Adhesion Complexes Research Articles

Shear stress-stimulated AMPK couples endothelial cell mechanics, metabolism, and vasodilation.

Endothelial cells respond to mechanical force by stimulating cellular signaling, but how these pathways are linked to elevations in cell metabolism and whether metabolism supports the mechanical response remains poorly understood. Here, we show that the application of force to endothelial cells stimulates VE-cadherin to activate liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis. VE-cadherin stimulated AMPK increases eNOS activity and localization to the plasma membrane, reinforcement of the actin cytoskeleton and cadherin adhesion complex, and glucose uptake. We present evidence for the increase in metabolism being necessary to fortify the adhesion complex, actin cytoskeleton, and cellular alignment. Together these data extend the paradigm for how mechanotransduction and metabolism are linked to include a connection to vasodilation, thereby providing new insight into how diseases involving contractile, metabolic, and vasodilatory disturbances arise.

A universal resist-assisted metal transfer method for 2D semiconductor contacts

Abstract With the explosive exploration of two-dimensional (2D) semiconductors for device applications, ensuring effective electrical contacts has become critical for optimizing device performance. Here, we demonstrate a universal resist-assisted metal transfer method for creating nearly free-standing metal electrodes on the SiO2/Si substrate, which can be easily transferred onto 2D semiconductors to form van der Waals (vdW) contacts. In this method, polymethyl methacrylate (PMMA) serves both as an electron resist for electrode patterning and as a sacrificial layer. Contacted with our transferred electrodes, MoS₂ exhibits tunable Schottky barrier heights and a transition from n-type dominated to ambipolar conduction with increasing metal work functions, while InSe shows pronounced ambipolarity. Additionally, using α-In2Se3 as an example, we demonstrate that our transferred electrodes enhance resistance switching in ferroelectric memristors. Finally, the universality of our method is evidenced by the successful transfer of various metals with different adhesion forces and complex patterns.

Cell-cell junctions in focus - imaging junctional architectures and dynamics at high resolution.

Studies utilizing electron microscopy and live fluorescence microscopy have significantly enhanced our understanding of the molecular mechanisms that regulate junctional dynamics during homeostasis, development and disease. To fully grasp the enormous complexity of cell-cell adhesions, it is crucial to study the nanoscale architectures of tight junctions, adherens junctions and desmosomes. It is important to integrate these junctional architectures with the membrane morphology and cellular topography in which the junctions are embedded. In this Review, we explore new insights from studies using super-resolution and volume electron microscopy into the nanoscale organization of these junctional complexes as well as the roles of the junction-associated cytoskeleton, neighboring organelles and the plasma membrane. Furthermore, we provide an overview of junction- and cytoskeletal-related biosensors and optogenetic probes that have contributed to these advances and discuss how these microscopy tools enhance our understanding of junctional dynamics across cellular environments.

Advances in mechanotransduction and sonobiology: effects of audible acoustic waves and low-vibration stimulations on mammalian cells

AbstractIn recent decades, research on mechanotransduction has advanced considerably, focusing on the effects of audible acoustic waves (AAWs) and low-vibration stimulation (LVS), which has propelled the field of sonobiology forward. Taken together, the current evidence demonstrates the influence of these biosignals on key cellular processes, such as growth, differentiation and migration in mammalian cells, emphasizing the determining role of specific physical parameters during stimulation, such as frequency, sound pressure level/amplitude and exposure time. These mechanical waves interact with various cellular elements, including ion channels, primary cilia, cell–cell adhesion receptors, cell–matrix and extracellular matrix proteins, and focal adhesion complexes. These components connect with the cytoskeletal fibre network, enabling the transmission of mechanical stimuli towards the nucleus. The nucleus, in turn, linked to the cytoskeleton via the linkers of the nucleoskeleton and cytoskeleton complex, acts as a mechanosensitive centre, not only responding to changes in cytoskeletal stiffness and nuclear tension but also regulating gene expression through the transcriptional co-activator YAP/TAZ and interactions between chromatin and the nuclear envelope. This intricate chain of mechanisms highlights the potential of sonobiology in various fields, including dentistry, regenerative medicine, tissue engineering and cancer research. However, progress in these fields requires the establishment of standardized measurement methodologies and biocompatible experimental setups to ensure the reproducibility of results.

