During development, cells differentiate and form specific adhesive contacts with other cells in order to create tissues. Defects in this process give rise to developmental abnormalities and metastatic cancers. The strength and mechanical properties of cell-cell adhesions govern the mechanical strength and integrity of tissues subject to mechanical stress, such as epithelia. Despite these important physiological outcomes, mechanisms underlying the response of cells to external mechanical forces remain poorly studied. Gaining insight into these mechanisms is crucial to our understanding of normal development, wound healing, disease processes and tissue engineering therapies.The proposed project integrates the expertise of 4 teams, on 3 continents to examine how mechanical forces influence the formation and properties of cell-cell adhesions.
This collaborative project brings together expertise in the design of: (i) devices to accurately measure forces at the molecular and cellular scale (Lim & Ladoux); (ii) nano and micro-fabricated environments (Ladoux); (iii) molecular and cellular tools and (iv) biochemical characterization of molecules in cell-cell contacts (Nelson and Mège). Additionally, the interface between these groups provides a novel training platform for postdoctoral fellows, and undergraduate and graduate students in team building and multi-disciplinary research that crosses boundaries between engineering, biophysics, biochemistry and cell biology. The ability to examine the effects of forces on both cellular and molecular scales offers a powerful way to define the relations between molecular and structural intracellular organization, cell mechanical properties and cell behavior.
The specific goals of the proposal are: 1. Design and validate new tools that enable experimental procedures to be more quantitative in the context of mechanics of cell-cell junctions in epithelial tissues, and develop well-defined microenvironments to control cell adhesion and obtain quantitative measurements of cell behaviors ; 2. Investigate how cells regulate cell-cell junctions in coordination with cytoskeleton dynamics; 3. Investigate the mechano-sensitivity of cadherin-associated proteins at the single-molecule level. These studies will define the mechanisms of tissue cohesion at molecular, subcellular and multicellular scales, and provide a mechanistic framework of the effects of external mechanical forces on epithelial cell-cell contacts.

During development, cells differentiate and form specific adhesive contacts with other cells in order to create tissues. Defects in this process give rise to developmental abnormalities and metastatic cancers. The strength and mechanical properties of cell-cell adhesions govern the mechanical strength and integrity of tissues subject to mechanical stress, such as epithelia. Despite these important physiological outcomes, mechanisms underlying the response of cells to external mechanical forces remain poorly studied. Gaining insight into these mechanisms is crucial to our understanding of normal development, wound healing, disease processes and tissue engineering therapies.The proposed project integrates the expertise of 4 teams, on 3 continents to examine how mechanical forces influence the formation and properties of cell-cell adhesions.
This collaborative project brings together expertise in the design of: (i) devices to accurately measure forces at the molecular and cellular scale (Lim & Ladoux); (ii) nano and micro-fabricated environments (Ladoux); (iii) molecular and cellular tools and (iv) biochemical characterization of molecules in cell-cell contacts (Nelson and Mège). Additionally, the interface between these groups provides a novel training platform for postdoctoral fellows, and undergraduate and graduate students in team building and multi-disciplinary research that crosses boundaries between engineering, biophysics, biochemistry and cell biology. The ability to examine the effects of forces on both cellular and molecular scales offers a powerful way to define the relations between molecular and structural intracellular organization, cell mechanical properties and cell behavior.
The specific goals of the proposal are: 1. Design and validate new tools that enable experimental procedures to be more quantitative in the context of mechanics of cell-cell junctions in epithelial tissues, and develop well-defined microenvironments to control cell adhesion and obtain quantitative measurements of cell behaviors ; 2. Investigate how cells regulate cell-cell junctions in coordination with cytoskeleton dynamics; 3. Investigate the mechano-sensitivity of cadherin-associated proteins at the single-molecule level. These studies will define the mechanisms of tissue cohesion at molecular, subcellular and multicellular scales, and provide a mechanistic framework of the effects of external mechanical forces on epithelial cell-cell contacts.

