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.

Small regulatory RNAs carry out diverse functions in both bacterial and eukaryotic cell physiology. Despite a known role for sRNAs in ribosome biogenesis in eukaryotes, the potential role for sRNAs in bacterial ribosome biogenesis and/or (pre-) ribosome degradation and storage under stress conditions is unknown. Based on their functional properties and versatilities I hypothesize that small RNAs are involved in bacterial ribosome biogenesis. Using a combination of bioinformatic, cell biological and biochemical techniques in vitro and in vivo, I aim to identify small RNAs with physiologically relevant functions in ribosome assembly either as assembly co-factors or as regulators of ribosome biogenesis in response to environmental changes. Moreover, I will investigate at which conditions the identified sRNAs regulate ribosome biogenesis, which interaction partners are involved, which mechanisms are employed and which consequences result from sRNA function. To determine the influence of sRNAs on folding and processing of rRNA, I will use an in-vitro co-transcriptional assembly system.
In parallel, I aim to identify artificial small RNAs that could be used as effectors of ribosome biogenesis or as probes to monitor co-transcriptional folding of ribosomal RNA and thus the kinetics of ribosome assembly in mechanistic detail. The work will provide physiological and mechanistic insight into the function and regulation by sRNAs in ribosome biogenesis as well as into co-transcriptional rRNA folding.

While the fundamental functions of sleep remain unknown, deficits in learning and memory after sleep loss are shared between humans and genetic model systems, including the fruit fly. My past research has found that sleep strongly modulates the consolidation of recently learned associations into long-term memories - sleep deprivation after learning prevents memory consolidation, and acutely inducing sleep enhances the consolidation of newly acquired memories that would typically be forgotten. While these data demonstrate an important role for sleep in memory processing, the molecular processes that occur during sleep to support memory consolidation remain unknown. To date, the complexity and parallel structures of memory-encoding and sleep control circuits in mammalian systems prevent researchers from properly investigating these mechanisms. In the fruit fly, however, memory-encoding circuits in the Mushroom Body and sleep control neurons in the dorsal Fan-shaped Body are accessible for in vivo whole cell recordings and genetically addressable at the resolution of individual neurons. The proposed project will 1) use in vivo patch clamp recordings to examine the neural circuits that promote sleep during memory consolidation, and 2) profile actively translated mRNAs in memory-encoding circuits to identify proteins that facilitate memory consolidation during sleep. Understanding and controlling these mechanisms will not only provide insight into the neural functions of sleep, but support the development of tools to offset the cognitive consequences of sleep loss and to influence whether recently acquired memories are retained or forgotten.

Viral entry into its host cell may results in either successful or abortive infection. Abortive infection of permissive cells, which are able to support viral replication, is rarely discussed but is quite abundant. What makes one cell resist to infection while its neighbor succumb to it is the subject of this research proposal. This is important as it could lead to the discovery of novel cellular anti-viral modalities and deepen our understanding of host-virus interactions.
My goal is to characterize the anti-viral response to viral infections in single cells, its variability among genetically identical cells and how this relates to infection outcome (successful or abortive).
To do so, I will learn and implement new technologies developed by the Tay lab. The success of the project relies on using a combination of live-cell imaging with in-lab fabricated microfluidics devices that allow the retrieval of single cells with known fates for further analyses, such as single-cell RNA-sequencing. By using viruses that express fluorescent markers I will be able to monitor infection in living cells in real-time, extract and molecularly profile cells in specific time points, for example in the process of aborting viral infection (when fluorescent proteins level stop raising or begin to drop). The results from this screening approach will be validated using standard cell-biology and virology techniques. I also plan to use this technology to investigate the response of the known anti-viral pathways.
I expect to reveal new molecular participants in the anti-viral response as well as to explore the variability in the response among single cells and how it affects infection outcome.

