Seabirds have the longest migrations on earth and can travel 8 million km in a lifetime, yet how they navigate across a seemingly featureless ocean is still one of the greatest puzzles in nature. Evidence from mammalian and insect systems shows that animals adjust their behavior in response to infrasound and a handful of studies have suggested pigeons may use infrasound for navigation. These low frequency sound waves can propagate over hundreds of kilometers, creating “hills” and “valleys” of an infrasoundscape that birds may use to navigate, like a topological map. When combined with meteorological and oceanographic models, these maps can be modeled to create real time soundscapes that individual seabirds could use in movement decisions over spatial scales. By combining a network of 60 international atmospheric infrasound and hydro-acoustic monitoring stations that detect signals from around the globe with a database of over 15,000 seabird movement tracks, we will have a unique opportunity to explore the role of atmospheric and oceanic infrasound in navigation, respectively for aerial and aquatic species. The mechanisms allowing animals to detect low frequency sound has been identified in other taxa, and our study will examine how seabird sensory organs may capture infrasound. The development of an innovative movement framework grounded in landscape ecology will allow us to assess determinants of large-scale movement, notably the effect of infrasound in directing migration and commuting trips in the open ocean. Furthermore, novel biologging devices, which can detect sound and meteorological parameters, will be used to simultaneously capture movement, infrasound and weather conditions to examine individual movement decisions at fine scale. Finally, interspecific comparisons will assess the relative importance of infrasound for seabird navigation, with respect to phenotypical and phylogenetic differences, thus offering a complete assessment of the physiology, behavior and physics underpinning the use of infrasound in navigation.

This work leverages cutting-edge genomic and shape analysis methods to study the evolution of mammalian cooperative societies. Current approaches to studying vertebrate cooperative behavior work in the classical, but mechanism-free, frameworks of behavioral ecology and life history theory. Thus, while cooperation itself is of long-standing interest, we know little about how animals that occupy distinct roles in cooperative societies differ at the molecular level.
Here, we propose to integrate new molecular and computational approaches with a 24-year field study of the most cooperative nonhuman mammal yet described, the meerkat. Like cooperative insects, meerkat societies are characterized by a division of social roles: adults are dominant breeders or morphologically and physiologically distinct helpers, who feed and guard the breeders’ young. However, all helpers retain the capacity to transition to breeder throughout life. Meerkats thus present an exceptional opportunity to study alternative phenotypes in cooperative societies. We will first investigate how helpers and breeders are differentiated at the gene regulatory level, including whether steroid hormone signaling generates these differences. Second, we will test social role-driven differences in growth and immune defense. We will track growth by developing computational geometry-based approaches to perform 3D skeletal reconstructions from X-ray data and immune defense using experimental pathogen challenges. Finally, we will test the hypothesis that the competing demands of growth, reproduction, and immune defense create “competition” at the transcriptional level, which is resolved differently by helpers and breeders. Our experiments will not only quantify phenotypic transition-associated trade-offs, but also identify the genes and pathways that mediate them.
The methods required for these analyses either do not exist or will need to be generalized to field studies for the first time. Thus, this study requires collaboration across behavioral ecology, genomics, immunology, and computational image analysis. Together, it will contribute a powerful model for applying modern tools to long-standing puzzles in evolution and behavior. It will also yield new insight into the molecular changes involved in the evolution of cooperative societies—a subject of fascination and controversy for almost two centuries.

What underlies the ability to count and where did it come from? This project tests the broad hypothesis that the ability to represent the number of objects in a set (numerosity) has an evolutionarily conserved neural basis, and identifies the cell and molecular processes involved using multidisciplinary analysis in zebrafish. Although a wide range of species are able to estimate numerosities, only in primates has a neural mechanism homologous to humans’ been demonstrated and the underlying cellular processes are unknown. Using automated operant conditioning we train zebrafish to perform numerical tasks and identify lines of fish with differential abilities (e.g. mutants in candidate genes identified from human studies generated using CRISPR). We use 2 photon light sheet imaging of neural activity as wildtype and mutant larvae discriminate numerosities to identify circuits involved. Behavioural analysis will establish the ontogeny and extent of zebrafish’ numerosity. Genetic analysis tests the hypothesis that numerosity has an evolutionarily conserved basis. Neural imaging will test the hypothesis that “number neurons” exist in fish as in primates and indicate the circuits involved.

