A family of NADPH/NADP biosensors reveals in vivo dynamics of central redox metabolism across eukaryotes.

The NADPH/NADP redox couple is central to metabolism and redox signalling. NADP redox state is differentially regulated by distinct enzymatic machineries at the subcellular compartment level. Nonetheless, a detailed understanding of subcellular NADP redox dynamics is limited by the availability of appropriate tools.

Decisive role of mDia-family formins in cell cortex function of highly adherent cells.

Cortical formins, pivotal for the assembly of linear actin filaments beneath the membrane, exert only minor effects on unconfined cell migration of weakly and moderately adherent cells. However, their impact on migration and mechanostability of highly adherent cells remains poorly understood. Here, we demonstrate that loss of cortical actin filaments generated by the formins mDia1 and mDia3 drastically compromises cell migration and mechanics in highly adherent fibroblasts.

A molecular optomechanics approach reveals functional relevance of force transduction across talin and desmoplakin.

Many mechanobiological processes that govern development and tissue homeostasis are regulated on the level of individual molecular linkages, and a number of proteins experiencing piconewton-scale forces in cells have been identified. However, under which conditions these force-bearing linkages become critical for a given mechanobiological process is often still unclear. Here, we established an approach to revealing the mechanical function of intracellular molecules using molecular optomechanics.

Multiplexed Molecular Tension Sensor Measurements Using PIE-FLIM.

Genetically encoded Förster Resonance Energy Transfer (FRET)-based tension sensors were developed to enable the quantification of piconewton (pN)-scale forces that act across distinct proteins in living cells and organisms. An important extension of this technology is the multiplexing of tension sensors to monitor several independent FRET probes in parallel. Here we describe how pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) can be implemented to enable the analysis of two co-expressed tension sensor constructs.

Genetically encoded dual fluorophore reporters for graded oxygen-sensing in light microscopy.

Hypoxia is an essential regulator of cell metabolism, affects cell migration and angiogenesis during development and contributes to a wide range of pathological conditions. Multiple techniques to assess hypoxia through oxygen-imaging have been developed. However, significant limitations include low spatiotemporal resolution, limited tissue penetration of exogenous probes and non-dynamic signals due to irreversible probe-chemistry.

Piezo1 and Piezo2 foster mechanical gating of K channels.

Mechanoelectrical transduction is mediated by the opening of different types of force-sensitive ion channels, including Piezo1/2 and the TREK/TRAAK K channels. Piezo1 curves the membrane locally into an inverted dome that reversibly flattens in response to force application. Moreover, Piezo1 forms numerous preferential interactions with various membrane lipids, including cholesterol.

Peptide-PAINT Enables Investigation of Endogenous Talin with Molecular Scale Resolution in Cells and Tissues.

Talin is a cell adhesion molecule that is indispensable for the development and function of multicellular organisms. Despite its central role for many cell biological processes, suitable methods to investigate the nanoscale organization of talin in its native environment are missing. Here, we overcome this limitation by combining single-molecule resolved PAINT (points accumulation in nanoscale topography) imaging with the IRIS (image reconstruction by integrating exchangeable single-molecule localization) approach, enabling the quantitative analysis of genetically unmodified talin molecules in cells.

Molecular Force Measurement with Tension Sensors.

The ability of cells to generate mechanical forces, but also to sense, adapt to, and respond to mechanical signals, is crucial for many developmental, postnatal homeostatic, and pathophysiological processes. However, the molecular mechanisms underlying cellular mechanotransduction have remained elusive for many decades, as techniques to visualize and quantify molecular forces across individual proteins in cells were missing. The development of genetically encoded molecular tension sensors now allows the quantification of piconewton-scale forces that act upon distinct molecules in living cells and even whole organisms.

PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin.

Leukocyte extravasation is an essential step during the immune response and requires the destabilization of endothelial junctions. We have shown previously that this process depends in vivo on the dephosphorylation of VE-cadherin-Y731. Here, we reveal the underlying mechanism.

Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion.

Single-molecule localization microscopy (SMLM) enabling the investigation of individual proteins on molecular scales has revolutionized how biological processes are analysed in cells. However, a major limitation of imaging techniques reaching single-protein resolution is the incomplete and often unknown labeling and detection efficiency of the utilized molecular probes. As a result, fundamental processes such as complex formation of distinct molecular species cannot be reliably quantified.

Metavinculin modulates force transduction in cell adhesion sites.

Vinculin is a ubiquitously expressed protein, crucial for the regulation of force transduction in cells. Muscle cells express a vinculin splice-isoform called metavinculin, which has been associated with cardiomyopathies. However, the molecular function of metavinculin has remained unclear and its role for heart muscle disorders undefined.

A small proportion of Talin molecules transmit forces at developing muscle attachments in vivo.

Cells in developing organisms are subjected to particular mechanical forces that shape tissues and instruct cell fate decisions. How these forces are sensed and transmitted at the molecular level is therefore an important question, one that has mainly been investigated in cultured cells in vitro. Here, we elucidate how mechanical forces are transmitted in an intact organism.

Genetically Encoded FRET-Based Tension Sensors.

