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FIP200 or Atg5 depletion increases fibronectin-induced cell adhesion and spreading. (A) Western blot analysis of cell lysates harvested along the time course of fibronectin-induced MCF10A wild-type cell spreading. Immunoblotting was performed against the indicated proteins. Quantitation of p62 expression was normalized to actin and represented as the fold change from the zero time point (cells in suspension). Results represent at least 3 independent experiments. (B) Immunofluorescence microscopy of MCF10A cells following attachment and spreading on fibronectin for indicated time points. Cells were immunostained for p62 and LC3. Scale bar = 10 μm. Quantitation was performed on multiple images from random fields of view. The number of p62 or LC3 puncta (indicated with arrowheads) were quantified from 100 cells for p62 and 13–15 cells for LC3 across 3 independent experiments. Results represent the average number of p62 or LC3 puncta per cell. Error bars represent SEM. (C) Brightfield images across the time course of adhesion to fibronectin of MCF10A wild-type, FIP200 KO, and Atg5 KO cells. Higher magnification regions illustrate phenotypic differences and dotted line represents representative cell diameter. Scale bar = 100 μm. (D) Quantitation of individual cell area represented as square pixels were plotted across the time course of fibronectin adhesion. Results represent 50–100 cells per experiment, repeated at least 3 independent times. Black bar represents the mean and error bars are the SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.
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Autophagy is an essential catabolic intracellular pathway that maintains homeostasis by degrading long-lived proteins, damaged organelles, and provides an energy source during nutrient starvation. It is now understood that autophagy has discrete functions as a selective lysosomal degradation pathway targeting large cytosolic structural and signalin...
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Oral cancer is a very aggressive disease with high rates of recurrence and metastasis. This study aimed at addressing how efficiently tongue cancer is suppressed after carbon ion irradiation. Here, the close relationship between upregulated expression of focal adhesion kinase (FAK) and high metastatic status in tongue squamous cell carcinoma patien...
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... Various stress conditions such as starvation induce autophagy, in which autophagosomes (double-membrane organelles) engulf cargo and subsequently fuse with lysosomes for degradation [6]. In breast cancer cells, autophagy supports cell migration by facilitating the degradation of FA complexes [7][8][9]. Interestingly, distinct molecular mechanisms were uncovered in different cell lines, suggesting that autophagy-mediated FA turnover is context-dependent. Our understanding of how autophagy degrades FAs in primary cells, particularly in primary human brain endothelial cells, remains largely unknown. ...
Focal adhesions (FAs) play a crucial role in cell spreading and adhesion, and their autophagic degradation is an emerging area of interest. This study investigates the role of Thrombospondin Type 1 Domain-Containing Protein 1 (THSD1) in regulating autophagy and FA stability in brain endothelial cells, shedding light on its potential implications for cerebrovascular diseases. Our research reveals a physical interaction between THSD1 and FAs. Depletion of THSD1 significantly reduces FA numbers, impairing cell spreading and adhesion. The loss of THSD1 also induces autophagy independently of changes in mTOR and AMPK activation, implying that THSD1 primarily governs FA dynamics rather than serving as a global regulator of nutrient and energy status. Mechanistically, THSD1 negatively regulates Beclin 1, a central autophagy regulator, at FAs through interactions with focal adhesion kinase (FAK). THSD1 inactivation diminishes FAK activity and relieves its inhibitory phosphorylation on Beclin 1. This, in turn, promotes the complex formation between Beclin 1 and ATG14, a critical event for the activation of the autophagy cascade. In summary, our findings identify THSD1 as a novel regulator of autophagy that degrades FAs in brain endothelial cells. This underscores the distinctive nature of THSD1-mediated, cargo-directed autophagy and its potential relevance to vascular diseases due to the loss of endothelial FAs. Investigating the underlying mechanisms of THSD1-mediated pathways holds promise for discovering novel therapeutic targets in vascular diseases.
... We treated primary astrocytes derived from the brains of Vlgr1-deficient Vlgr1/DrumB mutant mice or wild type (WT) mice with either DMSO or nocodazole (NDZ) (10 μM) in DMSO. Consistent with previous reports, 21,25,26 NDZ treatment induced microtubule depolymerization leading to cellcycle synchronization and accumulation of FAs (Figure 2A,B). After replacing the NDZ medium by a fresh medium without NDZ (NDZ washout) a phase of rapid FA disassembly was introduced which rested for approximately 45 min (Figure 2A,B). ...
... In the NDZ washout assay, FAs accumulated in response to NDZ-induced microtubule depolymerization are first rapidly degraded after NDZ washout, before FAs are reassembled. 21,25,26 Our results show that the time course of the later FA de novo assembly, but not the disassembly phase, is significantly delayed to an equal extent in the primary brain astrocytes of both Vlgr1-deficient mouse models studied. (Figure 2A-C; Figure S3A,B). ...
VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest adhesion G protein-coupled receptor aGPCRs. Mutations in VLGR1/ADGRV1 are associated with human Usher syndrome, the most common form of deaf-blindness, and also with epilepsy in humans and mice. VLGR1 is expressed almost ubiquitously, but is mainly found in the CNS and in the sensory cells of the eye and inner ear. Little is known about the pathogenesis of the diseases related to VLGR1. We previously identified VLGR1 as a vital component of focal adhesions (FA) serving as a metabotropic mechanoreceptor controls cell spreading and migration. FAs are highly dynamic and turnover in response to internal and external signals. Here, we aimed to elucidate how VLGR1 participates in FA turnover. Nocodazole washouts and live cell imaging of paxillin-DsRed2 consistently showed that FA disassembly was not altered, .but de novo assembly of FA was significantly delayed in Vlgr1-deficient astrocytes, indicating that VLGR1 is enrolled in FA assembly. In FRAP experiments recovery rates were significantly reduced in Vlgr1-deficient FAs, indicating reduced turnover kinetics in VLGR1-deficient FAs. We showed that VLGR1 regulates cell migration by controlling the FA turnover during their assembly and expect novel insights into pathomechanisms related to pathogenic dysfunctions of VLGR1.
... Additionally, a recent study shows that the deletion of RB1CC1 or ATG5 leads to different FA morphologies, but they both result in reduced cell motility [87]. However, during energy starvation, RB1CC1 is activated by ULK1 and inhibits focal adhesion kinase PTK2/FAK, leading to reduced cell motility and tumor metastasis [88]. ...
Autophagy, a process of cellular self-digestion, delivers intracellular components including superfluous and dysfunctional proteins and organelles to the lysosome for degradation and recycling and is important to maintain cellular homeostasis. In recent decades, autophagy has been found to help fight against a variety of human diseases, but, at the same time, autophagy can also promote the procession of certain pathologies, which makes the connection between autophagy and diseases complex but interesting. In this review, we summarize the advances in understanding the roles of autophagy in human diseases and the therapeutic methods targeting autophagy and discuss some of the remaining questions in this field, focusing on cancer, neurodegenerative diseases, infectious diseases and metabolic disorders.
... 34 Additionally, loss of ATG5 function made the expression of paxillin reduced, resulting in stable adhesion plaques and reduced directed cell motility, and autophagy enhanced FAK signaling and degraded paxillin to promote FA disassembly to suppress cell adhesion then resulted in less cell spreading. 35 The present study demonstrated that inhibition of paxillin was found in the SphK1 knockdown CRC cells in vitro and vivo, moreover, suppression of autophagy by 3MA reversed the expression of paxillin and its phosphorylation in SphK1overexpressed CRC cells. SphK1-driven autophagy may induce the assembly and disassembly of FA via regulating paxillin and its phosphorylation. ...
Invasion and metastasis are the main causes of colorectal cancer (CRC)‐related death. Accumulating evidence suggested that sphingosine kinase 1 (SphK1) promoted the metastasis of CRC and autophagy played an important role in SphK1 promoting the metastasis of malignancy. However, the mechanism by which SphK1‐driven autophagy promotes invasion and metastasis in CRC remains to be clarified. In the present study, immunohistochemical detection showed the expression of SphK1 and paxillin was higher in human CRC tissues than those of normal colorectal mucosal tissues, they were both associated with TNM staging, lymphatic, and distance metastasis. In addition, study of in situ tumor transplantation model in nude mice showed that the suppression of SphK1 inhibited the growth of colonic orthotopic implantation tumors and the expression of paxillin, p‐paxillin, LC3 in the tumor. So, SphK1 may promote CRC metastasis via inducing the expression of paxillin expression and its phosphorylation, in vivo. Furthermore, results of CCK8 assay, transwell and wound healing assays showed that SphK1 promoted the viability, invasion, and metastasis of CRC cells. Transmission electron microscopy detection showed that SphK1 is the key factor in autophagy induction in CRC cells. Moreover, western blot examination indicated that the expression of LC3Ⅱ/Ⅰ, paxillin, p‐paxillin, MMP‐2, and vimentin was enhanced in SphK1‐overexpressed CRC cells and suppressed in SphK1 knockdown CRC cells, meanwhile, the expression of E‐cadherin was suppressed in SphK1‐overexpressed CRC cells and enhanced in SphK1 knockdown CRC cells. Suppression of autophagy by 3MA reversed the expression of paxillin and its phosphorylation in SphK1‐overexpressed CRC cells, indicated that SphK1‐driven autophagy induced the expression of paxillin and its phosphorylation in CRC cells. Together, these findings reveal that SphK1‐driven autophagy may promote the invasion and metastasis of CRC via promoting the expression of focal adhesion paxillin and its phosphorylation. SphK1‐driven autophagy may promote the invasion and metastasis of CRC via promoting of the expression of focal adhesion paxillin and its phosphorylation.
