Microtubule organization plays a central role in cell differentiation, orchestrating essential processes such as cell polarization, mechanotransduction, organelle positioning and intracellular transport. A hallmark of many differentiated cells is the transition from a centrosomal to a non-centrosomal microtubule-organizing center (MTOC). Here, we demonstrate that both centrosomal and nuclear envelope (NE)-associated MTOCs coexist in osteoclasts. We show that the key players for NE-MTOC formation, the AKAP6 and nesprin-1 (SYNE1) isoforms AKAP6β and nesprin-1α, previously considered muscle specific, are upregulated during osteoclast differentiation, suggesting a conserved role in NE-MTOC assembly across cell types. Targeted depletion of AKAP6 in RAW264.7-derived osteoclasts led to the displacement of the Golgi and MTOC-associated proteins PCM1, pericentrin and CDK5RAP2 from the NE, while their centrosomal localization remained intact. This selectively impaired microtubule nucleation from the NE without disrupting centrosomal microtubule activity, enabling a functional dissection of the two MTOCs. Loss of NE-MTOC activity, through AKAP6 depletion, impaired podosome formation and significantly reduced bone resorption capacity, highlighting the distinct and essential role of NE-derived microtubules in osteoclast function.

Adhesion G protein-coupled receptors (aGPCRs) constitute a structurally and functionally distinct group within the superfamily of GPCRs. In 2015, the International Union of Pharmacology invited the Adhesion GPCR Consortium to publish a comprehensive review about aGPCRs and establish a unified nomenclature. Since then, substantial progress has been made in delineating the biological roles, molecular architecture, biochemical properties, expression profiles, ligand repertoire, and activation and signaling strategies of aGPCRs. Commensurate with these advances, their relevance to human pathophysiology has become increasingly apparent. In a coordinated effort, the Adhesion GPCR Consortium has reviewed recent progress in this field and provides a comprehensive assessment of the current understanding of aGPCR biology, including a focus on human and mammalian aGPCRs, their evolutionary origins, methodological approaches, and model systems for their investigation, as well as emerging approaches for their therapeutic targeting. SIGNIFICANCE STATEMENT: Adhesion G protein-coupled receptors are versatile cell-surface proteins that integrate structural, biochemical, and physiological functions, with major roles in health and disease. This review summarizes current knowledge of their molecular features, functions in diverse model systems, and emerging opportunities for therapeutic targeting, providing a comprehensive resource that connects basic biology with translational applications across multiple scientific disciplines.

How adhesion G protein-coupled receptors (aGPCRs) control development remains unclear. aGPCR Adgrg6/Gpr126 has been associated with heart trabeculation. Defects in this process cause cardiomyopathies and cardiac dysfunction. How cardiomyocytes attain trabecular identity is poorly understood. Here, we show that different domains of Gpr126 distinctly regulate compact wall integrity and trabecular identity. Maternal zygotic (MZ) gpr126stl47 early truncation mutants exhibit hypotrabeculation, whereby N-cadherin distributes randomly along apical/basal/lateral membranes of compact layer cardiomyocytes. In contrast, zygotic and MZ gpr126st49 mutants, expressing a N-terminal fragment lacking the GPS motif (NTFΔGPS), exhibit a multilayered ventricular wall containing polarized cardiomyocytes with normal N-cadherin localization and increased Notch activity. Notably, endocardially expressed gpr126 C-terminal fragment (CTF) reinstates trabeculation in gpr126st49 mutants. Collectively, our data reveal domain-specific roles of Gpr126 during trabeculation, whereby the NTF is required for maintaining cell-cell adhesion and compact wall integrity, whereas the CTF is essential to provide trabecular identity.

Microtubule organization plays a central role in cell differentiation, orchestrating essential processes such as cell polarization, mechanotransduction, organelle positioning and intracellular transport. A hallmark of many differentiated cells is the transition from a centrosomal to a non-centrosomal microtubule-organizing center (MTOC). Here, we demonstrate that both centrosomal and nuclear envelope (NE)-associated MTOCs coexist in osteoclasts. We show that the key players for NE-MTOC formation, the AKAP6 and nesprin-1 (SYNE1) isoforms AKAP6(3 and nesprin-1 alpha, previously considered muscle specific, are upregulated during osteoclast differentiation, suggesting a conserved role in NE-MTOC assembly across cell types. Targeted depletion of AKAP6 in RAW264.7-derived osteoclasts led to the displacement of the Golgi and MTOC-associated proteins PCM1, pericentrin and CDK5RAP2 from the NE, while their centrosomal localization remained intact. This selectively impaired microtubule nucleation from the NE without disrupting centrosomal microtubule activity, enabling a functional dissection of the two MTOCs. Loss of NEMTOC activity, through AKAP6 depletion, impaired podosome formation and significantly reduced bone resorption capacity, highlighting the distinct and essential role of NE-derived microtubules in osteoclast function.