During development, cells differentiate and form specific adhesive contacts with other cells in order to create tissues. Defects in this process give rise to developmental abnormalities and metastatic cancers. The strength and mechanical properties of cell-cell adhesions govern the mechanical strength and integrity of tissues subject to mechanical stress, such as epithelia. Despite these important physiological outcomes, mechanisms underlying the response of cells to external mechanical forces remain poorly studied. Gaining insight into these mechanisms is crucial to our understanding of normal development, wound healing, disease processes and tissue engineering therapies.The proposed project integrates the expertise of 4 teams, on 3 continents to examine how mechanical forces influence the formation and properties of cell-cell adhesions.
This collaborative project brings together expertise in the design of: (i) devices to accurately measure forces at the molecular and cellular scale (Lim & Ladoux); (ii) nano and micro-fabricated environments (Ladoux); (iii) molecular and cellular tools and (iv) biochemical characterization of molecules in cell-cell contacts (Nelson and Mège). Additionally, the interface between these groups provides a novel training platform for postdoctoral fellows, and undergraduate and graduate students in team building and multi-disciplinary research that crosses boundaries between engineering, biophysics, biochemistry and cell biology. The ability to examine the effects of forces on both cellular and molecular scales offers a powerful way to define the relations between molecular and structural intracellular organization, cell mechanical properties and cell behavior.
The specific goals of the proposal are: 1. Design and validate new tools that enable experimental procedures to be more quantitative in the context of mechanics of cell-cell junctions in epithelial tissues, and develop well-defined microenvironments to control cell adhesion and obtain quantitative measurements of cell behaviors ; 2. Investigate how cells regulate cell-cell junctions in coordination with cytoskeleton dynamics; 3. Investigate the mechano-sensitivity of cadherin-associated proteins at the single-molecule level. These studies will define the mechanisms of tissue cohesion at molecular, subcellular and multicellular scales, and provide a mechanistic framework of the effects of external mechanical forces on epithelial cell-cell contacts.

During development, cells differentiate and form specific adhesive contacts with other cells in order to create tissues. Defects in this process give rise to developmental abnormalities and metastatic cancers. The strength and mechanical properties of cell-cell adhesions govern the mechanical strength and integrity of tissues subject to mechanical stress, such as epithelia. Despite these important physiological outcomes, mechanisms underlying the response of cells to external mechanical forces remain poorly studied. Gaining insight into these mechanisms is crucial to our understanding of normal development, wound healing, disease processes and tissue engineering therapies.The proposed project integrates the expertise of 4 teams, on 3 continents to examine how mechanical forces influence the formation and properties of cell-cell adhesions.
This collaborative project brings together expertise in the design of: (i) devices to accurately measure forces at the molecular and cellular scale (Lim & Ladoux); (ii) nano and micro-fabricated environments (Ladoux); (iii) molecular and cellular tools and (iv) biochemical characterization of molecules in cell-cell contacts (Nelson and Mège). Additionally, the interface between these groups provides a novel training platform for postdoctoral fellows, and undergraduate and graduate students in team building and multi-disciplinary research that crosses boundaries between engineering, biophysics, biochemistry and cell biology. The ability to examine the effects of forces on both cellular and molecular scales offers a powerful way to define the relations between molecular and structural intracellular organization, cell mechanical properties and cell behavior.
The specific goals of the proposal are: 1. Design and validate new tools that enable experimental procedures to be more quantitative in the context of mechanics of cell-cell junctions in epithelial tissues, and develop well-defined microenvironments to control cell adhesion and obtain quantitative measurements of cell behaviors ; 2. Investigate how cells regulate cell-cell junctions in coordination with cytoskeleton dynamics; 3. Investigate the mechano-sensitivity of cadherin-associated proteins at the single-molecule level. These studies will define the mechanisms of tissue cohesion at molecular, subcellular and multicellular scales, and provide a mechanistic framework of the effects of external mechanical forces on epithelial cell-cell contacts.

The last two stages in eukaryotic translation, termination and ribosome recycling, are very distinct from the corresponding, well-characterized stages in bacteria. Thus, eukaryotic termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition and peptidyl-tRNA hydrolysis by eRF1, whereas eukaryotic ribosome recycling is mediated by ABCE1, a unique highly conserved member of the ATP cassette family of proteins that contains an iron-sulfur cluster domain, and which does not exist in bacteria. Moreover, in contrast to bacteria, eukaryotic termination and ribosome recycling are intimately linked because eRF1 plays a key role in both processes. Although these two stages of eukaryotic protein synthesis are of major importance in regulation of eukaryotic translation, and are linked to downstream events such as reinitiation and mRNA surveillance, their mechanisms have only recently begun to attract attention. However, future progress in elucidating the molecular mechanism of eukaryotic termination and ribosome recycling is strongly compromised by the lack of structural data. The primary aim of this proposal is therefore to obtain a comprehensive overview of the architecture of defined intermediate states in termination and ribosome recycling and to model the transition between them. To achieve this goal, we shall integrate the complementary expertise of four leading laboratories in cryo-EM, X-ray crystallography, computational biology and functional studies.
The specific aims of the proposed research include: (i) elucidation of the molecular basis for the omnipotent decoding capacity of eRF1 using X-ray studies of modeled eRF1-associated 80S ribosomal complexes containing different stop codons in the A-site, (ii) determination by cryo-EM of the structures of eukaryotic ribosomal complexes corresponding to different stages of termination, from initial ribosomal attachment of release factors through GTP hydrolysis by eRF3 to the final stage corresponding to post-termination complexes containing deacylated tRNA in the P-site, (iii) visualization by cryo-EM of ABCE1 in post-termination complexes and identification of its critical functional interactions with 80S ribosomes and eRF1, (iv) computational analysis and dynamic simulation of ribosomal transition between individual stages in termination and ribosome recycling based on X-ray and cryo-EM results, and (v) functional investigation of the mechanisms of mammalian reinitiation, regarding its importance as a regulatory post-termination event. In summary, the results that are anticipated to emerge from the proposed studies will for the first time provide an integrated structural and mechanistic outline of the last two stages in eukaryotic translation.

This project seeks to understand how cells migrate in complex environments. While cell migration on flat substrates has been thoroughly studied, cells migrating in vivo are faced with substrates of varying geometry, molecular composition and mechanical properties. Here, the cells show a much richer phenotype and are known to adapt their internal machinery to the extracellular environment. Different cell types exhibit specific migration strategies such that some of them use predominantly actin polymerization-powered protrusions, while others use motility based on pressure-driven blebbing as a default. Furthermore, cells have been observed to switch between these two different migration strategies upon manipulation of the actin polymerization dynamics within the cell, but also upon modification of such substrate properties as rigidity and adhesiveness. We will employ zebrafish germ cells and mouse leukocytes as in vivo model systems that represent the “professionally” blebbing vs. the actin polymerization driven phenotype. In vitro, these and other model cell types will be subjected to genetic, pharmacological and most importantly environmental manipulations that will uncover their range of plasticity regarding force generation and transduction. We will study how the cellular protrusive and contractile activities respond to variations of the geometry, adhesiveness and stiffness of the external substrate and to the presence of external loads. Readouts will include the dynamic visualization of cytoskeletal components, morphological response of the cell and actual motility, while forces exerted by the cell will be recorded quantitatively. Our data will be analyzed in the light of physical models to understand the mechanisms by which internal and external factors affect the strategy of cell motility, and to identify the key physical parameters governing the efficiency of actin-powered protrusions and bleb-based motility. The proposed research will establish novel techniques to quantitatively measure forces and visualize cytokeletal dynamics in a wide range of engineered as well as physiological settings. The integration of experimental data and theoretical modeling should ultimately lead to the development of a universal phase diagram that allows to predict the migratory strategy of a cell as a function of internal and environmental conditions.

As mechanical systems, cells control their shape via tight coordination of the dynamic self-assembly of cytoskeletal polymers (e.g. actin filaments) and modulation of intracellular tension exerted among these filaments by molecular motor proteins (e.g. myosins). Highly dynamic and contractile actomyosin-based structures, such as stress fibers of non-muscle cells and sarcomeres of muscle tissue, lie at the heart of fundamental cellular processes including morphogenesis, establishment of polarity and overall motility. Although it is well established that stress fibers play central roles in cell biology, the chemical and physical principles driving the formation of these highly organized contractile structures are largely unknown. The identification of the biochemical mechanisms and biophysical laws that govern the macroscopic organization of the cytoskeleton in cells is rendered difficult by the complexity of the systems. Simplified experimental systems in which actin dynamics can be reconstituted in the presence of regulatory factors and motor proteins are needed to understand the mechanism of stress fiber formation and the consequences of myosin-dependent force generation on the organization and stability of complex actin structures. We will use a multi-disciplinary approach to reconstitute the assembly, turnover, and contractility of stress fibers in vitro and determine how the mechanical and contractile properties of intermediate and assembled structures depend on the individual protein components. Our approach will integrate a new versatile micropatterning method capable of imposing spatial boundary condition to the growth of actin filaments, the use of diverse molecular motors with unique biochemical properties, and the use of AFM and laser trap nanometry to obtain reliable determination of the generated contractile fiber structures and mechanical properties. Theoretical biophysical modeling and computer simulation will complement these experimental works. General principles regarding cell mechanics and the hierarchy of molecular components will emerge from this multi-disciplinary work.

The architecture of cells and subcellular structures can show remarkable variability between tissues and organisms, but there is currently little understanding of the evolutionary basis of this diversity. Thus it is unclear why metaphase spindles in different Eukaryotes exhibit a range of morphologies and the volumes of these spindles vary over one thousand fold. Here we propose a comparative study to investigate the evolutionary forces shaping the spindle. Using one cell stage nematode embryos as a model system, we will characterize spindle structure in several strains of ~60 species exhibiting a range of cell sizes, karyotypes, and life histories. A more detailed analysis of microtubule dynamics and spindle architecture from ~10 select species will reveal the biophysical basis of variation in spindle morphology. This comparative data will be interpreted using phylogenies and quantitative models of phenotypic evolution. We will then test the developed biophysical and evolutionary models with perturbation. This unique comparative approach combines molecular methods, quantitative microscopy, and mathematical models, to gain insight into how the mitotic spindle is shaped through evolution by selection, drift, and biophysical constraints.

Folding of epithelial sheets is one of the most ancient mechanisms leading to the formation of three-dimensional structures in developing tissues. In a highly simplified picture, epithelial morphogenesis can be separated into two steps. First, inductive signals establish two-dimensional patterns of gene expression across the epithelia. At the next step, these patterns are converted into spatial patterns of force generation and mechanical properties of cells, thus controlling the folding of a sheet into a target morphology. Previous studies of epithelial morphogenesis focused on single genes and small networks, but a systems-level model of morphogenesis in any given experimental context is yet to be developed. In developing such a picture, the key questions are related to the number and identities of involved genes, diversity and dynamics of their expression patterns, mechanisms of pattern formation, and connection between patterning and morphogenesis. We will investigate these questions in the context of the formation of the Drosophila eggshell, an established genetic model of epithelial morphogenesis.

This will be the first time that genetics, systems biology, cell biology, nonlinear dynamics, and continuum mechanics will be brought together to study patterning and morphogenesis in a system highly amenable to genetic manipulations. We will establish a two-dimensional atlas for the expression of dozens of genes involved in eggshell morphogenesis and formulate quantitative models for the formation of these patterns by signaling pathways. Based on live imaging and genetic experiments, we will formulate the hypotheses regarding the connection between patterns of gene expression and the mechanical properties of patterned epithelia. We will explore these hypotheses computationally, using a continuum mechanics approach, and experimentally, using live imaging. The result of this integrative approach will be the first experimentally validated systems-level model for two-dimensional epithelial patterning and resulting folding into a target three-dimensional morphology.

