Given its anatomical organization, it is clear that Layer (L)1 of the neocortex plays a key role in cortical function. L1 is composed almost exclusively of afferent axons originating from distal brain areas and the dendrites of their postsynaptic targets. Thus, L1 acts as an integration hub of distant and local activity. Despite the vast amount of neocortical recordings amassed over the past decades, L1 remains largely uncharacterized. Existing methods are unable to record afferent activity from the acellular L1 neuropil. Genetically encoded calcium indicators (GECIs) allow recording activity from presynaptic structures of defined neuronal populations. However, current sensors diffuse poorly to distant synapses or photobleach significantly when imaged in small compartments, reducing the signal-to-noise ratio. Here, we propose to develop novel specialized sensors for studying the connections linking distant brain regions, such as L1 afferents. First, we will develop genetic strategies for the efficient targeting of GECIs to presynaptic compartments while preserving their sensitivity and photostability during synaptic optical recordings. Second, we will develop methods to optically record activity from synapses with known connectivity. Combining the expertise of each team we will rapidly test these novel sensors in vivo and ex vivo in axons representing the three main classes of L1 inputs. The sensitivity of the different sensors is going to be measured by imaging L1 axonal boutons in acute brain slices. The performance of the synaptic sensors in vivo is going to be characterized by imaging L1 axonal boutons in awake behaving mice. By applying these novel sensors we will describe the basic functional organization of L1. We will characterize afferent activity from cholinergic and thalamic projections to L1 in behaving animals with synaptic resolution. In addition we are going to determine whether functionally diverse cortico-cortical signals intermingled in L1 target different pyramidal neurons. Our project will shed light on the functional organization of L1. Importantly, the tools to be generated will have wide applications in neuroscience by allowing recordings from afferents of any length scale and relating their function with connectivity.

The exquisitely complex mechanism of the human feet encompasses over a quarter of the body’s bones. How and why did the foot structure evolve to this current form? How do the demands of walking and running stably with low energy consumption affect foot evolution? The arched human foot reduces energy consumption by storing and releasing elastic energy like a bow and string. But does the foot’s elasticity also aid in maintaining stability of gait on uneven terrains? We have assembled an international team of researchers spanning the fields of physics, mathematics, and biomechanics to address how the foot has evolved and how it is controlled to balance the needs for stability and energy consumption during locomotion.
A soft foot smoothes out the ground’s uneven features, which may help stabilize running on realistic, uneven terrains. We expect that the neural system actively controls the softness of the foot by modulating the geometry of its arched form. We verify this hypothesis using a combination of measurements on human subjects running on uneven terrains and mathematical analysis of the foot’s elasticity. Our experiments will measure the spatial distribution of 3D forces under the foot using a newly developed high resolution photoelastic technique. These simultaneous and detailed physiological and mechanical measurements give us the ability to detect subtle changes in the mechanics of the foot, thus enabling the development of an anatomically faithful, mathematically rigorous model of foot function. These models then help us glean invariant principles of foot function used to mathematically design feet that optimally balance stability and energy consumption based on the terrain unevenness. We use these theories to also augment physical replicas of ancestral foot skeletons with muscle-like springs and motors in order to assess their functional capabilities against human performance. We hope, this comparative analysis will explain the time course of foot evolution, from the advent of regular walking 3.6 million years ago, to regular endurance running and emergence of a human-like form 1.5 million years ago. Our results also have implications to the design of prosthetic and robotic feet that could help in striking the right balance between energy storage and stability on real world uneven terrains.

Our goal is to study physiologically important components within the voltage-gated sodium (Nav) channel signaling complex that may cause disorders when mutated, and to resolve their mechanisms of action at the molecular level. In particular, we will focus on vital members of the Nav channel signaling complex that do not contribute to the ion-conducting pore. Reflecting their medical importance, mutations found in several of these proteins have been implicated in various epilepsy syndromes, long-QT syndrome (LQTS), LQTS-associated Sudden Infant Death Syndrome, and Brugada syndrome, possibly through dysregulation of the Nav channel signaling complex in the brain and the heart, respectively. Little is known about the underlying machinery that governs the influence of these elements and their mutants on the functional properties of Nav channels. To elucidate the mechanisms by which these proteins influence Nav channel function, we will combine innovative biophysical techniques with molecular biology and animal toxin-based pharmacology, state-of-the-art biochemical approaches, mouse genetics, and X-ray crystallography. Successful completion of our aims will help define the function of the larger Nav channel signaling complex as found in humans, which is essential for developing new strategies that can correct for abnormal behaviors.

