Machine learning interpretable models of cell mechanics from protein images.

Cellular form and function emerge from complex mechanochemical systems within the cytoplasm. Currently, no systematic strategy exists to infer large-scale physical properties of a cell from its molecular components. This is an obstacle to understanding processes such as cell adhesion and migration. Here, we develop a data-driven modeling pipeline to learn the mechanical behavior of adherent cells. We first train neural networks to predict cellular forces from images of cytoskeletal proteins. Strikingly, experimental images of a single focal adhesion (FA) protein, such as zyxin, are sufficient to predict forces and can generalize to unseen biological regimes. Using this observation, we develop two approaches-one constrained by physics and the other agnostic-to construct data-driven continuum models of cellular forces. Both reveal how cellular forces are encoded by two distinct length scales. Beyond adherent cell mechanics, our work serves as a case study for integrating neural networks into predictive models for cell biology.

c-Src-induced vascular malformations require localised matrix degradation at focal adhesions.

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.

Actomyosin-mediated cellular tension promotes Yap nuclear translocation and myocardial proliferation through α5 integrin signaling.

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state results in the loss of regenerative potential of the mammalian heart shortly after birth. Nonmuscle myosin IIB (NM IIB)-mediated actomyosin contractility regulates cardiomyocyte cytokinesis in the embryonic heart, and NM IIB levels decline after birth, suggesting a role for cellular tension in the regulation of cardiomyocyte cell cycle activity in the postnatal heart. To investigate the role of actomyosin contractility in cardiomyocyte cell cycle arrest, we conditionally activated ROCK2 kinase domain (ROCK2:ER) in the murine postnatal heart. Here, we show that α5/ß1 integrin and fibronectin matrix increase in response to actomyosin-mediated tension. Moreover, activation of ROCK2:ER promotes nuclear translocation of Yap, a mechanosensitive transcriptional co-activator, and enhances cardiomyocyte proliferation. Finally, we show that reduction of myocardial α5 integrin rescues the myocardial proliferation phenotype in ROCK2:ER hearts. These data demonstrate that cardiomyocytes respond to increased intracellular tension by altering their intercellular contacts in favor of cell-matrix interactions, leading to Yap nuclear translocation, thus uncovering a function for nonmuscle myosin contractility in promoting cardiomyocyte proliferation in the postnatal heart.

Spatial organization of lysosomal exocytosis relies on membrane tension gradients.

Lysosomal exocytosis is involved in many key cellular processes but its spatiotemporal regulation is poorly known. Using total internal reflection fluorescence microscopy (TIRFM) and spatial statistics, we observed that lysosomal exocytosis is not random at the adhesive part of the plasma membrane of RPE1 cells but clustered at different scales. Although the rate of exocytosis is regulated by the actin cytoskeleton, neither interfering with actin or microtubule dynamics by drug treatments alters its spatial organization. Exocytosis events partially co-appear at focal adhesions (FAs) and their clustering is reduced upon removal of FAs. Changes in membrane tension following a hypo-osmotic shock or treatment with methyl-ß-cyclodextrin were found to increase clustering. To investigate the link between FAs and membrane tension, cells were cultured on adhesive ring-shaped micropatterns, which allow to control the spatial organization of FAs. By using a combination of TIRFM and fluorescence lifetime imaging microscopy (FLIM), we revealed the existence of a radial gradient in membrane tension. By changing the diameter of micropatterned substrates, we further showed that this gradient as well as the extent of exocytosis clustering can be controlled. Together, our data indicate that the spatial clustering of lysosomal exocytosis relies on membrane tension patterning controlled by the spatial organization of FAs.

Protein kinase A is a functional component of focal adhesions.

Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and the actin cytoskeleton and also transmit signals that regulate cell adhesion, cytoskeletal dynamics, and cell migration. While many of these signals are rooted in reversible tyrosine phosphorylation, phosphorylation of FA proteins on Ser/Thr residues is far more abundant yet its mechanisms and consequences are far less understood. The cAMP-dependent protein kinase (protein kinase A; PKA) has important roles in cell adhesion and cell migration and is both an effector and regulator of integrin-mediated adhesion to the ECM. Importantly, subcellular localization plays a critically important role in specifying PKA function. Here, we show that PKA is present in isolated FA-cytoskeleton complexes and active within FAs in live cells. Furthermore, using kinase-catalyzed biotinylation of isolated FA-cytoskeleton complexes, we identify 53 high-stringency candidate PKA substrates within FAs. From this list, we validate tensin-3 (Tns3)-a well-established molecular scaffold, regulator of cell migration, and a component of focal and fibrillar adhesions-as a novel direct substrate for PKA. These observations identify a new pathway for phospho-regulation of Tns3 and, importantly, establish a new and important niche for localized PKA signaling and thus provide a foundation for further investigation of the role of PKA in the regulation of FA dynamics and signaling.

