The current Zika virus epidemic attracted public attention to the problem of mosquito-borne viral infections. Vector control is an essential element to prevent disease transmission due to the absence of arbovirus-specific drugs and limited availability of vaccines. Historical vector control methods such as the use of insecticides and environmental control are facing challenges due to the wide spread of insecticide resistance throughout natural mosquito populations and the complexity of breeding site elimination in the modern urban environment. Novel genetics-based strategies are emerging as promising complement to historical mosquito control methods. One idea is to genetically-manipulate the vectors so that they become unable to support pathogen infection, replication or transmission. The development of these novel transmission-blocking interventions requires in-depth insights into how mosquito vectors interact with and transmit arboviruses. It is thought that the immune system influences the efficiency by which mosquitoes transmit specific viruses. Recently, a novel immune response was identified that recognizes viral RNA and breaks it down into small fragments. In this project, the investigators will study whether mosquitoes from different locations across the globe differ in this immune response. Moreover, they will analyze how these differences influence transmission of the epidemic Dengue and Zika viruses. The multi-disciplinary research team includes experts in mosquito evolution and genomics, entomology, and virology, allowing a complementary approach to address the research aims. The proposed project will have immediate and profound implications for public health and may lead to novel mosquito control strategies.

The nucleolus is a large, complex, and dynamic nuclear body, whose structure is directly related to its role in producing ribosomes. It consists of three morphologically and compositionally distinct sublayers with liquid like properties, which hierarchically self-organize around transcriptionally active rRNA genes in a process that is poorly understood. Moreover, despite the significance of ribosome biogenesis, little is known about the order and loci of ribosome assembly, and the role ribosomal intermediates play in shaping nucleolar organization. A major difficulty in studying these processes lays in the absence of stable physical boundaries in between the sublayers, which facilitate a continuous flow of pre-ribosomal intermediates and hampers single layer isolation for in vitro analysis. Here I propose to recapitulate immiscible liquid phases of purified nucleolar components onto core particles with tethered rRNA in order to seed nuclei with isolated nucleolar sublayers. I will monitor the recruitment of sublayer specific components from the nucleoplasm into the synthetic layers and study ribosome biogenesis using the immobilized rRNA. The ability to seed nucleolar sublayers will be used to study nucleolar self-organization and to probe compositional changes in each sublayer separately, and to link them to concomitantly occurring ribosome production steps. Furthermore, it offers a means for studying ribosome biogenesis in vivo while restricting ribosome production steps only to those occurring within a specific sublayer, synchronizing the initial rRNA level of assembly, and sequestering ribosomal intermediates.

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 proposed project will investigate the origin of animal cell contractility through both comparative and experimental evolution approaches in the choanoflagellate Salpingoeca rosetta. The project will address three scales of biological organization: (1) Gene expression. The differential transcriptomic profile of contractile and non-contractile S. rosetta cell types will be determined by single-cell RNAseq. In parallel, I will investigate the function in S. rosetta of the three key myogenic transcription factors of animals (Mef2, SRF and Myocardin) by ChIP-seq and CRISPR/Cas9-based genome editing, and I will determine their interaction partners by pulldown/mass spectrometry. (2) Cellular phenotype. Choanoflagellates show an intense contractile activity during settlement. I will test whether this contractility is controlled by membrane depolarization and calcium influx by live imaging of choanoflagellates expressing calcium/voltage sensors, pharmacological assays, and microelectrode voltage measurements. (3) Multicellular phenotype. S. rosetta develops into spherical colonies of up to 50 cells. In an experimental evolution approach, I will assess whether simple (actomyosin-mediated) departures from this spherical shape can be brought about by consistently applying, over hundreds of generations, selection pressures thought to have favored the early evolution of animal morphogenesis: selection for larger colonies or for benthic feeding from bacterial biofilms.

Due to its high prevalence among birds and high human fatality rate, avian influenza represents a serious and continuing pandemic threat, in particular via mutations that facilitate human infection, resulting in pathogenic strains. Mutations associated with human infection are concentrated in the PB2 subunit of influenza polymerase. This subunit is imported into the nucleus, where viral replication and transcription occurs, via a selective interaction with importin-alpha. Once in the nucleus, interaction of PB2 with protein ANP32A has been proposed to play an essential species-specific regulatory role.