The purpose of this proposal is to recreate an ancient living system, an organism that uses RNA catalysts, ribozymes, as part of its translation apparatus. We will achieve this grand goal by progressively reintroducing ribozymes into cellular metabolism, ultimately replacing protein enzymes. To this end, Hiroaki Suga will develop novel ribozymes, Flexizymes, that can charge tRNAs with amino acids. Andy Ellington will adapt Flexizymes for use in cells, and replace cognate aminoacyl-tRNA synthetases. Michael Jewett will adapt Flexizyme charging to an orthogonal ribosome that can specifically utilize Flexizymes. Both Ellington and Jewett will hand organisms containing Flexizymes and orthogonal ribosomes to Philippe Marliere for high-throughput evolutionary adaptation and optimization. Ultimately, this will result in the creation of an organism that serves as a doppelganger for ancient living systems in transition from an RNA to a protein world. This ribo-organism will be unique in that it will have two translation apparatuses operating side-by-side, one of which has the normal complement of cellular machinery, and one of which has Flexizymes, orthogonal tRNAs, an orthogonal ribosome, and a new genetic code. We will initially carry out our goals by altering the machinery for the incorporation of histidine, but eventually expand the genetic code to unnatural amino acid analogues of histidine, 1,2,4-triazole-3-alanine (T3A) and alpha-hydroxy histidine (AHH), in order to highlight potential biotechnology applications. The incorporation of these new amino acids into the genetic code, both as 21st amino acids (via suppression) and in competition with an existing amino acid, histidine (via missense incorporation), should serve as a modern experimental surrogate for the ancient establishment of the genetic code. This project is also notable for its broad interdisciplinary flavor, in that it spans from a chemist (Suga) to a biochemist (Ellington) to a bioengineer (Jewett) to an evolutionary biologist (Marliere). Each individual field touches on the other, but the overall arc traverses a much broader swath of science and technology than would otherwise be possible. The intermingling of distinct cultures is also apparent in the scope of the work, which for the first time attempts multi-scale evolutionary optimization of an entire cellular sub-system, translation.

Mammals have the ability to generate an infinite variety of motor behaviors, from simple actions such as walking to highly complex movements like object manipulation or speech. The flexibility of the motor repertoire is essential for animal survival because it enables the generation of goal-directed movements adapted to environmental constrains. Some motor patterns are present at birth while new motor skills can be acquired through training and experience. Not surprisingly, communication among numerous brain areas is needed to ensure accurate acquisition and execution of motor programs. The classical model for motor learning proposes that only a subset of structures along the motor command pathway within forebrain and cerebellum are subjected to activity-dependent adjustments and become reorganized during acquisition of a new skill. In contrast, downstream motor regions located in the brainstem are considered to be simple executive centers for stereotyped motor behaviors. Here I challenge this model and propose that learning-induced plasticity in brainstem motor circuits is essential to adapt the executive motor signal to drive new skills. To evaluate my hypothesis I will combine cutting-edge viral and mouse genetic tools together with in vivo and ex vivo electrophysiology to characterize how learning-induced signals are encoded in the brainstem. This proposal supports a new conceptual framework in which plasticity mechanisms along the entire motor command pathway including brainstem circuits, underlie the formation of motor memories.

The segmented body pattern of vertebrates is established in the early development through the periodic formation of somites, the vertebrae precursors, from the presomitic mesoderm (PSM). The rhythmic formation of somites is under control of a molecular oscillator, the segmentation clock, which controls the cyclic transcription of a group of genes within the PSM.
Previous microarray analyses and candidate gene approaches identify many cyclic genes in the PSM, mostly genes associated to the Wnt, Notch and FGF-signaling pathways. Yet, a comprehensive, dynamic and quantitative analysis of the transcriptional dynamics and regulation of cyclic genes is still lacking, mostly due to the challenge to obtain samples with unequivocal time resolution. By using a microfluidic system that allows controlling the rhythm and, thus, synchronizing samples in relation to an external reference time, we are now able to obtain nearly unlimited PSM cells accurately staged. With a combination of high-throughput sequencing methods, we plan to identify unknown cyclic genes and regulatory elements (enhancers/promoter) with a role in somitogenesis. We aim to establish a temporal relationship between genes and their regulatory regions, and identify molecular mechanisms that control the rhythmic formation of somites. For example we may discover that cyclic expression relies on the periodic interplay of multiple enhancers. Or, that it is caused by cyclic epigenomic modification within regulatory elements. Additionally, by identifying sequence motifs within these elements we will be able to connect signaling pathways to oscillatory gene expression.

Although the basis of nucleotide binding and oligomerization domain (Nod) receptors signaling has been discussed for several years, the molecular details that underlie this process are still being uncovered. Moreover its presence on brain neurons hasn’t been described yet. The role of Nod2 receptor goes beyond simply sensing the presence of a potential pathogen and activating the immune system. Its activation has been linked to stem cells cytoprotection, analgesic effects, protection from cerebral ischemia-reperfusion damage and altered sleeping patterns. Moreover, mutations on this gene have been associated with bipolar disorder and schizophrenia in humans. The cytosolic Nod2 receptor can be activated by muramyl dipeptides a product of the degradation of peptidoglycan, a polymer present in almost all bacteria cell wall and increasing evidence demonstrate that endogenous ligands like damage-associated molecular patters, can also activate Nod receptors. Particularly, not much is known about the impact of Nod2 activation on behavior or the mechanisms and the specific effects on brain physiology. Understanding the role of Nod2 on brain neurons can lead to better understanding of many neurological disorders. Preliminary data from Dr. Lledo lab have shown, the presence of Nod2 receptors in neurons from the striatal and thalamic areas of the brain. They also observed that Nod2 knock-outs show less anxiety, more social aggressiveness and lack of discrimination of olfactory cues in social interactions and that Nod2 signaling can selectively disrupt paradoxical sleep. Therefore the purpose of this proposal is to assess the impact of Nod2 signaling on brain activity and behavior.

Polyploid cells, which contain more than two pairs of homologous chromosomes, are widely present across protozoa, plants and animals. Polyploidization occurs in many cell and tissue types as part of a developmental program, and is crucial for increases in metabolic output, cell mass and cell size. Two cell cycle variations, endoreplication and endomitosis, are known to give rise to polyploid cells. Although the function and molecular regulation of endoreplication has been extensively studied, the molecular factors that control endomitosis are largely unknown. Specifically, it is unclear how endomitotic cycles are initiated and executed, and what the advantages are of endomitosis over endoreplication. The C. elegans intestine provides an ideal system to study these questions, as transitions between canonical, endomitotic and endoreplicative cycles all take place at defined moments during development. In this study, we will identify the molecular regulators of endomitosis in the C. elegans intestine by performing single-cell sequencing and RNA Tomography sequencing (Tomo-seq) of intestinal cells at specific cell-cycle stages. Moreover, we will uncover the physiological importance of endomitosis and the functional difference between endomitosis and endoreplication by generating tissue-specific C. elegans mutants with alterations in the intestinal cell cycles and analyzing their intestinal function. Together, these approaches will provide an in-depth analysis of endomitosis mechanism and function, providing insights into how tissues specify cell-cycle programs and what the importance is of specific forms of polyploidization.

While cancer has been shown to rely on the accumulation of multiple mutations, conditions such as injury have been also shown to trigger cancer. Consistently, a recurrent mutation in Squamous Cell Carcinoma (SCC) in the Ras oncogene is not sufficient to trigger SCC unless either injury or a second mutation occurs. Yet, it remains elusive how wound-triggered behaviors lead to SCC. This proposal aims to identify wound-induced epithelial and niche cell behaviors that are required for cancer initiation. My post-doctoral lab has recently established an imaging approach to study how epithelial stem cell behaviors are coordinated to close a wound in live mice. I will build on this understanding and combine the study of wound repair in the context of a mouse model expressing activated-Ras mutation in epithelial stem cells. Specifically, I will determine what changes in cell behaviors, such as proliferation and migration, occur in the Ras model and I will use drug-based and genetic manipulation models to determine the functional impact of examined mutant behaviors towards SCC initiation. Furthermore, I will interrogate and manipulate the immune response, a central component of both the wound repair process and SCC, in the epithelial Ras mutant model to dissect the immune cell role during SCC insurgence. Our innovative live imaging approaches to track my populations of interest in combination with manipulative approaches will allow me to identify the relevant cells and behaviors towards cancer initiation. All together my project will provide new insights into how the physiological process of wound repair can be hijacked into a process of malignant transformation.

My goal is to understand how the cadherin-catenin complex, a force-responsive protein assembly that links neighboring cells, transduces extracellular forces into intracellular signaling events. Thus far, absolute force magnitude has been considered the main control parameter in mechanotransduction; but because mechanical cues in tissues and organs are subject to tight temporal control, it is likely that force timescales complement force magnitude to modulate junctional remodeling and downstream signal transduction. For example, during Drosophila embryogenesis regular contraction forces mediate the formation of the ventral furrow and gastrulation. In this project, I will characterize, for the first time to my knowledge, the response of intercellular adhesions to not only force amplitude, but also its temporal variation. Specifically, I will combine single-molecule force spectroscopy and single-molecule fluorescence microscopy techniques to directly quantify the kinetics of individual cadherin-catenins complexes in cells upon temporally controlled mechanical stimuli. Using this approach, I will determine how the cadherin-catenin complex integrates temporal cues into the mechanotransduction process. As a working model, I propose that cadherin clustering responds both the force magnitude and timescale, and that clustering is the key event that drives downstream signal transduction. My long-term goal as an independent investigator is to use the approaches outlined in this proposal to understand how macromolecular complexes produce, detect and respond to forces to accomplish biological processes such as mitosis, cell migration and mechanotransduction.

Life involves a myriad of inter-related chemical processes, almost none of which would proceed at adequate rates without the assistance of enzymes. Enzymes are “Nature’s catalysts”, accelerating the rates of these reactions by up to ~20 orders of magnitude, thus making life as we know it possible. No consensus has yet emerged about the ultimate origin of the "catalytic power" of enzymes, with a rather wide variety of hypotheses put forward over the years. In addition, the limitations in our understanding of enzyme catalysis are highlighted by our inability to reproduce the catalytic power of the best naturally occurring enzymes in any human made catalyst (including de novo designed enzymes); as Feynman famously pointed out, "What I cannot create, I do not understand". This lack of a definitive understanding in the origins of enzyme catalysis is most likely related to the fact that natural enzymes are the complex outcome of natural selection operating over vast expanses of time in an evolutionary process that may be determined to some extent by contingency. Here, we posit that an in-depth understanding of the catalytic power of enzymes will only be possible when we have at our disposal a procedure that routinely reproduces the emergence of new functions from non-catalytic (or minimally catalytic) scaffolds in the laboratory. Therefore, during the course of this project, we will:
1) use phylogenetic analysis to reconstruct sequences of ancestral proteins from a structure having high potential to serve as a scaffold for generating novel functionalities;
2) prepare the encoded proteins in the lab and test them not just experimentally but also computationally for the biophysical and biochemical properties that confer high evolvability on a protein scaffold;
3) synthesize very large libraries of up to ten trillion (10E13) variants using highly evolvable ancestral proteins as scaffolds;
4) use ultra-high-throughput in vitro and in vivo methodologies to screen these libraries for non-natural functions;
5) use both experimental and multiscale modeling tools to characterize the resulting de novo enzymes for the biophysical, biochemical and structural features relevant for efficient catalysis;
6) use the above characterization to test different hypotheses about the evolutionary and molecular origin of enzyme catalysis.

While there is growing realization that sleep is important for memory consolidation, most evidence remains correlative and the causal role of specific sleep events remains unclear. Moreover, there is a substantial gap between detailed electrophysiological animal research and non-invasive cognitive human research – where recording neural activity in the medial temporal lobe is particularly challenging. The proposed research aims to understand the causal role that sleep plays in memory consolidation. Upon informed consent, epilepsy patients at UCLA implanted with intracranial electrodes for clinical monitoring will participate in learning/memory paradigms combined with recordings and intracranial electrical stimulation during sleep. I will address two specific aims: (1) Determine which sleep activities correlate best with learning and memory improvements: I hypothesize that for declarative memory, NREM sleep will be best correlated with learning, and that coordinated coupling between neocortical slow waves, sleep spindles and hippocampal ripples, co-occurring with reactivation of neuronal ensembles that were selectively engaged in the learning task, will be maximally correlated with successful learning. (2) Elucidate the causal mechanisms through intracranial electrical stimulation. I hypothesize that electrical stimulation in the neocortex that mimics or locks to endogenous slow wave up-states and hippocampal ripples will be effective in promoting memory consolidation. This proposal has the potential to generate a breakthrough in our understanding of the coordination between neocortex and hippocampus during sleep and how sleep causally aids human memory consolidation.

Cell motility plays an important role in a multitude of biological processes, including embryogenesis, wound healing, and cancer metastasis. As cells move, they exert forces onto their environment and become increasingly polarized, resulting in an elongated shape and signaling and cytoskeletal components that become localized in the anterior or posterior of the cell. It is currently unclear how force generation is correlated with the distribution of key signaling components and how cells regulate their polarity. To address these questions requires a careful and precise control of external cues and the ability to link molecular events to the cell's macroscopic behavior and the generation of forces. In this project, I will quantify the establishment and reversal of cell polarity during chemotaxis using innovative microfluidic devices together with traction force microscopy (TFM) and confocal microscopy. Specifically, I will expose unpolarized Dictyostelium discoideum cells to carefully controlled chemoattractant gradients generated in microfluidic devices. I will quantify the kinetics of several key signaling components and of traction forces using confocal microscopy and TFM, respectively. Furthermore, I will examine how polarity is reversed by rapidly changing the gradient experienced by cells that migrate in specially designed 1D micropatterned tracks. By correlating the dynamics of the markers with the observed traction force patterns, I will be able to determine how cell polarity is established and reversed. Our experiments should result in a more comprehensive understanding of cell polarity, a crucial component of cell motility in many eukaryotic cell types.