We propose to bring together an international and intercontinental team of biophysicists, cell biologists and neuroscientists to investigate the cellular and molecular mechanisms by which large proteinaceous aggregates can cross biological membranes. We propose three specific research aims. First, we will use state-of-the-art technology to understand the properties of protein aggregates that endow them with membrane-penetrating properties. The second aim seeks to determine the extent to which internalized aggregates can move between cells within a tissue and the role of endocytic, autophagic and cytoskeletal processes. Third, we propose to assess the extent to which protein aggregates can nucleate the aggregation of endogenous proteins within the brains of specially engineered mouse lines, and determine the role of motor-based axonal transport in this process. Together, this highly collaborative project seeks to understand the fundamental cellular and molecular basis for a newly discovered phenomenon that may play a critical role in the pathogenesis of diverse conformational diseases.

The control of cerebral vessel diameter is of fundamental importance in maintaining healthy brain function because it is critical to match cerebral blood flow to the metabolic demand of active neurons. The hypothesis that we will test is that astrocytes are essential communicators between blood vessels and neurons to regulate cerebral blood flow. Although there are numerous data indicating that astrocytes have critical roles in these processes there are still outstanding questions and critical gaps in our knowledge. A key shortcoming in previous studies is the difficulty to selectively activate neurons versus astrocytes. This grant will fund research to develop and apply the necessary tools for two photon uncaging and imaging in vivo. This team will apply novel two photon uncaging approaches in the cortex in vivo to address fundamental questions of how astrocytes signaling can alter cerebral blood vessels. A major question concerning the roles of astrocytes in the regulation of cerebral blood flow is the mechanism by which astrocytes are induced to cause either constrictions or dilations as a result of calcium transients. It is clear that astrocytes have the ability to induce either response in adjacent blood vessels. A recent study from MacVicar’s lab using a new two photon sensitive caged calcium (DMNPE-4) from Ellis-Davis’ lab has shown that the metabolic state of the surrounding tissue determines the polarity of the astrocyte influence on blood vessel diameter. This was determined in studies in brain slices in which oxygen levels and metabolic controls could be rigorously controlled and manipulated. The major pressing issue is to test these mechanisms described in brain slice studies using in vivo experiments. This collaborative effort will provide the expertise to do this. The Charpak lab has years of experience examining two photon signals in the olfactory bulb in vivo. The MacVicar and Charpak labs will combine efforts to examine astrocyte effects on blood vessel dynamics in the cortex and olfactory bulb in vivo. They will use new caged probes developed by the Ellis-Davis’ lab. New probes are necessary to provide the sensitivity that is required for in vivo photolysis and to provide new compounds to be uncaged for these investigations.

Textbooks present motor memory as monolithic: once acquired, never forgotten. They dissociate it from declarative memory by suggesting that motor memory does not have a short-term form. However, recent theoretical and experimental results have overturned this view, suggesting that motor memory is supported by processes that have multiple timescales: fast processes that learn quickly but decay rapidly with time, and slow processes that are less sensitive to movement errors but show better retention. Indeed, it appears that formation of motor memory may be a gradual transformation from the fast to the slow processes, and this transformation may depend on time and the statistical properties of the learner’s performance errors. We propose to use this idea to shed new light on one of the fundamental problems in neuroscience: the distinct functions of the cortical motor structures vs. the cerebellum in learning and retention of skilled movements.

The unsolved problem is illustrated by two examples: whereas neurophysiological and neuroimaging experiments have found correlates of adaptation in the primary motor cortex (M1), functional disruption of this area by transcranial magnetic stimulation (TMS) appears to produce no effects on acquisition, yet produces post-adaptation amnesia, i.e., rapid forgetting. In contrast, while the cerebellum is crucial for adaptation, damage to this structure appears to spare some skills that were acquired before the damage. Our basic hypothesis is that the faster timescales of memory are dependent on the cerebellum, effectively learning to predict the consequences of motor commands and correcting the motor commands via internal feedback, and the slower timescales are dependent on the cerebral cortex, effectively learning to produce the motor commands appropriate for the specific conditions of the task.

Our project combines computational, psychophysical, neuropsychological, and neurophysiological approaches to investigate how time and statistics of performance. We will use common eye and arm movement paradigms in behavioral and neurophysiological experiments to explore how controlled changes in sensory and motor noise effect motor control and learning. This will enable us to directly relate high level behavior to neural mechanisms