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Autophagy Apr 2024Activated transmembrane receptors continue to signal following endocytosis and are only silenced upon ESCRT-mediated internalization of the receptors into intralumenal...
Activated transmembrane receptors continue to signal following endocytosis and are only silenced upon ESCRT-mediated internalization of the receptors into intralumenal vesicles (ILVs) of the endosomes. Accordingly, endosomes with dysfunctional receptor internalization into ILVs can cause sustained receptor signaling which has been implicated in cancer progression. Here, we describe a surveillance mechanism that allows cells to detect and clear physically intact endosomes with aberrant receptor accumulation and elevated signaling. Proximity biotinylation and proteomics analyses of ESCRT-0 defective endosomes revealed a strong enrichment of the ubiquitin-binding macroautophagy/autophagy receptors SQSTM1 and NBR1, a phenotype that was confirmed in cell culture and fly tissue. Live cell microscopy demonstrated that loss of the ESCRT-0 subunit HGS/HRS or the ESCRT-I subunit VPS37 led to high levels of ubiquitinated and phosphorylated receptors on endosomes. This was accompanied by dynamic recruitment of NBR1 and SQSTM1 as well as proteins involved in autophagy initiation and autophagosome biogenesis. Light microscopy and electron tomography revealed that endosomes with intact limiting membrane, but aberrant receptor downregulation were engulfed by phagophores. Inhibition of autophagy caused increased intra- and intercellular signaling and directed cell migration. We conclude that dysfunctional endosomes are surveyed and cleared by an autophagic process, simaphagy, which serves as a failsafe mechanism in signal termination. AKT: AKT serine/threonine kinase; APEX2: apurinic/apyrimidinic endodoexyribonuclease 2; ctrl: control; EEA1: early endosome antigen 1; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HGS/HRS: hepatocyte growth factor-regulated tyrosine kinase substrate; IF: immunofluorescence; ILV: intralumenal vesicle; KO: knockout; LIR: LC3-interacting region; LLOMe: L-leucyl-L-leucine methyl ester (hydrochloride); MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; NBR1: NBR1 autophagy cargo receptor; PAG10: Protein A-conjugated 10-nm gold; RB1CC1/FIP200: RB1 inducible coiled-coil 1; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TUB: Tubulin; UBA: ubiquitin-associated; ULK1: unc-51 like autophagy activating kinase 1; VCL: Vinculin; VPS37: VPS37 subunit of ESCRT-I; WB: western blot; WT: wild-type.
Topics: Endosomes; Humans; Endosomal Sorting Complexes Required for Transport; Autophagy; Signal Transduction; Animals; Intracellular Signaling Peptides and Proteins; Sequestosome-1 Protein; Autophagosomes; Endocytosis; HeLa Cells; Cell Movement
PubMed: 37840274
DOI: 10.1080/15548627.2023.2267958 -
PLoS Computational Biology Oct 2023Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which...
Cells interact with the extracellular matrix (ECM) via cell-ECM adhesions. These physical interactions are transduced into biochemical signals inside the cell which influence cell behaviour. Although cell-ECM interactions have been studied extensively, it is not completely understood how immature (nascent) adhesions develop into mature (focal) adhesions and how mechanical forces influence this process. Given the small size, dynamic nature and short lifetimes of nascent adhesions, studying them using conventional microscopic and experimental techniques is challenging. Computational modelling provides a valuable resource for simulating and exploring various "what if?" scenarios in silico and identifying key molecular components and mechanisms for further investigation. Here, we present a simplified mechano-chemical model based on ordinary differential equations with three major proteins involved in adhesions: integrins, talin and vinculin. Additionally, we incorporate a hypothetical signal molecule that influences adhesion (dis)assembly rates. We find that assembly and disassembly rates need to vary dynamically to limit maturation of nascent adhesions. The model predicts biphasic variation of actin retrograde velocity and maturation fraction with substrate stiffness, with maturation fractions between 18-35%, optimal stiffness of ∼1 pN/nm, and a mechanosensitive range of 1-100 pN/nm, all corresponding to key experimental findings. Sensitivity analyses show robustness of outcomes to small changes in parameter values, allowing model tuning to reflect specific cell types and signaling cascades. The model proposes that signal-dependent disassembly rate variations play an underappreciated role in maturation fraction regulation, which should be investigated further. We also provide predictions on the changes in traction force generation under increased/decreased vinculin concentrations, complementing previous vinculin overexpression/knockout experiments in different cell types. In summary, this work proposes a model framework to robustly simulate the mechanochemical processes underlying adhesion maturation and maintenance, thereby enhancing our fundamental knowledge of cell-ECM interactions.