Our proposal is aimed at discovering the molecular mechanisms underlying the remarkable force-sensing and responsive properties of cellular attachment to the extracellular matrix. Many proteins contribute to the intracellular machinery that links the cytoplasmic domains of the transmembrane integrin adhesion receptors to the actomyosin contractile apparatus within the cell. These integrin adhesion complexes (IACs) provide a paradigm for a distinctive class of subcellular protein complex. Rather than assembling a structure of fixed stoichiometry (e.g. ribosome, centriole) via exclusive interactions, evidence is emerging that IACs engage a dynamic set of heterogeneous interactions that evolve from IAC initiation through maturation to achieve their signaling and mechanical functions.
Thus, we hypothesize that a key feature of IACs is their ability to exchange multivalent interactions between components, so changing their composition in response to diverse inputs, including force, developmental history and location within the organism. We have selected a few pivotal components of the IACs as the focus for our project: namely talin, kindlins, the IPP sub-complex (integrin-linked-kinase (ILK), PINCH, parvin), and vinculin.
To test this hypothesis we will combine Giannone's expertise in live single protein tracking and super-resolution microscopy with Brown's expertise in Drosophila developmental genetics. First we will advance existing methods to achieve the challenging task of quantitative super-resolution imaging within IACs in living tissues. Second, we will develop new tools to image interacting proteins, study their dynamic behavior and alter the interactions. Discovering the regulation of IAC rearrangement will greatly improve our understanding not only of mechanisms mediating Integrin adhesion but also of dynamic macromolecular protein complexes.
By bringing together the contrasting approaches of the two applicants we will gain an exceptional view of how the molecular machinery at integrin adhesion sites has evolved to be able to respond diverse environments and activities within the organism. We anticipate that this will lead to an understanding of general principles directing the progressive formation of macromolecular complexes.

The X-ray free-electron laser (XFEL) promises the study of systems that cannot be crystallized and the ability to follow the evolution of structures undergoing reactions or other dynamic processes, overcoming limitations of crystallography (which requires crystals) and cryo-electron microscopy (which requires cooled samples). Both of those methods are fundamentally constrained by the problem of radiation damage, which sets a limit to the exposure that can be tolerated by the sample. The XFEL breaks this limit with very intense and brief X-ray pulses that are shorter than the time atoms can move on the atomic scale, even though the sample is ultimately vaporized. This enhanced dose tolerance has been well demonstrated by high-intensity experiments using protein nanocrystals, where diffraction patterns are collected at many thousands of times higher exposures than is possible otherwise. However, even at these extreme intensities, the diffraction signal of non-crystalline objects is low, comparable to the achievable signals of biological molecules in cryo-electron microscopy (cryo-EM).
We propose to develop radically new methods to image single uncrystallized biological molecules at atomic resolution by XFEL diffraction of nano-engineered samples. By attaching DNA origami structures to the sample we obtain stronger signal than from the molecule alone. The structural information of the sample is built up from millions of diffraction patterns from such samples, collected one at a time at the repetition rate of the XFEL. These patterns can only be fully interpreted to give a three-dimensional (3D) structure of the molecule if they are individually registered to each other in all three orientations (similar to cryo-EM). The flexibility of DNA nanotechnology will be exploited to build structures that align in a flowing jet or orient on a membrane substrate such as graphene. The unknown rotation of the object about the alignment axis will be obtained from signatures based on the designed DNA structure. In addition to boosting the diffraction signal and orienting the molecule, the known DNA origami structure provides a holographic reference to phase the aggregated diffraction intensities and give the 3D electron density map. Our general-purpose technique will be applied to obtain atomic resolution imaging of biological molecules that do not readily crystallize.