Genetically encoded Förster resonance energy transfer (FRET)-based tension sensors measure piconewton-scale forces across individual molecules in living cells or whole organisms. These biosensors show comparably high FRET efficiencies in the absence of tension, but FRET quickly decreases when forces are applied. In this article, we describe how such biosensors can be generated for a specific protein of interest, and we discuss controls to confirm that the observed differences in FRET efficiency reflect changes in molecular tension.

Mechanical loading of desmosomes depends on the magnitude and orientation of external stress.

Desmosomes are intercellular adhesion complexes that connect the intermediate filament cytoskeletons of neighboring cells, and are essential for the mechanical integrity of mammalian tissues. Mutations in desmosomal proteins cause severe human pathologies including epithelial blistering and heart muscle dysfunction. However, direct evidence for their load-bearing nature is lacking.

Unforgettable force - crosstalk and memory of mechanosensitive structures.

The ability of cells to sense and respond to mechanical stimuli is crucial for many developmental and homeostatic processes, while mechanical dysfunction of cells has been associated with numerous pathologies including muscular dystrophies, cardiovascular defects and epithelial disorders. Yet, how cells detect and process mechanical information is still largely unclear. In this review, we outline major mechanisms underlying cellular mechanotransduction and we summarize the current understanding of how cells integrate information from distinct mechanosensitive structures to mediate complex mechanoresponses.

Multiplexing molecular tension sensors reveals piconewton force gradient across talin-1.

Förster resonance energy transfer (FRET)-based tension sensor modules (TSMs) are available for investigating how distinct proteins bear mechanical forces in cells. Yet, forces in the single piconewton (pN) regime remain difficult to resolve, and tools for multiplexed tension sensing are lacking. Here, we report the generation and calibration of a genetically encoded, FRET-based biosensor called FL-TSM, which is characterized by a near-digital force response and increased sensitivity at 3-5 pN.

Sensing the mechano-chemical properties of the extracellular matrix.

The ability of cells to adhere and sense their mechano-chemical environment is key to many developmental, postnatal homeostatic and pathological processes; however, the underlying molecular mechanisms are still poorly understood. Here, we summarize recent progress that indicates how cell adhesion, mechanotransduction and chemical signaling are coordinated in cells, and we discuss how the combination of novel experimental approaches with theoretical studies is currently utilized to unravel the molecular mechanisms governing mechano-chemical coupling during cell adhesion.

The Piconewton Force Awakens: Quantifying Mechanics in Cells.

The development of calibrated Förster resonance energy transfer (FRET)-based tension sensors has allowed the first analyses of mechanical processes with piconewton (pN) sensitivity in cells. Here, we introduce the working principle of this emerging microscopy method and discuss how it has been utilized to obtain quantitative insights into the mechanisms of intracellular force transduction in cell-matrix adhesions, cell-cell junctions, and at the cell cortex. These examples demonstrate that genetically encoded tension sensors are powerful tools to unravel force transduction mechanisms, but also indicate current limitations.

Investigating piconewton forces in cells by FRET-based molecular force microscopy.

The ability of cells to sense and respond to mechanical forces is crucial for a wide range of developmental and pathophysiological processes. The molecular mechanisms underlying cellular mechanotransduction, however, are largely unknown because suitable techniques to measure mechanical forces across individual molecules in cells have been missing. In this article, we highlight advances in the development of molecular force sensing techniques and discuss our recently expanded set of FRET-based tension sensors that allows the analysis of mechanical forces with piconewton sensitivity in cells.

Extracellular rigidity sensing by talin isoform-specific mechanical linkages.

The ability of cells to adhere and sense differences in tissue stiffness is crucial for organ development and function. The central mechanisms by which adherent cells detect extracellular matrix compliance, however, are still unknown. Using two single-molecule-calibrated biosensors that allow the analysis of a previously inaccessible but physiologically highly relevant force regime in cells, we demonstrate that the integrin activator talin establishes mechanical linkages following cell adhesion, which are indispensable for cells to probe tissue stiffness.

How to Measure Molecular Forces in Cells: A Guide to Evaluating Genetically-Encoded FRET-Based Tension Sensors.

The ability of cells to sense and respond to mechanical forces is central to a wide range of biological processes and plays an important role in numerous pathologies. The molecular mechanisms underlying cellular mechanotransduction, however, have remained largely elusive because suitable methods to investigate subcellular force propagation were missing. Here, we review recent advances in the development of biosensors that allow molecular force measurements.

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

Background: 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.

The small heat shock protein Hsp27 affects assembly dynamics and structure of keratin intermediate filament networks.

The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance.

Generation and analysis of biosensors to measure mechanical forces within cells.

The inability to measure mechanical forces within cells has been limiting our understanding of how mechanical information is processed on the molecular level. In this chapter, we describe a method that allows the analysis of force propagation across distinct proteins within living cells using Förster resonance energy transfer (FRET)-based biosensors.

The role of integrin-linked kinase in the molecular architecture of focal adhesions.

Integrin-mediated focal adhesions (FAs) are large, multi-protein complexes that link the actin cytoskeleton to the extracellular matrix and take part in adhesion-mediated signaling. These adhesions are highly complex and diverse at the molecular level; thus, assigning particular structural or signaling functions to specific components is highly challenging. Here, we combined functional, structural and biophysical approaches to assess the role of a major FA component, namely, integrin-linked kinase (ILK), in adhesion formation.