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).

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.

Monomeric actin is readily detectable in the nucleus of mammalian somatic cells. Whether this pool of actin forms filaments has been debated for decades. Recent advancements have established that, indeed, nuclear actin can assemble dynamic filamentous structures in response to external stimuli. However, how nuclear actin filaments exert their biological function remains an unanswered question in cell biology. This is particularly relevant given the highly dynamic nature of nuclear actin filaments, which reside within the intricately organised nucleus that harbours the genome in the form of chromatin. Thus, through this collaboration, we aim to address this by developing and applying novel approaches for optogenetics, advanced cell-imaging and next generation sequencing. Accordingly, we will employ tools to spatiotemporally control nuclear actin filaments with light, and determine their role in regulating chromatin organisation and nuclear structure. Further, we will determine the cell and biological consequences of actin filament-driven chromatin organisation/re-organisation in relation to cell division and cellular reprogramming. By completing these studies, we envisage to advance our understanding of the biological functions of nuclear actin filaments in a manner that will encompass nuclear actin polymerisation as a key mediator of chromatin organisation and regulation. Beyond this, findings from our studies may have translational potential in areas of regenerative medicine, and may delineate new mechanisms that underlie ageing and degeneration.

The adult mammalian stomach epithelium is constantly renewed by proliferative isthmus stem/progenitor cells (ISPCs) and maintains its homeostasis under hostile luminal conditions. In the gastric corpus epithelium, differentiated Troy+ chief cells were identified as a novel stem cell population with high plasticity. Interestingly, Troy+ stem cells (TroySCs) can switch between a dormant and active state. TroySCs proliferate very slowly in homeostasis, whereas they rapidly divide upon ISPC loss facilitated by 5-FU treatment. This suggests its role as a reserve stem cell for the ISPC. However, TroySCs are also activated upon parietal cell loss despite the presence of actively cycling ISPCs. Thus, the role of TroySCs still remains vague in homeostasis and regeneration from various injuries. Here, I propose to investigate a potential role of TroySCs, beyond that of the reserve stem cells, by combining state-of-the-art mouse genetics, single cell transcriptome analysis, and mathematical modeling. First, I will characterize the population dynamics of the corpus epithelial cells under homeostasis and regeneration by lineage tracing and single cell analysis in mice. Second, based on the cell population data, I will construct a mathematical model for the corpus epithelium and theoretically investigate the function of TroySCs by perturbing their dynamics. Third, I will experimentally examine the role of TroySCs using in vivo and in vitro functional genetics. This work will reveal an essential role of quiescent stem cells in homeostasis and pathogenesis and shed light on a fundamental question about why quiescent and active stem cell populations coexist in various adult tissues.

Life as we know it depends on the membrane compartmentalization of the cell. Membrane proteins and their organizing scaffolding proteins maintain the flow of information and materials across the membrane. This project will advance the characterization of membrane and membrane associated proteins by developing novel electron microscopy and biochemical experiments enabled by new nanomaterials.
We focus on gephyrin, the major scaffolding protein in inhibitory synapses, and the glycine receptor (GlyR), which is one of the channels that gephyrin anchors to the synapse. Currently there is no detailed molecular mechanism to how gephyrin polymerizes and forms a scaffold, although the proteins and post-translational modifications that influence clustering in cells have been identified. A major puzzle is the variability and apparent geometric inconsistency of the scaffold. For instance, gephyrin’s hexagonal/trimeric binding geometry does not match the geometry of the binding sites within GlyR, which are arranged within a pentamer. GlyR dependent synapses have important regulatory functions in the brain stem and pain pathway and thus are potential drug targets. The emergence of a recent structure of a GlyR homopentamer has left us with questions about the structure of a physiologically relevant heteropentamer channel, the cytosolic part of the protein, and most importantly about conformational states of the channel that have not been visualized. These gaps in knowledge have hampered efforts to understand gephyrin-GlyR binding as well as to design new GlyR drugs.
This project will develop i) a nanosheet synthetic scaffold that can present gephyrin with its binding motif in a precisely defined geometry (Lau:Materials Science) and ii) nanoparticles functionalized with GlyR drug targets that provide high electron contrast labeling and bi-functional crosslinkers for the structure-function measurements (Yameen:Organic Chemistry). These nanomaterials will enable assays of gephyrin scaffold formation and GlyR complex formation, and electron microscopy functional mapping of GlyR (Mim:Structural Biology). The proposed research will provide insight into gephyrin-GlyR structure-function relationship and demonstrate new tools for biological characterization.

New genetic diversity originates in each generation through genome misreplication during germline development. Despite the fundamental nature of this process, observations of germline mutation have been indirect until recently, with the advent of coupled parent-offspring sequencing. However, because germline mutations are rare, and families are inherently limited in size, statistically robust estimates of germline mutation rates need to aggregate data from hundreds of distinct pedigrees, potentially integrating over biologically meaningful inter- and intra-individual mutational heterogeneity. Here, we propose a novel approach to study the individualistic component of germline mutation, using single-cell sequencing of gametes from a single individual. To provide proof of principle, will employ the model organism Ciona intestinalis, a hermaphroditic marine invertebrate with a small genome, which enables us to observe, with unprecedented depth, mutations occurring in both the male and female germlines in a single genetic background. By also sequencing offspring from the same individual, we will test the hypothesis that gamete sequencing can be used to measure mutations entering subsequent generations. Large samples of gametes will also enable us to study intra-individual mutational heterogeneity. Mutations shared by numerous gametes will be analyzed phylogenetically to provide a portrait of the lineage tree of germline stem cells, and of the dynamic nature of mutational processes along this tree. By extending this approach to the population scale, it should be possible to examine the patterns, and ultimately, the causes of inter-individual variation in germline mutation.

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

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

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