Except for the nucleus, mitochondria are the only organelles in our cells that contain their own genome. Acquired genomic aberrations in mtDNA lead to mitochondrial dysfunction, a chief cause of neurological and aging diseases. mtDNA mutations, ranging from single-base substitutions to large-scale deletions, are also found in high frequency in many tumors, and recent experiments have established their role in driving metastasis. While the underlying basis for mtDNA deletions is unknown, two scenarios can explain their formation – infidelity of DNA replication machinery or errors in DNA double-strand break (DSB) repair.
The overall goal of our proposed study is to elucidate the molecular mechanism of mtDNA instability by deciphering both facets of mtDNA metabolism – replication and repair – and test if their deregulation causes deletions in the mitochondrial genome. Then, we will directly study the impact of the most common mtDNA deletion on cancer progression.
We will combine a single-molecule DNA combing approach with super-resolution microscopy to unravel the replication profile of mtDNA and highlight specific regions that might stall replication forks, causing replicative slippage and subsequent sequence loss. Biophysical analysis will be carried out to emphasize the structural nature of these deletion-prone regions. Using these molecular and biophysical tools, we will then address the genetic basis for misregulation of mtDNA replication in cancer cells.
The prevalence of short repetitive sequences at the junction of mtDNA deletions prompted us to ask if they are due the activity of alt-NHEJ, a recently identified pathway of DNA DSB repair that is predominantly driven by sequence-homology and often associated with significant sequence loss. To address this question we plan to manipulate the mitochondrial genome using TALE-technology (Transcription Activator-Like effector) both to incorporate reporter plasmids into its DNA and induce targeted DSBs. This innovative approach will allow us to investigate the DSB repair pathway(s) operating in the mitochondria and test if alt-NHEJ is the predominant pathway in deletion-bearing cells. Once the basis of the deletions is underscored, we will recapitulate the most common mtDNA deletion in human cells by coordinated cleavage with two TALE-nuclease and directly assess its contribution to tumor progression.
Despite the profound impact of aberrant mtDNA on cellular function, mitochondrial genomic instability remains largely an uncharted territory of cancer research. Our proposed study surpasses the abundance of descriptive literature to firmly establish the role of deletions in tumor progression. More importantly, it will highlight the underlying mechanism behind their formation, a critical step towards predicting and preventing their accumulation in cancer cells.

The contractile cortex is a thin actomyosin network that subtends the membrane of animal cells. The cortex is the main determinant of animal cell shape. As such, it plays a key role in processes such as cell division, where morphogenesis is driven by cortex remodelling in response to cell-cycle dependent signalling. In spite of its physiological importance, our knowledge of the cortex remains poor. In the first grant, we have established a multidisciplinary collaboration that allowed us to uncovered the proteic composition of the cortex and identify the two key proteins that nucleate actin in the cortex: the Arp2/3 complex and the formin Diaph1. This led to the proposal that the cortex is a composite network, which mechanics can be fine-tuned by regulating the relative contribution of the two main nucleators.
The aim of this project is to build on these findings to investigate the control of cortex mechanics during the cell cycle. Taking a mutiscale approach, we will explore how changes in the activity and abundance of cortical proteins during the cell cycle modulate the network architecture of the cortex, and how this, in turn, gives rise to whole cell mechanics. Building on the first grant, we propose to systematically examine how cell-cycle dependent signalling influences cortex composition and cortical protein activity, and ultimately cortex mechanical properties.
As the cortex is an intrinsically mechanical structure, its biological properties cannot be understood in isolation from its mechanics. In particular, our preliminary results indicate that the dramatic cell shape changes occurring during the cell cycle are driven by changes in cortical mechanics. Cell cycle-dependent modulation of the activity of actin nucleators provides a potential mechanism. Therefore, having identified the cortical actin nucleators, we will determine how changes in their activity affect cortex architecture and mechanics. Coupled biochemical and proteomic studies will search for signalling pathways altering nucleator activity. In vitro studies will examine how actin networks assembled by different nucleators interact and if tension can modulate nucleation activity. Electron microscopy and subdiffraction imaging techniques will enable us to characterise cortex architecture, while Atomic Force Microscopy will be used to characterise cortex mechanics. Furthermore, we will use proteomics to identify proteins whose abundance and activity vary most during the cell cycle. Using these results, we will determine key cortex regulators by systematically depleting/overactivating each in turn and measuring the resulting changes in cortex architecture and mechanics. Our integrated interdisciplinary approach will allow us to uncover how signalling regulates cell mechanics through modulating cortex architecture.