The behavior of PB2 in solution reveals a high level of conformational flexibility that is essential to function. In addition intrinsically disordered domains of both ANP32A and importin-alpha are thought to play important roles in the interaction with PB2. This uncommonly high level of disorder presents particular challenges for structural studies, requiring development of state-of-the-art NMR-based technology.

My aim is to investigate the molecular basis of avian influenza adaptation to nuclear import and replication in human host. I will characterize structurally and dynamically key interactions between human-adapted avian influenza polymerase and human host proteins to contribute to a deeper understanding of viral replication, providing the basic information to facilitate design of innovative drugs.

Viruses of the positive-sense RNA ((+)ssRNA) type are a major class of pathogens. In humans they cause diseases ranging from hepatitis C to viral myocarditis, Zika, Dengue fever and Chikungunya. The extracellular stage of these pathogens, the virus particles, have been studied in great detail often resulting in 3D structures at atomic resolution. Here, we propose to study an intracellular stage of (+)ssRNA viruses which has been more elusive to structural and mechanistic studies. Upon entering a target cell, (+)ssRNA viruses create dedicated organelles called replication complexes (RCs) , in which the viral genome is copied and hidden from detection until it is packaged into new virus particles. Since RCs form on cellular membranes as inseparable parts of the infected cell, they have largely resisted detailed characterisation by established methods of structural and molecular biology.

To overcome this challenge, we will study (+)ssRNA virus RCs using cryo-electron tomography (cET). cET can create 3D images (tomograms) of the interior of cells with a resolution high enough to identify individual macromolecular complexes. These tomograms will reveal in unprecedented detail how replication complexes are constructed from virus proteins and membranes inside the infected cell.

In parallel, we will create synthetic replication complexes from their individual protein-, RNA- and lipid components. The formation and subsequent RNA synthesis by such in vitro reconstituted replication complexes will be monitored by fluorescence microscopy, allowing us to make detailed observations about their molecular mechanisms.

The intestinal epithelium has the complex task of transporting water, ions and nutrients, while providing an efficient barrier to microbes and other potentially harmful luminal constituents. This delicate balance is notably altered in chronic inflammatory conditions of the gut, such as Crohn’s disease.
Luminal nutrients such as glucose have long been recognized as regulators of epithelial permeability. Recently, a role of the enteric nervous system in the control of barrier function has been proposed. Indeed, alterations to either enteric neurons or enteric glial cells leads to dysregulation of epithelial permeability. The structural basis for an interaction between the epithelium, glial cells and nerves has recently been provided, but neither the detailed nature nor the integration of this communication have been examined.
My project aims to investigate how nutrient-sensitive epithelial cells called enteroendocrine cells communicate with enteric glia and nerves in the mucosa to regulate epithelial permeability.
My approach will be to evaluate the signal integration, from the luminal stimulus to the epithelial response. We will use a combination of live cell imaging techniques and fluorescent reporters to visualize the propagation of the signals in living tissues. We will selectively block or release enteroendocrine cell mediators, inhibit enteric glial cells and/or block or stimulate enteric nerves to dissect the various components of this crosstalk.
My project will lead to a comprehensive understanding of signal integration in the intestine between the epithelium and the enteric nervous system, extending the concept of neural control of intestinal barrier function.

Extracellular interactome networks consisting of secreted and cell surface proteins coordinate complex biological processes, including embryonic development, homeostatic regulation and cancer progression. Identification of extracellular protein-protein interactions (PPIs) is central to biomedical science and therapeutics. For example, multiple cell surface and secreted proteins are targets for FDA-approved drugs. However, a description of extracellular PPIs for many of these proteins remains either unknown or incompletely known. We are particularly interested in Wnt signaling that controls the central nervous system (CNS) vascular development and blood-brain-barrier (BBB) formation and maintenance. The CNS vascular dysfunction including BBB breakdown is associated with brain trauma, stroke, multiple sclerosis and neurodegenerative disorders. So far information of extracellular PPIs of Wnt signaling for the development and regulation of CNS vasculature is limited. Here, we aim to utilize and improve an innovative technology for systematic and unbiased methods to identify novel extracellular PPIs related to Wnt signaling for the CNS vascularization and the BBB integrity. We expect to validate novel candidate PPIs using biochemical and biophysical assays. Additional experiments will assess these PPIs and their significance for CNS vascularization using cell culture systems and mouse genetic models. The information obtained from these studies will expand our knowledge of CNS vascular biology and bring a new approach to the study of extracellular interactomes.

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.

The Hsp90 molecular chaperone helps fold and activate 10% of the proteome and 60% of the human kinome. Together with the kinase specific co-chaperone Cdc37 it facilitates the folding and regulation of its client-kinases. This is accompanied by transitions between open and closed Hsp90 conformations triggered by ATP hydrolysis. Recently the closed Hsp90ß:Cdc37:Cdk4 complex was solved by the Agard lab using cryoEM. It reveals that Hsp90ß inactivates Cdk4, through clamping an unfolded ß-sheet and thereby separating the two kinase lobes. I aim to elucidate the biochemical and structural basis for kinase capture and refolding by the chaperone machinery. This might either occur directly by kinase binding to a very different “open” Hsp90ß:Cdc37 complex or involve unfolding by Hsp70 and subsequent transfer to Hsp90ß via a “transfer complex” of Hsp70, Hop, Hsp90ß, Cdc37 and the kinase. I will reconstitute the chaperone system in-vitro to identify key steps and necessary protein components. The kinase folding state will be monitored using ligand/inhibitor binding as well as FRET between the lobes. Using this system, I will also study the role of several co-chaperones including FKBP51, Aha1 and PP5. To allow structural characterization of the important dynamic intermediates by cryoEM, I will trap these states using a variety of strategies and determine their structures to atomic resolution by state-of-the-art cryoEM. In vivo relevance will be assessed by disrupting the underlying interactions in cancer cell lines. This will offer the future perspective to pharmacologically target the identified structural states relevant in many human cancers.

Cardiac hypertrophy from different causes represents a major culprit for premature sudden cardiac death in humans. The infrequently feeding Burmese python exhibits a dramatic change in metabolism after a large meal that requires organs to grow, including physiologic cardiac hypertrophy much as is seen in athletes after exercise. Most interestingly, such growth is reversible and cyclically occurs at every meal. This could be clinically important since regression is not seen in most pathological hypertrophy. With this project, we aim to define the mechanisms that allow the python heart to revert hypertrophy, in the attempt to provide novel insights for regression of cardiac growth in mammals. We hypothesize the presence of one or more factors promoting regression in the python plasma after the peak of digestion. We will test this hypothesis by treating hypertrophied neonatal rat myocytes with python plasma and evaluate cellular dimension as a marker for regression of growth. The sponsor’s laboratory has established this protocol. Once we have defined the trigger responsible for regression of hypertrophy, we will study the mechanisms employed by the python to reduce cardiac mass. We envision the involvement of complex signalling cascades leading to apoptosis and autophagy that are needed to reduce cardiac mass in a controlled manner. Moreover, we will extend our research on established mammalian pathological models of cardiac hypertrophy. We will attempt to activate the same signalling pathways in mammalian models in order to reduce cardiac mass. The results of this project are expected to unveil novel pathways that may lead to therapeutics for cardiovascular diseases.

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.

Aminoacyl-tRNA synthetases (aaRS) are the major translators of the genetic code by assigning the correct amino acids to the corresponding tRNAs and are thus of indisputable importance for protein biosynthesis. Additionally, several aaRS moonlight as inducer of cell signaling pathways. Recently, aaRS splice variants have been identified, which display unique cytokine-like properties. These splice variants are often truncated in their N- or C-terminus, thereby creating new termini. Terminal amine isotopic labeling of substrates (TAILS) was developed to map protease cleavage patterns. In the project suggested here, I will utilize TAILS to enrich and identify splice variants of AARS, especially of Arginyl-tRNA synthetase (RARS). Surprisingly, RARS signaling has not been studied yet, despite experimental evidence that RARS is present in the nucleus, signaling elicited by a RARS-splice variant, and the regulatory role of arginine in the immune system and cancer. Establishment of a timeline for RARS splicing events upon stimulation will decipher the underlying biological function and elucidate possible signaling functions of RARS. Investigation of RARS-induced signaling, interaction partners, and cellular dynamics will corroborate these results. As RARS splice variants lack export sequences, paracrine signaling could be mediated by exosomes. Dynamic regulation of RARS variants upon stimulation may lead to the identification of upstream regulators initiating splicing. Together, I suggest a new method for precise and quantitative detection of splice variants on protein level with a direct biological application for an enzyme class of very fundamental importance.

