Working a second job: Cell adhesion proteins that moonlight in the nucleus.

Cells are adept at sensing changes in their environment, transmitting signals internally to coordinate responses to external stimuli, and thereby influencing adaptive changes in cell states and behavior. Often, this response involves modulation of gene expression in the nucleus, which is seen largely as a physically separated process from the rest of the cell. Mechanosensing, whereby a cell senses physical stimuli, and integrates and converts these inputs into downstream responses including signaling cascades and gene regulatory changes, involves the participation of several macromolecular structures. Of note, the extracellular matrix (ECM) and its constituent macromolecules comprise an essential part of the cellular microenvironment, allowing cells to interact with each other, and providing both structural and biochemical stimuli sensed by adhesion transmembrane receptors. This highway of information between the ECM, cell adhesion proteins, and the cytoskeleton regulates cellular behavior, the disruption of which results in disease. Emerging evidence suggests a more direct role for some of these adhesion proteins in chromatin structure and gene regulation, RNA maturation and other non-canonical functions. While many of these discoveries were previously limited to observations of cytoplasmic-nuclear transport, recent advances in microscopy, and biochemical, proteomic and genomic technologies have begun to significantly enhance our understanding of the impact of nuclear localization of these proteins. This review will briefly cover known cell adhesion proteins that migrate to the nucleus, and their downstream functions. We will outline recent advances in this very exciting yet still emerging field, with impact ranging from basic biology to disease states like cancer.

Molecular basis of conformational changes and mechanics of integrins.

Integrin, as a mechanotransducer, establishes the mechanical reciprocity between the extracellular matrix (ECM) and cells at integrin-mediated adhesion sites. This study used steered molecular dynamics (SMD) simulations to investigate the mechanical responses of integrin αvß3 with and without 10th type III fibronectin (FnIII10) binding for tensile, bending and torsional loading conditions. The ligand-binding integrin confirmed the integrin activation during equilibration and altered the integrin dynamics by changing the interface interaction between ß-tail, hybrid and epidermal growth factor domains during initial tensile loading. The tensile deformation in integrin molecules indicated that fibronectin ligand binding modulates its mechanical responses in the folded and unfolded conformation states. The bending deformation responses of extended integrin models reveal the change in behaviour of integrin molecules in the presence of Mn2+ ion and ligand based on the application of force in the folding and unfolding directions of integrin. Furthermore, these SMD simulation results were used to predict the mechanical properties of integrin underlying the mechanism of integrin-based adhesion. The evaluation of integrin mechanics provides new insights into understanding the mechanotransmission (force transmission) between cells and ECM and contributes to developing an accurate model for integrin-mediated adhesion. This article is part of a discussion meeting issue 'Supercomputing simulations of advanced materials'.

Analysis of Integrin-Dependent Melanoblast Migration During Development.

The neural crest is a transient embryonic structure that gives rise to a number of important cell types and tissues, including most of the peripheral and enteric nervous systems, pigment-producing skin cells known as melanocytes, and many craniofacial structures. Melanoblasts, the precursors of melanocytes, are derived from the so-called trunk neural crest cells. These cells delaminate and migrate along a dorsolateral pathway to colonize their final destination in the skin, and consequently, defects in melanoblast migration result in pigmentation defects. Studying melanocyte migration is a topic of great interest due to the involvement of melanocytes in highly metastatic skin cancer. A role for integrin-mediated adhesion is well established in neural crest migration, and our recent work has provided direct evidence for a key role for integrin-based adhesion in melanocyte migration. Imaging of melanoblast migration in the context of intact skin has proven to be a particularly powerful tool to study integrin-based adhesion during melanoblast migration. Here, we describe the use of skin explants combined with genetically encoded markers for melanocytes and high-resolution live imaging as a powerful and informative approach to analyze melanoblast migration in an ex vivo context.

Crosstalk with keratinocytes causes GNAQ oncogene specificity in melanoma.