Loss of Cell-Cell Contact Inhibits Cellular Differentiation of α-Catenin Knock Out P19 Embryonal Carcinoma Cells and Their Colonization into the Developing Mouse Embryos.

Cadherin-catenin cell-cell adhesion complexes, composed of cadherin, β-catenin or plakoglobin, and α-catenin (α-cat) molecules, are crucial for maintaining cell-cell contact and are commonly referred to as "adherens junctions (AJs)." Inactivating this system leads to loss of cell-cell contact and developmental arrest in early embryos. However, it remains unclear whether the loss of cell-cell contact affects the differentiation of embryonic cells. In this study, we explored the use of a murine embryonal carcinoma cell line, P19, as an in vitro model for early embryogenesis. P19 cells easily form embryoid bodies (EBs) and are susceptible to cellular differentiation in response to retinoic acid (RA) and teratoma formation. Using CRISPR/Cas9 technology to disrupt the endogenous α-cat gene in P19 cells, we generated α-cat knockout (KO) cells that exhibited a loss of cell-cell contact. When cultivated on non-coated dishes, these α-cat KO cells formed EBs, but their structures were labile. In the RA-containing medium, the α-cat KO EBs failed to produce differentiated cells on their outer layer and continued to express SSEA-1, an antigen specific to pluripotent cells. Teratoma formation assays revealed an absence of overt differentiated cells in tumors derived from α-cat KO P19 cells. Aggregation assays revealed the inability of the KO cells to colonize into the zona pellucida-denuded 8-cell embryos. These findings suggest that the AJs are essential for promoting the early stages of cellular differentiation and for the colonization of early-developing embryos.

A lytic transglycosylase connects bacterial focal adhesion complexes to the peptidoglycan cell wall

The Gram-negative bacterium Myxococcus xanthus glides on solid surfaces. Dynamic bacterial focal adhesion complexes (bFACs) convert proton motive force from the inner membrane into mechanical propulsion on the cell surface. It is unclear how the mechanical force transmits across the rigid peptidoglycan (PG) cell wall. Here, we show that AgmT, a highly abundant lytic PG transglycosylase homologous to Escherichia coli MltG, couples bFACs to PG. Coprecipitation assay and single-particle microscopy reveal that the gliding motors fail to connect to PG and thus are unable to assemble into bFACs in the absence of an active AgmT. Heterologous expression of E. coli MltG restores the connection between PG and bFACs and thus rescues gliding motility in the M. xanthus cells that lack AgmT. Our results indicate that bFACs anchor to AgmT-modified PG to transmit mechanical force across the PG cell wall.

A lytic transglycosylase connects bacterial focal adhesion complexes to the peptidoglycan cell wall.

The Gram-negative bacterium Myxococcus xanthus glides on solid surfaces. Dynamic bacterial focal adhesion complexes (bFACs) convert proton motive force from the inner membrane into mechanical propulsion on the cell surface. It is unclear how the mechanical force transmits across the rigid peptidoglycan (PG) cell wall. Here, we show that AgmT, a highly abundant lytic PG transglycosylase homologous to Escherichia coli MltG, couples bFACs to PG. Coprecipitation assay and single-particle microscopy reveal that the gliding motors fail to connect to PG and thus are unable to assemble into bFACs in the absence of an active AgmT. Heterologous expression of E. coli MltG restores the connection between PG and bFACs and thus rescues gliding motility in the M. xanthus cells that lack AgmT. Our results indicate that bFACs anchor to AgmT-modified PG to transmit mechanical force across the PG cell wall.