Biofabrication represents a promising technique for creating tissues for regeneration or as models for drug testing. Collagen-based hydrogels are widely used as suitable matrix owing to their biocompatibility and tunable mechanical properties. However, one major challenge is that the encapsulated cells interact with the collagen matrix causing construct shrinkage. Here, we present a hydrogel with high shape fidelity, mimicking the major components of the extracellular matrix. We engineered a composite hydrogel comprising gallic acid (GA)-functionalized hyaluronic acid (HA), collagen I, and HA-coated multiwall carbon nanotubes (MWCNT). This hydrogel supports cell encapsulation, exhibits shear-thinning properties enhancing injectability and printability, and importantly significantly mitigates shrinkage when loaded with human fibroblasts compared to collagen I hydrogels (∼20 % vs. > 90 %). 3D-bioprinted rings utilizing human fibroblast-loaded inks maintain their shape over 7 days in culture. Furthermore, inclusion of HAGA into collagen I hydrogels increases mechanical stiffness, radical scavenging capability, and tissue adhesiveness. Notably, the here developed hydrogel is also suitable for human induced pluripotent stem cell-derived cardiomyocytes and allows printing of functional heart ventricles responsive to pharmacological treatment. Cardiomyocytes behave similar in the newly developed hydrogels compared to collagen I, based on survival, sarcomere appearance, and calcium handling. Collectively, we developed a novel material to overcome the challenge of post-fabrication matrix shrinkage conferring high shape fidelity.

Non-centrosomal microtubule-organizing centers (ncMTOCs) are important for the function of differentiated cells. Yet, ncMTOCs are poorly understood. Previously, several components of the nuclear envelope (NE)-MTOC have been identified. However, the temporal localization of MTOC proteins and Golgi to the NE and factors controlling the switch from a centrosomal MTOC to a ncMTOC remain elusive. Here, we utilized the in vitro differentiation of C2C12 mouse myoblasts as a model system to study NE-MTOC formation. We find based on longitudinal co-immunofluorescence staining analyses that MTOC proteins are recruited in a sequential and gradual manner to the NE. AKAP9 localizes with the Golgi to the NE after the recruitment of MTOC proteins. Moreover, siRNA-mediated depletion experiments revealed that Mbnl2 is required for proper NE-MTOC formation by regulating the expression levels of AKAP6β. Finally, Mbnl2 depletion affects Pcnt isoform expression. Taken together, our results shed light on how mammals post-transcriptionally control the switch from a centrosomal MTOC to an NE-MTOC and identify Mbnl2 as a novel modulator of ncMTOCs in skeletal muscle cells.

Cardiovascular diseases pose a significant global health challenge, driving ongoing efforts to develop effective treatments. Various biofabrication technologies utilizing numerous materials have been employed to design functional cardiac tissues. Choosing the right material is crucial to support cardiac cell growth, proliferation, tissue maturation and functionality. 3D printing enables the fabrication of structures that mimic the hierarchical organization of native cardiac tissue, further enhancing its function. Electrospinning produces nanofibrous scaffolds with a high surface area and porosity, mimicking the extracellular matrix and promoting the cell behaviors required for tissue formation. Although typically employed independently, combining these technologies can enable the fabrication of patches with properties closely resembling those of native cardiac tissues. Recent research focuses on the use of electroconductive materials, which enhance cell-to-cell communication and promote the maturation of cardiomyocytes, thereby preventing arrhythmic contractions and improving the functionality of engineered cardiac tissues. In this review, recent studies showcasing the applications of electroconductive biopolymer-based fibrous materials and hydrogels designed using 3D printing and/or electrospinning for cardiac tissue engineering are discussed. Furthermore, the review evaluates the synergistic effects of biopolymer-based materials and electrical components in 3D printed electroconductive hydrogels. It also discusses the challenges faced in fabricating these hydrogels and explores their future prospects for biomedical applications.

