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. Leukocyte-induced stimulation of PECAM-1 triggers dissociation of the phosphatase SHP2 which then directly targets VE-cadherin-Y731. The binding site of PECAM-1 for SHP2 is needed for VE-cadherin dephosphorylation and subsequent endocytosis. Importantly, the contribution of PECAM-1 to leukocyte diapedesis in vitro and in vivo was strictly dependent on the presence of Y731 of VE-cadherin. In addition to SHP2, dephosphorylation of Y731 required Ca2+ -signaling, non-muscle myosin II activation, and endothelial cell tension. Since we found that ß-catenin/plakoglobin mask VE-cadherin-Y731 and leukocyte docking to endothelial cells exert force on the VE-cadherin-catenin complex, we propose that leukocytes destabilize junctions by PECAM-1-SHP2-triggered dephosphorylation of VE-cadherin-Y731 which becomes accessible by actomyosin-mediated mechanical force exerted on the VE-cadherin-catenin complex.

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. We studied Drosophila muscle attachment sites, which experience high mechanical forces during development and require integrin-mediated adhesion for stable attachment to tendons. Therefore, we quantified molecular forces across the essential integrin-binding protein Talin, which links integrin to the actin cytoskeleton. Generating flies expressing 3 Förster resonance energy transfer (FRET)-based Talin tension sensors reporting different force levels between 1 and 11 piconewton (pN) enabled us to quantify physiologically relevant molecular forces. By measuring primary Drosophila muscle cells, we demonstrate that Drosophila Talin experiences mechanical forces in cell culture that are similar to those previously reported for Talin in mammalian cell lines. However, in vivo force measurements at developing flight muscle attachment sites revealed that average forces across Talin are comparatively low and decrease even further while attachments mature and tissue-level tension remains high. Concomitantly, the Talin concentration at attachment sites increases 5-fold as quantified by fluorescence correlation spectroscopy (FCS), suggesting that only a small proportion of Talin molecules are mechanically engaged at any given time. Reducing Talin levels at late stages of muscle development results in muscle-tendon rupture in the adult fly, likely as a result of active muscle contractions. We therefore propose that a large pool of adhesion molecules is required to share high tissue forces. As a result, less than 15% of the molecules experience detectable forces at developing muscle attachment sites at the same time. Our findings define an important new concept of how cells can adapt to changes in tissue mechanics to prevent mechanical failure in vivo.

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. We demonstrate that a previously reported peptide can be utilized to specifically label the two major talin isoforms expressed in mammalian tissues with a localization precision of <10 nm. Our experiments show that the methodology performs equally well as state-of-the-art single-molecule localization techniques, and the first applications reveal a thus far undescribed cell adhesion structure in differentiating stem cells. Furthermore, we demonstrate the applicability of this peptide-PAINT technique to mouse tissues paving the way to single-protein imaging of endogenous talin proteins under physiologically relevant conditions.

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. In addition, we present a method allowing the simultaneous evaluation of coexpressed tension sensor constructs using two-color fluorescence lifetime microscopy. Finally, we introduce a procedure to calculate the fraction of mechanically engaged molecules within cells. Application of these techniques to new talin biosensors reveals an intramolecular tension gradient across talin-1 that is established upon integrin-mediated cell adhesion. The tension gradient is actomyosin- and vinculin-dependent and sensitive to the rigidity of the extracellular environment.

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. We also discuss the concept of mechanical memory and describe how cells store information on previous mechanical events for different periods of time.

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. In addition, we provide a theoretical framework for the design of additional tension sensor modules with adjusted force sensitivity.

Dynamic molecular processes mediate cellular mechanotransduction.

Cellular responses to mechanical forces are crucial in embryonic development and adult physiology, and are involved in numerous diseases, including atherosclerosis, hypertension, osteoporosis, muscular dystrophy, myopathies and cancer. These responses are mediated by load-bearing subcellular structures, such as the plasma membrane, cell-adhesion complexes and the cytoskeleton. Recent work has demonstrated that these structures are dynamic, undergoing assembly, disassembly and movement, even when ostensibly stable. An emerging insight is that transduction of forces into biochemical signals occurs within the context of these processes. This framework helps to explain how forces of varying strengths or dynamic characteristics regulate distinct signalling pathways.

Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics.

Mechanical forces are central to developmental, physiological and pathological processes. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent. We show that tension across vinculin in stable FAs is approximately 2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.

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. We show here that ILK plays a key role in the formation of focal complexes, early forms of integrin adhesions, and confirm its involvement in the assembly of fibronectin-bound fibrillar adhesions. Examination of ILK-null fibroblasts by cryo-electron tomography pointed to major structural changes in their FAs, manifested as disarray of the associated actin filaments and an increase in the packing density of FA-related particles. Interestingly, adhesion of the mutant cells to the substrate required a higher ligand density than in control cells. These data indicate that ILK has a key role in integrin adhesion assembly and sub-structure, and in the regulation of the FA-associated cytoskeleton.

Mutations in the paxillin-binding site of integrin-linked kinase (ILK) destabilize the pseudokinase domain and cause embryonic lethality in mice.

Integrin-linked kinase (ILK) localizes to focal adhesions (FAs) where it regulates cell spreading, migration, and growth factor receptor signaling. Previous reports showed that overexpressed ILK in which Val(386) and Thr(387) were substituted with glycine residues (ILK-VT/GG) could neither interact with paxillin nor localize to FA in cells expressing endogenous wild-type ILK, implying that paxillin binding to ILK is required for its localization to FAs. Here, we show that introducing this mutation into the germ line of mice (ILK-VT/GG) caused vasculogenesis defects, resulting in a general developmental delay and death at around embryonic day 12.5. Fibroblasts isolated from ILK-VT/GG mice contained mutant ILK in FAs, showed normal adhesion to and spreading on extracellular matrix substrates but displayed impaired migration. Biochemical analysis revealed that VT/GG substitutions decreased ILK protein stability leading to decreased ILK levels and reduced binding to paxillin and α-parvin. Because paxillin depletion did not affect ILK localization to FAs, the embryonic lethality and the in vitro migration defects are likely due to the reduced levels of ILK-VT/GG and diminished binding to parvins.

Induction of membrane circular dorsal ruffles requires co-signalling of integrin-ILK-complex and EGF receptor.

Integrin and receptor tyrosine kinase signalling networks cooperate to regulate various biological functions. The molecular details underlying the integration of both signalling networks remain largely uncharacterized. Here we identify a signalling module composed of a fibronectin-α5ß1-integrin-integrin-linked-kinase (ILK) complex that, in concert with epidermal growth factor (EGF) cues, cooperatively controls the formation of transient actin-based circular dorsal ruffles (DRs) in fibroblasts. DR formation depends on the precise spatial activation of Src at focal adhesions by integrin and EGF receptor signals, in an ILK-dependent manner. In a SILAC-based phosphoproteomics screen we identified the tumour-suppressor Cyld as being required for DR formation induced by α5ß1 integrin and EGF receptor co-signalling. Furthermore, EGF-induced Cyld tyrosine phosphorylation is controlled by integrin-ILK and Src as a prerequisite for DR formation. This study provides evidence for a novel function of integrin-ILK and EGF signalling crosstalk in mediating Cyld tyrosine phosphorylation and fast actin-based cytoskeletal rearrangements.

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. Our knowledge about the molecular and physical mechanisms defining the network structure remains scarce, especially for the intermediate filament cytoskeleton. Here, we investigate the effect of small heat shock proteins on the keratin 8/18 intermediate filament cytoskeleton using a well-controlled model system of reconstituted keratin networks. We demonstrate that Hsp27 severely alters the structure of such networks by changing their assembly dynamics. Furthermore, the C-terminal tail domain of keratin 8 is shown to be essential for this effect. Combining results from fluorescence and electron microscopy with data from analytical ultracentrifugation reveals the crucial role of kinetic trapping in keratin network formation.

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. Our protocol covers all essential steps from biosensor expression and live cell PIE image acquisition to lifetime calculations.

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. When applied to the integrin activator talin, the technique provides direct evidence that its role as a mechanical linker is indispensable for the maintenance of cell-matrix adhesions and overall cell integrity. Applying the technique to desmoplakin shows that mechanical engagement of desmosomes to intermediate filaments is expendable under homeostatic conditions yet strictly required for preserving cell-cell adhesion under stress. These results reveal a central role of talin and desmoplakin as mechanical linkers in cell adhesion structures and demonstrate that molecular optomechanics is a powerful tool to investigate the molecular details of mechanobiological processes.

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. First genetically-encoded reporters only partly overcame these limitations as the green and red fluorescent proteins (GFP/RFP) families require molecular oxygen for fluorescence. For the herein presented ratiometric and FRET-FLIM reporters dUnORS and dUnOFLS, we exploited oxygen-dependent maturation in combination with the hypoxia-tolerant fluorescent-protein UnaG. For ratiometric measurements, UnaG was fused to the orange large Stokes Shift protein CyOFP1, allowing excitation with a single light-source, while fusion of UnaG with mOrange2 allowed FRET-FLIM analysis. Imaging live or fixed cultured cells for calibration, we applied both reporters in spheroid and tumor transplantation-models and obtained graded information on oxygen-availability at cellular resolution, establishing these sensors as promising tools for visualizing oxygen-gradients in-vivo.

Integrin-linked kinase is required for epidermal and hair follicle morphogenesis.

Integrin-linked kinase (ILK) links integrins to the actin cytoskeleton and is believed to phosphorylate several target proteins. We report that a keratinocyte-restricted deletion of the ILK gene leads to epidermal defects and hair loss. ILK-deficient epidermal keratinocytes exhibited a pronounced integrin-mediated adhesion defect leading to epidermal detachment and blister formation, disruption of the epidermal-dermal basement membrane, and the translocation of proliferating, integrin-expressing keratinocytes to suprabasal epidermal cell layers. The mutant hair follicles were capable of producing hair shaft and inner root sheath cells and contained stem cells and generated proliferating progenitor cells, which were impaired in their downward migration and hence accumulated in the outer root sheath and failed to replenish the hair matrix. In vitro studies with primary ILK-deficient keratinocytes attributed the migration defect to a reduced migration velocity and an impaired stabilization of the leading-edge lamellipodia, which compromised directional and persistent migration. We conclude that ILK plays important roles for epidermis and hair follicle morphogenesis by modulating integrin-mediated adhesion, actin reorganization, and plasma membrane dynamics in keratinocytes.

Integrin-linked kinase: integrin's mysterious partner.

Integrin-mediated cell adhesion regulates a vast number of biological processes including migration, survival and proliferation of cells. It is therefore not surprising that defects in integrin function are often rate-limiting for development and profoundly affect the progression of several diseases. The functions of integrins are mediated through the recruitment of cytoplasmic plaque proteins. One of these is integrin-linked kinase, which connects integrins to the actin cytoskeleton and transduces signals through integrins to the extracellular matrix and from integrins to various subcellular compartments.

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. In this review, we discuss the physical principles, advantages, and limitations of this increasingly popular method. By highlighting current examples from the literature, we demonstrate how molecular tension sensors can be utilized to obtain access to previously unappreciated biophysical parameters that define the propagation of mechanical forces on molecular scales. We discuss how the methodology can be further developed and provide a perspective on how the technique could be applied to uncover entirely novel aspects of mechanobiology in the future.

Piezo1 and Piezo2 foster mechanical gating of K2P channels.

Mechanoelectrical transduction is mediated by the opening of different types of force-sensitive ion channels, including Piezo1/2 and the TREK/TRAAK K2P 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. Whether this structural architecture influences the functionality of neighboring membrane proteins is unknown. Here, we show that Piezo1/2 increase TREK/TRAAK current amplitude, slow down activation/deactivation, and remove inactivation upon mechanical stimulation. These findings are consistent with a mechanism whereby Piezo1/2 cause a local depletion of membrane cholesterol associated with a prestress of TREK/TRAAK channels. This regulation occurs in mouse fibroblasts between endogenous Piezo1 and TREK-1/2, both channel types acting in concert to delay wound healing. In conclusion, we demonstrate a community effect between different structural and functional classes of mechanosensitive ion channels.

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. Here, we establish a super-resolution microscopy framework, called quantitative single-molecule colocalization analysis (qSMCL), which permits the identification of absolute molecular quantities and thus the investigation of molecular-scale processes inside cells. The method combines multiplexed single-protein resolution imaging, automated cluster detection, in silico data simulation procedures, and widely applicable experimental controls to determine absolute fractions and spatial coordinates of interacting species on a true molecular level, even in highly crowded subcellular structures. The first application of this framework allowed the identification of a long-sought ternary adhesion complex-consisting of talin, kindlin and active ß1-integrin-that specifically forms in cell-matrix adhesion sites. Together, the experiments demonstrate that qSMCL allows an absolute quantification of multiplexed SMLM data and thus should be useful for investigating molecular mechanisms underlying numerous processes in cells.