Intestinal Tuft Cells Are Enriched With Protocadherins.

Intestinal tuft cells are rare cells that regulate diverse functions. They harbor chemosensory receptors and signal to the mucosal immune system in response to external stimuli, though their full function and structure remain unclear. Named for their apical "tuft" of long actin-rich microvilli, tuft cells facilitate chemoreception and other physiological responses. In enterocytes, microvilli are stabilized by intermicrovillar adhesion complexes (IMACs) composed of several proteins, including cadherin-related family member-2 (CDHR2) and cadherin-related family member-5 (CDHR5), Myosin 7b, and Usher syndrome type 1 C (USH1C). We hypothesized that IMACs would be enriched in tuft cells to regulate microvillar organization. Immunostaining of murine intestinal tissue revealed that CDHR2 and CDHR5 colocalize with the tuft cell markers, DCLK1, phospho-EGFR, advillin, and cytokeratin 18. CDHR2 was dispersed throughout murine tuft cells, while CDHR5 was concentrated on the apical surface. USH1C and Myosin 7b were present in tuft cells, but at lower levels. Human single-cell RNA sequencing revealed robust CDHR2 and CDHR5 expression in tuft cells in the small intestine and colon. Immunostaining of human intestinal tissue confirmed CDHR2 and CDHR5 localization to the apical surface of tuft cells. Our findings demonstrate that protocadherins are key components of murine and human intestinal tuft cells.

The role and regulation of integrins in cell migration and invasion.

Integrin receptors are the main molecular link between cells and the extracellular matrix (ECM) as well as mediating cell-cell interactions. Integrin-ECM binding triggers the formation of heterogeneous multi-protein assemblies termed integrin adhesion complexes (IACs) that enable integrins to transform extracellular cues into intracellular signals that affect many cellular processes, especially cell motility. Cell migration is essential for diverse physiological and pathological processes and is dysregulated in cancer to favour cell invasion and metastasis. Here, we discuss recent findings on the role of integrins in cell migration with a focus on cancer cell dissemination. We review how integrins regulate the spatial distribution and dynamics of different IACs, covering classical focal adhesions, emerging adhesion types and adhesion regulation. We discuss the diverse roles integrins have during cancer progression from cell migration across varied ECM landscapes to breaching barriers such as the basement membrane, and eventual colonization of distant organs.

Nonequilibrium cluster-cluster aggregation in the presence of anchoring sites.

Nonequilibrium cluster-cluster aggregation of particles diffusing in or at the cell membrane has been hypothesized to lead to domains of finite size in different biological contexts, such as lipid rafts, cell adhesion complexes, or postsynaptic domains in neurons. In this scenario, the desorption of particles balances a continuous flux to the membrane, imposing a cutoff on possible aggregate sizes and giving rise to a stationary size distribution. Here, we investigate the case of nonequilibrium cluster-cluster aggregation in two dimensions where diffusing particles and/or clusters remain fixed in space at specific anchoring sites, which should be particularly relevant for synapses but may also be present in other biological or physical systems. Using an effective mean-field description of the concentration field around anchored clusters, we derive an expression for their average size as a function of parameters such as the anchoring site density. We furthermore propose and solve appropriate rate equationsthat allow us to predict the size distributions of both diffusing and fixed clusters. We confirm our results with particle-based simulations and discuss potential implications for biological and physical systems.

Cadherin adhesion complexes direct cell aggregation in the epithelial transition of Wnt-induced nephron progenitor cells.