Background Organs and tissues need to be vascularized during development. Similarly, vascularization is required to engineer thick tissues. How vessels are formed during organogenesis is not fully understood, and vascularization of engineered tissues remains a significant challenge. Methods and Results Here, we show that the extracellular matrix protein nephronectin is required for vascularization during zebrafish development as well as adult fin regeneration and is sufficient to promote mammalian vessel formation and maturation. Nephronectin a morphants and mutants exhibit diminished axial vein sprouting and posterior intersegmental vessel growth. Notably, the angiogenesis‐associated integrins itgav and itgb3.1 are coexpressed with nephronectin a in the region of the caudal vein plexus and posterior somites; nephronectin binds to integrin alpha‐V/integrin beta‐3.1 (ITGAV/ITGB3.1), and itgav morphants phenocopy nephronectin a mutants. In addition, nephronectin a mutants showed decreased vessel maturation compared with wild‐type siblings during caudal fin regeneration in adult zebrafish. Moreover, nephronectin promotes mammalian endothelial cell migration and tube formation in 2D and 3‐dimensional in vitro tissue culture. Further, nephronectin enhances vascular endothelial growth factor‐induced periaortic vascular capillary interconnectivity, vessel diameter, and vessel stability. Conclusions Collectively, our results identify nephronectin as a proangiogenic factor during embryonic development, which can be used to improve the vascularization of engineered tissues.

Tissue engineering enables the production of tissues and organ-like structures as models for drug testing and mechanistical studies or functional replacements for injured tissues. Available cytocompatible materials are limited in number, suffer from insufficient mechanical properties, and cells interacting with them often cause construct shrinkage. As shape is important for function, identifying cytocompatible, shrink-resistant materials are a major aim. Here, it is shown that hydrogels made of interpenetrating networks of collagen-I and recombinant spider silk protein eADF4(C16)-RGD nanofibrils exhibit synergistic and tunable mechanical properties. Composite hydrogels allow cell adhesion and spreading and are resistant to shrinkage mediated by fibroblasts, C2C12 myoblasts, and human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. Myoblasts differentiate and fuse into myotubes, and hiPSC-cardiomyocytes can be cultured long-term, show spontaneous contractions, and remain drug responsive. Collectively, a novel composite material is developed to overcome the challenge of post-fabrication matrix shrinkage conferring high shape fidelity suitable for tissue engineering.

AIMS:Activation of Protease Activated Receptor 2 (PAR2) has been shown to be involved in regulation of injury-related processes including inflammation, fibrosis and hypertrophy. In this study we will investigate the role of PAR2 in cardiac injury in a mouse model of hypertension using continuous infusion with angiotensin II. METHODS:Hypertension was induced in 12 weeks old wildtype (wt, n = 8) and PAR2 deficient mice (n = 9) by continuous infusion with angiotensin II for 4 weeks using osmotic minipumps. At the end, hearts were collected for analysis of left ventricular hypertrophy (LVH), myocardial capillary supply, fibrosis and localization of PAR2 expression using histological, immunohistological and mRNA expression analysis techniques. In addition, rat cardiac fibroblasts were treated with angiotensin II and PAR2 was inhibited by a blocking antibody and the PAR2 inhibitor AZ3451. RESULTS:Cardiac PAR2 mRNA expression was downregulated by 40±20% in wt mice treated with AngII compared to untreated controls. Four weeks after AngII treatment, LVH was significantly increased in AngII-treated wt mice compared to similarly treated PAR2-deficient animals as determined by relative heart weight, left ventricular cross-sectional area, and analysis of ventricular lumen area determined on sections. Treatment of wt mice resulted in an approximately 3-fold increase in cardiac expression of FGF23, which was 50% lower in PAR2-deficient animals compared to wt animals and therefore no longer significantly different from expression levels in untreated control mice. In contrast, cardiac interstitial fibrosis was significantly higher in PAR2-deficient mice compared to similar treated wt controls, as assessed by Sirius Red staining (>3-fold) and collagen IV staining (>2-fold). Additional experiments with isolated cardiac fibroblasts showed induction of pro-fibrotic genes when treated with PAR2 inhibitors. CONCLUSION:In angiotensin II-induced cardiac injury, PAR2 deficiency has an ambivalent effect, enhancing fibrosis on the one hand, but reducing LVH on the other.

Therapy resistance and metastasis, the most fatal steps in cancer, are often triggered by a (partial) activation of the epithelial-mesenchymal transition (EMT) programme. A mesenchymal phenotype predisposes to ferroptosis, a cell death pathway exerted by an iron and oxygen-radical-mediated peroxidation of phospholipids containing polyunsaturated fatty acids. We here show that various forms of EMT activation, including TGF beta stimulation and acquired therapy resistance, increase ferroptosis susceptibility in cancer cells, which depends on the EMT transcription factor Zeb1. We demonstrate that Zeb1 increases the ratio of phospholipids containing pro-ferroptotic polyunsaturated fatty acids over cyto-protective monounsaturated fatty acids by modulating the differential expression of the underlying crucial enzymes stearoyl-Co-A desaturase 1 (SCD), fatty acid synthase (FASN), fatty acid desaturase 2 (FADS2), elongation of very long-chain fatty acid 5 (ELOVL5) and long-chain acyl-CoA synthetase 4 (ACSL4). Pharmacological inhibition of selected lipogenic enzymes (SCD and FADS2) allows the manipulation of ferroptosis sensitivity preferentially in high-Zeb1-expressing cancer cells. Our data are of potential translational relevance and suggest a combination of ferroptosis activators and SCD inhibitors for the treatment of aggressive cancers expressing high Zeb1. Schwab, Rao et al. report that Zeb1 mediates enhanced ferroptosis sensitivity in cancer cells after EMT activation, associated with altered expression of selected lipogenic enzymes and an subsequent increase in the PUFA:MUFA ratio.

Myocardial infarction (MI) causes cell death, disrupts electrical activity, triggers arrhythmia, and results in heart failure, whereby 50-60% of MI-associated deaths manifest as sudden cardiac deaths (SCD). The most effective therapy for SCD prevention is implantable cardioverter defibrillators (ICDs). However, ICDs contribute to adverse remodeling and disease progression and do not prevent arrhythmia. This work develops an injectable collagen-PEDOT:PSS (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate) hydrogel that protects infarcted hearts against ventricular tachycardia (VT) and can be combined with human induced pluripotent stem cell (hiPSC)-cardiomyocytes to promote partial cardiac remuscularization. PEDOT:PSS improves collagen gel formation, micromorphology, and conductivity. hiPSC-cardiomyocytes in collagen-PEDOT:PSS hydrogels exhibit near-adult sarcomeric length, improved contractility, enhanced calcium handling, and conduction velocity. RNA-sequencing data indicate enhanced maturation and improved cell-matrix interactions. Injecting collagen-PEDOT:PSS hydrogels in infarcted mouse hearts decreases VT to the levels of healthy hearts. Collectively, collagen-PEDOT:PSS hydrogels offer a versatile platform for treating cardiac injuries.

The complexity in structure and function of the nervous system, as well as its slow rate of regeneration, makes it more difficult to treat it compared to other tissues. Neural tissue engineering aims to create an appropriate environment for nerve cell proliferation and differentiation. Fibrous scaffolds with suitable morphology and topography and better mimicry of the extracellular matrix have been promising for the alignment and migration of neural cells. On this premise, to improve the properties of the scaffold, we combined montmorillonite (MMT) with chitosan (CS) polymer and created microfibers with variable diameters and varied concentrations of MMT using microfluidic technology and tested its suitability for the rat pheochromocytoma cell line (PC12). According to the findings, CS/MMT 0.1 % compared to CS/MMT 0 % microfibers showed a 201 MPa increase in Young's modulus, a 68 mS/m increase in conductivity, and a 1.4-fold increase in output voltage. Analysis of cell mitochondrial activity verified the non-toxicity, resulting in good cell morphology with orientation along the microfiber. Overall, the results of this project showed that with a low concentration of MMT, the properties of microfibers can be significantly improved and a suitable scaffold can be designed for neural tissue engineering.

