Cell-cell Connections Research Articles

Pnn deficiency alters expressions of calcium handling proteins and impairs nuclear envelope structure in cardiomyocytes

Abstract Pnn is a multi-functional protein playing roles in the regulation of gene transcription, mRNA alternative splicing, as well as cell-cell connection. Previous studies suggested an association between Pnn genetic defects and arrhythmogenic right ventricular cardiomyopathy (ARVC) in human. In this study, we applied an inducible cardiomyocyte-specific Pnn depletion mouse model (Myh6-CreERT2, Pnnflox/flox) to determine the impact of loss of Pnn on cardiac structure and function in mice. Six weeks after inducing Pnn gene disruption, electrocardiographic abnormalities, ventricular dilatation, and reduced left ventricular ejection fraction (LVEF) were observed in mice with loss of Pnn in cardiomyocytes. Histopathological examinations showed that the proliferation of cardiac fibroblasts and expression of α-smooth muscle actin were increased with Pnn depletion, while the cell-cell connection between cardiomyocytes were impaired. In addition to impaired intercalated discs, loss of Pnn also altered the distribution and expression of nuclear envelope proteins and the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins, such as nuclear lamins, Emerin, and SUNs, in cardiomyocytes. Microarray analysis as well as subsequent RT-PCR and Western blots also indicated that loss of Pnn regulated myocardial expressions of genes responsible for calcium handling, such as RYR2, Atp2a2, Slc8a1, and Kcnj3. Furthermore, infiltrations of macrophages and neutrophiles were observed in the myocardium with Pnn depletion, while expression levels of oxidative stress-associated proteins were also significantly regulated by Pnn depletion. In conclusion, Pnn plays an essential role in the maintenance of intercalated disc integrity and nuclear envelope structure of cardiomyocytes, while cardiomyocyte-specific loss of Pnn impairs the calcium handling and leads to arrhythmogenic dilated cardiomyopathy in mice.Cardiac MRI of mice with Pnn depletionMyocardial expression of SUN2 and Emerin

Virulence-linked adhesin drives mutualist colonization of the bee gut via biofilm formation.

Bacterial biofilms are stable multicellular structures that can enable long term host association. Yet, the role of biofilms in supporting gut mutualism is still not fully understood. Here, we investigate Snodgrassella alvi , a beneficial bacterial symbiont of honey bees, and find that biofilm formation is required for its colonization of the bee gut. We constructed fifteen S. alvi mutants containing knockouts of genes known to promote colonization with putative roles in biofilm formation. Genes required for colonization included staA and staB , encoding trimeric autotransporter adhesins (TAAs) and mltA , encoding a lytic transglycosylase. Intriguingly, TAAs are considered virulence factors in pathogens but support mutualism by the symbiont S. alvi. In vitro , biofilm formation was reduced in Δ staB cells and abolished in the other two mutants. Loss of staA also reduced auto-aggregation and cell-cell connections. Based on structural predictions, StaA/B are massive (>300 nm) TAAs with many repeats in their stalk regions. Further, we find that StaA/B are conserved across Snodgrassella species, suggesting that StaA/B-dependent colonization is characteristic of this symbiont lineage. Finally, staA deletion increases sensitivity to bactericidal antimicrobials, suggesting that the biofilm indirectly buffers against antibiotic stress. In all, the inability of two biofilm-deficient strains (Δ staA and Δ mltA ) to effectively mono-colonize bees indicates that S. alvi biofilm formation is required for colonization of the bee gut. We envision the bee gut system as a genetically tractable model for studying the physical basis of biofilm-mutualist-gut interactions.

Myotube Guidance: Shaping up the Musculoskeletal System.

Myofibers are highly specialized contractile cells of skeletal muscles, and dysregulation of myofiber morphogenesis is emerging as a contributing cause of myopathies and structural birth defects. Myotubes are the myofiber precursors and undergo a dramatic morphological transition into long bipolar myofibers that are attached to tendons on two ends. Similar to axon growth cones, myotube leading edges navigate toward target cells and form cell-cell connections. The process of myotube guidance connects myotubes with the correct tendons, orients myofiber morphology with the overall body plan, and generates a functional musculoskeletal system. Navigational signaling, addition of mass and volume, and identification of target cells are common events in myotube guidance and axon guidance, but surprisingly, the mechanisms regulating these events are not completely overlapping in myotubes and axons. This review summarizes the strategies that have evolved to direct myotube leading edges to predetermined tendon cells and highlights key differences between myotube guidance and axon guidance. The association of myotube guidance pathways with developmental disorders is also discussed.

Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8+ T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

Modeling doxorubicin-induced cardiotoxicity with human myocardial tissue slices: insights on genetic susceptibility and cardioprotection

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): PSIDER and HARVEY (ZonMW) Introduction Cardiotoxicity poses a significant challenge in drug development, often leading to the withdrawal of approved drugs. To address this issue, there is a critical need for improved models to predict cardiac adverse effects. In line with the imperative for animal-free preclinical testing, this study aimed to develop a novel model using human myocardial tissue slices to get a deeper understanding and potential prevention of doxorubicin-Induced cardiotoxicity. Methods Human myocardial tissue slices of 300 μm thick were derived from end-stage heart failure patients explanted hearts, including those harbouring likely or pathogenic cardiovascular mutations (class 4 and 5) (N=3) and non-genetic cardiovascular etiology (N=3). Myocardial tissue slices were cultured under physiological mechanical and electrical conditions. Slices were exposed to doxorubicin (1 µM) over a 10-day culture period, and their responses were compared to DMSO-vehicle controls, by recording contractile force and Ca2+ transients. Additionally, a subset of slices was treated with a cardioprotective agent, dexrazoxane (100 µM), 1 day prior to and during doxorubicin exposure to assess potential cardioprotection. Results Doxorubicin-exposed slices with a pathogenic mutation exhibited reduced mechanical force, increased threshold potential, and impaired ability to follow pacing at higher frequencies. Additionally, aberrations in calcium handling, manifested as arrhythmia-like disconnection of muscle fibers, were notably observed in these slices. Instead, slices from patients without pathogenic mutations showed minimal cardiotoxicity when exposed to doxorubicin. Encouragingly, pretreatment with dexrazoxane demonstrated a protective effect against doxorubicin-induced cardiotoxicity in the genetically susceptible group. Our functional findings corroborated our structural data, highlighting the breakdown of sarcomere structure and cell-cell connections and increased DNA damage, particularly in patients harbouring a genetic cardiovascular mutation. Conclusion In summary, human myocardial tissue slices faithfully recapitulate doxorubicin-induced cardiotoxicity. Importantly, our data suggests the utility of employing genetic testing for known cardiovascular mutations as a valuable step before initiating doxorubicin treatment. Additionally, we demonstrate for the first time the in vitro cardioprotective potential of dexrazoxane in a human tissue model. These findings pave the way for using ex vivo myocardial tissue slices to evaluate cardiotoxicity and their value as a powerful tool for evaluating the efficacy and delivery approaches.

Human induced pluripotent cardiomyocytes as a model for cardiomyopathy with a Myozap gene mutation

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): 1- Central Department of Missions, Egyptian Ministry of Higher education and Scientific Research. 2- small fund from Tampere University. The contractility of the heart is based on cardiomyocytes (CM). These cardiomyocytes are connected through a complex network of proteins forming the intercalated disc (ID) which is crucial to ensure mechanical and electrochemical coupling. Dilated cardiomyopathy (DCM) is a disease of the myocardium that can lead to heart failure and life-threatening arrhythmias. Up to 30% of DCM cases are caused by mutations in genes encoding proteins of the sarcomere (the basic contractile unit) or desmosome (a cell-to-cell adhesion structure) and the structural cytoskeleton. Myozap (Myocardium-Enriched Zonula Occludens-1-Associated Protein) is a distinctive cardiac-enriched ID protein. It is crucial for biomechanical stress adaption in CMs due to its outstanding role in the Rho/SRF (Serum Response Factor) signalling pathway. In this study, we have identified two Finnish families with DCM and carrying a novel variant in myozap. Our aim is to characterize myozap gene mutation-both heterozygous and homozygous truncating variants-as well as its implication in DCM. We have established patient-specific human induced pluripotent stem-cell lines and derived cardiomyocyte (hiPSC-CM) to study the disease at the cellular level. Myozap expression, compared to the control cell line, was evaluated by immunohistochemistry. Electrophysiological methods, including microelectrode array (MEA) and patch clamping, were implemented to measure various parameters with emphasis on those ones related to cell-cell connection e.g., the conduction velocity. Moreover, metabolic stress indicators, including oxygen consumption rate (OCR), were estimated by Seahorse. Additionally, qPCR and ELISA were performed to measure the colocalised and closely related genes to ID and protein level of troponin, respectively. Immunohistochemistry revealed downregulation and absence of myozap expression in heterozygous and homozygous cell lines, respectively. Compared to the control one, differences in BPM, FPD were observed. Metabolic analysis of the myozap mutant elucidated mitochondrial impairment compared to the control. Some SRF and sarcomeric target genes, including MYH7, MYH6 and Myl-2 were downregulated. On the contrary, apoptotic genes (e.g., caspase 3) were upregulated. Tight junction protein (TJP) was upregulated, and this is very importance since it is closely related to the myozap in the ID proteins network. Moreover, troponin protein level, measured by ELISA, showed upregulation in mutant myozap. In conclusion, our study provides a new insight into understanding myozap’s significance in cardiac biology and in cardiomyopathy pathophysiology.

Abstract 111: COL8A1 Modulates Endothelial Phenotype During Inflammatory Endothelial-to-Mesenchymal Transition

Background: Endothelial-to-mesenchymal transition (EndMT), the dynamic process whereby endothelial cells (ECs) gradually lose endothelial identity while acquiring mesenchymal characteristics, plays significant roles in inflammatory cardiovascular pathologies such as atherosclerosis. The mechanisms of EC phenotypic modulation during EndMT remain widely unexplored. Understanding the factors driving the endothelial changes during EndMT may allow for the development of novel therapeutic targets to modulate this transition. Methods/Results: Inflammatory EndMT was induced in human aortic ECs (HAECs) by treatment with tumor necrosis factor-α (TNF-α), which showed a partial EndMT as evidenced by morphological changes, downregulation of endothelial markers, decreased angiogenic capacity, upregulation of mesenchymal markers, and increased cellular invasion. Results indicated that TNF-α treatment resulted in the loss of endothelial markers after short-term (24h) treatment. Interestingly, the expression of COL8A1 , which encodes for one of the two alpha chains of the non-fibrillar type VIII collagen, was selectively downregulated among collagens by TNF-a treatment at early time-points. In HAECs, overexpression of COL8A1 , as well as exposure to exogenous recombinant COL8A1, led to increased endothelial marker expression. COL8A1 knockdown potentiates TNF-α induced endothelial phenotype loss (reduced endothelial markers and decreased angiogenic capacity), while COL8A1 overexpression partially rescues TNF-α induced endothelial phenotype loss. RNA sequencing revealed that COL8A1 knockdown resulted in the decreased expression of genes related to endothelial function (e.g., focal adhesion, cell-cell connections). Gene Set Enrichment Analysis (GSEA) showed significant enrichment of the TNF-α/NFKB signaling geneset and cardiac epithelial-mesenchymal transition geneset following COL8A1 knockdown. Conclusion: Our findings suggest that COL8A1 mediates the EC transition during inflammatory EndMT by serving to maintain normal endothelial function, whereby TNF-α decreases COL8A1 expression to facilitate the loss of the EC phenotype.

Mesenchymal Stem Cells Increase Drug Tolerance of A431 Cells Only in 3D Spheroids, Not in 2D Co-Cultures.

Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell-cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC-cancer cell interactions and could help design new, better treatment options for tumors.

Epidermal growth factor receptor inhibition leads to cellular phenotype correction of DSP-mutated keratinocytes.

Desmoplakin (DSP) is a desmosomal component expressed in skin and heart, essential for desmosome stability and intermediate filament connection. Pathogenic variants in the DSP gene encoding DSP, lead to heterogeneous skin, adnexa and heart-related phenotypes, including skin fragility, woolly hair (WH), palmoplantar keratoderma (PPK) and arrhythmogenic/dilated cardiomyopathy (ACM/DCM). The ambiguity of computer-based prediction analysis of pathogenicity and effect of DSP variants, indicates a necessity for functional analysis. Here, we report a heterozygous DSP variant that was not previously described, NM_004415.4:c.3337C>T (NM_004415.4(NP_004406.2):p.(Arg1113*)) in a patient with PPK, WH and ACM. RNA and protein analysis revealed ~50% reduction of DSP mRNA and protein expression. Patient's keratinocytes showed fragile cell-cell connections and perinuclear retracted intermediate filaments. Epidermal growth factor receptor (EGFR) is a transmembrane protein expressed in the basal epidermal layer involved in proliferation and differentiation, processes that are disrupted in the development of PPK, and in the regulation of the desmosome. In skin of the abovementioned patient, evident EGFR upregulation was observed. EGFR inhibition in patient's keratinocytes strongly increased DSP expression at the plasma membrane, improved intermediate filament connection with the membrane edges and reduced the cell-cell fragility. This cell phenotypic recovery was due to a translocation of DSP to the plasma membrane together with an increased number of desmosomes. These results indicate a therapeutic potential of EGFR inhibitors for disorders caused by DSP haploinsufficiency.

Validation and identification of anoikis-related lncRNA signatures for improving prognosis in clear cell renal cell carcinoma.

Clear cell carcinoma (ccRCC) usually has a high metastasis rate and high mortality rate. To enable precise risk stratification, there is a need for novel biomarkers. As one form of apoptosis, anoikis results from the disruption of cell-cell connection or cell-ECM attachment. However, the impact of anoikis-related lncRNAs on ccRCC has not yet received adequate attention. The study utilized univariate Cox regression analysis in order to identify the overall survival (OS) associated anoikis-related lncRNAs (ARLs), followed by the LASSO algorithm for selection. On this basis, a risk model was subsequently established using five anoikis-related lncRNAs. To dig the inner molecular mechanism, KEGG, GO, and GSVA analyses were conducted. Additionally, the immune infiltration landscape was estimated using the ESTIMATE, CIBERSORT, and ssGSEA algorithms. The study constructed a novel risk model based on five ARLs (AC092611.2, AC027601.2, AC103809.1, AL133215.2, and AL162586.1). Patients categorized as low-risk exhibited significantly better OS. Notably, the study observed marked different immune infiltration landscapes and drug sensitivity by risk stratification. Additionally, the study preliminarily explored potential signal pathways associated with risk stratification. The study exhibited the crucial role of ARLs in the carcinogenesis of ccRCC, potentially through differential immune infiltration. Furthermore, the established risk model could serve as a valuable stratification factor for predicting OS prognosis.

Methylation Status of CDH1 Gene in Gastric Biopsy Specimens and Its Association with H. Pylori Infection.

Helicobacter pylori (H. pylori) have been accepted as having an etiologic role in gastro-duodenal diseases as chronic gastritis, peptic ulcer, and gastric carcinoma. Methylation of CGI has been correlated with the tumorigenic process since it can inactivate tumor suppressor genes. CDH1 is a tumor suppressor gene that encodes the E-cadherin protein, which is preserving cell-cell connections. Early stages of gastric carcinogenesis may be affected by the promoter methylation-mediated inactivation of this gene. This study aimed to investigate the methylation status of CDH1 using Methylation-Specific PCR (MSP) technique in clinical suspected patients with H. pylori infection who undergoing upper gastrointestinal endoscopy and correlated it with H. pylori detection by glmM PCR test. Fifty gastric mucosal biopsies were selected from one hundred and five samples included in this study. The detection of H. pylori was performed with the PCR primers specific to glmM gene. Bisulfite modification was done and the methylation status of the CDH1 gene was detected using MSP reaction. H. pylori was detected in 36% (18/50) of study population using glmM gene PCR test, 89% (16/18) of H. pylori positive cases were CDH1 methylated positive (chi-square, p-value=0.002). CDH1 methylation can be present in cancerous and noncancerous gastric mucosa, where 60% (18/30) of CDH1 methylation positive gastric mucosa showed gastritis as an endoscopy finding and gastric cancer in 6% (2/30). There was a significant correlation between and CDH1 methylation positive results and age group (P-value = 0.02). There was no significant correlation between CDH1 methylation positive results and participants gender (p-value=0.431) and clinical symptoms (all P-value > 0.05). This work suggested strong significance association between H. pylori infection and CDH1 methylation.