In the developing mammalian kidney, nephron formation is initiated by a subset of nephron progenitor cells (NPCs). Wnt input activates a β-catenin (Ctnnb1)-driven, transcriptional nephrogenic program and the mesenchymal to epithelial transition (MET) of NPCs. Using an in vitro mouse NPC culture model, we observed that activation of the Wnt pathway results in the aggregation of induced NPCs, which is an initiating step in the MET program. Genetic removal showed aggregation was dependent on β-catenin. Modulating extracellular Ca2+ levels showed cell-cell contacts were Ca2+ dependent, suggesting a role for cadherin (Cdh)-directed cell adhesion. Molecular analysis identified Cdh2, Cdh4 and Cdh11 in NPCs, and the β-catenin directed upregulation of Cdh3 and Cdh4 accompanying the MET of induced NPCs. Mutational analysis of β-catenin supported a role for a Lef/Tcf-β-catenin-mediated transcriptional response in the cell aggregation process. Genetic removal of all four cadherins, and independent removal of α-catenin or of β-catenin-α-catenin interactions, abolished aggregation, but not the inductive response to Wnt pathway activation. These findings, and data in an accompanying article highlight the role of β-catenin in linking transcriptional programs to the morphogenesis of NPCs in mammalian nephrogenesis.

Extracellular vesicles regulate metastable phenotypes of lymphangioleiomyomatosis cells via shuttling ATP synthesis to pseudopodia and activation of integrin adhesion complexes.

Pulmonary lymphangioleiomyomatosis (LAM) is metastatic sarcoma but mechanisms regulating LAM metastasis are unknown. Extracellular vesicle (EV) regulate cancer metastasis but their roles in LAM have not yet been investigated. Here, we report that EV biogenesis is increased in LAM and LAM EV cargo is enriched with lung tropic integrins, metalloproteinases, and cancer stem cell markers. LAM-EV increase LAM cell migration and invasion via the ITGα6/β1-c-Src-FAK-AKT axis. Metastable (hybrid) phenotypes of LAM metastasizing cells, pivotal for metastasis, are regulated by EV from primary tumor or metastasizing LAM cells via shuttling ATP synthesis to cell pseudopodia or activation of integrin adhesion complex, respectively. In mouse models of LAM, LAM-EV increase lung metastatic burden through mechanisms involving lung extracellular matrix remodeling. Collectively, these data provide evidence for the role of EV in promoting LAM lung metastasis and identify novel EV-dependent mechanisms regulating metastable phenotypes of tumor cells. Clinical impact of research is that it establishes LAM pathway as novel target for LAM therapy.

MultiBac System-based Purification and Biophysical Characterization of Human Myosin-7a.

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common and severe form of deaf-blindness in humans. It is hypothesized that myosin-7a forms a transmembrane adhesion complex with other Usher proteins, essential for the structural-functional integrity of photoreceptor and cochlear hair cells. However, due to the challenges in obtaining pure, intact protein, the exact functional mechanisms of human myosin-7a remain elusive, with limited structural and biomechanical studies available. Recent studies have shown that mammalian myosin-7a is a multimeric motor complex consisting of a heavy chain and three types of light chains: regulatory light chain (RLC), calmodulin, and calmodulin-like protein 4 (CALML4). Unlike calmodulin, CALML4 does not bind to calcium ions. Both the calcium-sensitive, and insensitive calmodulins are critical for mammalian myosin-7a for proper fine-tuning of its mechanical properties. Here, we describe a detailed method to produce recombinant human myosin-7a holoenzyme using the MultiBac Baculovirus protein expression system. This yields milligram quantities of high-purity full-length protein, allowing for its biochemical and biophysical characterization. We further present a protocol for assessing its mechanical and motile properties using tailored in vitro motility assays and fluorescence microscopy. The availability of the intact human myosin-7a protein, along with the detailed functional characterization protocol described here, paves the way for further investigations into the molecular aspects of myosin-7a in vision and hearing.

A conserved protein tyrosine phosphatase, PTPN-22, functions in diverse developmental processes in C. elegans.