Paxillin regulates liver fibrosis via actin polymerization and ERK activation in hepatic stellate cells.

Liver injury leads to fibrosis and cirrhosis. The primary mechanism underlying the fibrogenic response is the activation of hepatic stellate cells (HSCs), which are 'quiescent' in normal liver but become 'activated' after injury by transdifferentiating into extracellular matrix (ECM)-secreting myofibroblasts. Given that integrins are important in HSC activation and fibrogenesis, we hypothesized that paxillin, a key downstream effector in integrin signaling, might be critical in the fibrosis pathway. Using a cell-culture-based model of HSC activation and in vivo models of liver injury, we found that paxillin is upregulated in activated HSCs and fibrotic livers. Overexpression of paxillin (both in vitro and in vivo) led to increased ECM protein expression, and depletion of paxillin in a novel conditional mouse injury model reduced fibrosis. The mechanism by which paxillin mediated this effect appeared to be through the actin cytoskeleton, which signals to the ERK pathway and induces ECM protein production. These data highlight a novel role for paxillin in HSC biology and fibrosis.

Dynamically regulated focal adhesions coordinate endothelial cell remodelling in developing vasculature.

The assembly of a mature vascular network involves coordinated endothelial cell (EC) shape changes, including the process of EC elongation. How EC elongation is dynamically regulated in vivo is not fully understood. Here, we have generated a zebrafish mutant that is deficient for the integrin adaptor protein Talin 1 (Tln1). Using a new focal adhesion (FA) marker line expressing endothelial Vinculinb-eGFP, we demonstrate that EC FAs function dynamically and are lost in our tln1 mutants, allowing us to uncouple the primary roles of FAs in EC morphogenesis from the secondary effects that occur due to systemic vessel failure or loss of blood flow. Tln1 loss led to compromised F-actin rearrangements, perturbed EC elongation and disrupted cell-cell junction linearisation in vessel remodelling. Finally, chemical induction of actin polymerisation restored actin dynamics and EC elongation during vascular morphogenesis. Together, we identify that FAs are essential for EC elongation and junction linearisation in flow-pressured vessels and that they influence actin polymerisation in cellular morphogenesis. These observations can explain the severely compromised vessel beds and vascular leakage observed in mutant models that lack integrin signalling. This article has an associated 'The people behind the papers' interview.

Focal adhesion kinase regulates tendon cell mechanoresponse and physiological tendon development.

Tendons enable locomotion by transmitting high tensile mechanical forces between muscle and bone via their dense extracellular matrix (ECM). The application of extrinsic mechanical stimuli via muscle contraction is necessary to regulate healthy tendon function. Specifically, applied physiological levels of mechanical loading elicit an anabolic tendon cell response, while decreased mechanical loading evokes a degradative tendon state. Although the tendon response to mechanical stimuli has implications in disease pathogenesis and clinical treatment strategies, the cell signaling mechanisms by which tendon cells sense and respond to mechanical stimuli within the native tendon ECM remain largely unknown. Therefore, we explored the role of cell-ECM adhesions in regulating tendon cell mechanotransduction by perturbing the genetic expression and signaling activity of focal adhesion kinase (FAK) through both in vitro and in vivo approaches. We determined that FAK regulates tendon cell spreading behavior and focal adhesion morphology, nuclear deformation in response to applied mechanical strain, and mechanosensitive gene expression. In addition, our data reveal that FAK signaling plays an essential role in in vivo tendon development and postnatal growth, as FAK-knockout mouse tendons demonstrated reduced tendon size, altered mechanical properties, differences in cellular composition, and reduced maturity of the deposited ECM. These data provide a foundational understanding of the role of FAK signaling as a critical regulator of in situ tendon cell mechanotransduction. Importantly, an increased understanding of tendon cell mechanotransductive mechanisms may inform clinical practice as well as lead to the discovery of diagnostic and/or therapeutic molecular targets.

An ULK1/2-PXN mechanotransduction pathway suppresses breast cancer cell migration.