Agent-based approach for elucidating the release from collective arrest of cell motion in corneal epithelial cell sheet

Changes in cell fluidity have been observed in various cellular tissues and are strongly linked to biological phenomena such as self-organization. Recent studies suggested variety of mechanisms and factors, which are still being investigated. This study aimed to investigate changes in cell fluidity in multi-layered cell sheets, by exploring the collective arrest of cell motion and its release in cultures of corneal epithelial cells. We constructed mathematical models to simulate the behaviors of individual cells, including cell differentiation and time-dependent changes in cell-cell connections, which are defined by stochastic or kinetic rules. Changes in cell fluidity and cell sheet structures were expressed by simulating autonomous cell behaviors and interactions in tissues using an agent-based model. A single-cell level spatiotemporal analysis of cell state transition between migratable and non-migratable states revealed that the release from collective arrest of cell motion was initially triggered by a decreased ability to form cell-cell connections in the suprabasal layers, and was propagated by chain migration. Notably, the disruption of cell-cell connections and stratification occurred in the region of migratable state cells. Hence, a modeling approach that considers time-dependent changes in cell properties and behavior, and spatiotemporal analysis at the single-cell level can effectively delineate emergent phenomena arising from the complex interplay of cells.

Epigenetic regulator RNF20 underlies temporal hierarchy of gene expression to regulate postnatal cardiomyocyte polarization

Differentiated cardiomyocytes (CMs) must undergo diverse morphological and functional changes during postnatal development. However, the mechanisms underlying initiation and coordination of these changes remain unclear. Here, we delineate an integrated, time-ordered transcriptional network that begins with expression of genes for cell-cell connections and leads to a sequence of structural, cell-cycle, functional, and metabolic transitions in mouse postnatal hearts. Depletion of histone H2B ubiquitin ligase RNF20 disrupts this gene network and impairs CM polarization. Subsequently, assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis confirmed that RNF20 contributes to chromatin accessibility in this context. As such, RNF20 is likely to facilitate binding of transcription factors at the promoters of genes involved in cell-cell connections and actin organization, which are crucial for CM polarization and functional integration. These results suggest that CM polarization is one of the earliest events during postnatal heart development and provide insights into how RNF20 regulates CM polarity and the postnatal gene program.

Improvements in Maturity and Stability of 3D iPSC-Derived Hepatocyte-like Cell Cultures.

Induced pluripotent stem cell (iPSC) technology enables differentiation of human hepatocytes or hepatocyte-like cells (iPSC-HLCs). Advances in 3D culturing platforms enable the development of more in vivo-like liver models that recapitulate the complex liver architecture and functionality better than traditional 2D monocultures. Moreover, within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation and maintenance of hepatocyte metabolic function. Thus, models combining 3D culture and co-culturing of various cell types potentially create more functional in vitro liver models than 2D monocultures. Here, we report the establishment of 3D cultures of iPSC-HLCs alone and in co-culture with human umbilical vein endothelial cells (HUVECs) and adipose tissue-derived mesenchymal stem/stromal cells (hASCs). The 3D cultures were performed as spheroids or on microfluidic chips utilizing various biomaterials. Our results show that both 3D spheroid and on-chip culture enhance the expression of mature liver marker genes and proteins compared to 2D. Among the spheroid models, we saw the best functionality in iPSC-HLC monoculture spheroids. On the contrary, in the chip system, the multilineage model outperformed the monoculture chip model. Additionally, the optical projection tomography (OPT) and electrical impedance tomography (EIT) system revealed changes in spheroid size and electrical conductivity during spheroid culture, suggesting changes in cell-cell connections. Altogether, the present study demonstrates that iPSC-HLCs can successfully be cultured in 3D as spheroids and on microfluidic chips, and co-culturing iPSC-HLCs with NPCs enhances their functionality. These 3D in vitro liver systems are promising human-derived platforms usable in various liver-related studies, specifically when using patient-specific iPSCs.

Spatial proteomics of immune microenvironment in nonalcoholic steatohepatitis-associated hepatocellular carcinoma.

NASH-HCC is inherently resistant to immune checkpoint blockade, but its tumor immune microenvironment is largely unknown. We applied the imaging mass cytometry to construct a spatially resolved single-cell atlas from the formalin-fixed and paraffin-embedded tissue sections from patients with NASH-HCC, virus-HCC (HBV-HCC and HCV-HCC), and healthy donors. Based on 35 biomarkers, over 750,000 individual cells were categorized into 13 distinct cell types, together with the expression of key immune functional markers. Higher infiltration of T cells, myeloid-derived suppressor cell (MDSCs), and tumor-associated macrophages (TAMs) in HCC compared to controls. The distribution of immune cells in NASH-HCC is spatially heterogeneous, enriched at adjacent normal tissues and declined toward tumors. Cell-cell connections analysis revealed the interplay of MDSCs and TAMs with CD8 + T cells in NASH-HCC. In particular, exhausted programmed cell death 1 (PD-1 + )CD8 + T cells connected with programmed cell death-ligand 1 (PD-L1 + )/inducible T cell costimulator (ICOS + ) MDSCs and TAMs in NASH-HCC, but not in viral HCC. In contrast, CD4 + /CD8 + T cells with granzyme B positivity were reduced in NASH-HCC. Tumor cells expressed low PD-L1 and showed few connections with immune cells. Our work provides the first detailed spatial map of single-cell phenotypes and multicellular connections in NASH-HCC. We demonstrate that interactions between MDSCs and TAMs with effector T cells underlie immunosuppression in NASH-HCC and are an actionable target.

Micromorphological observation of HLE cells under knockdown of Fascin using LV-SEM.

Liver cancer is one of the most prevalent cancers in Japan with hepatocellular carcinoma (HCC) as the major histological subtype. Successful novel treatments for HCC have been reported; however, recurrences or metastasis may occur, which results in poor prognoses and high mortality of HCC patients. Fascin, an actin-bundling protein, regulates cell adhesion, migration, and invasion. Its overexpression positively correlates with poor prognosis of malignant tumors, and Fascin is considered as one of the tumor biomarkers and therapeutic target proteins. In this study, we attempted to reveal the relationship between Fascin and HCC using HLE, one of the human HCC cell lines. We performed the study with classical immunocytochemistry and recently developed techniques, such as wound-healing assay, spheroid cultivation, and low-vacuum scanning electron microscopy (LV-SEM). Non-Fascin-knockdown (FKD) cell spheroid had a regular spherical appearance with tight cell-cell connections, while FKD cell spheroid had an irregular shape with loose cell-cell connections. Cells of non-FKD spheroid presented fibrous protrusions on the cell surface, contrarily, cells of FKD spheroids showed bulbous-shaped protrusions. Morphological observation of FKD and non-FKD HLE spheroids were performed using LV-SEM. Our study may help to reveal the roles of Fascin in the process of HCC formation and its malignancy.

A novel classifier combining G protein-coupled receptors and the tumor microenvironment is associated with survival status in glioblastoma.

Background: Numerous studies have highlighted the crucial role of G protein-coupled receptors (GPCRs) in tumor microenvironment (TME) remodeling and their correlation with tumor progression. However, the association between GPCRs and the TME in glioblastoma (GBM) remains largely unexplored. Methods: In this study, we investigated the expression profile of GPCRs in GBM using integrated data from single-cell RNA sequencing and bulk sequencing. Surgical samples obtained from meningioma and GBM patients underwent single-cell RNA sequencing to examine GPCR levels and cell-cell interactions. Tumor microenvironment (TME) score is calculated by the infiltrated immune cells with CIBERSORT. Results: Our findings revealed a predominantly increased expression of GPCRs in GBM, and demonstrated that the classification of GPCRs and TME is an independent risk factor in GBM. Patients with high GPCR expression in the tumor tissue and low TME score exhibited the worst outcomes, suggesting a potentially aggressive tumor phenotype. On the other hand, patients with low GPCR expression in the tumor tissue and high TME score showed significantly better outcomes, indicating a potentially more favorable tumor microenvironment. Furthermore, the study found that T cells with high GPCR levels displayed extensive cell-cell connections with other tumor and immune cells in the single cell RNA analysis, indicating their potential involvement in immune escape. Conclusion: In conclusion, GPCRs in combination with TME classification can serve as prognostic markers for GBM. GPCRs play an essential role in tumor progression and the TME in GBM.

