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.

Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignaling in focal adhesions.

Transmembrane signalling receptors, such as integrins, organise as nanoclusters that are thought to provide several advantages including, increasing avidity, sensitivity (increasing the signal-to-noise ratio) and robustness (signalling above a threshold rather than activation by a single receptor) of the signal compared to signalling by single receptors. Compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, if nanoclusters function as signalling hubs remains poorly understood. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at sub-diffraction limited resolution of ~100nm length scale within a focal adhesion to examine the dynamics of diverse focal adhesion proteins. We show that (i) subregions of focal adhesions are enriched in immobile population of integrin ß3 organised as nanoclusters, which (ii) in turn serve to organise nanoclusters of associated key adhesome proteins- vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signalling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct dynamics dependent on function. (iv) long-lived nanoclusters function as signalling hubs- wherein phosphorylated FAK and paxillin formed stable nanoclusters in close proximity to immobile integrin nanoclusters which are disassembled in response to inactivation signal by phosphatase PTPN12 (v) signalling takes place in response to an external signal such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. Taken together, these results demonstrate that signalling downstream of transmembrane receptors is organised as hubs of signalling proteins (FAK, paxillin, vinculin) seeded by nanoclusters of the transmembrane receptor (integrin).

TiO2 Nano-Biopatterning Reveals Optimal Ligand Presentation for Cell-Matrix Adhesion Formation.

Nanoscale organization of transmembrane receptors is critical for cellular functions, enabled by the nanoscale engineering of bioligand presentation. Previously, a spatial threshold of ≤60 nm for integrin binding ligands in cell-matrix adhesion is demonstrated using monoliganded gold nanoparticles. However, the ligand geometric arrangement is limited to hexagonal arrays of monoligands, while plasmonic quenching limits further investigation by fluorescence-based high-resolution imaging. Here, these limitations are overcome with dielectric TiO2 nanopatterns, eliminating fluorescence quenching, thus enabling super-resolution fluorescence microscopy on nanopatterns. By dual-color super-resolution imaging, high precision and consistency among nanopatterns, bioligands, and integrin nanoclusters are observed, validating the high quality and integrity of both nanopattern functionalization and passivation. By screening TiO2 nanodiscs with various diameters, an increase in fibroblast cell adhesion, spreading area, and Yes-associated protein (YAP) nuclear localization on 100 nm diameter compared with smaller diameters was observed. Focal adhesion kinase is identified as the regulatory signal. These findings explore the optimal ligand presentation when the minimal requirements are sufficiently fulfilled in the heterogenous extracellular matrix network of isolated binding regions with abundant ligands. Integration of high-fidelity nano-biopatterning with super-resolution imaging allows precise quantitative studies to address early signaling events in response to receptor clustering and their nanoscale organization.

Recent Advancement in Elimination Strategies and Potential Rejuvenation Targets of Senescence.

Cellular senescence is a state of exiting the cell cycle, resisting apoptosis, and changing phenotype. Senescent cells (SCs) can be identified by large, distorted morphology and irreversible inability to replicate. In early development, senescence has beneficial roles like tissue patterning and wound healing, where SCs are cleared by the immune system. However, there is a steep rise in SC number as organisms age. The issue with SC accumulation stems from the loss of cellular function, alterations of the microenvironment, and secretions of pro-inflammatory molecules, consisting of cytokines, chemokines, matrix metalloproteinases (MMPs), interleukins, and extracellular matrix (ECM)-associated molecules. This secreted cocktail is referred to as the senescence-associated secretory phenotype (SASP), a hallmark of cellular senescence. The SASP promotes inflammation and displays a bystander effect where paracrine signaling turns proliferating cells into senescent states. To alleviate age-associated diseases, researchers have developed novel methods and techniques to selectively eliminate SCs in aged individuals. Although studies demonstrated that selectively killing SCs improves age-related disorders, there are drawbacks to SC removal. Considering favorable aspects of senescence in the body, this paper reviews recent advancements in elimination strategies and potential rejuvenation targets of senescence to bring researchers in the field up to date.

