Statistical Mechanics of Allosteric Enzymes

The concept of allostery in which macromolecules switch between two different conformations is a central theme in biological processes ranging from gene regulation to cell signaling to enzymology. Allosteric enzymes pervade metabolic processes, yet a simple and unified treatment of the effects of allostery in enzymes has been lacking. In this work, we take a step toward this goal by modeling allosteric enzymes and their interaction with two key molecular players—allosteric regulators and competitive inhibitors. We then apply this model to characterize existing data on enzyme activity, comment on how enzyme parameters (such as substrate binding affinity) can be experimentally tuned, and make novel predictions on how to control phenomena such as substrate inhibition

Quantitative biology: where modern biology meets physical sciences

Quantitative methods and approaches have been playing an increasingly important role in cell biology in recent years. They involve making accurate measurements to test a predefined hypothesis in order to compare experimental data with predictions generated by theoretical models, an approach that has benefited physicists for decades. Building quantitative models in experimental biology not only has led to discoveries of counterintuitive phenomena but has also opened up novel research directions. To make the biological sciences more quantitative, we believe a two-pronged approach needs to be taken. First, graduate training needs to be revamped to ensure biology students are adequately trained in physical and mathematical sciences and vice versa. Second, students of both the biological and the physical sciences need to be provided adequate opportunities for hands-on engagement with the methods and approaches necessary to be able to work at the intersection of the biological and physical sciences. We present the annual Physiology Course organized at the Marine Biological Laboratory (Woods Hole, MA) as a case study for a hands-on training program that gives young scientists the opportunity not only to acquire the tools of quantitative biology but also to develop the necessary thought processes that will enable them to bridge the gap between these disciplines

Molecular Fingerprint and Developmental Regulation of the Tegmental GABAergic and Glutamatergic Neurons Derived from the Anterior Hindbrain

Tegmental nuclei in the ventral midbrain and anterior hindbrain control motivated behavior, mood, memory, and movement. These nuclei contain inhibitory GABAergic and excitatory glutamatergic neurons, whose molecular diversity and development remain largely unraveled. Many tegmental neurons originate in the embryonic ventral rhombomere 1 (r1), where GABAergic fate is regulated by the transcription factor (TF) Tal1. We used single-cell mRNA sequencing of the mouse ventral r1 to characterize the Tal1-dependent and independent neuronal precursors. We describe gene expression dynamics during bifurcation of the GABAergic and glutamatergic lineages and show how active Notch signaling promotes GABAergic fate selection in postmitotic precursors. We identify GABAergic precursor subtypes that give rise to distinct tegmental nuclei and demonstrate that Sox14 and Zfpm2, two TFs downstream of Tal1, are necessary for the differentiation of specific tegmental GABAergic neurons. Our results provide a framework for understanding the development of cellular diversity in the tegmental nuclei.Peer reviewe

AD-linked R47H-TREM2 mutation induces disease-enhancing microglial states via AKT hyperactivation

The hemizygous R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), a microglia-specific gene in the brain, increases risk for late-onset Alzheimer’s disease (AD). Using transcriptomic analysis of single nuclei from brain tissues of patients with AD carrying the R47H mutation or the common variant (CV)–TREM2, we found that R47H-associated microglial subpopulations had enhanced inflammatory signatures reminiscent of previously identified disease-associated microglia (DAM) and hyperactivation of AKT, one of the signaling pathways downstream of TREM2. We established a tauopathy mouse model with heterozygous knock-in of the human TREM2 with the R47H mutation or CV and found that R47H induced and exacerbated TAU-mediated spatial memory deficits in female mice. Single-cell transcriptomic analysis of microglia from these mice also revealed transcriptomic changes induced by R47H that had substantial overlaps with R47H microglia in human AD brains, including robust increases in proinflammatory cytokines, activation of AKT signaling, and elevation of a subset of DAM signatures. Pharmacological AKT inhibition with MK-2206 largely reversed the enhanced inflammatory signatures in primary R47H microglia treated with TAU fibrils. In R47H heterozygous tauopathy mice, MK-2206 treatment abolished a tauopathy-dependent microglial subcluster and rescued tauopathy-induced synapse loss. By uncovering disease-enhancing mechanisms of the R47H mutation conserved in human and mouse, our study supports inhibitors of AKT signaling as a microglial modulating strategy to treat AD

