measurement-force-sensitive-protein-dynamics-living-cells-using

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Duke University

The vacuole within: how cellular organization dictates notochord function.

Intervertebral disc (IVD) disorders are a major contributor to disability and societal health care costs. Nucleus pulposus (NP) cells of the IVD exhibit changes in both phenotype and morphology with aging-related IVD degeneration that may impact the onset and progression of IVD pathology. Studies have demonstrated that immature NP cell interactions with their extracellular matrix (ECM) may be key regulators of cellular phenotype, metabolism and morphology. The objective of this article is to review our recent experience with studies of NP cell-ECM interactions that reveal how ECM cues can be manipulated to promote an immature NP cell phenotype and morphology. Findings demonstrate the importance of a soft (&lt;700 Pa), laminin-containing ECM in regulating healthy, immature NP cells. Knowledge of NP cell-ECM interactions can be used for development of tissue engineering or cell delivery strategies to treat IVD-related disorders. 

The role of extracellular matrix elasticity and composition in regulating the nucleus pulposus cell phenotype in the intervertebral disc: a narrative review.

Actomyosin-based contractility acts on cadherin junctions to support tissue integrity and morphogenesis. The actomyosin apparatus of the epithelial zonula adherens (ZA) is built by coordinating junctional actin assembly with Myosin II activation. However, the physical interaction between Myosin and actin filaments that is necessary for contractility can induce actin filament turnover, potentially compromising the contractile apparatus itself.

Tension-sensitive actin assembly supports contractility at the epithelial zonula adherens.

Cells are exquisitely sensitive to the mechanical nature of their environment, including applied force and the stiffness of the extracellular matrix (ECM). Recent evidence has shown that these variables are critical regulators of diverse processes mediating embryonic development, adult tissue physiology, and many disease states, including cancer, atherosclerosis, and myopathies. Often, detection of mechanical stimuli is mediated by the structures that link cells that surround ECM, the focal adhesions (FAs). FAs are intrinsically force sensitive and display altered dynamics, structure, and composition in response to applied load. While much progress has been made in determining the proteins that localize to and regulate the formation of these structures, less is known about the role of tension across specific proteins in this process. A recently developed class of force-sensitive biosensors is enabling a greater understanding of the molecular bases of cellular mechanosensitivity and cell migration. 

The detection and role of molecular tension in focal adhesion dynamics.

Due to an increased appreciation for the importance of mechanical stimuli in many biological contexts, an interest in measuring the forces experienced by specific proteins in living cells has recently emerged. The development and use of Förster resonance energy transfer (FRET)-based molecular tension sensors has enabled these types of studies and led to important insights into the mechanisms those cells utilize to probe and respond to the mechanical nature of their surrounding environment. The process for creating and utilizing FRET-based tension sensors can be divided into three main parts: construction, imaging, and analysis. First we review several methods for the construction of genetically encoded FRET-based tension sensors, including restriction enzyme-based methods as well as the more recently developed overlap extension or Gibson Assembly protocols. Next, we discuss the intricacies associated with imaging tension sensors, including optimizing imaging parameters as well as common techniques for estimating artifacts within standard imaging systems. Then, we detail the analysis of such data and describe how to extract useful information from a FRET experiment. Finally, we provide a discussion on identifying and correcting common artifacts in the imaging of FRET-based tension sensors. 

Construction, imaging, and analysis of FRET-based tension sensors in living cells.

Mechanical stimuli are known to be potent regulators of the form and function of cells and organisms. Although biological regulation has classically been understood in terms of principles from solution biochemistry, advancements in many fields have led to the development of a suite of techniques that are able to reveal the interplay between mechanical loading and changes in the biochemical properties of proteins in systems ranging from single molecules to living organisms. Here, we review these techniques and highlight the emergence of a new molecular-scale understanding of the mechanisms mediating the detection and response of cells to mechanical stimuli, a process termed mechanotransduction. Specifically, we focus on the role of subcellular adhesion structures in sensing the stiffness of the surrounding environment because this process is pertinent to applications in tissue engineering as well the onset of several mechanosensitive disease states, including cancer. 

Molecular-Scale Tools for Studying Mechanotransduction.

Cadherin-based cell-cell adhesions are a primary determinant of tissue structure. For several decades, it had been thought that the primary function of these ubiquitous structures was to resist external mechanical loads. Here we review recent evidence that cadherins also couple together the force-generating actomyosin cytoskeletons of neighbouring cells, serve as potent regulators of the actomyosin cytoskeleton, and activate diverse signalling pathways in response to applied load. In considering the force sensitivity of the molecular-scale processes that mediate these events, we propose a dynamic picture of the force-sensitive processes in cell-cell contacts. This quantitative and physical understanding of the mechanobiology of cadherin cell-cell junctions will aid endeavours to study the fundamental processes mediating the development and maintenance of tissue structure.

Towards a Dynamic Understanding of Cadherin-Based Mechanobiology.