Intrinsic self-organization of integrin nanoclusters within focal adhesions is required for cellular mechanotransduction.

Upon interaction with the extracellular matrix, the integrin receptors form nanoclusters as a first biochemical response to ligand binding. Here, we uncover a critical biodesign principle where these nanoclusters are spatially self-organized, facilitating effective mechanotransduction. Mouse Embryonic Fibroblasts (MEFs) with integrin ß3 nanoclusters organized themselves with an intercluster distance of ~550 nm on uniformly coated fibronectin substrates, leading to larger focal adhesions. We determined that this spatial organization was driven by cell-intrinsic factors since there was no pre-existing pattern on the substrates. Altering this spatial organization using cyclo-RGD functionalized Titanium nanodiscs (of 100 nm, corroborating to the integrin nanocluster size) spaced at intervals of 300 nm (almost half), 600 nm (normal) or 1000 nm (almost double) resulted in abrogation in mechanotransduction, indicating that a new parameter i.e., an optimal intercluster distance is necessary for downstream function. Overexpression of α-actinin, which induces a kink in the integrin tail, disrupted the establishment of the optimal intercluster distance, while simultaneous co-overexpression of talin head with α-actinin rescued it, indicating a concentration-dependent competition, and that cytoplasmic activation of integrin by talin head is required for the optimal intercluster organization. Additionally, talin head-mediated recruitment of FHOD1 that facilitates local actin polymerization at nanoclusters, and actomyosin contractility were also crucial for establishing the optimal intercluster distance and a robust mechanotransduction response. These findings demonstrate that cell-intrinsic machinery plays a vital role in organizing integrin receptor nanoclusters within focal adhesions, encoding essential information for downstream mechanotransduction signalling.

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.

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.

Force- and cell state-dependent recruitment of Piezo1 drives focal adhesion dynamics and calcium entry.

Mechanosensing is an integral part of many physiological processes including stem cell differentiation, fibrosis, and cancer progression. Two major mechanosensing systems-focal adhesions and mechanosensitive ion channels-can convert mechanical features of the microenvironment into biochemical signals. We report here unexpectedly that the mechanosensitive calcium-permeable channel Piezo1, previously perceived to be diffusive on plasma membranes, binds to matrix adhesions in a force-dependent manner, promoting cell spreading, adhesion dynamics, and calcium entry in normal but not in most cancer cells tested except some glioblastoma lines. A linker domain in Piezo1 is needed for binding to adhesions, and overexpression of the domain blocks Piezo1 binding to adhesions, decreasing adhesion size and cell spread area. Thus, we suggest that Piezo1 is a previously unidentified component of focal adhesions in nontransformed cells that catalyzes adhesion maturation and growth through force-dependent calcium signaling, but this function is absent in most cancer cells.

Cancer cells can be killed mechanically or with combinations of cytoskeletal inhibitors.

For over two centuries, clinicians have hypothesized that cancer developed preferentially at the sites of repeated damage, indicating that cancer is basically "continued healing." Tumor cells can develop over time into other more malignant types in different environments. Interestingly, indefinite growth correlates with the depletion of a modular, early rigidity sensor, whereas restoring these sensors in tumor cells blocks tumor growth on soft surfaces and metastases. Importantly, normal and tumor cells from many different tissues exhibit transformed growth without the early rigidity sensor. When sensors are restored in tumor cells by replenishing depleted mechanosensory proteins that are often cytoskeletal, cells revert to normal rigidity-dependent growth. Surprisingly, transformed growth cells are sensitive to mechanical stretching or ultrasound which will cause apoptosis of transformed growth cells (Mechanoptosis). Mechanoptosis is driven by calcium entry through mechanosensitive Piezo1 channels that activate a calcium-induced calpain response commonly found in tumor cells. Since tumor cells from many different tissues are in a transformed growth state that is, characterized by increased growth, an altered cytoskeleton and mechanoptosis, it is possible to inhibit growth of many different tumors by mechanical activity and potentially by cytoskeletal inhibitors.

Tumor Suppressor DAPK1 Catalyzes Adhesion Assembly on Rigid but Anoikis on Soft Matrices.

Cancer cells normally grow on soft surfaces due to impaired mechanosensing of the extracellular matrix rigidity. Upon restoration of proper mechanosensing, cancer cells undergo apoptosis on soft surfaces (anoikis) like most normal cells. However, the link between mechanosensing and activation of anoikis is not clear. Here we show that death associated protein kinase 1 (DAPK1), a tumor suppressor that activates cell death, is directly linked to anoikis activation through rigidity sensing. We find that when rigidity sensing is decreased through inhibition of DAPK1 activity, cells are transformed for growth on soft matrices. Further, DAPK1 catalyzes matrix adhesion assembly and is part of adhesions on rigid surfaces. This pathway involves DAPK1 phosphorylation of tropomyosin1.1, the talin1 head domain, and tyrosine phosphorylation of DAPK1 by Src. On soft surfaces, DAPK1 rapidly dissociates from the adhesion complexes and activates apoptosis as catalyzed by PTPN12 activity and talin1 head. Thus, DAPK1 is important for adhesion assembly on rigid surfaces and the activation of anoikis on soft surfaces through its binding to rigidity-sensing modules.

Ligand functionalization of titanium nanopattern enables the analysis of cell-ligand interactions by super-resolution microscopy.

The spatiotemporal aspects of early signaling events during interactions between cells and their environment dictate multiple downstream outcomes. While advances in nanopatterning techniques have allowed the isolation of these signaling events, a major limitation of conventional nanopatterning methods is its dependence on gold (Au) or related materials that plasmonically quench fluorescence and, thus, are incompatible with super-resolution fluorescence microscopy. Here we describe a novel method that integrates nanopatterning with single-molecule resolution fluorescence imaging, thus enabling mechanistic dissection of molecular-scale signaling events in conjunction with nanoscale geometry manipulation. Our method exploits nanofabricated titanium (Ti) whose oxide (TiO2) is a dielectric material with no plasmonic effects. We describe the surface chemistry for decorating specific ligands such as cyclo-RGD (arginine, glycine and aspartate: a ligand for fibronectin-binding integrins) on TiO2 nanoline and nanodot substrates, and demonstrate the ability to perform dual-color super-resolution imaging on these patterns. Ti nanofabrication is similar to other metallic materials like Au, while the functionalization of TiO2 is relatively fast, safe, economical, easy to set up with commonly available reagents, and robust against environmental parameters such as humidity. Fabrication of nanopatterns takes ~2-3 d, preparation for functionalization ~1.5-2 d, and functionalization 3 h, after which cell culture and imaging experiments can be performed. We suggest that this method may facilitate the interrogation of nanoscale geometry and force at single-molecule resolution, and should find ready applications in early detection and interpretation of physiochemical signaling events at the cell membrane in the fields of cell biology, immunology, regenerative medicine, and related fields.

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.

When PIP2 Meets p53: Nuclear Phosphoinositide Signaling in the DNA Damage Response.

The mechanisms that maintain genome stability are critical for preventing tumor progression. In the past decades, many strategies were developed for cancer treatment to disrupt the DNA repair machinery or alter repair pathway selection. Evidence indicates that alterations in nuclear phosphoinositide lipids occur rapidly in response to genotoxic stresses. This implies that nuclear phosphoinositides are an upstream element involved in DNA damage signaling. Phosphoinositides constitute a new signaling interface for DNA repair pathway selection and hence a new opportunity for developing cancer treatment strategies. However, our understanding of the underlying mechanisms by which nuclear phosphoinositides regulate DNA damage repair, and particularly the dynamics of those processes, is rather limited. This is partly because there are a limited number of techniques that can monitor changes in the location and/or abundance of nuclear phosphoinositide lipids in real time and in live cells. This review summarizes our current knowledge regarding the roles of nuclear phosphoinositides in DNA damage response with an emphasis on the dynamics of these processes. Based upon recent findings, there is a novel model for p53's role with nuclear phosphoinositides in DNA damage response that provides new targets for synthetic lethality of tumors.

