also with phospholipids. However, the mechanisms and biological roles of these lipid-interactions are largely unknown. We will collaboratively examine the molecular principles of filopodia assembly, with a specific focus on the roles of membrane curvature sensing and phosphoinositide clustering in this process. We will use advanced cell biology methods (genome editing combined with live-cell imaging approaches) to identify the mechanisms by which central filopodial proteins are recruited to these membrane protrusions in cells. Phosphoinositide and curvature sensing properties as well as potential synergetic effects of these proteins will be quantitatively measured by in vitro experiments (optical tweezers, confocal microscopy, FCS) using model membrane systems (membrane nanotubes pulled from Giant Unilamellar Vesicles) with controlled curvature, and also in silico (multiscale simulations) and in vivo with relevant mutant proteins. These computationally efficient simulations are based on systematic coarse-graining methods, in which the molecular resolution is reduced but the effects of key molecular features retained, thus adding insight into the cooperative processes underlying filopodia formation. Such insight will also help to connect the results of the in vivo and in vitro experiments. Finally, once a minimal set of proteins responsible for this synergy will have been identified, we will include actin and reconstruct a synthetic filopodium. Additional positive feedback is eventually expected to take place due to actin polymerizing against the membrane. Thus, by virtue of reconstituted systems, we will aim to identify the essential physical mechanisms underlying the very first steps of filopodia formation.

The cohesin-complex mediates sister chromatid cohesion from S-phase until mitosis and is involved in the formation of higher-order chromatin structure. To fulfill these vital functions, cohesin is loaded and positioned in the genome by mechanisms that are only poorly understood. In vitro, loading of cohesin on DNA only requires ATP and a loading-complex formed by Scc2-Scc4, while loading in vivo on chromatin is regulated by additional factors. For example, in Xenopus laevis oocytes, cohesin loading strictly depends on pre-replication complexes (pre-RCs), which are formed in telophase/G1.
Mechanistic studies are required to understand how cohesin-loading occurs at the molecular level. I will first determine the mechanism by which Scc2-Scc4 loads cohesin on DNA. Using single-molecule FRET and optical tweezers, I will monitor the effect of Scc2-Scc4 on conformational changes of cohesin as it is loaded on a DNA template. After characterizing this minimal loading reaction, I will reconstitute cohesin-loading during telophase/G1 using a purified system. With these experiments I will address why and how loading of cohesin is regulated by the formation of pre-RCs.

The capacity of cells to move and migrate is fundamental to their viability, whether they originate from multicellular or single-celled organisms. This is exemplified in the process of infection, such as that by the the protozoan parasite Plasmodium, the causative agent of malaria disease in humans. During an infection, cell migration for both the human immune cell or the malaria parasite both rely on force generation from structures within each that link them to, and propel them across, the extracellular environment. For the immune cell, its amoeboid-like movement is the product of polymerising actin filaments combined with force generation from a myosin motor, which together drive changes in cell shape propelling the cell at speeds of several micron/min. In contrast, while relying on very same actin-myosin proteins, the malaria parasite does not change its shape, yet can move at speeds of >1 micron/sec, an order of magnitude above our fastest cells. Whilst a great deal is understood about amoeboid migration, we know little about how malaria parasites achieve directional motility or such great speed.
Underpinning Plasmodium cell migration across its lifecycle, whether in the liver, the blood circulatory system or mosquito, is an unconventional myosin (XIV), lacking many of the canonical features associated with myosin motors. Together with dynamic parasite actin filaments these somehow generate a force that drives the parasite forwards, however, the mechanics of how this actually works is far from understood. Here, building on insights we gained into actin regulation and organisation in the malaria parasite from our first HFSP program, we turn our attention firmly on myosin to understanding how it interacts with actin inside the cell to produce directional cell migration. Combining the state-of-the-art in biochemical methods, molecular and cellular parasitology, biophysics and structural biology (including cryoelectron microscopy), we aim to dissect at every level - from single molecule to whole cell - how motor organisation inside the malaria parasite leads to directional, fast cell movement. This will uncover profound insights into the workings of an ancient, supremely fast cell migration machine, and may potentially reveal weaknesses that could be targeted to cure one of mankind’s greatest diseases