Topics: Focal Adhesions; Vinculin; Actins; Integrins; Extracellular Matrix; Cell Adhesion; Talin
PubMed: 37801464
DOI: 10.1371/journal.pcbi.1011500 -
BMC Genomics Oct 2023Phenotypic plasticity is a crucial adaptive mechanism that enables organisms to modify their traits in response to changes in their environment. Predator-induced...
Comparative transcriptome analysis of two Daphnia galeata genotypes displaying contrasting phenotypic variation induced by fish kairomones in the same environment of the Han River, Korea.
BACKGROUND
Phenotypic plasticity is a crucial adaptive mechanism that enables organisms to modify their traits in response to changes in their environment. Predator-induced defenses are an example of phenotypic plasticity observed across a wide range of organisms, from single-celled organisms to vertebrates. In addition to morphology and behavior, these responses also affect life-history traits. The crustacean Daphnia galeata is a suitable model organism for studying predator-induced defenses, as it exhibits life-history traits changes under predation risk. To get a better overview of their phenotypic plasticity under predation stress, we conducted RNA sequencing on the transcriptomes of two Korean Daphnia galeata genotypes, KE1, and KB11, collected in the same environment.
RESULTS
When exposed to fish kairomones, the two genotypes exhibited phenotypic variations related to reproduction and growth, with opposite patterns in growth-related phenotypic variation. From both genotypes, a total of 135,611 unigenes were analyzed, of which 194 differentially expressed transcripts (DETs) were shared among the two genotypes under predation stress, which showed consistent, or inconsistent expression patterns in both genotypes. Prominent DETs were related to digestion and reproduction and consistently up-regulated in both genotypes, thus associated with changes in life-history traits. Among the inconsistent DETs, transcripts encode vinculin (VINC) and protein obstructor-E (OBST-E), which are associated with growth; these may explain the differences in life-history traits between the two genotypes. In addition, genotype-specific DETs could explain the variation in growth-related life-history traits between genotypes, and could be associated with the increased body length of genotype KE1.
CONCLUSIONS
The current study allows for a better understanding of the adaptation mechanisms related to reproduction and growth of two Korean D. galeata genotypes induced by predation stress. However, further research is necessary to better understand the specific mechanisms by which the uncovered DETs are related with the observed phenotypic variation in each genotype. In the future, we aim to unravel the precise adaptive mechanisms underlying predator-induced responses.
Topics: Animals; Transcriptome; Daphnia; Pheromones; Rivers; Genotype; Fishes; Predatory Behavior; Biological Variation, Population; Gene Expression Profiling; Republic of Korea
PubMed: 37784038
DOI: 10.1186/s12864-023-09701-x -
ACS Sensors Oct 2023Integrin tensions are critical for cell mechanotransduction. By converting force to fluorescence, molecular tension sensors image integrin tensions in live cells with a...
Integrin tensions are critical for cell mechanotransduction. By converting force to fluorescence, molecular tension sensors image integrin tensions in live cells with a high resolution. However, the fluorescence signal intensity results collectively from integrin tension magnitude, tension dwell time, integrin density, sensor accessibility, and so forth, making it highly challenging to specifically monitor the molecular force level of integrin tensions. Here, a ratiometric tension sensor (RTS) was developed to exclusively monitor the integrin tension magnitude. The RTS consists of two tension-sensing units that are coupled in series and always subject to the same integrin tension. These two units are activated by tension to fluoresce in separate spectra and with different activation rates. The ratio of their activation probabilities, reported by fluorescence ratiometric measurement, is solely determined by the local integrin tension magnitude. RTS responded sensitively to the variation of integrin tension magnitude in platelets and focal adhesions due to different cell plating times, actomyosin inhibition, or vinculin knockout. At last, RTS confirmed that integrin tension magnitude in platelets and focal adhesions decreases monotonically with the substrate rigidity, verifying the rigidity dependence of integrin tensions in live cells and suggesting that integrin tension magnitude could be a key biomechanical factor in cell rigidity sensing.
Topics: Integrins; Mechanotransduction, Cellular; Focal Adhesions; Mechanical Phenomena; Actin Cytoskeleton
PubMed: 37738233
DOI: 10.1021/acssensors.3c00606 -
Cancers Sep 2023Breast cancer (BC) is the most common malignancy among women worldwide. Around 15-25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is...
Breast cancer (BC) is the most common malignancy among women worldwide. Around 15-25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is associated with a worse prognosis and shortened disease-free survival. Therefore, anti-HER2 therapies have been developed, such as monoclonal antibodies (trastuzumab, Tz), antibody-drug conjugates (ado-trastuzumab emtansine, T-DM1), and pharmacological inhibitors of tyrosine kinase activity (lapatinib, Lp). Although Tz, the standard treatment, has significantly improved the prognosis of patients, resistance still affects a significant population of women and is currently a major challenge in clinical oncology. Therefore, this study aims to identify potential biomarkers to predict disease progression (prognostic markers) and the efficacy of Tz treatment (predictive markers) in patients with HER2+ BC. We hypothesize that proteins involved in cell motility are implicated in Tz-resistance. We aim to identify alterations in Tz-resistant cells to guide more efficient oncologic decisions. By bioinformatics, we selected candidate proteins and determined how their expression, localization, and the process they modulate were affected by anti-HER2 treatments. Next, using HER2+ BC patients' data, we assessed these proteins as prognostic and predictive biomarkers. Finally, using Tz-resistant cells, we evaluated their roles in Tz response. We identified deregulated genes associated with cell motility in Tz/T-DM1-resistant vs. -sensitive cells. We showed that Tz, T-DM1, and Lp decrease cell viability, and their effect is enhanced in combinations. We determined synergism between Tz/T-DM1 and Lp, making possible a dose reduction of each drug to achieve the same therapeutic effect. We found that combinations (Tz/T-DM1 + Lp) efficiently inhibit cell adhesion and migration. Furthermore, we demonstrated the induction of FAK nuclear and cortactin peri-nuclear localization after T-DM1, Lp, and Tz/T-DM1 + Lp treatments. In parallel, we observed that combined treatments downregulate proteins essential for metastatic dissemination, such as SRC, FAK, and paxillin. We found that low vinculin (VCL) and cortactin (CTTN) mRNA expression predicts favorable survival rates and has diagnostic value to discriminate between Tz-sensible and Tz-resistant HER2+ BC patients. Finally, we confirmed that vinculin and cortactin are overexpressed in Tz-resistance cells, SKBR3-RTz. Moreover, we found that Tz plus FAK/paxillin/cortactin-silencing reduced cell adhesion/migration capacity in Tz-sensitive and -resistant cells. In conclusion, we demonstrate that combined therapies are encouraging since low doses of Tz/T-DM1 + Lp inhibit metastatic processes by downregulating critical protein expression and affecting its subcellular localization. We propose that vinculin and cortactin might contribute to Tz-sensibility/resistance in BC cells. Finally, we identify potential prognostic and predictive biomarkers that are promising for personalized BC management that would allow efficient patient selection in order to mitigate resistance and maximize the safety and efficacy of anti-HER2 therapies.
PubMed: 37686651
DOI: 10.3390/cancers15174374 -
Heliyon Sep 2023Emerging evidence indicates that fibroblasts play pivotal roles in immunoregulation by producing various proteins under health and disease states. In the present study,...
UNLABELLED
Emerging evidence indicates that fibroblasts play pivotal roles in immunoregulation by producing various proteins under health and disease states. In the present study, for the first time, we compared the proteomes of serum-starved human skin fibroblasts and peripheral blood mononuclear cells (PBMCs) using Nano-LC-ESI-tandem mass spectrometry. This analysis contributes to a better understanding of the underlying molecular mechanisms of chronic inflammation and cancer, which are intrinsically accompanied by growth factor deficiency.The proteomes of starved fibroblasts and PBMCs consisted of 307 and 294 proteins, respectively, which are involved in lymphocyte migration, complement activation, inflammation, acute phase response, and immune regulation. Starved fibroblasts predominantly produced extracellular matrix-related proteins such as collagen/collagenase, while PBMCs produced focal adhesion-related proteins like beta-parvin and vinculin which are involved in lymphocyte migration. PBMCs produced a more diverse set of inflammatory molecules like heat shock proteins, while fibroblasts produced human leukocytes antigen-G and -E that are known as main immunomodulatory molecules. Fifty-four proteins were commonly found in both proteomes, including serum albumin, amyloid-beta, heat shock cognate 71 kDa, and complement C3. GeneMANIA bioinformatic tool predicted 418 functions for PBMCs, including reactive oxygen species metabolic processes and 241 functions for starved fibroblasts such as antigen processing and presentation including non-classical MHC -Ib pathway, and negative regulation of the immune response. Protein-protein interactions network analysis indicated the immunosuppressive function for starved fibroblasts-derived human leucocytes antigen-G and -E. Moreover, in an model of allogeneic transplantation, the immunosuppressive activity of starved fibroblasts was experimentally documented.
CONCLUSION
Under serum starvation-induced metabolic stress, both PBMCs and fibroblasts produced molecules like heat shock proteins and amyloid-beta, which can have pathogenic roles in auto-inflammatory diseases such as rheumatoid arthritis, type 1 diabetes mellitus, systemic lupus erythematosus, aging, and cancer. However, starved fibroblasts showed immunosuppressive activity in an model of allogeneic transplantation, suggesting their potential to modify such adverse reactions by down-regulating the immune system.
PubMed: 37674821
DOI: 10.1016/j.heliyon.2023.e19238 -
European Journal of Cell Biology Dec 2023Podosomes have been known for several decades as micron-sized, F-actin-rich structures that play a pivotal role in cell migration and invasion, as they are able to...
Podosomes have been known for several decades as micron-sized, F-actin-rich structures that play a pivotal role in cell migration and invasion, as they are able to mediate both cell-matrix attachment as well as extracellular matrix degradation. Particularly in monocytic cells, podosomes have been shown to fulfill a variety of additional functions such as sensing of substrate rigidity and topography, or cell-cell fusion. Increasing evidence now points to the involvement of podosome-like structures also during phagocytosis by immune cells such as macrophages, dendritic cells, and neutrophils. Here, we compare the different cell models and experimental set ups where "phagocytic podosomes" have been described. We also discuss the composition and architecture of these structures, their potential involvement in mechanosensing and particle disruption, as well as the pros and cons for addressing them as bona fide podosomes.
Topics: Podosomes; Actin Cytoskeleton; Actins; Macrophages; Phagocytosis
PubMed: 37625234
DOI: 10.1016/j.ejcb.2023.151356 -
Journal of Cardiovascular Translational... Dec 2023Predictors of myocardial recovery in heart failure (HF) are poorly understood. We explored if vinculin (VCL) variants are associated with myocardial recovery in dilated...
Predictors of myocardial recovery in heart failure (HF) are poorly understood. We explored if vinculin (VCL) variants are associated with myocardial recovery in dilated cardiomyopathy (DCM). Six infants with DCM with a VCL loss-of-function (LOF) variant were identified. Median age at diagnosis was 2 months, median LV ejection fraction was 24%, and median LV end-diastolic diameter z-score was 10.8. All patients received HF medications. Five patients (83%) showed normalization of LV function at a median age of 2.7 years. One patient progressed to end-stage HF requiring heart transplant. This case series identified a unique phenotype of HF with reduced ejection fraction at presentation that evolved to HF with recovered EF in over 80% of infant DCM cases with LOF VCL variants. These findings have prognostic implications for counseling and management of VCL-associated DCM and highlight a possible genetic basis for HF with recovered ejection fraction.