α-Catenin links integrin adhesions to F-actin to regulate ECM mechanosensing and rigidity dependence.

Both cell-cell and cell-matrix adhesions are regulated by mechanical signals, but the mechanobiological processes that mediate the cross talk between these structures are poorly understood. Here we show that α-catenin, a mechanosensitive protein that is classically linked with cadherin-based adhesions, associates with and regulates integrin adhesions. α-Catenin is recruited to the edges of mesenchymal cells, where it interacts with F-actin. This is followed by mutual retrograde flow of α-catenin and F-actin from the cell edge, during which α-catenin interacts with vinculin within integrin adhesions. This interaction affects adhesion maturation, stress-fiber assembly, and force transmission to the matrix. In epithelial cells, α-catenin is present in cell-cell adhesions and absent from cell-matrix adhesions. However, when these cells undergo epithelial-to-mesenchymal transition, α-catenin transitions to the cell edge, where it facilitates proper mechanosensing. This is highlighted by the ability of α-catenin-depleted cells to grow on soft matrices. These results suggest a dual role of α-catenin in mechanosensing, through both cell-cell and cell-matrix adhesions.

Application of piconewton forces to individual filopodia reveals mechanosensory role of L-type Ca2+ channels.

Filopodia are ubiquitous membrane projections that play crucial role in guiding cell migration on rigid substrates and through extracellular matrix by utilizing yet unknown mechanosensing molecular pathways. As recent studies show that Ca2+ channels localized to filopodia play an important role in regulation of their formation and since some Ca2+ channels are known to be mechanosensitive, force-dependent activity of filopodial Ca2+ channels might be linked to filopodia's mechanosensing function. We tested this hypothesis by monitoring changes in the intra-filopodial Ca2+ level in response to application of stretching force to individual filopodia of several cell types using optical tweezers. Results show that stretching forces of tens of pN strongly promote Ca2+ influx into filopodia, causing persistent Ca2+ oscillations that last for minutes even after the force is released. Several known mechanosensitive Ca2+ channels, such as Piezo 1, Piezo 2 and TRPV4, were found to be dispensable for the observed force-dependent Ca2+ influx, while L-type Ca2+ channels appear to be a key player in the discovered phenomenon. As previous studies have shown that intra-filopodial transient Ca2+ signals play an important role in guidance of cell migration, our results suggest that the force-dependent activation of L-type Ca2+ channels may contribute to this process. Overall, our study reveals an intricate interplay between mechanical forces and Ca2+ signaling in filopodia, providing novel mechanistic insights for the force-dependent filopodia functions in guidance of cell migration.

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.

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.

Local contractions regulate E-cadherin rigidity sensing.

E-cadherin is a major cell-cell adhesion molecule involved in mechanotransduction at cell-cell contacts in tissues. Because epithelial cells respond to rigidity and tension in tissue through E-cadherin, there must be active processes that test and respond to the mechanical properties of these adhesive contacts. Using submicrometer, E-cadherin-coated polydimethylsiloxane pillars, we find that cells generate local contractions between E-cadherin adhesions and pull to a constant distance for a constant duration, irrespective of pillar rigidity. These cadherin contractions require nonmuscle myosin IIB, tropomyosin 2.1, α-catenin, and binding of vinculin to α-catenin. Cells spread to different areas on soft and rigid surfaces with contractions, but spread equally on soft and rigid without. We further observe that cadherin contractions enable cells to test myosin IIA-mediated tension of neighboring cells and sort out myosin IIA-depleted cells. Thus, we suggest that epithelial cells test and respond to the mechanical characteristics of neighboring cells through cadherin contractions.

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.