Macrophages play beneficial roles in protective immunity. However, they also favor the progression of several pathologies when they massively infiltrate diseased tissues. A present challenge in cancer, for instance, is to control macrophage tissue infiltration, which involves the mesenchymal motility. This motility is characterized by the ability of the cell to form protrusive structures called podosomes. A podosome constitutes a submicron core of actin filaments surrounded by a ring of integrin-based adhesion complexes. Our working model postulates a mechanical connection that counterbalances the actin-rich protrusive core by traction at the adhesive ring, likely embodied by acto-myosin contractile cables. Therefore core and cables would form a unique two-module protrusive force generator that balances forces at the level of a single podosome to ultimately contribute to the mechanics of macrophage migration.
Our objective is to build a sound experimental corpus to substantiate this two-module mechanism of force generation and determine its implications for macrophage 3D motility. To this end, we will characterize podosome molecular architecture and formulate its relationship to force generation. Furthermore, we will identify the mechanical role of key podosome components in cell migration.
We assembled a multidisciplinary team that combines cutting-edge expertise in: i) macrophage 3D migration, ii) 3D nanoscale imaging, iii) live super-resolution imaging, iv) cryo-electron tomography technologies allowing resolution of actin filaments within cellular networks, v) measurement of podosome protrusion forces and vi) mechanics of 3D cell migration in custom microfabricated environments.
Our ambitious research plan will deliver the nanoscale localization of podosome molecular components (applicants A1, A3), determine how the architecture evolves along with force dynamics (A2), investigate the mechanics of 3D migrating cells (A4) and reveal the role of podosome components in these uncharted features (A1-4). Thanks to this groundbreaking study, we will articulate mechanical and architectural insights into an integrated portrait of podosomes from the molecular scale up to the biological context of 3D cell migration and thus identify molecular means for the modulation of pathological macrophage tissue infiltration.

Neural oscillations, the byproduct of periodic fluctuations in synchronized brain activity, are a defining feature of neural circuits. While oscillatory activity is closely correlated with both an animal’s behavioral and cognitive state, it is unclear whether oscillations are causally involved in behavior and cognition. In this research proposal, we will focus on thalamo-cortical spindle oscillations, examining their possible causal role in memory consolidation. Our research proposal consists of two main research aims: 1) quantifying the causal relationship between thalamo-cortical spindles and memory-related oscillatory dynamics known as hippocampal sharp-wave ripples and 2) behaviorally measuring the causal relationship between spindle generation and memory consolidation. This will be accomplished by a synergistic merger of molecular-biology (Halassa lab) and a computational toolbox (Bendor lab), while performing large-scale neural recordings (Bendor and Halassa lab). Our combined, state of the art toolbox provides a more temporally and spatially precise method of recording, stimulating and blocking spindle activity, while directly addressing causal interactions using sophisticated Bayesian-based computational methods. Using these methods we will directly test two hypotheses, that thalamo-cortical spindles and hippocampal ripples have causal, bi-directionally interactions (aim 1), and that the amount of thalamo-cortical spindle activity is related to the strength of the encoded memory, with this effect being both modality-specific and sensitive to the timing of hippocampal ripples (aim 2).