The remodeling and stiffening of the extracellular matrix (ECM) is a well‐recognized modulator of breast cancer progression. How changes in the mechanical properties of the ECM are converted into biochemical signals that direct tumor cell migration and metastasis remain poorly characterized. Here, we describe a new role for the autophagy‐inducing serine/threonine kinases ULK1 and ULK2 in mechanotransduction. We show that ULK1/2 activity inhibits the assembly of actin stress fibers and focal adhesions (FAs) and as a consequence impedes cell contraction and migration, independent of its role in autophagy. Mechanistically, we identify PXN/paxillin, a key component of the mechanotransducing machinery, as a direct binding partner and substrate of ULK1/2. ULK‐mediated phosphorylation of PXN at S32 and S119 weakens homotypic interactions and liquid–liquid phase separation of PXN, impairing FA assembly, which in turn alters the mechanical properties of breast cancer cells and their response to mechanical stimuli. ULK1/2 and the well‐characterized PXN regulator, FAK/Src, have opposing functions on mechanotransduction and compete for phosphorylation of adjacent serine and tyrosine residues. Taken together, our study reveals ULK1/2 as important regulator of PXN‐dependent mechanotransduction.
The Metazoan complexity arises from a primary building block, the epithelium, which comprises a layer of polarized cells that divide the organism into compartments. Most of these body compartments are organs formed by epithelial tubes that enclose an internal hollow space or lumen. Over the last decades, multiple studies have unmasked the paramount events required to form this lumen de novo. In epithelial cells, these events mainly involve recognizing external clues, establishing and maintaining apicobasal polarity, endo-lysosomal trafficking, and expanding the created lumen. Although canonical autophagy has been classically considered a catabolic process needed for cell survival, multiple studies have also emphasized its crucial role in epithelial polarity, morphogenesis and cellular homeostasis. Furthermore, non-canonical autophagy pathways have been recently discovered as atypical secretory routes. Both canonical and non-canonical pathways play essential roles in epithelial polarity and lumen formation. This review addresses how the molecular machinery for epithelial polarity and autophagy interplay in different processes and how autophagy functions influence lumenogenesis, emphasizing its role in the lumen formation key events.
Membrane binding and unbinding dynamics play a crucial role in the biological activity of several non-integral membrane proteins, which have to be recruited to the membrane in order to perform their functions. By localizing to the membrane, these proteins are able to induce downstream signal amplification in their respective signaling pathways. Here we present a 3D computational approach using reaction-diffusion equations to investigate the relation between membrane localization of Focal Adhesion Kinase (FAK), Ras homolog family member A (RhoA) and signal amplification of the YAP/TAZ signaling pathway. Our results show that the theoretical scenarios, in which FAK is membrane-bound, yield robust and amplified YAP/TAZ nuclear translocation signals. Moreover, we predict that the amount of YAP/TAZ nuclear translocation increases with cell spreading, confirming the experimental findings in the literature. In summary, our in silico predictions show that when the cell membrane interaction area with the underlying substrate increases, for example through cell spreading, this leads to more encounters between membrane-bound signaling partners and downstream signal amplification. Since membrane activation is a motif common to many signaling pathways, this study has important implications for understanding the design principles of signaling networks.
Aberrant epigenetic alterations play a decisive role in cancer initiation and propagation via the regulation of key tumor suppressor genes and oncogenes or by modulation of essential signaling pathways. Autophagy is a highly regulated mechanism required for the recycling and degradation of surplus and damaged cytoplasmic constituents in a lysosome dependent manner. In cancer, autophagy has a divergent role. For instance, autophagy elicits tumor promoting functions by facilitating metabolic adaption and plasticity in cancer stem cells (CSCs) and cancer cells. Moreover, autophagy exerts pro-survival mechanisms to these cancerous cells by influencing survival, dormancy, immunosurveillance, invasion, metastasis, and resistance to anti-cancer therapies. In addition, recent studies have demonstrated that various tumor suppressor genes and oncogenes involved in autophagy, are tightly regulated via different epigenetic modifications, such as DNA methylation, histone modifications and non-coding RNAs. The impact of epigenetic regulation of autophagy in cancer cells and CSCs is not well-understood. Therefore, uncovering the complex mechanism of epigenetic regulation of autophagy provides an opportunity to improve and discover novel cancer therapeutics. Subsequently, this would aid in improving clinical outcome for cancer patients. In this review, we provide a comprehensive overview of the existing knowledge available on epigenetic regulation of autophagy and its importance in the maintenance and homeostasis of CSCs and cancer cells.