Electrotransfection is a widely used method for delivering genes into cells with electric pulses. Although different hypotheses have been proposed, the mechanism of electrotransfection remains controversial. Previous studies have indicated that uptake and intracellular trafficking of plasmid DNA (pDNA) are mediated by endocytic pathways, but it is still unclear which pathways are directly involved in the delivery. To this end, the present study investigated the dependence of electrotransfection on macropinocytosis. Data from the study demonstrated that electric pulses induced cell membrane ruffling and actin cytoskeleton remodeling. Using fluorescently labeled pDNA and a macropinocytosis marker (i.e., dextran), the study showed that electrotransfected pDNA co-localized with dextran in intracellular vesicles. Furthermore, electrotransfection efficiency could be decreased significantly by reducing temperature or treatment of cells with a pharmacological inhibitor of Rac1 and could be altered by changing Rac1 activity. Taken together, the findings suggested that electrotransfection of pDNA involved Rac1-dependent macropinocytosis.

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection.

Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells.

Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells.

Cells have evolved into complex sensory machines that communicate with their microenvironment via mechanochemical signaling. Extracellular mechanical cues trigger complex biochemical pathways in the cell, which regulate various cellular processes. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes, also known as the integrin adhesome, that link the extracellular matrix (ECM) to the actin cytoskeleton, and are part of powerful intracellular machinery orchestrating mechanotransduction pathways. As forces are transmitted across FAs, individual proteins undergo structural and functional changes that involve a conversion of chemical to mechanical energy. The local composition of early adhesions likely defines the regional stress levels and determines the type of newly recruited proteins, which in turn modify the local stress distribution. Various approaches have been used for detecting and exploring molecular mechanisms through which FAs are spatiotemporally regulated, however, many aspects are yet to be understood. Current knowledge on the molecular mechanisms of mechanosensitivity in adhesion proteins is discussed herein along with important questions yet to be addressed, are discussed.

The "Stressful" Life of Cell Adhesion Molecules: On the Mechanosensitivity of Integrin Adhesome.

The nucleus has long been postulated to play a critical physical role during cell polarization and migration, but that role has not been defined or rigorously tested. Here, we enucleated cells to test the physical necessity of the nucleus during cell polarization and directed migration. Using enucleated mammalian cells (cytoplasts), we found that polarity establishment and cell migration in one dimension (1D) and two dimensions (2D) occur without the nucleus. Cytoplasts directionally migrate toward soluble (chemotaxis) and surface-bound (haptotaxis) extracellular cues and migrate collectively in scratch-wound assays. Consistent with previous studies, migration in 3D environments was dependent on the nucleus. In part, this likely reflects the decreased force exerted by cytoplasts on mechanically compliant substrates. This response is mimicked both in cells with nucleocytoskeletal defects and upon inhibition of actomyosin-based contractility. Together, our observations reveal that the nucleus is dispensable for polarization and migration in 1D and 2D but critical for proper cell mechanical responses.

Enucleated cells reveal differential roles of the nucleus in cell migration, polarity, and mechanotransduction.

Cell migration is a complex process, requiring coordination of many subcellular processes including membrane protrusion, adhesion, and contractility. For efficient cell migration, cells must concurrently control both transmission of large forces through adhesion structures and translocation of the cell body via adhesion turnover. Although mechanical regulation of protein dynamics has been proposed to play a major role in force transmission during cell migration, the key proteins and their exact roles are not completely understood. Vinculin is an adhesion protein that mediates force-sensitive processes, such as adhesion assembly under cytoskeletal load. Here, we elucidate the mechanical regulation of vinculin dynamics. Specifically, we paired measurements of vinculin loads using a Förster resonance energy transfer-based tension sensor and vinculin dynamics using fluorescence recovery after photobleaching to measure force-sensitive protein dynamics in living cells. We find that vinculin adopts a variety of mechanical states at adhesions, and the relationship between vinculin load and vinculin dynamics can be altered by the inhibition of vinculin binding to talin or actin or reduction of cytoskeletal contractility. Furthermore, the force-stabilized state of vinculin required for the stabilization of membrane protrusions is unnecessary for random migration, but is required for directional migration along a substrate-bound cue. These data show that the force-sensitive dynamics of vinculin impact force transmission and enable the mechanical integration of subcellular processes. These results suggest that the regulation of force-sensitive protein dynamics may have an underappreciated role in many cellular processes.

Vinculin Force-Sensitive Dynamics at Focal Adhesions Enable Effective Directed Cell Migration.

Molecular tension sensors have contributed to a growing understanding of mechanobiology. However, the limited dynamic range and inability to specify the mechanical sensitivity of these sensors has hindered their widespread use in diverse contexts. Here, we systematically examine the components of tension sensors that can be altered to improve their functionality. Guided by the development of a first principles model describing the mechanical behavior of these sensors, we create a collection of sensors that exhibit predictable sensitivities and significantly improved performance . Utilized in the context of vinculin mechanobiology, a trio of these new biosensors with distinct force- and extension-sensitivities reveal that an extension-based control paradigm regulates vinculin loading in a variety of mechanical contexts. To enable the rational design of molecular tension sensors appropriate for diverse applications, we predict the mechanical behavior, in terms of force and extension, of additional 1020 distinct designs.

Brenton D. Hoffman

Department of Biomedical Engineering

Duke University

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Brenton D. Hoffman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Engineering

Duke University

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Department of Biomedical Engineering