Sophisticated alien-like systems in squids, octopuses, and other cephalopods capture the human imagination, and are of growing research interest. Some represent unique innovations, like dynamic camouflage, high pressure jet propulsion, and stretchable arms with tasting suckers that grip. Other features are convergent traits that are surprisingly similar, but molecularly different, from familiar biological systems in ourselves and our vertebrate cousins, including large brains, sophisticated eyes, and a muscular heart that drives a high pressure circulatory system. Parallel evolution of these complex systems in cephalopods and vertebrates is likely due to an ancient evolutionary competition to dominant as large, active swimming, visual predators in early oceans, causing both groups to engineer their own genetic, cellular, anatomical and physiological solutions to similar environmental challenges. This matchless competition profoundly shaped complexity in both lineages, and offers researchers today an extraordinary and uniquely powerful opportunity to distill basic principles and reveal novel solutions of how to make a brain, complex eyes, and a heart with sophisticated cardiovascular regulation. Here, through detailed comparisons between cephalopods vs vertebrates, together with state-of-the-art technologies, we will decipher mechanisms and uncover alternative solutions: how to design a circulatory system with rhythmic heartbeats?
To characterize, reverse engineer and control novel types of circulatory systems, powerful genetic tools will be developed. We will establish, for the first time, targeted genome editing and light-based genetic tools to control activity in the world’s smallest cephalopod - the pygmy squid Idiosepius – a novel revolutionary model for biomedicine. Second, using this transparent marine organism, we will produce a genomic portrait of the entire circulatory system at single-cell resolution. Finally, with real-time imaging technologies and sophisticated genetic controls, we will develop new ways to regulate, not one, but three cephalopod hearts and their pacemakers. As a result, our international team will transform the pygmy squid into a cephalopod ‘lab rat’, and discover fundamental principles that led to the origins of high-pressure circulatory systems, providing new materials and ideas for synthetic biology and bioengineering.

Touch is one of the primary sensory percepts. Although most well-studied in vertebrates, it is critical to most organism including insects which rely on touch to navigate through the world around them. Moths use a long proboscis, a specialized mouthpart to actively acquire tactile information from flowers during feeding. They unfurl their proboscis and probe the flower surface in a manner reminiscent of how rats use their whiskers to sense their environment, an extensively studied active sensing system. However unlike whiskers that have mechanosensors only the base, the proboscis has sensors along its entire length. Moreover, the mechanical properties of the proboscis (diameter and stiffness) can also be actively controlled by the moth during feeding. Thus we see a novel sensory system that can be tuned on the fly.
Moths also acquire rapid mechanosensory information about the relative position of flower from the proboscis to stabilize flight and feed even in windy conditions. They combine this tactile information with the proprioceptive information from the neck and proboscis to form a representation of flower shape and position. Despite the central role of touch in the plant-pollinator interaction, little is known about the proboscis neuromechanics. Using a combination of behavioral, biomechanical and electrophysiological and data analytic studies on the nocturnal hawkmoth, Manduca sexta, I aim to understand how proboscis mechanics govern the interaction with flowers, how the proboscis is actuated to acquire relevant information, and in turn, how tactile information is encoded by sensors to enable this essential ecosystem service.

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.

The discovery that the cytoskeletons are not unique to eukaryotes has raised several important questions about the origin and functional role of ancient cytoskeletal proteins and their evolution into modern eukaryotic cytoskeletal networks. Toward this goal, we propose to better understand the synergies required between proteins and lipids by developing synthetic cells that combine the complexity and evolved sophistication of biological processes with the control and robustness of modern synthetic and materials chemistry.
We will develop a bottom-up approach towards the design of a fully synthetic functional cytoskeleton. Our project will implement supramolecular peptides, de novo artificial lipid synthesis, and protein channel engineering. These artificial building blocks (peptides, lipids, proteins) will be symbiotically self-assembled to enable the self-regulation of size and shape of synthetic cells.
The specific objective of this proposal will be to integrate artificial cytoskeletal peptides with bioorthogonal membranes and stimuli-responsive transport proteins to create hybrid “cells” capable of mimicking the function of natural cells but with improved control, stability, and simplicity. This strategy could dramatically improve our understanding of the physical requirements needed to couple simple early proteins with lipid membranes and help us understand the fundamental reasons for how and why cytoskeletal proteins evolved to control cell division and morphology.
The potential discoveries will allow a better understanding of the origin and development of the cytoskeletal network in cells. Furthermore, the creation of hybrid synthetic cells will enable a myriad of studies that will dramatically expand our understanding of how complex living cells evolved on our planet.