Protein tyrosine phosphatases non-receptor type (PTPNs) have been studied extensively in the context of the adaptive immune system; however, their roles beyond immunoregulation are less well explored. Here we identify novel functions for the conserved C. elegans phosphatase PTPN-22, establishing its role in nematode molting, cell adhesion, and cytoskeletal regulation. Through a non-biased genetic screen, we found that loss of PTPN-22 phosphatase activity suppressed molting defects caused by loss-of-function mutations in the conserved NIMA-related kinases NEKL-2 (human NEK8/NEK9) and NEKL-3 (human NEK6/NEK7), which act at the interface of membrane trafficking and actin regulation. To better understand the functions of PTPN-22, we carried out proximity labeling studies to identify candidate interactors of PTPN-22 during development. Through this approach we identified the CDC42 guanine-nucleotide exchange factor DNBP-1 (human DNMBP) as an in vivo partner of PTPN-22. Consistent with this interaction, loss of DNBP-1 also suppressed nekl-associated molting defects. Genetic analysis, co-localization studies, and proximity labeling revealed roles for PTPN-22 in several epidermal adhesion complexes, including C. elegans hemidesmosomes, suggesting that PTPN-22 plays a broad role in maintaining the structural integrity of tissues. Localization and proximity labeling also implicated PTPN-22 in functions connected to nucleocytoplasmic transport and mRNA regulation, particularly within the germline, as nearly one-third of proteins identified by PTPN-22 proximity labeling are known P granule components. Collectively, these studies highlight the utility of combined genetic and proteomic approaches for identifying novel gene functions.

The unfolded protein response sensor PERK mediates mechanical stress-induced maturation of focal adhesion complexes in glioblastoma cells.

Stiffening of the brain extracellular matrix (ECM) in glioblastoma promotes tumor progression. Previously, we discovered that protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) plays a role in glioblastoma stem cell (GSC) adaptation to matrix stiffness through PERK/FLNA-dependent F-actin remodeling. Here, we examined the involvement of PERK in detecting stiffness changes via focal adhesion complex (FAC) formation. Compared to control GSCs, PERK-deficient GSCs show decreased vinculin and tensin expression, while talin and integrin-β1 remain constant. Furthermore, vimentin was also reduced while tubulin increased, and a stiffness-dependent increase of the differentiation marker GFAP expression was absent in PERK-deficient GSCs. In conclusion, our study reveals a novel role for PERK in FAC formation during matrix stiffening, which is likely linked to its regulation of F-actin remodeling.

A lytic transglycosylase connects bacterial focal adhesion complexes to the peptidoglycan cell wall.

The Gram-negative bacterium Myxococcus xanthus glides on solid surfaces. Dynamic bacterial focal adhesion complexes (bFACs) convert proton motive force from the inner membrane into mechanical propulsion on the cell surface. It is unclear how the mechanical force transmits across the rigid peptidoglycan (PG) cell wall. Here we show that AgmT, a highly abundant lytic PG transglycosylase homologous to Escherichia coli MltG, couples bFACs to PG. Coprecipitation assay and single-particle microscopy reveal that the gliding motors fail to connect to PG and thus are unable to assemble into bFACs in the absence of an active AgmT. Heterologous expression of E. coli MltG restores the connection between PG and bFACs and thus rescues gliding motility in the M. xanthus cells that lack AgmT. Our results indicate that bFACs anchor to AgmT-modified PG to transmit mechanical force across the PG cell wall.

Solvent-induced phase separation and Hofmeister effect enhanced strong, tough, and adhesive polyion complex hydrogels