Believe it or not, the white shark on “Shark Week” is at a disadvantage: whereas your bones house a microscopic universe of cells, working in concert to fix microscopic wear and tear we cause every day, the cartilage of shark skeletons can be added to, but not repaired. Think of it this way: with bone, you upgrade components just as they go out of style or fail, but with cartilage you’re stuck working with the same old model you started with (explaining why my knees hurt). But sharks and other cartilaginous fishes use a curious work-around: although their entire skeleton is cartilage —not unlike what you’d find on spareribs at the dinner table— it’s reinforced with a thin, outer shell. The shell itself is striking under a microscope: thousands of geometric blocks called ‘tesserae’ made of the same mineral as in our bones and fitting together like floor tiles to wrap the entire skeleton. Although tesserae were first observed more than 100 years ago, we still have no idea what they are actually doing as these spectacular fish swim, bite, and glower on our TV screens. We’re curious about the mechanical role tesserae play in skeletal performance. To get a true feel for how tesserae manage and distribute forces, we plan to make our own shark bits: imitation skeletons scaled up for easier mechanical testing. Using high-resolution engineering and materials science tools, we will characterize geometries and tissue properties that define tesserae of a model species, and then use those features and a state-of-the-art 3D, multi-material printer —typically for industrial applications and capable of printing physical models with rigid and flexible parts— to manufacture tesseral mats that can be pushed, pulled, and fractured in ways that echo biological conditions. Like any design process, when the model doesn’t work, we’ll return to the source (the fish) for a deeper understanding of the template; once the parameters are clear, we can apply our models to the wide range of tesserae shapes and sizes in different species with different ecologies. This will shine a light on the functional role of this biological tiling, but also point to generalizable features useful for manmade tiled composites. Given that sharks live long lives and have existed for millions of years with irreparable, tiled skeletons, we have much to learn: why replace the old model if it never breaks?

The vast majority (>95%) of human proteins is thought to be affected by alternative splicing. In fact, alternative splicing is one of the mechanisms thought to be underlying the increased organismal complexity of higher mammals. Without a doubt, the combinatorial complexity enabled by alternative splicing is mediated by changes on the protein level. However, only few studies thus far have addressed the consequences of alternative splicing on protein function, e.g. in the context of interaction networks. Particularly, large-scale interactome network mapping so far ignores the increased complexity and dynamic modulation of network connectivity by differential splicing. This project aims to fill this gap and identify protein interactions that are regulated by tissue-specific alternative splicing. We will use a bioinformatic modeling approach and analysis of deep-sequencing transcriptional profiling data to identify physiologically relevant splice isoforms that are likely to affect physical protein-protein interactions. Subsequently, we will experimentally identify differential interaction partners of the identified proteins and their isoforms using high-quality high-throughput interactome mapping by screening the different isoforms for interactions against a genome-wide set of ORFs and subsequent systematic verification. In the third part of the project we aim to genetically demonstrate the biological role of experimentally validated differentially-interacting-splice-isoforms using phenotypic screens and biological follow-up studies for selected high-confidence candidates. All information will be integrated with existing network maps and other biological information.

The development of the central nervous system (CNS) is one of the most spectacular processes in biology. Key aspects include the formation of neuronal axons, their subsequent growth and guidance through thick layers of nerve tissue, and the folding of the brain. All these processes involve motion and must thus be driven by forces. However, while our understanding of the biochemical and molecular control of these processes is increasing rapidly, the contribution of the dynamic interplay between cellular forces and tissue elasticity remains poorly understood – mostly due to a lack of suitable measurement techniques. In this highly interdisciplinary project, we will integrate international expertise in optics, biophysics and neurobiology to investigate the role of mechanics in CNS development. We will develop a novel photonic toolbox for in situ, label-free and non-contact tracking of mechanical force and elasticity at cellular and intra-cellular levels with microscopic spatial and real-time temporal resolution. Force sensing will be based on a radically new approach that will enable the continued measurement of cellular forces down to the pN range without the need for detaching cells from their substrate as required in traction force microscopy. To measure the elasticity of cells and extra-cellular matrix material with 3D microscopic resolution, we will develop Brillouin-scattering based ElAsticity Mapping (BEAM). Unlike standard methods for cellular level elasticity measurements (eg atomic force microscopy), which are usually either invasive or limited to surfaces, BEAM will provide a non-contact, non-perturbative probe. These innovative experimental techniques will be applied to illuminate how forces exerted by neurons contribute to axon formation and neuronal guidance. Ultimately, we will investigate the involvement of mechanics in brain morphogenesis. Our approach will allow to look at these crucial biological processes from a new angle and shed new light on CNS development. Beyond this, our novel platform will be broadly applicable to areas far beyond the boundaries of neurobiology, as it provides unique cell-microenvironment metrics to relate mechanical interactions to cell behavior.