Manipulations microfluidiques de gouttelettes pour l'évolution dirigée des protéines

La compartimentalisation de la "soupe primordiale" dans des vésicules est considérée comme l'un des principaux facteurs ayant permis l'émergence de la vie. Ces gouttelettes de quelques micromètres créent un lien entre génotype et phénotype, et grâce à laThe compartmentalization of the primordial soup into vesicles is thought to be one of the key features in the early emergence of life. These tiny micrometer-sized droplets provided a linkage between phenotype and genotype, and through division, a mechani

Manipulations microfluidiques de gouttelettes pour l'évolution dirigée des protéines

La compartimentalisation de la "soupe primordiale" dans des vésicules est considérée comme l'un des principaux facteurs ayant permis l'émergence de la vie. Ces gouttelettes de quelques micromètres créent un lien entre génotype et phénotype, et grâce à la division, un mécanisme pour l'hérédité et l'évolution, qui a conduit à l'émergence des cellules actuelles. De tels microcompartiments, peuvent être créés au laboratoire sous la forme d'une émulsion composée de millions de gouttelettes contenant des gènes et tous les ingrédients nécessaires pour leur expression in vitro. Ces émulsions miment ainsi des populations de cellules artificielles qui peuvent être sélectionnées pour un phénotype donné sous des conditions strictement contrôlées non réalisables dans des systèmes in vivo. Cette thèse de doctorat présente le développement de systèmes microfluidiques pour l'évolution dirigée. Les résultats obtenus montrent qu il est possible de produire des gouttelettes hautement monodisperses pouvant être manipulées de manière précise et contrôlable, ce qui était jusqu à présent impossible pour des émulsions réalisées par des méthodes classiques. En utilisant un ensemble de nouveaux dispositifs microfluidiques et une composition adéquate d'huile porteuse, des gènes uniques ont été amplifiés et leur expression in vitro mesurée en microgouttelettes. Ces dispositifs ont ensuite été utilisés pour réaliser et analyser des réactions biologiques complexes et multi-étapes. Une technique originale de fusion passive de paires de gouttelettes a également été développée. Ces travaux constituent les premiers pas vers la création de plate-formes microfluidiques intégrées et totalement in vitro.The compartmentalization of the primordial soup into vesicles is thought to be one of the key features in the early emergence of life. These tiny micrometer-sized droplets provided a linkage between phenotype and genotype, and through division, a mechanism for heredity and evolution, which gave rise to modern cells. Man-made compartments, in the form of an emulsion, can also provide a tool of linking genotype to phenotype. Composed of millions of droplets containing genes with all ingredients necessary for in vitro expression, emulsions mimic populations of artificial cells that can be selected for a particular phenotype under strictly controlled conditions not feasible in living systems. The research described in this doctoral thesis focuses on the development of droplet-based microfluidics for protein evolution and presents the first steps toward an integrated and completely in vitro microfluidics platform. The results obtained in this work show that it is possible to produce highly monodisperse picoliter volume droplets (CV<1%) that can be manipulated in a precise and controllable manner, previously impossible in bulk emulsions. Using a set of novel microfluidic devices and an adequate composition of carrier oil single genes in droplets were amplified and their in vitro expression measured. The same microfluidic system was also used to perform multiple operations in order to analyze complex and sequential biological reactions in droplets. Moreover, a new passive droplet fusion technique has been developed, which can be used for preparation of monodisperse emulsions composed of pairwise fused droplets

Manipulations microfluidiques de gouttelettes pour l'évolution dirigée des protéines