The reuse of research software is central to research efficiency and academic exchange. The application of software enables researchers to reproduce, validate, and expand upon study findings. The analysis of open-source code aids in the comprehension, comparison, and integration of approaches. Often, however, no further use occurs because relevant software cannot be found or is incompatible with existing research processes. This results in repetitive software development, which impedes the advancement of individual researchers and entire research communities. In this article, the DataDesc (Data Description) framework is presented—an approach to describing data models of software interfaces with machine-actionable metadata. In addition to a specialized metadata schema, an exchange format and support tools for easy collection and the automated publishing of software documentation are introduced. This approach practically increases the FAIRness, i.e., findability, accessibility, interoperability, and reusability, of research software as well as effectively promotes its impact on research.

Uncovering the function of understudied G protein-coupled receptors (GPCRs) provides a wealth of untapped therapeutic potential. The poorly understood adhesion GPCR Gpr126 (Adgrg6) is widely expressed in developing kidneys. In adulthood, Gpr126 expression is enriched in parietal epithelial cells (PECs) and epithelial cells of the collecting duct and urothelium. Whether Gpr126 plays a role in kidney disease remains unclear. Here, we characterized Gpr126 expression in diseased kidneys in mice, rats, and humans. RT-PCR data show that Gpr126 expression is altered in kidney disease. A quantitative RNAscope® analysis utilizing cell type-specific markers revealed that Gpr126 expression upon tubular damage is mainly increased in cell types expressing Gpr126 under healthy conditions as well as in cells of the distal and proximal tubules. Upon glomerular damage, an increase was mainly detected in PECs. Notably, Gpr126 expression was upregulated in an ischemia/reperfusion model within hours, while upregulation in a glomerular damage model was only detected after weeks. An analysis of kidney microarray data from patients with lupus nephritis, IgA nephropathy, focal segmental glomerulosclerosis (FSGS), hypertension, and diabetes as well as single-cell RNA-seq data from kidneys of patients with acute kidney injury and chronic kidney disease indicates that GPR126 expression is also altered in human kidney disease. In patients with FSGS, an RNAscope® analysis showed that GPR126 mRNA is upregulated in PECs belonging to FSGS lesions and proximal tubules. Collectively, we provide detailed insights into Gpr126 expression in kidney disease, indicating that GPR126 is a potential therapeutic target.

The mechanical interplay between contractility and mechanosensing in striated muscles is of fundamental importance for tissue morphogenesis, load adaptation, and disease progression, but remains poorly understood. In this study, we investigate the dependence of contractile force generation of cardiac and skeletal muscle on environmental stiffness. Using in vitro engineered muscle micro-tissues that are attached to flexible elastic pillars, we vary the stiffness of the microenvironment over three orders of magnitude and study its effect on contractility. We find that the active contractile force upon electrical stimulation of both cardiac and skeletal micro-tissues increases with environmental stiffness according to a strong power-law relationship. To explore the role of adhesion-mediated mechanotransduction processes, we deplete the focal adhesion protein β-parvin in skeletal micro-tissues. This reduces the absolute contractile force but leaves the mechanoresponsiveness unaffected. Our findings highlight the influence of external stiffness on the adaptive behavior of muscle tissue and shed light on the complex mechanoadaptation processes in striated muscle.### Competing Interest StatementThe authors have declared no competing interest.

Abstract Long-acting passive immunization strategies are needed to protect immunosuppressed vulnerable groups from infectious diseases. To further explore this concept for COVID-19, we constructed Adeno-associated viral (AAV) vectors encoding the human variable regions of the SARS-CoV-2 neutralizing antibody, TRES6, fused to murine constant regions. An optimized vector construct was packaged in hepatotropic (AAV8) or myotropic (AAVMYO) AAV capsids and injected intravenously into syngeneic TRIANNI-mice. The highest TRES6 serum concentrations (511 µg/ml) were detected 24 weeks after injection of the myotropic vector particles and mean TRES6 serum concentrations remained above 100 µg/ml for at least one year. Anti-drug antibodies or TRES6-specific T cells were not detectable. After injection of the AAV8 particles, vector mRNA was detected in the liver, while the AAVMYO particles led to high vector mRNA levels in the heart and skeletal muscle. The analysis of the Fc-glycosylation pattern of the TRES6 serum antibodies revealed critical differences between the capsids that coincided with different binding activities to murine Fc-γ-receptors. Concomitantly, the vector-based immune prophylaxis led to protection against SARS-CoV-2 infection in K18-hACE2 mice. High and long-lasting expression levels, absence of anti-drug antibodies and favourable Fc-γ-receptor binding activities warrant further exploration of myotropic AAV vector-based delivery of antibodies and other biologicals.