Inorganic polyphosphate (polyP) is a biopolymer that serves multiple critical functions in biology. PolyP has been mainly studied in bacteria and shown to be involved in such diverse processes as the bacterial stress response, biofilm formation, plasmid uptake and antibiotic resistance. In comparison, its functions in mammals are significantly less well understood. Even so, polyP has been linked to multiple important physiological phenomena, such as blood clotting, chaperone function, posttranslational protein modification and neuronal signaling.
While in bacteria the enzymes that generate polyP are known (polyphosphate kinases Ppks), the mammalian enzymes remain elusive to date. In fact, we know that polyP is present in different cell types and that it is enriched in different organelles, but we do not know how and where it is made and how it is shuttled. Moving polyP research forward will critically require identification of the elusive enzymes that make and traffic polyP. Currently, a dearth of chemical tools has precluded such deeper insight into mammalian polyP physiology.
Our proposed research will culminate in new technologies required to make, modify, analyze, transport and deliver polyP both ex and in vivo. We will develop and combine multiple novel synthetic approaches to generate a range of modified polyP analogs that are not yet available. Furthermore, we will develop technologies to deliver and release those synthetic analogs in a controlled way outside and inside of cells. Together, these technologies will provide valuable novel insight into how cells make, regulate and distribute polyP. We will use these tools to identify the mammalian enzymes involved in polyP turnover, investigate how polyP levels are affected by other signaling molecules such as inositol pyrophosphates, and try to understand how polyP affects different biological processes. For example, an in-depth evaluation of the role of polyP in blood clotting will be achieved using our novel approaches. These studies will lay the foundation for further research into polyP physiology. By virtue of the combination of synthetic organic chemistry, drug delivery technology, and cell and animal biology, we will be able to develop and apply powerful novel tools dedicated to the elucidation of polyP physiology in mammals.

Genome architecture is emerging as a key epigenetic property providing either positional or topological cues that regulate gene expression. By following certain folding principles and controlling gene positioning within the nucleus or chromosomal interactions, the three-dimensional organization of chromatin seems to exert a pivotal role in coordinating the emergence of unique cellular phenotypes. During early mouse embryo development, the nuclear volume decreases ten times, while encompassing the same amount of genetic material. At the same time, transcriptional networks undergo drastic changes, switching from maternally-controlled to zygote-specific programs. While these expression patterns must unfold in a robust manner from one stage of development to the next, they also need to incorporate some degree of flexibility and cell-to-cell variability, eventually making every cell unique throughout differentiation. Deciphering how the interplay between genome organization and expression operates during this highly dynamic period in a multicellular organism’s lifespan is a formidable challenge. Here, I propose to establish a pioneering framework for assessing various aspects of genome organization while monitoring gene expression profiles in exactly the same single blastomeres, tracing their astounding dynamics while the embryo is developing into a blastocyst. By combining multi-assay measurements in the same single blastomeres with high-dimensional correlative data analyses, I aim at identifying general design principles governing DNA folding, as well as the crosstalk between genome architecture and gene expression.

Tuberculosis is an ancient disease caused by Mycobacterium tuberculosis that still exerts a massive burden on public health in many parts of the world. The success of M. tuberculosis is primarily due to its ability to survive within macrophages for months and even years in an asymptomatic latent state. It is still largely unknown what makes this pathogenic invader successful, or what determines if the immune cells eliminate the assault or succumb to it. Thus far studies of host-pathogen encounters were performed at the population level, averaging molecular signatures, while masking any cell-to-cell variation. The heterogeneous, stochastic, and dynamic nature of both host and pathogen suggest that descriptions of average behavior may fail to accurately characterize these interactions. Here, I propose to take a systematic quantitative approach to study the process of infection by M. tuberculosis, establishing a fluorescent method to specifically differentiate the outcomes of infection, and combining it with single-cell RNA-seq. Using this method I will examine whether variation in transcriptional profile of individual M. tuberculosis-macrophage encounters translate to distinct infection outcomes. I will apply this method to determine the degree to which genetic diversity among clinical strains is reflected in cell-to-cell variability of the transcriptional profile and translates to distinct of infection outcomes among these strains. Collectively my studies will expose mechanisms by which transcriptional heterogeneity affects the outcome of infection, and provide a holistic view on the process of M. tuberculosis infection.