Different melanoma subtypes exhibit specific and non-overlapping sets of oncogene and tumor suppressor mutations, despite a common cell of origin in melanocytes. For example, activation of the Gαq/11 signaling pathway is a characteristic initiating event in primary melanomas that arise in the dermis, uveal tract, or central nervous system. It is rare in melanomas arising in the epidermis. The mechanism for this specificity is unknown. Here, we present evidence that in the mouse, crosstalk with the epidermal microenvironment actively impairs the survival of melanocytes expressing the GNAQQ209L oncogene. We found that GNAQQ209L, in combination with signaling from the interfollicular epidermis (IFE), stimulates dendrite extension, leads to actin cytoskeleton disorganization, inhibits proliferation, and promotes apoptosis in melanocytes. The effect was reversible and paracrine. In contrast, the epidermal environment increased the survival of wildtype and BrafV600E expressing melanocytes. Hence, our studies reveal the flip side of Gαq/11 signaling, which was hitherto unsuspected. In the future, the identification of the epidermal signals that restrain the GNAQQ209L oncogene could suggest novel therapies for GNAQ and GNA11 mutant melanomas.

Precise coordination of cell-ECM adhesion is essential for efficient melanoblast migration during development.

Melanoblasts disperse throughout the skin and populate hair follicles through long-range cell migration. During migration, cells undergo cycles of coordinated attachment and detachment from the extracellular matrix (ECM). Embryonic migration processes that require cell-ECM attachment are dependent on the integrin family of adhesion receptors. Precise regulation of integrin-mediated adhesion is important for many developmental migration events. However, the mechanisms that regulate integrin-mediated adhesion in vivo in melanoblasts are not well understood. Here, we show that autoinhibitory regulation of the integrin-associated adapter protein talin coordinates cell-ECM adhesion during melanoblast migration in vivo Specifically, an autoinhibition-defective talin mutant strengthens and stabilizes integrin-based adhesions in melanocytes, which impinges on their ability to migrate. Mice with defective talin autoinhibition exhibit delays in melanoblast migration and pigmentation defects. Our results show that coordinated integrin-mediated cell-ECM attachment is essential for melanoblast migration and that talin autoinhibition is an important mechanism for fine-tuning cell-ECM adhesion during cell migration in development.

A survey-based analysis of the academic job market.

Many postdoctoral researchers apply for faculty positions knowing relatively little about the hiring process or what is needed to secure a job offer. To address this lack of knowledge about the hiring process we conducted a survey of applicants for faculty positions: the survey ran between May 2018 and May 2019, and received 317 responses. We analyzed the responses to explore the interplay between various scholarly metrics and hiring outcomes. We concluded that, above a certain threshold, the benchmarks traditionally used to measure research success - including funding, number of publications or journals published in - were unable to completely differentiate applicants with and without job offers. Respondents also reported that the hiring process was unnecessarily stressful, time-consuming, and lacking in feedback, irrespective of outcome. Our findings suggest that there is considerable scope to improve the transparency of the hiring process.

Dynamic protein hydrogels with reversibly tunable stiffness regulate human lung fibroblast spreading reversibly.

We report the engineering of a protein-based dynamic hydrogel that can be reversibly tuned between stiff and soft states via a redox reaction. When cultured on this hydrogel surface, human lung fibroblasts can dynamically and reversibly change their morphology in response to changes in hydrogel stiffness.

Direct binding of Talin to Rap1 is required for cell-ECM adhesion in Drosophila.

Attachment of cells to the extracellular matrix (ECM) via integrins is essential for animal development and tissue maintenance. The cytoplasmic protein Talin (encoded by rhea in flies) is necessary for linking integrins to the cytoskeleton, and its recruitment is a key step in the assembly of the adhesion complex. However, the mechanisms that regulate Talin recruitment to sites of adhesion in vivo are still not well understood. Here, we show that Talin recruitment to, and maintenance at, sites of integrin-mediated adhesion requires a direct interaction between Talin and the GTPase Rap1. A mutation that blocks the direct binding of Talin to Rap1 abolished Talin recruitment to sites of adhesion and the resulting phenotype phenocopies that seen with null alleles of Talin. Moreover, we show that Rap1 activity modulates Talin recruitment to sites of adhesion via its direct binding to Talin. These results identify the direct Talin-Rap1 interaction as a key in vivo mechanism for controlling integrin-mediated cell-ECM adhesion.

Talin Autoinhibition Regulates Cell-ECM Adhesion Dynamics and Wound Healing In Vivo.

Cells in multicellular organisms are arranged in complex three-dimensional patterns. This requires both transient and stable adhesions with the extracellular matrix (ECM). Integrin adhesion receptors bind ECM ligands outside the cell and then, by binding the protein talin inside the cell, assemble an adhesion complex connecting to the cytoskeleton. The activity of talin is controlled by several mechanisms, but these have not been well studied in vivo. By generating mice containing the activating point mutation E1770A in talin (Tln1), which disrupts autoinhibition, we show that talin autoinhibition controls cell-ECM adhesion, cell migration, and wound healing in vivo. In particular, blocking autoinhibition gives rise to more mature, stable focal adhesions that exhibit increased integrin activation. Mutant cells also show stronger attachment to ECM and decreased traction force. Overall, these results demonstrate that modulating talin function via autoinhibition is an important mechanism for regulating multiple aspects of integrin-mediated cell-ECM adhesion in vivo.

Matrix metalloproteinase-14 is a mechanically regulated activator of secreted MMPs and invasion.

Matrix metalloproteinases (MMPs) are extracellular matrix (ECM) degrading enzymes and have complex and specific regulation networks. This includes activation interactions, where one MMP family member activates another. ECM degradation and MMP activation can be initiated by several different stimuli including changes in ECM mechanical properties or intracellular contractility. These mechanical stimuli are known enhancers of metastatic potential. MMP-14 facilitates local ECM degradation and is well known as a major mediator of cell migration, angiogenesis and invasion. Recently, function blocking antibodies have been developed to specifically block MMP-14, providing a useful tool for research as well as therapeutic applications. Here we utilize a selective MMP-14 function blocking antibody to delineate the role of MMP-14 as an activator of other MMPs in response to changes in cellular contractility and ECM stiffness. Inhibition using function blocking antibodies reveals that MMP-14 activates soluble MMPs like MMP-2 and -9 under various mechanical stimuli in the pancreatic cancer cell line, Panc-1. In addition, inhibition of MMP-14 abates Panc-1 cell extension into 3D gels to levels seen with non-specific pan-MMP inhibitors at higher concentrations. This strengthens the case for MMP function blocking antibodies as more potent and specific MMP inhibition therapeutics.

Cellular contractility and extracellular matrix stiffness regulate matrix metalloproteinase activity in pancreatic cancer cells.

The pathogenesis of cancer is often driven by local invasion and metastasis. Recently, mechanical properties of the tumor microenvironment have been identified as potent regulators of invasion and metastasis, while matrix metalloproteinases (MMPs) are classically known as significant enhancers of cancer cell migration and invasion. Here we have been able to sensitively measure MMP activity changes in response to specific extracellular matrix (ECM) environments and cell contractility states. Cells of a pancreatic cancer cell line, Panc-1, up-regulate MMP activities between 3- and 10-fold with increased cell contractility. Conversely, they down-regulate MMP activities when contractility is blocked to levels seen with pan-MMP activity inhibitors. Similar, albeit attenuated, responses are seen in other pancreatic cancer cell lines, BxPC-3 and AsPC-1. In addition, MMP activity was modulated by substrate stiffness, collagen gel concentration, and the degree of collagen cross-linking, when cells were plated on collagen gels ranging from 0.5 to 5 mg/ml that span the physiological range of substrate stiffness (50-2000 Pa). Panc-1 cells showed enhanced MMP activity on stiffer substrates, whereas BxPC-3 and AsPC-1 cells showed diminished MMP activity. In addition, eliminating heparan sulfate proteoglycans using heparinase completely abrogated the mechanical induction of MMP activity. These results demonstrate the first functional link between MMP activity, contractility, and ECM stiffness and provide an explanation as to why stiffer environments result in enhanced cell migration and invasion.

Mixed-surface, lipid-tethered quantum dots for targeting cells and tissues.

Quantum dots (QDs), with their variable luminescent properties, are rapidly transcending traditional labeling techniques in biological imaging and hold vast potential for biosensing applications. An obstacle in any biosensor development is targeted specificity. Here we report a facile procedure for creating QDs targeted to the cell membrane with the goal of cell-surface protease biosensing. This procedure generates water-soluble QDs with variable coverage of lipid functional groups. The resulting hydrophobicity is quantitatively controlled by the molar ratio of lipids per QD. Appropriate tuning of the hydrophobicity ensures solubility in common aqueous cell culture media, while providing affinity to the lipid bilayer of cell membranes. The reaction and exchange process was directly evaluated by measuring UV absorption spectra associated with dithiocarbamate formation. Cell membrane binding was assessed using flow cytometry and total internal reflection fluorescence imaging with live cells, and tissue affinity was measured using histochemical staining and fluorescence imaging of frozen tissue sections. Increases in cell and tissue binding were found to be regulated by both QD hydrophobicity and surface charge, underlying the importance of QD surface properties in the optimization of both luminescence and targeting capability.