La compartimentalisation de la "soupe primordiale" dans des vésicules est considérée comme l'un des principaux facteurs ayant permis l'émergence de la vie. Ces gouttelettes de quelques micromètres créent un lien entre génotype et phénotype, et grâce à la division, un mécanisme pour l hérédité et l évolution, qui a conduit à l'émergence des cellules actuelles. De tels microcompartiments, peuvent être créés au laboratoire sous la forme d une émulsion composée de millions de gouttelettes contenant des gènes et tous les ingrédients nécessaires pour leur expression in vitro. Ces émulsions miment ainsi des populations de cellules artificielles qui peuvent être sélectionnées pour un phénotype donné sous des conditions strictement contrôlées non réalisables dans des systèmes in vivo. Cette thèse de doctorat présente le développement de systèmes microfluidiques pour l évolution dirigée. Les résultats obtenus montrent qu il est possible de produire des gouttelettes hautement monodisperses pouvant être manipulées de manière précise et contrôlable, ce qui était jusqu à présent impossible pour des émulsions réalisées par des méthodes classiques. En utilisant un ensemble de nouveaux dispositifs microfluidiques et une composition adéquate d huile porteuse, des gènes uniques ont été amplifiés et leur expression in vitro mesurée en microgouttelettes. Ces dispositifs ont ensuite été utilisés pour réaliser et analyser des réactions biologiques complexes et multi-étapes. Une technique originale de fusion passive de paires de gouttelettes a également été développée. Ces travaux constituent les premiers pas vers la création de plate-formes microfluidiques intégrées et totalement in vitro.The compartmentalization of the primordial soup into vesicles is thought to be one of the key features in the early emergence of life. These tiny micrometer-sized droplets provided a linkage between phenotype and genotype, and through division, a mechanism for heredity and evolution, which gave rise to modern cells. Man-made compartments, in the form of an emulsion, can also provide a tool of linking genotype to phenotype. Composed of millions of droplets containing genes with all ingredients necessary for in vitro expression, emulsions mimic populations of artificial cells that can be selected for a particular phenotype under strictly controlled conditions not feasible in living systems. The research described in this doctoral thesis focuses on the development of droplet-based microfluidics for protein evolution and presents the first steps toward an integrated and completely in vitro microfluidics platform. The results obtained in this work show that it is possible to produce highly monodisperse picoliter volume droplets (CV<1%) that can be manipulated in a precise and controllable manner, previously impossible in bulk emulsions. Using a set of novel microfluidic devices and an adequate composition of carrier oil single genes in droplets were amplified and their in vitro expression measured. The same microfluidic system was also used to perform multiple operations in order to analyze complex and sequential biological reactions in droplets. Moreover, a new passive droplet fusion technique has been developed, which can be used for preparation of monodisperse emulsions composed of pairwise fused droplets.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

A fast and efficient microfluidic system for highly selective one-to-one droplet fusion

International audienc

Multi-step microfluidic droplet processing: kinetic analysis of an in vitro translated enzyme

International audienc

High-throughput single-cell antibody secretion quantification and enrichment using droplet microfluidics-based FRET assay

High-throughput screening and enrichment of antibody-producing cells have many important applications. Herein, we present a droplet microfluidic approach for high-throughput screening and sorting of antibody-secreting cells using a Förster resonance electron transfer (FRET)-based assay. The FRET signal is mediated by the specific binding of the secreted antibody to two fluorescently labeled probes supplied within a droplet. Functional hybridoma cells expressing either membrane-bound or secreted monoclonal antibodies (mAbs), or both, were efficiently differentiated in less than 30 min. The antibody secretion rate by individual hybridoma cells was recorded in the range of 14,000 Abs/min, while the density of membrane-bound fraction was approximately 100 Abs/μm2. Combining the FRET assay with droplet-based single-cell sorting, an 800-fold enrichment of antigen-specific cells was achieved after one round of sorting. The presented system overcomes several key limitations observed in conventional FACS-based screening methods and should be applicable to assaying various other secreted proteins.ISSN:2589-004