The Targeted Deletion of Genes Responsible for Expression of the Mth60 Fimbriae Leads to Loss of Cell-Cell Connections in Methanothermobacter thermautotrophicus ΔH.

This study is a continuation by the Environmental Biotechnology Group of the University of Tübingen in memoriam to Reinhard Wirth, who initiated the work on Mth60 fimbriae at the University of Regensburg. Growth in biofilms or biofilm-like structures is the prevailing lifestyle for most microbes in nature. The first crucial step to initiate biofilms is the adherence of microbes to biotic and abiotic surfaces. Therefore, it is crucial to elucidate the initial step of biofilm formation, which is generally established through cell-surface structures (i.e., cell appendages), such as fimbriae or pili, that adhere to biotic and abiotic surfaces. The Mth60 fimbriae of Methanothermobacter thermautotrophicus ΔH are one of only a few known archaeal cell appendages that do not assemble via the type IV pili assembly mechanism. Here, we report the constitutive expression of Mth60 fimbria-encoding genes from a shuttle-vector construct and the deletion of the Mth60 fimbria-encoding genes from the genomic DNA of M. thermautotrophicus ΔH. For this, we expanded our system for genetic modification of M. thermautotrophicus ΔH using an allelic-exchange method. While overexpression of the respective genes increased the number of Mth60 fimbriae, deletion of the Mth60 fimbria-encoding genes led to a loss of Mth60 fimbriae in planktonic cells of M. thermautotrophicus ΔH compared to the wild-type strain. This, either increased or decreased, number of Mth60 fimbriae correlated with a significant increase or decrease of biotic cell-cell connections in the respective M. thermautotrophicus ΔH strains compared to the wild-type strain. IMPORTANCE Methanothermobacter spp. have been studied for the biochemistry of hydrogenotrophic methanogenesis for many years. However, a detailed investigation of certain aspects, such as regulatory processes, was impossible due to the lack of genetic tools. Here, we amend our genetic toolbox for M. thermautotrophicus ΔH with an allelic exchange method. We report the deletion of genes that encode the Mth60 fimbriae. Our findings provide the first genetic evidence of whether the expression of these genes underlies regulation and reveal a role of the Mth60 fimbriae in the formation of cell-cell connections of M. thermautotrophicus ΔH.

Adherens junction: the ensemble of specialized cadherin clusters.

The cell-cell connections in adherens junctions (AJs) are mediated by transmembrane receptors, type I cadherins (referred to here as cadherins). These cadherin-based connections (or trans bonds) are weak. To upregulate their strength, cadherins exploit avidity, the increased affinity of binding between cadherin clusters compared with isolated monomers. Formation of such clusters is a unique molecular process that is driven by a synergy of direct and indirect cis interactions between cadherins located at the same cell. In addition to their role in adhesion, cadherin clusters provide structural scaffolds for cytosolic proteins, which implicate cadherin into different cellular activities and signaling pathways. The cluster lifetime, which depends on the actin cytoskeleton, and on the mechanical forces it generates, determines the strength of AJs and their plasticity. The key aspects of cadherin adhesion, therefore, cannot be understood at the level of isolated cadherin molecules, but should be discussed in the context of cadherin clusters.

Nuclear SUN1 stabilizes endothelial cell junctions via microtubules to regulate blood vessel formation.

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here, we show that the nuclear-localized linker of the nucleoskeleton and cytoskeleton (LINC) complex protein SUN1 regulates vascular sprouting and endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted junction function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts, and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and junction integrity.