Birds are paedomorphic dinosaurs: they exhibit in their adult skeleton some traits that were present in the juveniles of their dinosaur ancestors. The mechanisms underlying this paedomorphosis are largely unknown. We hypothesize that it is caused by differences in the rates of skeletal growth and differentiation, which are known to be higher in birds than in dinosaurs. The aim of this research proposal is to investigate the mechanisms that control how fast skeletal cells differentiate, and how their rates of differentiation influence the morphology of the skeleton. Most of the vertebrate skeleton develops by replacement of cartilage extracellular matrix by bone, a process called endochondral ossification. We propose to undertake this investigation by comparing endochondral ossification in embryos of two species of birds with different rates of development – quails and zebra finches – to embryos of alligators, which will be used as out-group. We will use two novel experimental approaches: (1) the adaptation of recently developed whole-mount techniques and cutting edge imaging technologies (micro-computed tomography and confocal light microscopy) to the study of skeletal development, and (2) the compared analysis of in vitro culture of cartilage cells obtained from the three species. Those experiments will allow us to characterize, in each species, the temporal and spatial expression of molecular regulators of endochondral ossification, its relation to changes in the shape and growth of the skeleton, the relative duration of cell cycle progression, and the rates of chondrocyte differentiation.

Upon antigen recognition, T cells switch from a fast migratory to a stationary state. T-cell receptor (TCR) recognises specific peptide bound to major histocompatibility complex (pMHC) molecules on the surface of antigen-presenting cells (APCs). TCR interaction with pMHC of high affinity leads to a migration stop and robust TCR signalling. However, how a molecular recognition event can coordinate global cellular changes remains unclear. Here, I propose that membrane tension is the cellular coordinator that translates antigen recognition into a stop signal and ultimately enables T cell activation. I hypothesize that TCR engagement in motile cells creates mechanical stress that increases membrane tension, leading to extracellular calcium entry through mechanosensitive ion channels, which results in arrest of locomotion, cytoskeletal rearrangement and TCR signalling. Using a FRET-based force sensor and micro-fabricated migration channels, I will quantify the mechanical forces that act on the TCR when motile T cells encounter immobilised pMHC molecules. I will then measure membrane tension with optical tweezers and membrane topography with 3D super-resolution microscopy as a function of pMHC affinity. Next, I will investigate the role of mechanosensitive ion channels and image cytoskeletal remodelling and trafficking of signalling vesicles in T cell during the transition phase. Taken together, the proposed project will provide a better understanding of how mechanical forces derived from cell motility mediate antigen-induced T cell arrest and activation.

Although many of the molecules that determine animal body plans have been uncovered in recent years, we still cannot predict the final design of an animal from a diagram of how these molecules interact. This limitation stems in great part from the fact that current technologies to visualize development rely on dead embryos or employ reporter systems that cannot capture the dynamics that underlie developmental processes. Measurements of these dynamics constitute just one step toward predictability; in order to sharpen our understanding, theoretical models that generate quantitative predictions must accompany such measurements. Our interdisciplinary team, which consists of researchers with extensive expertise in developmental biology (Andrew Oates, Francis Crick Institute), the theory and measurement of gene expression (Hernan Garcia, UC Berkeley), and protein engineering (Roberto Chica, University of Ottawa), is uniquely positioned to establish a predictive understanding of the gene regulatory networks that govern vertebrate body plans. We will focus on the segmentation clock in zebrafish, in which the length of body segments is thought to be determined by a network of oscillating genes. We will develop new technology to simultaneously monitor mRNA and protein concentrations in real time during segmentation of the zebrafish embryo. Furthermore, we will develop theoretical models that will leverage this new information to predict how molecular interactions lead to oscillations with a prescribed period and amplitude. These predictions will be tested via the creation of synthetic oscillators with engineered dynamical properties that will be used to generate embryos with altered body plans. We anticipate that these iterations of model and experiment will set the stage for a new paradigm in synthetic biology that enables the rational design of multicellular organisms.