Topics: Infant; Humans; Child, Preschool; Stroke Volume; Ventricular Function, Left; Vinculin; Heart Failure; Prognosis
PubMed: 37548861
DOI: 10.1007/s12265-023-10421-6 -
Journal of Clinical Medicine Jul 2023In the present study, we measured the serum vascular endothelial cadherin (VEC) and vinculin (Vcn) concentrations in patients with type B acute aortic dissection (TBAD)...
BACKGROUND
In the present study, we measured the serum vascular endothelial cadherin (VEC) and vinculin (Vcn) concentrations in patients with type B acute aortic dissection (TBAD) to evaluate their diagnostic value for this condition.
METHODS
A total of 100 patients with TBAD and 90 matched controls were included in the study. The serum concentrations of VEC and Vcn were measured using enzyme-linked immunosorbent assays.
RESULTS
The serum VEC and Vcn concentrations were significantly higher in participants with TBAD than in healthy controls. Compared with patients with acute myocardial infarction (AMI), the serum concentrations of VEC and Vcn in patients with TBAD were higher, and the Vcn showed significant difference, with statistical significance. Receiver operating characteristic analysis generated areas under the curves for VEC and Vcn that were diagnostic for TBAD (0.599 and 0.655, respectively). The optimal cut-off values were 3.975 ng/μL and 128.1 pg/mL, the sensitivities were 43.0% and 35.0%, and the specificities were 73.3% and 90.0%, respectively. In addition, the use of a combination of serum VEC and Vcn increased the AUC to 0.661, with a sensitivity of 33.0% and a specificity of 93.33%. A high serum Vcn concentration was associated with a higher risk of visceral malperfusion in participants with TBAD (odds ratio (OR) = 1.007, 95% confidence interval [CI]: 1.001-1.013, = 0.014). In participants with refractory pain, the adjusted OR for the serum VEC concentration increased to 1.172 (95% CI: 1.010-1.361; = 0.036), compared with participants without refractory pain.
CONCLUSION
This study is the first to show the diagnostic value of serum VEC and Vcn for AAD and their relationships with the clinical characteristics of patients with TBAD. Thus, VEC and Vcn are potential serum markers of TBAD.
PubMed: 37510844
DOI: 10.3390/jcm12144730 -
ELife Jul 2023Integrin-mediated cell attachment rapidly induces tyrosine kinase signaling. Despite years of research, the role of this signaling in integrin activation and focal...
Integrin-mediated cell attachment rapidly induces tyrosine kinase signaling. Despite years of research, the role of this signaling in integrin activation and focal adhesion assembly is unclear. We provide evidence that the Src-family kinase (SFK) substrate Cas (Crk-associated substrate, p130Cas, BCAR1) is phosphorylated and associated with its Crk/CrkL effectors in clusters that are precursors of focal adhesions. The initial phospho-Cas clusters contain integrin β1 in its inactive, bent closed, conformation. Later, phospho-Cas and total Cas levels decrease as integrin β1 is activated and core focal adhesion proteins including vinculin, talin, kindlin, and paxillin are recruited. Cas is required for cell spreading and focal adhesion assembly in epithelial and fibroblast cells on collagen and fibronectin. Cas cluster formation requires Cas, Crk/CrkL, SFKs, and Rac1 but not vinculin. Rac1 provides positive feedback onto Cas through reactive oxygen, opposed by negative feedback from the ubiquitin proteasome system. The results suggest a two-step model for focal adhesion assembly in which clusters of phospho-Cas, effectors and inactive integrin β1 grow through positive feedback prior to integrin activation and recruitment of core focal adhesion proteins.
Topics: Phosphorylation; Focal Adhesions; Phosphoproteins; Integrin beta1; Crk-Associated Substrate Protein; Protein-Tyrosine Kinases; Integrins; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesion Kinase 1
PubMed: 37489578
DOI: 10.7554/eLife.90234