When plants and animals grow they often develop patterns, such as the spiral arrangement of leaves around a stem or the overlapping pattern of scales on a snake. Some of these patterns are controlled by genes acting to shape the cells, and these patterns have been well studied. However, many biological patterns arise simply from physical forces. These patterns depend on the chemistry of the materials that plants and animals are made of, and on the forces that arise as these materials grow. We hypothesise that a single set of rules governs this mechanical pattern formation, and that these rules will relate to how tissues grow, what they are built of, and how stiff they are. By defining and understanding these rules we will be able to explain a great deal of the diversity of living organisms.
We have chosen to study the formation of a particular type of pattern – buckling, or wrinkling, of layers of the skin. Our team comprises a plant biologist, who will study buckling of the petal surface of Hibiscus trionum, an animal biologist, who will study buckling of the skin of corn snakes, and a polymer engineer, who will model buckling in artificial systems and generate rules and predictions. The two biologists will test these predictions in their different models, and the team will work iteratively to refine the models. The two biologists will also share tools and techniques to enable them to measure the same properties of their different systems.
Our findings are poised to provide new understanding of the universal principles that apply to life, and specifically growth processes in both plants and animals. They will help evolutionary biologists to explain the great diversity of plant and animal form, and will underpin many future applications in which engineers use bioinspiration to generate new materials and structures.

Asymmetric cell fate assignation during asymmetric division relies on the biased dispatch of fate determinants between the two daughter cells. I recently discovered a novel mechanism of asymmetric dispatch of fate determinants in Drosophila. In our system, molecular motors on Sara endosomes amplify the asymmetry of the central spindle to induce robust asymmetric segregation of endosomes. Since Sara endosomes contain Notch and its ligand Delta, this asymmetric dispatch contributes to polarized signaling and asymmetric fate in the bristle lineage. This synthetic biology proposal aims at reconstituting asymmetric cell division in non-polarized, symmetrically dividing cells. The physics of the system is such that I can do this either by controlling central spindle asymmetry (i.e. modify the writing), or by controlling the biophysical properties of the motors carrying the fate determinants (i.e. the reading). Strategically, this project capitalizes on the physical understanding of the system and on pioneering pluridisciplinary assays. In particular, I will i) use opto-nanobodies to reshape the asymmetry of the central spindle in space and time in vivo, ii) use multiprotein micropatterning on glass to cluster bivalent transmembrane nanobodies in cells and thereby create cortical polarity cues in naïve cells, and iii) engineer a motor designed to exponentially amplify the low, stochastic asymmetry of the central spindle in non-polarized cells. This synthetic biology project will challenge our current view of asymmetric cell division and pave the way towards restoring asymmetric cell fate in mammalian cells in situations where it has been lost, such as tumorigenesis or ageing.

When we navigate and make decisions in familiar environments we rely on an internal, mental map of the features and general shape of the environment. Remarkably, work over the last four decades has revealed how different locations in space and different navigational plans are represented as patterns of activity in thousands of neurons in the brains of rodents. It stands to reason that this 'neural representation' of an environment should remain stable as long as an animal is using it, to allow the animal the reliably navigate. However, we found that the neural activity that makes up this map changes over time so that different neurons represent specific features of a familiar environment after several days. We do not have a theory to explain how the brain can build and use such a continually changing mental map, nor do we know why the map changes, or what purpose this continual change might serve. In this project we will build and experimentally test new theories and models of how the brain represents environments, potentially changing our understanding of how the brain works.

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

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

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

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

To understand how cellular machineries work, we typically rely on reconstituted systems that often do not represent the complexity existing in vivo. We lack innovative methods to describe protein-protein interactions at high-resolution, specially the very transient ones, in their physiological environment. Chromatin is a good example of a system whose complexity cannot be fully reconstituted in vitro. Indeed chromatin is regulated by hundreds of chromatin remodelling enzymes and hundreds of possible combinations of histone post-translational modifications (PTMs) and variants. This complexity cannot be fully reconstituted in vitro.
We propose a novel combination of synthetic biology, mass spectrometry and high-resolution imaging to define the molecular details of how proteins function on chromatin in their physiological environment at high resolution. The challenge is to use photochemical traps installed by genetic code expansion in histones of living cells to “freeze” interactions of proteins bound to chromatin, especially the very transient ones that could be disrupted or missed by conventional purification or bulk chemical cross-linking. The trapped protein-chromatin complexes will be analysed by cryo-electron microscopy. By mass spectrometry we will map the interactions between remodelers and histones in vivo and we will quantitatively describe all chromatin PTMs associated to specific remodelers. This way, we will be able to analyse the spatio-temporal activity of chromatin-bound complexes at high-resolution at specific time points or upon specific stimuli.

The spectacular manoeuvres of flocking birds and schooling fish are among the most dramatic and mysterious sights in the natural world. How can hundreds or thousands of individuals coordinate their movements so perfectly, behaving almost as a single super-organism? The answer to this puzzle began to be uncovered through mathematical models showing that collective order can emerge as a by-product if all individuals within a group follow simple rules to align with and stay close to their neighbours. However, unlike the simulated agents in these models, real animals are not identical, and can differ both in their individual characteristics and in their relationships with one another. A group’s composition is therefore likely to affect its overall structure and cohesion as well as its ability to reach consensus decisions when responding to the environment. Understanding these effects has important implications, from determining how animal groups respond to threats, to mitigating the impacts of crop pests, managing crowd safety and developing intelligent systems in robotics. We will use mixed-species flocks of rooks and jackdaws (birds of the crow family, or corvids) to understand the effects of group composition on collective behaviour in nature. Combining field experiments with cutting-edge imaging and computational techniques, we will produce 3D reconstructions of the movements of every bird within flocks of varying composition and examine how a flock’s composition affects its structure and movements, and its responsiveness when avoiding or mobbing predators. Are more homogeneous groups better able to respond as a coherent unit, or does diversity enhance group responses as in human social institutions? Our 3D reconstructions will also allow us to determine the fine-scale internal structure of flocks. Do corvid flocks, like human crowds, contain sub-groups, reflecting flock members' social preferences? Finally, we will use our data to understand how individuals' flight decisions are influenced by who their neighbours are. By building mathematical models based on these measurements and testing the models using flocks of robot-controlled drones, we can find out how local interactions and social preferences among neighbours generate both internal sub-structure and collective order in complex societies.

Precise and coordinated regulation of tyrosine phosphatases and kinases is crucial to mount an effective immune response and to prevent autoimmune diseases. Lyp, a lymphoid-specific phosphatase, is a negative regulator of T-cell receptor (TCR) signaling. Some studies point to a gain-of-function when Lyp interacts with the tyrosine kinase Csk, while others suggest that Lyp/Csk complex limits its activity. This project aims to elucidate how the spatiotemporal coordinated arrangement of Lyp, Csk, and the transmembrane adaptor phosphoprotein PAG to which both associate, down-regulates TCR signaling by exploiting the potential of super-resolution fluorescence microscopy. Super-resolution imaging has already proven to be a powerful tool to study the immunological synapse. Still, multiplexed interrogation of distinct target proteins remains challenging. I will implement a recently reported multiplexed super-resolution imaging technique named DNA-PAINT (Points Accumulation for Imaging in Nanoscale Topography) to image the target proteins with high spatial and temporal precision during T-cell activation using a single-laser. I will further develop a 3-color co-localization clustering analysis for unveiling the complex pathways involved in signaling termination. Given the strong association of a single-nucleotide polymorphism in the gene encoding Lyp with various autoimmune disorders, I will also address the regulatory mechanism involved in the disease-associated Lyp variant.

Neurons are characterized by their unique morphology, which requires efficient sorting and transport of cargoes over long distances. One class of molecules that affect neuronal development, function and survival are neurotrophins. Neurotrophic growth factors are target tissue-derived cues that bind to their cognate receptors on the pre-synaptic membrane and are transported retrogradely to the soma, where they induce signalling events which control transcriptional programs governing neuronal differentiation and survival. Despite their importance in neuronal homeostasis, the regulation of uptake and transport of neurotrophins and related growth factors is presently unknown. Recently, the host laboratory reported that the tetanus neurotoxin requires the synaptic basement membrane protein nidogen for its entry and axonal transport in mammalian motor neurons (Bercsenyi K, et al., 2014). Importantly, the tetanus toxin is known to enter the same intracellular trafficking pathway entered by neurotrophins (Salinas S, et al., 2010). In this context, this proposal is aimed at investigating the question: Does the synaptic basement membrane play a role in regulating the uptake, transport and biological activity of neurotrophins and related growth factors? As part of the research plan, I intend to identify growth factors that bind to nidogens and other components of the basement membrane at the mammalian neuromuscular junction, genetically modulate this binding and study its effects on neurotrophic factor endocytosis and trafficking, with a view to understanding the physiological relevance of this biological process.