For a cell to maintain protein homeostasis (proteostasis) extensive networks of components are needed to safeguard and rapidly correct unwanted proteomic changes. The performance of these proteostasis networks may be reduced under stress and during aging, which results in misfolding of proteins, their aggregation and/or loss of functionality. Such instability of the proteome manifests in age-dependent neurodegenerative diseases, like Alzheimer’s and Parkinson’s and other medical disorders.
Currently there is a shortfall of capacity to quantitatively measure how well the networks maintaining proteostasis operate. The central objective of this project is to build a new biosensor system that can quantitatively measure how efficiently proteostasis is managed. We will probe proteostasis in space (i.e. spatially inside mammalian cells) and over time in mammalian cells as well as in the whole organism context using an animal model (the nematode) that is ideal for the study of age-dependent changes in proteostasis. Success in this central objective will enable us to measure the latent buffering capacity of the quality control networks under normal and stressed conditions. In turn this will enable us to gain insight into how cells respond dynamically to stresses, how resilient cells are to such stresses, and how quality control systems become degraded or overwhelmed in disease contexts and upon ageing.
Our team bridges four disciplines needed to bring this system to light. Hatters (Australia) brings experience in biosensor design and molecular biology. Ebbinghaus (Germany) is the co-inventor of a temperature jump microscope, which will be employed to extract in-cell kinetic and thermodynamic folding information about the biosensor that will be used to understand proteostasis efficiencies. Dickson (United States) is a computational expert in protein-chaperone interactions and will build coarse-grained models of proteostasis. Nicholas (Australia) has experience in nematode biology to implement the biosensors in the nematode to study proteostasis upon ageing.
Our specific goals are to:
1. Build a biosensor and mathematical framework that can measure proteostasis buffering capacity in living cells.
2. Define the proteostasis buffering capacity within different organelles of mammalian cells.
3. Quantitate proteostasis changes upon ageing in a nematode model.

According to the World Health Organization, about 600 million people around the world are obese. Not only does obesity affect developed countries, it is also becoming a major problem for low and middle-income countries. Obesity results from an increased accumulation of lipids within adipose tissue. Triglycerides are stored in lipid droplets, ultimately leading to adipocytes (ADs) hypertrophy, altered hormonal release and adipose inflammation. Hence, obesity contributes in a major way to the burden of diabetes and cardiovascular diseases (metabolic syndrome).
Growing evidence indicates that static stretching promotes adipogenesis, possibly relevant to sedentary lifestyle, while dynamic stretching or vibrations, as occurring for instance during exercise, have the opposite effect. Altogether, these observations suggest that mechanical force has a major impact on the ability of ADs to accumulate lipids, with differential responses to specific types of mechanical stress. ADs are characterized by a unique ability for volume expansion upon triglyceride accumulation, increasing their size by more than 30-fold with a marked enhancement in effective cell stiffness. Consequently, within adipose depots, hypertrophic ADs generate a mechanical stress transmitted to resident cells. We postulate that a positive mechanical feedback loop acts in the process of adipogenesis and influences hormonal production. Using a combination of transdisciplinary approaches, including soft matter physics, cell biology, biophysics, physiology, pharmacology and clinical observations we will investigate the molecular basis of adipose cells mechanosensitivity. Important questions need to be addressed: What are the mechanical forces at play in adipose tissue? Can we measure tension generated within adipose depots in vivo? How is mechanical stress transduced at the molecular level in adipose cells? Does mechanical stress impact hormonal production and adipose inflammation? We will investigate the functional role for candidate mechanosensors, including the mechanosensitive ion channel Piezo1, the adhesion molecules integrins/talins, as well as the nuclear protein lamin-A in adipose plasticity and function. In conclusion, we will provide novel insights into the mechanobiology of adipose tissue, with expected practical perspectives for the treatment of obesity.