This proposal seeks to answer fundamental questions about cytoskeletal filaments, which are central regulators of cell biology. Our current understanding of bacteria is that these organisms use dynamic cytoskeletal systems to organize and regulate essential cellular processes. The results of our previous HFSP grant broke new ground by demonstrating that the number of these systems extends beyond homologs of the three primary eukaryotic cytoskeletons (i.e., actin, tubulin, and intermediate filaments). Two representative examples are our discovery of bactofilins, which are a bacteria-specific family of proteins, and of CTP synthase, an enzyme that is widely conserved across all kingdoms of life. These cytoskeletal proteins play key roles in regulating cell shape, metabolism, and intracellular compartmentalization, and their identification has had a transformative impact on the field of bacterial cell biology.
Building on our successful previous approaches, our renewal proposal is designed to characterize these novel cytoskeletons and, in addition, identify new cytoskeletal proteins. Apart from using a wide range of genetic, biochemical, and biophysical techniques, a central theme in this proposal is the identification, characterization, and application of small molecules to analyze protein function in vivo and in vitro. Small molecules can be powerful tools for studying bacterial and eukaryotic cytoskeletons, as they enable the rapid perturbation of protein function in vivo. Nevertheless, only few small molecules are currently available for specifically targeting cytoskeletal elements in bacteria. The discovery of chemical probes is poised to further our understanding of bacterial cell biology, while new classes of small molecules that emerge from these studies may provide starting points for developing new classes of antibacterial agents. Compounds that regulate widely conserved proteins may also inspire the development of therapeutic compounds for cancer and immune research and treatment.
In the first three years of the HFSP grant, we established a successful formula for productive collaboration between our labs. For the renewal, we have modified this formula to match the goals of the next phase of this project by focusing the expertise and capabilities of an interdisciplinary group of young scientists: Zemer Gitai uses high-throughput methods to study the bacterial cytoskeleton, Justin Kollman uses electron microscopy to study the biophysics of cytoskeletal assembly, Martin Thanbichler studies the biochemistry and genetics of the bacterial cytoskeleton, and Doug Weibel uses chemistry and engineering to study bacterial physiology. The salient features of this collaborative and interdisciplinary project will facilitate intellectual and cultural cross-pollination, and the scope and high-risk-high-reward merit of this proposal are designed specifically for an international funding mechanism such as the HFSP.

Most neurons in the mammalian brain are born during embryogenesis and persist throughout the life of the animal. The mammalian olfactory bulb is a notable exception in that it receives many thousands of newly-born inhibitory granule cells (GCs) throughout adulthood. GCs provide recurrent and lateral inhibition to mitral cells, principal cells in the olfactory bulb, but the precise functional role of adult neurogenesis is unknown. Here we propose a collaborative and innovative project to test the hypothesis that adult-born GCs play a unique and essential role in odor learning, combining optogenetics, intersectional genetics, in vivo two-photon calcium imaging and behavioral tasks.
To enable selective characterization and manipulation of adult-born GCs, we will develop a genetic method to selectively target adult-born GCs. This strategy combines two independent recombinases for intersectional specificity and an inducible recombination system. With this genetic method, we will express a genetically encoded calcium indicator selectively in adult-born GCs and use in vivo two-photon calcium imaging to characterize their odor response properties in awake, head-fixed mice. Next, to test the effect of suppressing the activity of adult-born GCs, we will develop optogenetic tools that suppress neuronal activity acutely and reversibly. The strategy includes modifications of pre-existing opsins to develop novel, inhibitory step-function opsins (iSFOs). iSFO-expressing neurons will be suppressed upon the delivery of a brief pulse of activating wavelength of light for extended periods of time even in the absence of sustained illumination until the delivery of an inactivating wavelength of light. We will express iSFO selectively in adult-born GCs using the genetic method described above. We will use two-photon calcium imaging to monitor in vivo the odor responses of mitral cells when adult-born GCs are suppressed and unsuppressed on alternating trials with iSFO in awake, head-fixed mice. We will determine relative contributions of local dendritic activity and global, action potential-dependent activity of adult-born GCs by targeting iSFO in specific subcellular compartments. Finally, we will test the role of adult-born GCs in odor learning, by training mice for odor discrimination tasks and determining if the suppression of adult-born GC activity by iSFO affects the animal’s learning.
Together, this collaboration combines complementary expertise and cutting-edge techniques of three co-PIs to understand the functional role of adult neurogenesis, one of the most dramatic forms of plasticity in the adult brain.