Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a major class of lipid-anchored plasma membrane proteins. GPI-APs form nanoclusters generated by cortical acto-myosin activity. While our understanding of the physical principles governing this process is emerging, the molecular machinery and functional relevance of GPI-AP nanoclustering are unknown. Here, we first show that a membrane receptor signaling pathway directs nanocluster formation. Arg-Gly-Asp motif-containing ligands bound to the ß1-integrin receptor activate src and focal adhesion kinases, resulting in RhoA signaling. This cascade triggers actin-nucleation via specific formins, which, along with myosin activity, drive the nanoclustering of membrane proteins with actin-binding domains. Concurrently, talin-mediated activation of the mechano-transducer vinculin is required for the coupling of the acto-myosin machinery to inner-leaflet lipids, thereby generating GPI-AP nanoclusters. Second, we show that these nanoclusters are functional; disruption of their formation either in GPI-anchor remodeling mutants or in vinculin mutants impairs cell spreading and migration, hallmarks of integrin function.

A Tale of Two States: Normal and Transformed, With and Without Rigidity Sensing.

For many years, major differences in morphology, motility, and mechanical characteristics have been observed between transformed cancer and normal cells. In this review, we consider these differences as linked to different states of normal and transformed cells that involve distinct mechanosensing and motility pathways. There is a strong correlation between repeated tissue healing and/or inflammation and the probability of cancer, both of which involve growth in adult tissues. Many factors are likely needed to enable growth, including the loss of rigidity sensing, but recent evidence indicates that microRNAs have important roles in causing the depletion of growth-suppressing proteins. One microRNA, miR-21, is overexpressed in many different tissues during both healing and cancer. Normal cells can become transformed by the depletion of cytoskeletal proteins that results in the loss of mechanosensing, particularly rigidity sensing. Conversely, the transformed state can be reversed by the expression of cytoskeletal proteins-without direct alteration of hormone receptor levels. In this review, we consider the different stereotypical forms of motility and mechanosensory systems. A major difference between normal and transformed cells involves a sensitivity of transformed cells to mechanical perturbations. Thus, understanding the different mechanical characteristics of transformed cells may enable new approaches to treating wound healing and cancer.

Appreciating force and shape­the rise of mechanotransduction in cell biology.

Although the shapes of organisms are encoded in their genome, the developmental processes that lead to the final form of vertebrates involve a constant feedback between dynamic mechanical forces, and cell growth and motility. Mechanobiology has emerged as a discipline dedicated to the study of the effects of mechanical forces and geometry on cell growth and motility­for example, during cell-matrix adhesion development­through the signalling process of mechanotransduction.

Transcription-independent functions of p53 in DNA repair pathway selection.

Recently discovered transcription-independent features of p53 involve the choice of DNA damage repair pathway after PARylation, and p53's complex formation with phosphoinositide lipids, PI(4,5)P2 . PARylation-mediated rapid accumulation of p53 at DNA damage sites is linked to the recruitment of downstream repair factors and tumor suppression. This links p53's capability to sense damaged DNA in vitro and its relevant functions in cells. Further, PI(4,5)P2 rapidly accumulates at damage sites like p53 and complexes with p53, while it is required for ATR recruitment. These findings help explain how p53 and PI(4,5)P2 maintain genome stability by directing DNA repair pathway choice. Additionally, there is a strong correlation between p53 sequence homology, genome mutation rates as well as lifespans across various mammalian species. Further investigation is required to better understand the connections between genome stability, tumor suppression, longevity and the transcriptional-independent function of p53.

Rapid recruitment of p53 to DNA damage sites directs DNA repair choice and integrity.

SignificanceOur work focuses on the critical longstanding question of the nontranscriptional role of p53 in tumor suppression. We demonstrate here that poly(ADP-ribose) polymerase (PARP)-dependent modification of p53 enables rapid recruitment of p53 to damage sites, where it in turn directs early repair pathway selection. Specifically, p53-mediated recruitment of 53BP1 at early time points promotes nonhomologous end joining over the more error-prone microhomology end-joining. Similarly, p53 directs nucleotide excision repair by mediating DDB1 recruitment. This property of p53 also correlates with tumor suppression in vivo. Our study provides mechanistic insight into how certain transcriptionally deficient p53 mutants may retain tumor-suppressive functions through regulating the DNA damage response.

Cooperative regulation of adherens junction expansion through epidermal growth factor receptor activation.

The mechanisms controlling the dynamics of expansion of adherens junctions are significantly less understood than those controlling their static properties. Here, we report that for suspended cell aggregates, the time to form a new junction between two cells speeds up with the number of junctions that the cells are already engaged in. Upon junction formation, the activation of epidermal growth factor receptor (EGFR) distally affects the actin turnover dynamics of the free cortex of the cells. The 'primed' actin cortex results in a faster expansion of the subsequent new junctions. In such aggregates, we show that this mechanism results in a cooperative acceleration of the junction expansion dynamics (kinetype) but does not alter the cell contractility, and hence the final junction size (phenotype). This article has an associated First Person interview with the first author of the paper.

Membrane Tilt Drives Phase Separation of Adhesion Receptors.

Cell adhesion receptors are transmembrane proteins that bind cells to their environment. These proteins typically cluster into disk-shaped or linear structures. Here, we show that such clustering patterns spontaneously emerge when the receptor senses the membrane deformation gradient, for example, by reaching a lower-energy conformation when the membrane is tilted relative to the underlying binding substrate. Increasing the strength of the membrane gradient-sensing mechanism first yields isolated disk-shaped clusters and then long linear structures. Our theory is coherent with experimental estimates of the parameters, suggesting that a tilt-induced clustering mechanism is relevant in the context of cell adhesion.

Optogenetic control of YAP cellular localisation and function.

YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attach a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression and cell proliferation. Similarly, optofYap can be used in zebrafish embryos to modulate target genes. We demonstrate that optoYAP can override a cell's response to substrate stiffness to generate anchorage-independent growth. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.

Small, Dynamic Clusters of Tir-Intimin Seed Actin Polymerization.

The understanding of actin pedestal formation by enteropathogenic Escherichia coli (EPEC) relies mainly on static ensemble information obtained from cell lysates. Here, the dynamic nature of signaling components on the subsecond timescale, which resemble phase condensates, is demonstrated. Unlike in vitro phase condensates, transfected intimin receptor (Tir) and downstream component form clusters 200 nm in diameter that are spaced ≈500 nm on average, indicating cellular regulation. On supported lipid bilayers with diffusive intimin, Tir-expressing fibroblasts formed Tir-intimin clusters even without Tir tyrosines, although Tir tyrosine phosphorylation is necessary for actin polymerization from clusters. Single-molecule tracking showed that Tir is diffusive in the clusters and exchanges with Tir in the plasma membrane. Further, Nck and N-WASP bind to the clusters and exchange with cytoplasmic molecules. Tir has a similar cluster lifetime to Nck, but longer than that of N-WASP. Actin polymerization from the clusters requires N-WASP binding, involved Arp2/3 activation, and stabilized N-WASP clusters. These dynamic properties are distinct from larger in vitro systems and do not depend significantly upon crosslinking. Thus, Tir-intimin clusters in the plasma membrane are limited in size by exchange and enhance signaling needed for actin polymerization that enables strong and stable bacterial attachment to host cells.

A new component in synaptic plasticity: upregulation of kinesin in the neurons of the gill-withdrawal reflex.

To explore how gene products, required for the initiation of synaptic growth, move from the cell body of the sensory neuron to its presynaptic terminals, and from the cell body of the motor neuron to its postsynaptic dendritic spines, we have investigated the anterograde transport machinery in both the sensory and motor neurons of the gill-withdrawal reflex of Aplysia. We found that the induction of long-term facilitation (LTF) by repeated applications of serotonin, a modulatory transmitter released during learning in Aplysia, requires upregulation of kinesin heavy chain (KHC) in both pre- and postsynaptic neurons. Indeed, upregulation of KHC in the presynaptic neurons alone is sufficient for the induction of LTF. However, KHC is not required for the persistence of LTF. Thus, in addition to transcriptional activation in the nucleus and local protein synthesis at the synapse, our studies have identified a third component critical for long-term learning-related plasticity: the coordinated upregulation of kinesin-mediated transport.

Cell response to substrate rigidity is regulated by active and passive cytoskeletal stress.

Morphogenesis, tumor formation, and wound healing are regulated by tissue rigidity. Focal adhesion behavior is locally regulated by stiffness; however, how cells globally adapt, detect, and respond to rigidity remains unknown. Here, we studied the interplay between the rheological properties of the cytoskeleton and matrix rigidity. We seeded fibroblasts onto flexible microfabricated pillar arrays with varying stiffness and simultaneously measured the cytoskeleton organization, traction forces, and cell-rigidity responses at both the adhesion and cell scale. Cells adopted a rigidity-dependent phenotype whereby the actin cytoskeleton polarized on stiff substrates but not on soft. We further showed a crucial role of active and passive cross-linkers in rigidity-sensing responses. By reducing myosin II activity or knocking down α-actinin, we found that both promoted cell polarization on soft substrates, whereas α-actinin overexpression prevented polarization on stiff substrates. Atomic force microscopy indentation experiments showed that this polarization response correlated with cell stiffness, whereby cell stiffness decreased when active or passive cross-linking was reduced and softer cells polarized on softer matrices. Theoretical modeling of the actin network as an active gel suggests that adaptation to matrix rigidity is controlled by internal mechanical properties of the cytoskeleton and puts forward a universal scaling between nematic order of the actin cytoskeleton and the substrate-to-cell elastic modulus ratio. Altogether, our study demonstrates the implication of cell-scale mechanosensing through the internal stress within the actomyosin cytoskeleton and its coupling with local rigidity sensing at focal adhesions in the regulation of cell shape changes and polarity.

Steps in Mechanotransduction Pathways that Control Cell Morphology.

It is increasingly clear that mechanotransduction pathways play important roles in regulating fundamental cellular functions. Of the basic mechanical functions, the determination of cellular morphology is critical. Cells typically use many mechanosensitive steps and different cell states to achieve a polarized shape through repeated testing of the microenvironment. Indeed, morphology is determined by the microenvironment through periodic activation of motility, mechanotesting, and mechanoresponse functions by hormones, internal clocks, and receptor tyrosine kinases. Patterned substrates and controlled environments with defined rigidities limit the range of cell behavior and influence cell state decisions and are thus very useful for studying these steps. The recently defined rigidity sensing process provides a good example of how cells repeatedly test their microenvironment and is also linked to cancer. In general, aberrant extracellular matrix mechanosensing is associated with numerous conditions, including cardiovascular disease, aging, and fibrosis, that correlate with changes in tissue morphology and matrix composition. Hence, detailed descriptions of the steps involved in sensing and responding to the microenvironment are needed to better understand both the mechanisms of tissue homeostasis and the pathomechanisms of human disease.

Competition for shared downstream signaling molecules establishes indirect negative feedback between EGFR and EphA2.

Cells sense a variety of extracellular growth factors and signaling molecules through numerous distinct receptor tyrosine kinases (RTKs) on the cell surface. In many cases, the same intracellular signaling molecules interact with more than one type of RTK. How signals from different RTKs retain the identity of the triggering receptor and how (or if) different receptors may synergize or compete remain largely unknown. Here we utilize an experimental strategy, combining microscale patterning and single-molecule imaging, to measure the competition between ephrin-A1:EphA2 and epidermal growth factor (EGF):EGF receptor (EGFR) ligand-receptor complexes for the shared downstream signaling molecules, Grb2 and SOS. The results reveal a distinct hierarchy, in which newly formed EGF:EGFR complexes outcompete ephrin-A1:EphA2 for Grb2 and SOS, revealing a type of negative crosstalk interaction fundamentally controlled by chemical mass action and protein copy number limitations.

Micro-stepping extended focus reduces photobleaching and preserves structured illumination super-resolution features.

Despite progress made in confocal microscopy, even fast systems still have insufficient temporal resolution for detailed live-cell volume imaging, such as tracking rapid movement of membrane vesicles in three-dimensional space. Depending on the shortfall, this may result in undersampling and/or motion artifacts that ultimately limit the quality of the imaging data. By sacrificing detailed information in the Z-direction, we propose a new imaging modality that involves capturing fast 'projections' from the field of depth and shortens imaging time by approximately an order of magnitude as compared to standard volumetric confocal imaging. With faster imaging, radiation exposure to the sample is reduced, resulting in less fluorophore photobleaching and potential photodamage. The implementation minimally requires two synchronized control signals that drive a piezo stage and trigger the camera exposure. The device generating the signals has been tested on spinning disk confocal and instant structured-illumination-microscopy (iSIM) microscopes. Our calibration images show that the approach provides highly repeatable and stable imaging conditions that enable photometric measurements of the acquired data, in both standard live imaging and super-resolution modes.This article has an associated First Person interview with the first author of the paper.

The Likelihood of an Occult Fracture in Skeletal Surveys Obtained in Children More Than 2 Years Old With Concerns of Physical Abuse.

OBJECTIVES: Skeletal surveys are necessary in the evaluation for physical abuse in children less than 2 years old, but when to obtain a skeletal survey in older children is less clear. METHODS: A retrospective study of patients older than 2 years who underwent skeletal survey over a 3-year period after implementation of an electronic health record physical abuse order set was conducted. Data were analyzed using descriptive statistics and compared with data from a cohort before order set implementation. The radiation dose of a skeletal survey in a 5-year old was calculated using a previously published technique. RESULTS: There were 325 skeletal surveys, a marked increase in the rate of skeletal surveys compared with before order set implementation. Less than 2% (6/325) of skeletal surveys demonstrated an occult fracture. Of the 6 patients with occult fractures, 4 were physically abused; in each case, the diagnosis of abuse was evident before the skeletal survey. The other 2 patients fell from windows. The radiation exposure was 0.34 mSv. CONCLUSIONS: The rate of occult fractures on skeletal survey is significantly lower than previously reported. This is likely because our population included all children who underwent skeletal survey and not the subset referred to a child abuse pediatrician. In addition, our data demonstrate that in children older than 2 years, skeletal surveys are unlikely to assist in making a diagnosis of physical abuse. The radiation exposure in a 5-year-old is 70% greater than in an infant, but still a dose, which represents a negligible health risk.

Stopping transformed cancer cell growth by rigidity sensing.

A common feature of cancer cells is the alteration of kinases and biochemical signalling pathways enabling transformed growth on soft matrices, whereas cytoskeletal protein alterations are thought to be a secondary issue. However, we report here that cancer cells from different tissues can be toggled between transformed and rigidity-dependent growth states by the absence or presence of mechanosensory modules, respectively. In various cancer lines from different tissues, cells had over tenfold fewer rigidity-sensing contractions compared with normal cells from the same tissues. Restoring normal levels of cytoskeletal proteins, including tropomyosins, restored rigidity sensing and rigidity-dependent growth. Further depletion of other rigidity sensor proteins, including myosin IIA, restored transformed growth and blocked sensing. In addition, restoration of rigidity sensing to cancer cells inhibited tumour formation and changed expression patterns. Thus, the depletion of rigidity-sensing modules through alterations in cytoskeletal protein levels enables cancer cell growth on soft surfaces, which is an enabling factor for cancer progression.

Integrin nanoclusters can bridge thin matrix fibres to form cell-matrix adhesions.

Integrin-mediated cell-matrix adhesions are key to sensing the geometry and rigidity of extracellular environments and influence vital cellular processes. In vivo, the extracellular matrix is composed of fibrous arrays. To understand the fibre geometries that are required for adhesion formation, we patterned nanolines of various line widths and arrangements in single, crossing or paired arrays with the integrin-binding peptide Arg-Gly-Asp. Single thin lines (width ≤30 nm) did not support cell spreading or formation of focal adhesions, despite the presence of a high density of Arg-Gly-Asp, but wide lines (>40 nm) did. Using super-resolution microscopy, we observed stable, dense integrin clusters formed on parallel (within 110 nm) or crossing thin lines (mimicking a matrix mesh) similar to those on continuous substrates. These dense clusters bridged the line pairs by recruiting activated but unliganded integrins, as verified by integrin mutants unable to bind ligands that coclustered with ligand-bound integrins when present in an active extended conformation. Thus, in a fibrous extracellular matrix mesh, stable integrin nanoclusters bridge between thin (≤30 nm) matrix fibres and bring about downstream consequences of cell motility and growth.

Integrin and cadherin clusters: A robust way to organize adhesions for cell mechanics.

Recent studies at the nanometer scale have revealed that relatively uniform clusters of adhesion proteins (50-100 nm) constitute the modular units of cell adhesion sites in both cell-matrix and cell-cell adhesions. Super resolution microscopy and membrane protein diffusion studies both suggest that even large focal adhesions are formed of 100 nm clusters that are loosely aggregated. Clusters of 20-50 adhesion molecules (integrins or cadherins) can support large forces through avidity binding interactions but can also be disassembled or endocytosed rapidly. Assembly of the clusters of integrins is force-independent and involves gathering integrins at ligand binding sites where they are stabilized by cytoplasmic adhesion proteins that crosslink the integrin cytoplasmic tails plus connect the clusters to the cell cytoskeleton. Cooperative-signaling events can occur in a single cluster without cascading to other clusters. Thus, the clusters appear to be very important elements in many cellular processes and can be considered as a critical functional module.

Matrix mechanics controls FHL2 movement to the nucleus to activate p21 expression.

Substrate rigidity affects many physiological processes through mechanochemical signals from focal adhesion (FA) complexes that subsequently modulate gene expression. We find that shuttling of the LIM domain (domain discovered in the proteins, Lin11, Isl-1, and Mec-3) protein four-and-a-half LIM domains 2 (FHL2) between FAs and the nucleus depends on matrix mechanics. In particular, on soft surfaces or after the loss of force, FHL2 moves from FAs into the nucleus and concentrates at RNA polymerase (Pol) II sites, where it acts as a transcriptional cofactor, causing an increase in p21 gene expression that will inhibit growth on soft surfaces. At the molecular level, shuttling requires a specific tyrosine in FHL2, as well as phosphorylation by active FA kinase (FAK). Thus, we suggest that FHL2 phosphorylation by FAK is a critical, mechanically dependent step in signaling from soft matrices to the nucleus to inhibit cell proliferation by increasing p21 expression.

Nuclear transport of paxillin depends on focal adhesion dynamics and FAT domains.

The nuclear transport of paxillin appears to be crucial for paxillin function but the mechanism of transport remains unclear. Here, we show that the nuclear transport of paxillin is regulated by focal adhesion turnover and the presence of FAT domains. Focal adhesion turnover was controlled using triangular or circular fibronectin islands. Circular islands caused higher focal adhesion turnover and increased the nuclear transport of paxillin relative to triangular islands. Mutating several residues of paxillin had no effect on its nuclear transport, suggesting that the process is controlled by multiple domains. Knocking out FAK (also known as PTK2) and vinculin caused an increase in nuclear paxillin. This could be reversed by rescue with wild-type FAK but not by FAK with a mutated FAT domain, which inhibits paxillin binding. Expressing just the FAT domain of FAK not only brought down nuclear levels of paxillin but also caused a large immobile fraction of paxillin to be present at focal adhesions, as demonstrated by fluorescence recovery after photobleaching (FRAP) studies. Taken together, focal adhesion turnover and FAT domains regulate the nuclear localization of paxillin, suggesting a possible role for transcriptional control, through paxillin, by focal adhesions.