Adipose tissue is now appreciated to be an endocrine organ, owing to the discovery of hormones such as leptin, which functions as an negative feedback neuroendocrine signal that keep weight in a narrow window of variation. The sympathetic nervous system (SNS) innervates all known organs, but the neuroanatomical origin of adipose innervation and its functional role in regulating adipose tissue phenotype and systemic metabolism remain largely unstudied.
Brain mapping owes its existence to ease of brain dissection and serial slicing. In contrast to this methodological ease, the anatomical location of the SNS along the anterior side of the verterbrae poses a difficult challenge for conventional histology, preventing a systematic study of these neurons. We here propose to perform whole body imaging using optoacoustic tomography, coupled with transgenic viral tracing for mapping subsets of SNS neurons projecting to brown (BAT), beige and white adipose tissue (WAT). These neurons will be functionally probed using chemogenic techniques.
We assembled a multidisciplinary research team that will work together closely on each phase of the project: Paul Cohen is a molecular biologist with expertise in white, beige and brown adipose biology. Ana Domingos is a neurobiologist with expertise in optogenetics who recently demonstrated the existence of neuro-adipose junctions. Daniel Razansky is a bio-engineer who has developed novel non-invasive methods for high performance molecular imaging, particularly opto-acoustic technologies.

Biological systems have evolved protein self-assemblies to generate efficient high-order nanoscale architectures and molecular machines. Translating biological organizational principles directly into synthetic protein cage-like nanostructures capable of sensing and acting on the environment in a programmable way, would constitutes a unprecedented scientific achievement and a formidable opportunity to interface and interrogate biological systems. However, it remains extremely challenging to unveil fundamental processes guiding three dimensional self-assembly of protein building blocks. Despite intense scrutiny, bottom-up methodologies enabling the de novo design and construction of tailored protein nanocages with programmable structure, dynamics and functionalities, are inexistent. The intention of this project is to take advantage of the recent progress of computational protein design to examine how complex and precise functional protein nanocage architectures can be rationally built. Specifically, I hypothesize that novel computational frameworks can be developed to engineer self-assembling nanorobots with important design space, capable of executing programmed tasks, such as triggered bioactuation, packaging and delivery of information to target structures. To this aim, we will expand protein-protein docking and interface modeling in order to establish libraries of modular and robust multifunctional components with specified architectures size and metastability, which will be experimentally validated in vitro. Establishing protein nanorobot programming as a universal framework would bring tremendous possibilities for basic research as well as biomedical applications.

Ethanol, a toxic substance consumed in many cultures, affects microbiota composition and chronic excessive consumption can lead to severe end-organ damage. Curiously, the role of microbiota in ethanol metabolism remains under-studied. Moreover, the selective pressure of ethanol on the microbiota, as well as its impact on the gut immune defences is under-investigated. I hypothesise that specific microbial clades are critical players in ethanol metabolism and that ethanol-driven microbial alterations are caused by ethanol resistance and by changes in the oxygen levels in intestinal lumen. Furthermore, these shifts and specifically microbially-generated ethanol metabolites impact gut and hepatic immune defences. Using isotopically labelled ethanol, I will determine the role of gut microbes in ethanol metabolism by ethanol metabolic flux analyses and identify the microbes that use ethanol as a carbon source. Next, I will investigate if anoxic-to-oxic shifts in the gut lumen and ethanol resistance drive the observed microbial shifts by employing cultivation devices with oxygen or ethanol gradients. I will also assess if chronic ethanol consumption results in intestinal angiogenesis. Finally, I will evaluate the impact of chronic ethanol consumption on gut immune responses using flow cytometry and confocal microscopy with a focus on innate immune cell population and function. Use of gnotobiotic mice with defined microbial communities informed by my microbiome-profiling experiments will further enlighten my immunologic analyses. This multidisciplinary approach will provide mechanistic insight for future studies aimed at reducing ethanol toxicity.