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.

Hic-5 regulates extracellular matrix-associated gene expression and cytokine secretion in cancer associated fibroblasts.

The focal adhesion protein, Hic-5 plays a key role in promoting extracellular matrix deposition and remodeling by cancer associated fibroblasts within the tumor stroma to promote breast tumor cell invasion. However, whether stromal matrix gene expression is regulated by Hic-5 is still unknown. Utilizing a constitutive Hic-5 knockout, Mouse Mammary Tumor Virus-Polyoma Middle T-Antigen spontaneous breast tumor mouse model, bulk RNAseq analysis was performed on cancer associated fibroblasts isolated from Hic-5 knockout mammary tumors. Functional network analysis highlighted a key role for Hic-5 in extracellular matrix organization, with both structural matrix genes, as well as matrix remodeling genes being differentially expressed in relation to Hic-5 expression. The subcellular distribution of the MRTF-A transcription factor and expression of a subset of MRTF-A responsive genes was also impacted by Hic-5 expression. Additionally, cytokine array analysis of conditioned media from the Hic-5 and Hic-5 knockout cancer associated fibroblasts revealed that Hic-5 is important for the secretion of several key factors that are associated with matrix remodeling, angiogenesis and immune evasion. Together, these data provide further evidence of a central role for Hic-5 expression in cancer associated fibroblasts in regulating the composition and organization of the tumor stroma microenvironment to promote breast tumor progression.

Antagonistic control of active surface integrins by myotubularin and phosphatidylinositol 3-kinase C2ß in a myotubular myopathy model.

X-linked centronuclear myopathy (XLCNM) is a severe human disease without existing therapies caused by mutations in the phosphoinositide 3-phosphatase MTM1. Loss of MTM1 function is associated with muscle fiber defects characterized by impaired localization of ß-integrins and other components of focal adhesions. Here we show that defective focal adhesions and reduced active ß-integrin surface levels in a cellular model of XLCNM are rescued by loss of phosphatidylinositiol 3-kinase C2ß (PI3KC2ß) function. Inactivation of the Mtm1 gene impaired myoblast differentiation into myotubes and resulted in reduced surface levels of active ß1-integrins as well as corresponding defects in focal adhesions. These phenotypes were rescued by concomitant genetic loss of Pik3c2b or pharmacological inhibition of PI3KC2ß activity. We further demonstrate that a hitherto unknown role of PI3KC2ß in the endocytic trafficking of active ß1-integrins rather than rescue of phosphatidylinositol 3-phosphate levels underlies the ability of Pik3c2b to act as a genetic modifier of cellular XLCNM phenotypes. Our findings reveal a crucial antagonistic function of MTM1 and PI3KC2ß in the control of active ß-integrin surface levels, thereby providing a molecular mechanism for the adhesion and myofiber defects observed in XLCNM. They further suggest specific pharmacological inhibition of PI3KC2ß catalysis as a viable treatment option for XLCNM patients.

Receptor-binding domain of SARS-CoV-2 is a functional αv-integrin agonist.

Among the novel mutations distinguishing SARS-CoV-2 from similar coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2-binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. Here, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared with RGD-containing, integrin-binding fragments of fibronectin. We determined that S1-RBD supported adhesion of fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells, while RBD-coated microparticles attached to epithelial monolayers in a cation-dependent manner. Cell adhesion to S1-RBD was RGD dependent and inhibited by blocking antibodies against αv and ß3 but not α5 or ß1 integrins. Similarly, we observed direct binding of S1-RBD to recombinant human αvß3 and αvß6 integrins, but not α5ß1 integrins, using surface plasmon resonance. S1-RBD adhesion initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin; triggered Akt activation; and supported cell proliferation. Thus, the RGD sequence of S1-RBD can function as an αv-selective integrin agonist. This study provides evidence that cell surface αv-containing integrins can respond functionally to spike protein and raises the possibility that S1-mediated dysregulation of extracellular matrix dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.

Phosphorylated paxillin and phosphorylated FAK constitute subregions within focal adhesions.

