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.

Endothelial knockdown of the tumor suppressor, WWOX, increases inflammation in ventilator-induced lung injury.

Chronic cigarette smoke exposure decreases lung expression of WWOX which is known to protect the endothelial barrier during infectious models of acute respiratory distress syndrome (ARDS). Proteomic analysis of WWOX-silenced endothelial cells (ECs) was done using tandem mass tag mass spectrometry (TMT-MS). WWOX-silenced ECs as well as those isolated from endothelial cell Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 h). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. These were repeated with dual silencing of WWOX and zyxin. Control and EC Wwox KO mice were subjected to high tidal volume ventilation. Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histological assays. TMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1ß, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 mitogen-activated kinases (MAPK) phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion but not in IL-6. Loss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears, in part, to be dependent on the upregulation of zyxin.NEW & NOTEWORTHY Prior tobacco smoke exposure is associated with an increased risk of acute respiratory distress syndrome (ARDS) during critical illness. Our laboratory is investigating one of the gene expression changes that occurs in the lung following smoke exposure: WWOX downregulation. Here we describe changes in protein expression associated with WWOX knockdown and its influence on ventilator-induced ARDS in a mouse model.

Decreased expression of p53 is associated with down expression of zyxin in breast cancer.

Background and Aims: Breast cancer (BC) is considered one of the most common malignant tumors leading to death in women, and genetic factors have a crucial role in BC pathogenesis. Zyxin (ZYX) is one of these factors that may be important in p53 level and function. Thus, the present work aimed to investigate the ZYX gene and protein expression in tumor tissue and matched margin tissue and its correlation with the p53 expression. Methods: In a present case-control study, 30 tumors and 30 matched margin tissues were obtained from Iran Tumor Bank/Tehran University of Medical Sciences. Real-time polymerase chain reaction and western blot analysis techniques were applied to evaluate the genes and protein expression, respectively. Results: The data showed that expression of the ZYX gene in tumor tissues significantly decreased (p = 0.0274) compared to matched margin tissues. In contrast, the p53 gene expression in tumor tissues had no significant difference with matched margin tissues. Additionally, we observed that ZYX and p53 genes expression in tumor tissues of estrogen receptor-positive patients had significant elevation than estrogen receptor-negative patients (p < 0.001, p < 0.001, respectively). The data of the western blot analysis technique showed that protein expression of ZYX (p = 0.0024) and P53 protein (p = 0.0218) in tumor tissues was significantly reduced compared to matched margin tissues. Additionally, our analysis showed a direct and significant correlation between the expression of ZYX and p53 proteins (r = 0.7797, p = 0.0126) and expression of ZYX and p53 genes (r = 0.3079, p = 0.0187). Conclusion: Based on our observation, ZYX might have a tumor suppressor role and is associated with p53.

The role of zyxin in signal transduction and its relationship with diseases.

This review highlighted the pivotal role of zyxin, an essential cell focal adhesions protein, in cellular biology and various diseases. Zyxin can orchestrate the restructuring and dynamic alterations of the cellular cytoskeleton, which is involved in cell proliferation, adhesion, motility, and gene transcription. Aberrant zyxin expression is closely correlated with tumor cell activity and cardiac function in both tumorigenesis and cardiovascular diseases. Moreover, in fibrotic and inflammatory conditions, zyxin can modulate cellular functions and inflammatory responses. Therefore, a comprehensive understanding of zyxin is crucial for deciphering signal transduction networks and disease pathogenesis. Investigating its role in diseases holds promise for novel avenues in early diagnosis and therapeutic strategies. Nevertheless, targeting zyxin as a therapeutic focal point presents challenges in terms of specificity, safety, drug delivery, and resistance. Nonetheless, in-depth studies on zyxin and the application of precision medicine could offer new possibilities for personalized treatment modalities.

Force-activated zyxin assemblies coordinate actin nucleation and crosslinking to orchestrate stress fiber repair.

As the cytoskeleton sustains cell and tissue forces, it incurs physical damage that must be repaired to maintain mechanical homeostasis. The LIM-domain protein zyxin detects force-induced ruptures in actin-myosin stress fibers, coordinating downstream repair factors to restore stress fiber integrity through unclear mechanisms. Here, we reconstitute stress fiber repair with purified proteins, uncovering detailed links between zyxin's force-regulated binding interactions and cytoskeletal dynamics. In addition to binding individual tensed actin filaments (F-actin), zyxin's LIM domains form force-dependent assemblies that bridge broken filament fragments. Zyxin assemblies engage repair factors through multi-valent interactions, coordinating nucleation of new F-actin by VASP and its crosslinking into aligned bundles by ɑ-actinin. Through these combined activities, stress fiber repair initiates within the cores of micron-scale damage sites in cells, explaining how these F-actin depleted regions are rapidly restored. Thus, zyxin's force-dependent organization of actin repair machinery inherently operates at the network scale to maintain cytoskeletal integrity.

Leveraging biotin-based proximity labeling to identify cellular factors governing early alphaherpesvirus infection.

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system in vivo. To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection.

Estudo da expressão e participação de VASP e Zyxin na diferenciação hematopoiética, na leucemia mieloide crônica e na via de sinalização BCR-ABL / VASP and Zyxin expression and participation in hematopoietic differentiation, in chronic myeloid leukemia and BCR-ABL signaling pathway

VASP (Vasodilator-stimulated phosphoprotein) e Zyxin são proteínas reguladoras de actina que controlam a adesão célula-célula. Zyxin dirige a montagem da actina através da interação e recrutamento da VASP a sítios específicos da adesão. A fosforilação da VASP ou da Zyxin altera suas atividades nas junções aderentes. PKA fosforila VASP em serina 157, regulando, assim, importantes funções celulares de VASP. VASP interage com ABL e é substrato da oncoproteína BCR-ABL. A presença da proteína BCR-ABL promove a oncogênese em pacientes com leucemia mieloide crônica (LMC) devido à ativação constitutiva da atividade tirosina quinase. Apesar de já descrita alteração da expressão de VASP e Zyxin em diferentes tumores epiteliais, o papel de VASP e Zyxin na LMC, na via de sinalização BCR-ABL e a participação destas proteínas na hematopoiese são desconhecidos. Desta maneira, demonstramos aqui ausência de p-VASP ser157 em células de medula óssea de pacientes com LMC, em contraste com a presença de p-VASP ser157 em doadores saudáveis. Pacientes com LMC em remissão, responsivos a inibidores de tirosina quinase, apresentam p-VASP ser157, enquanto os pacientes resistentes não expressam p-VASP ser157. Utilizando células K562 inibidas para VASP ou Zyxin, observamos que VASP e Zyxin modulam as proteínas anti-apoptóticas BCL-2 e BCL-XL da via de sinalização do BCR-ABL. Em adição, células K562 silenciadas para a VASP apresentam diminuição na atividade de FAK y925 e demonstramos que VASP interage com FAK. A expressão de VASP e Zyxin e de suas formas ativas aumenta durante a diferenciação megacariocítica e a inibição de VASP implica em diminuição na expressão do marcador CD61. Identificamos no presente estudo a participação de VASP e Zyxin na via do BCR-ABL, regulando a expressão de proteínas efetoras anti-apoptóticas e, também, na diferenciação megacariocítica...

VASP (vasodilator-stimulated phosphoprotein) and Zyxin are actin regulatory proteins that control cell-cell adhesion. Zyxin directs actin assembly by interacting and recruiting VASP to specific sites of adhesion. The phosphorylation of VASP and Zyxin modifies their activity in cell-cell junctions. PKA phosphorylates VASP at serine 157 regulating VASP cellular functions. VASP interacts with ABL and VASP is a substrate of BCR-ABL oncoprotein. The presence of BCR-ABL protein drives oncogenesis in patients with chronic myeloid leukemia (CML) due to a constitutive activation of tyrosine kinase activity. It has been described an altered expression of VASP and Zyxin in different types of tumor; however the function of VASP and Zyxin in CML, in BCR-ABL pathway and in hematopoiesis remains unknown. We describe here the absence of p-VASP ser157 in CML bone marrow cells, in contrast to p-VASP ser157 expression in healthy donors. Patients responsive to tyrosine kinase inhibitors present p-VASP ser157, while resistant patients do not have p-VASP ser157. In K562 cells we observed that VASP and Zyxin modulate anti-apoptotic proteins BCL-2 and BCL-XL. VASP depletion in K562 cells decreases FAK y925 activity and VASP interacts with FAK. Expression of VASP, p-VASP, Zyxin and p-Zyxin increases during megakaryocyte differentiation and VASP inhibition affects this differentiation through reduced CD61 expression in VASP depleted cells. We identify here the participation of VASP and Zyxin in BCR-ABL pathway affecting anti-apoptotic proteins and, also, in megakaryocyte differentiation. Then, the altered expression of VASP activity in CML patients may contribute to CML pathogenesis, affecting cellular differentiation or leukemic cell adhesion...