The presence of immune cells in decidua during early to mid-pregnancy creates a specific environment at maternal/fetal interface, which is crucial in preventing rejection of the fetus. Although unique receptors and interactions have been identified for decidual immune cells, the full spectrum of their responses is still not completely understood.
In my post-doctoral research, I will study the cell types, their transcriptional regulation, and their cellular interactions established in maternal/fetal immune cross-talk. This research will be addressed by analysing the transcriptomes of decidua-infiltrating immune cells at a single-cell level. With these results and using bioinformatics tools, I will reconstruct the specific niche required to ensure success at this first stage of pregnancy.
I will pay special attention to the unique cell-cell communication occurring in this particular context. To study cellular interactions, I will develop “CellCommDB”, a curated database of known ligands and receptor proteins involved in cell-cell communication. The innovative part of “CellCommDB” will be the integration of the multiple levels of complexity found in protein receptors, like the fact that many cell surface receptors consist of multiple different protein chains.
Overall, my results will help to decode the paradigm of maternal/fetal allogenic co-existence, and to design novel, targeted therapies to increase pregnancy success. Moreover, it will pave the path for further studies to analyze the unique cellular interactions that defines the function and regulation of tissues.

The cross-talk between integrin-based adhesions to the extracellular matrix and dynamic microtubules plays a crucial role in cell polarity and migration. Integrin-based adhesions can promote cortical microtubule stabilization in their vicinity. In turn, microtubules can strongly affect the formation and turnover of the adhesion sites. However, the molecular basis of these connections remains enigmatic.
Here, we propose to decipher the molecular chain of events induced by mechanotransduction at integrin adhesions that leads to cortical microtubule stabilization and analyze the morphogenetic impact of this process. To achieve this goal, we will bring together four distinct sets of expertise: cell biology of the cytoskeleton (Akhmanova); structural biology, biochemistry and biophysics of protein-protein interactions (Goult); mechanobiology and single molecule analysis of force dependence of protein-protein interactions (Yan) and genetic analysis of morphogenesis in flies (Tanentzapf).
We will use advanced cell manipulation assays to directly test whether mechanotransduction at adhesions affects microtubule capture and stabilization at the adjacent cortical sites. To uncover the molecular basis of this process, we will focus on a recently discovered connection between a cortical adaptor protein, which constitutes a part of the microtubule stabilization complex, and an adhesion component. At the same time, we will perform experiments, which will allow us to identify and explore additional links between focal adhesions and the microtubule-stabilizing complex, and thus generate a comprehensive functional map of these connections. Given that the investigated proteins are highly conserved, we will combine genetics, imaging and modeling to address their impact on morphogenesis and tissue maintenance in flies. Our work will shed light on how cellular mechanics and cytoskeletal dynamics are integrated at the molecular level.

Our body consists of trillions of cells, which derive from a single fertilized egg. The divisions that generate these cells constitute a genealogical tree, with a single root (the fertilized egg) and trillions of terminal branches (each of our cells). Knowing the shape and branching order of this tree is important because it provides vital information about how we developed; cells that are on the same branch shared the same cell precursors and a common history of developmental decisions. The cell lineage tree of an individual is also important for understanding cancer; its origins and history of colonizing the body.
Discovering the cell lineage of an organism is a huge challenge that has been solved only in the simplest cases. In the nematode worm Caenorhabditis, whose body consists of approximately 1000 cells, the complete cell lineage was painstakingly determined by direct observation of each cell division under the microscope. In larger organisms we can only infer partial cell lineages by direct observation, or through a method called clonal analysis in which genetic marks are used to label the progeny of individual cells. Each of these methods has its limitations. Direct observation is only applicable to transparent, tiny organisms and to events that occur within hours or days. Clonal analysis can inform us about the rough structure of the lineage tree, but its precise branching patterns are unresolved.
Here we propose a new strategy. It is based on the idea that random mutations, which accumulate in cells during the lifetime of an organism, can reveal the structure of its lineage tree: cells belonging to a major branch will uniquely share mutations that occurred in their common ancestors, those that lie together on a finer branch will share additional mutations, and so on. In principle, if a sufficient number of mutations were detected, we could infer the complete lineage tree of the individual. In practice, this approach is limited by our ability to find those rare somatic mutations within the genome of individual cells. Our strategy resolves this problem by relying on two new technologies: a method that allows us to generate mutations at specific sites in the genome, combined with a method for detecting these mutations in the organism, with single-cell precision.

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