Perceptual decision formation in human and non-human primates involves accumulation of noisy sensory evidence through a process known as evidence accumulation. While evidence in favor or against a choice accumulate, the decision systems provide a continuous flow of information to the motor system and prepare it for the probable outcome. During decision formation, neurons show evolving activity patterns that are thought to represent the progression of the decision toward a choice. The circuit mechanisms that produce these decision-related dynamics, however, remain unidentified. This proposal aims at identifying the circuit motifs behind such computations. I have previously demonstrated that during olfactory decisions flies engage in an evidence-accumulation process before committing to a choice. Reaction times in a trained odor discrimination task increase, and perceptual accuracy declines, as stimulus contrast is reduced, in quantitative agreement with a drift-diffusion model of evidence accumulation to threshold. I have also demonstrated that the mushroom bodies of Drosophila play an important role in decision formation. My proposal builds on these findings and aims to understand how neurons in the decision centers interact during odor discrimination tasks. I will use a single-fly-based assay and a virtual-reality arena to understand how behavioral outputs are generated in the fly brain during decision-making. Such insights will provide neurophysiological underpinnings for the theoretical models of decision-making.

The structure of large protein complexes is difficult to obtain, due to their large molecular weight and the strenuous task of finding crystallization conditions. Another challenge is the presence of heterogeneity in the conformation of the complex, which complicates studies by solid-state NMR and makes the particle orientation more difficult to determine in single-particle cryo-electron microscopy.
The technique of cryo-electron tomography (cryoET) benefits from additional geometrical information from the tilting of the specimen and can image one-of-a-kind 3D objects. The proposed project aims to produce structures at near-atomic resolution for different sub-states of desensitized AMPA-type glutamate receptor ion channels, as this sample exhibit a strong conformational heterogeneity.
The first project objective is to improve the resolution of cryoET for small systems which do not present conformational heterogeneity. We will use direct electron detectors to correct for sample movement, and new sample schemes to reduce radiation damage.
The second project objective is to develop data processing algorithms for the treatment of sample heterogeneity. We will use two strategies: the simultaneous determination of the particle class and particle orientation, and the treatment of rigid protein domains as independent sub-particles.
The third project objective is to investigate the relationship between the structural heterogeneity of a protein complex in non-crystalline amorphous ice and its dynamics and heterogeneity at ambient temperature in solution. We will compare cryoET reconstructions to measurements of the protein dynamics by solid-state NMR at ambient temperature.

Many microscopic building blocks of living organisms have well-defined handedness and this structural information can propagate across many length scales to establish macroscopic processes and structures that are inherently chiral. However, understanding how microscopic chirality leads to macroscopically chiral structures, such as the left-right symmetry breaking in embryo development, is inherently difficult in living organism due to their inherent complexity. We propose to study the emergence of chiral structures in a simplified reconstituted system of purified microtubule filaments with Kinesin-2 molecular motors, depletant agent and adenosine triphosphate (ATP). Our system will allow us to have an accurate control over the handedness of the molecular constituents and interactions at play. First, we will focus on the ability of this active gel to self-assemble and to produce spontaneous chiral flows under confinement, as seen in cytoplasmic streaming events. A second part will focus on the emergence of chiral flows resulting from asymmetric collective beating in reconstituted micro patterned cilia-like active microtubule bundles. Finally, we will assemble doublets and arrays of artificial active cilia to study how molecular chirality affects their beating synchronization. Taken together, the in vitro reconstitution of these cellular structures and behaviors will bring quantitatively understanding and control of the chiral molecular mechanisms at play both in reconstituted microtubule and kinesin networks and cilia-like beating of synthetic active microtubule bundles. This project will broaden our understanding of the emergence of asymmetry in developing organisms.