Integrin-mediated adhesions are convergence points for multiple signaling pathways. Their inner structure and diverse functions can be studied with super-resolution microscopy. Here, we examined the spatial organization within focal adhesions by analyzing several adhesion proteins with structured illumination microscopy (SIM). Paxillin (Pax) serves as a scaffold protein and signaling hub in focal adhesions, and focal adhesion kinase (FAK, also known as PTK2) regulates the dynamics of adhesions. We found that their phosphorylated forms, pPax and pFAK, form spot-like, spatially defined clusters within adhesions in several cell lines and confirmed these findings with additional super-resolution techniques. These clusters showed a more regular separation from each other compared with more randomly distributed signals for FAK or paxillin. Mutational analysis indicated that the active (open) FAK conformation is a prerequisite for the pattern formation of pFAK. Live-cell super-resolution imaging revealed that organization in clusters is preserved over time for FAK constructs; however, distance between clusters is dynamic for FAK, while paxillin is more stable. Combined, these data introduce spatial clusters of pPax and pFAK as substructures in adhesions and highlight the relevance of paxillin-FAK binding for establishing a regular substructure in focal adhesions.

Integrin α6ß4 requires plectin and vimentin for adhesion complex distribution and invasive growth.

Integrin α6ß4 binds plectin to associate with vimentin; however, the biological function remains unclear. Here, we utilized various integrin ß4 mutants and CRISPR-Cas9 editing to investigate this association. Upon laminin binding, integrin α6ß4 distinctly distributed peripherally as well as centrally, proximal to the nucleus. Upon fibronectin addition, integrin α6ß4 was centrally recruited to large focal adhesions (FAs) and enhanced Fak (also known as PTK2) phosphorylation. Integrin ß4 plectin-binding mutants or genetic deletion of plectin inhibited ß4 recruitment to FAs and integrin α6ß4-enhanced cell spreading, migration and three-dimensional invasive growth. Loss of the ß4 signaling domain (but retaining plectin binding) blocked migration and invasiveness but not cell spreading, recruitment to FAs or colony growth. Immunostaining revealed that integrin α6ß4 redistributed vimentin perinuclearly, where it colocalized with plectin and FAs. Depletion of vimentin completely blocked integrin ß4-enhanced invasive growth, Fak phosphorylation and proliferation in three dimensions but not two dimensions. In summary, we demonstrate the essential roles of plectin and vimentin in promoting an invasive phenotype downstream of integrin α6ß4. This article has an associated First Person interview with the first author of the paper.

Wnt signaling in cell adhesion, development, and colon cancer.

Wnt signaling is essential for embryonic development, influencing processes such as axis formation, cell proliferation and differentiation, cell fate decisions, and axon guidance. It also plays a role in maintaining tissue homeostasis in adult organisms. The loss of normal cell polarity and adhesion caused by Wnt signaling activation is a fundamental step for tumor progression and metastasis. Activating the canonical Wnt pathway is a driving force in many human cancers, especially colorectal, hepatocellular, and mammary carcinomas. Wnt causes the stabilization and nuclear transport of newly synthesized transcriptional regulator ß-catenin. The generally accepted view is that the canonical effects of Wnt growth factors are caused by the transcription of ß-catenin target genes. Here, we review recent findings that indicate Wnt is a regulator of many other cellular physiological activities, such as macropinocytosis, endosome trafficking, protein stability, focal adhesions, and lysosomal activity. Some of these regulatory responses occur within minutes and do not require new protein synthesis, indicating that there is much more to Wnt beyond the well-established transcriptional role of ß-catenin. The main conclusion that emerges from these studies is that in basal cell conditions, the activity of the key protein kinase GSK3, which is inhibited by Wnt pathway activation, normally represses the actin machinery that orchestrates macropinocytosis with implications in cancer. These contributions expand our understanding of the multifaceted roles of Wnt signaling in cellular processes, development, and cancer, providing insights into potential therapeutic targets and strategies.

TRPV4 regulates ß1 integrin-mediated cell-matrix adhesions and collagen remodeling.

Transient Receptor Potential Vanilloid-type 4 (TRPV4) is a mechanosensitive, Ca2+ -permeable plasma membrane channel that associates with focal adhesions, influences collagen remodeling, and is associated with fibrotic processes through undefined mechanisms. While TRPV4 is known to be activated by mechanical forces transmitted through collagen adhesion receptors containing the ß1 integrin, it is not understood whether TRPV4 affects matrix remodeling by altering ß1 integrin expression and function. We tested the hypothesis that TRPV4 regulates collagen remodeling through its impact on the ß1 integrin in cell-matrix adhesions. In cultured fibroblasts derived from mouse gingival connective tissues, which exhibit very rapid collagen turnover, we found that higher TRPV4 expression is associated with reduced ß1 integrin abundance and adhesion to collagen, reduced focal adhesion size and total adhesion area, and reduced alignment and compaction of extracellular fibrillar collagen. The reduction of ß1 integrin expression mediated by TRPV4 is associated with the upregulation of miRNAs that target ß1 integrin mRNA. Our data suggest a novel mechanism by which TRPV4 modulates collagen remodeling through post-transcriptional downregulation of ß1 integrin expression and function.

Physical aspects of epithelial cell-cell interactions: hidden system complexities.

The maintenance of homeostasis and the retention of ordered epithelial cell self-organization are essential for morphogenesis, wound healing, and the spread of cancer across the epithelium. However, cell-cell interactions in an overcrowded environment introduce a diversity of complications. Such interactions arise from an interplay between the cell compressive and shear stress components that accompany increased cell packing density. They can lead to various kinds of cell rearrangement such as: the epithelial-to-mesenchymal cell state transition; live cell extrusion; and cell jamming. All of these scenarios of cell rearrangement under mechanical stress relate to changes in the strengths of the cell-cell and cell-matrix adhesion contacts. The objective of this review study is twofold: first, to provide a comprehensive summary of the biological and physical factors influencing the effects of cell mechanical stress on cell-cell interactions, and the consequences of these interactions for the status of cell-cell and cell-matrix adhesion contacts; and secondly, to offer a bio-physical/mathematical analysis of the aforementioned biological aspects. By presenting these two approaches in conjunction, we seek to highlight the intricate nature of biological systems, which manifests in the form of complex bio-physical/mathematical equations. Furthermore, the juxtaposition of these apparently disparate approaches underscores the importance of conducting experiments to determine the multitude of parameters that contribute to the development of these intricate bio-physical/mathematical models.

Characterization of RACK1-depleted mammalian cells by a palette of microscopy approaches reveals defects in cell cycle progression and polarity establishment.

The Receptor for Activated C Kinase 1 (RACK1) is an evolutionarily conserved scaffold protein involved in the regulation of numerous cellular processes. Here, we used CRISPR/Cas9 and siRNA to reduce the expression of RACK1 in Madin-Darby Canine Kidney (MDCK) epithelial cells and Rat2 fibroblasts, respectively. RACK1-depleted cells were examined using coherence-controlled holographic microscopy, immunofluorescence, and electron microscopy. RACK1 depletion resulted in decreased cell proliferation, increased cell area and perimeter, and in the appearance of large binucleated cells suggesting a defect in the cell cycle progression. Our results show that the depletion of RACK1 has a pleiotropic effect on both epithelial and mesenchymal cell lines and support its essential role in mammalian cells.

Effect of Focal Adhesion Kinase and Vinculin Expression on Migration Parameters of Normal and Tumor Epitheliocytes.

Focal adhesions (FAs) are mechanosensory structures that transform physical stimuli into chemical signals guiding cell migration. Comprehensive studies postulate correlation between the FA parameters and cell motility metrics for individual migrating cells. However, which properties of the FAs are critical for epithelial cell motility in a monolayer remains poorly elucidated. We used high-throughput microscopy to describe relationship between the FA parameters and cell migration in immortalized epithelial keratinocytes (HaCaT) and lung carcinoma cells (A549) with depleted or inhibited vinculin and focal adhesion kinase (FAK) FA proteins. To evaluate relationship between the FA morphology and cell migration, we used substrates with varying stiffness in the model of wound healing. Cells cultivated on fibronectin had the highest FA area values, migration rate, and upregulated expression of FAK and vinculin mRNAs, while the smallest FA area and slower migration rate to the wound were specific to cells cultivated on glass. Suppression of vinculin expression in both normal and tumor cells caused decrease of the FA size and fluorescence intensity but did not affect cell migration into the wound. In contrast, downregulation or inactivation of FAK did not affect the FA size but significantly slowed down the wound closure rate by both HaCaT and A549 cell lines. We also showed that the FAK knockdown results in the FA lifetime decrease for the cells cultivated both on glass and fibronectin. Our data indicate that the FA lifetime is the most important parameter defining migration of epithelial cells in a monolayer. The observed change in the cell migration rate in a monolayer caused by changes in expression/activation of FAK kinase makes FAK a promising target for anticancer therapy of lung carcinoma.

THSD1 Suppresses Autophagy-Mediated Focal Adhesion Turnover by Modulating the FAK-Beclin 1 Pathway.

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.