Polyion complex (PIC) hydrogels possess excellent strength and toughness due to the dynamic and synergetic energy dissipation mechanism associated with the co-existence of both strong and weak bonds, ionic and hydrogen, in their networks. On the other hand, PIC hydrogels usually exhibit poor wet adhesion properties. Furthermore, the role such dynamic energy dissipation mechanism plays in determining interfacial binding is unknown. To address these challenges, here, we report the fabrication of an adhesive PIC hydrogel, poly(sodium p-styrenesulfonate) (NaSS)/poly(acryloyloxyethyltrimethyl ammonium chloride (DAC)–co-2-vinyl-4,6-diamino-1,3,5-triazine (VDT), i.e. PNaSS/P(DAC-co-VDT), by introducing a multi-hydrogen bonding capacity functional co-monomer, VDT, and solvent-induced gel phase separation into the fabrication process. The resulting hydrogel exhibited as high as 174 kPa adhesion strength, with retained high mechanical properties, typically 1.2 MPa tensile strength, 2.12 MPa Young’s modulus, and 452% strain. The mechanical properties of PNaSS/P(DAC-co-VDT) were tunable by adjusting the content of VDT and of the total monomer concentration in hydrogel precursor solutions (Cm), as well as by taking advantage of the Hofmeister effect. The dynamic modulus spectra of PNaSS/P(DAC-co-VDT) hydrogels at different Cm’s in extended frequency windows with time–temperature superposition method (TTS) were acquired and analyzed to explore the influence of polymer entanglement on the hydrogel network structure. The microscopic structure-macroscopic mechanical property relationship of PNaSS/P(DAC-co-VDT) hydrogels at different Cm’s and immersed in different salt solutions was further studied using the Mooney-Rivlin equation and by fitting the tensile data to Creton’s viscoelastic model. This study may provide new insights into designing and constructing strong and tough PIC hydrogel-based adhesives.

Adhesion of pancreatic tumor cell clusters by desmosomal molecules enhances early liver metastases formation

Desmosomes are intercellular adhesion complexes providing mechanical coupling and tissue integrity. Previously, a correlation of desmosomal molecule expression with invasion and metastasis formation in several tumor entities was described together with a relevance for circulating tumor cell cluster formation. Here, we investigated the contribution of the desmosomal core adhesion molecule desmoglein-2 (DSG2) to the initial steps of liver metastasis formation by pancreatic cancer cells using a novel ex vivo liver perfusion mouse model. We applied the pancreatic ductal adenocarcinoma cell line AsPC-1 with and without a knockout (KO) of DSG2 and generated mouse lines with a hepatocyte-specific KO of the known interacting partners of DSG2 (DSG2 and desmocollin-2). Liver perfusion with DSG2 KO AsPC-1 cells led to smaller circulating cell clusters and a reduced number of cells adhering to murine livers compared to control cells. While this was independent of the expression levels of desmosomal adhesion molecules in hepatocytes, we show that increased cluster size of cancer cells, which correlates with stronger cell–cell adhesion and expression of desmosomal molecules, is a major factor contributing to the early phase of metastatic spreading. In conclusion, impaired desmosomal adhesion results in reduced circulating cell cluster size, which is relevant for seeding and attachment of metastatic cells to the liver.

Inhibition of GSK3α,β rescues cognitive phenotypes in a preclinical mouse model of CTNNB1 syndrome

CTNNB1 syndrome is a rare monogenetic disorder caused by CTNNB1 de novo pathogenic heterozygous loss-of-function variants that result in cognitive and motor disabilities. Treatment is currently lacking; our study addresses this critical need. CTNNB1 encodes β-catenin which is essential for normal brain function via its dual roles in cadherin-based synaptic adhesion complexes and canonical Wnt signal transduction. We have generated a Ctnnb1 germline heterozygous mouse line that displays cognitive and motor deficits, resembling key features of CTNNB1 syndrome in humans. Compared with wild-type littermates, Ctnnb1 heterozygous mice also exhibit decreases in brain β-catenin, β-catenin association with N-cadherin, Wnt target gene expression, and Na/K ATPases, key regulators of changes in ion gradients during high activity. Consistently, hippocampal neuron functional properties and excitability are altered. Most important, we identify a highly selective inhibitor of glycogen synthase kinase (GSK)3α,β that significantly normalizes the phenotypes to closely meet wild-type littermate levels. Our data provide new insights into brain molecular and functional changes, and the first evidence for an efficacious treatment with therapeutic potential for individuals with CTNNB1 syndrome.

Depletion of calpain2 accelerates epithelial barrier establishment and reduces growth factor-induced cell scattering

Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, β-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes.