Cellular Cytoskeleton Research Articles

Examining prestructured β-actin peptides as substrates of histidine methyltransferase SETD3

The Nτ-His73 methylation of β-actin by histidine methyltransferase SETD3 is required for the integrity of the cellular cytoskeleton. Modulation of SETD3 activity in human cells facilitates cancer-like changes to the cell phenotype. SETD3 binds β-actin in an extended conformation, with a conserved bend-like motif surrounding His73. Here, we report on the catalytic specificity of SETD3 towards i, i + 3 stapled β-actin peptides possessing a limited conformational freedom surrounding the His73 substrate residue via positions Glu72 and Ile75. Stapled β-actin peptides were observed to be methylated less efficiently than the linear β-actin peptide. None of the stapled β-actin peptides efficiently inhibited the SETD3-catalyzed Nτ-His73 methylation reaction. Molecular dynamics simulations demonstrated that the unbound and SETD3-bound β-actin peptides display different backbone flexibility and bend-like conformations, highlighting their important role in substrate binding and catalysis. Overall, these findings suggest that reduced backbone flexibility of β-actin prevents the formation of optimal protein-peptide interactions between the enzyme and substrate, highlighting that the backbone flexibility needs to be considered when designing β-actin-based probes and inhibitors of biomedically important SETD3.

Protein kinase D1 mitigation against etoposide induced DNA damage in prostate cancer is associated with increased α-Catenin.

The E-cadherin, α- and β-Catenin interaction at the cell adherens junction plays a key role in cell adhesion; alteration in the expression and function of these genes are associated with disease progression in several solid tumors including prostate cancer. The membranous β-Catenin is dynamically linked to the cellular cytoskeleton through interaction with α-Catenin at amino acid positions threonine 120 (T120) to 151 of β-Catenin. Nuclear presence of α-Catenin modulates the sensitivity of cells to DNA damage. The objective of this study is to determine the role of α-Catenin and protein kinase D1 (PrKD1) in DNA damage response. Prostate cancer cells; LNCaP, LNCaP (Sh-PrKD1; silenced PrKD1), C4-2 and C4-2 PrKD1 were used for various sets of experiments to determine the role of DNA damage in PrKD1 overexpression and silencing cells. These cells were treated with compound-10 (100 nM) and Etoposide (30 µM), total cell lysates, cytosolic and nuclear fractions were prepared to observe various protein expressions. We performed single cell gel electrophoresis (COMET assay) to determine the etoposide induce DNA damage in C4-2 and C4-2 PrKD1 cells. The animal experiments were carried out to determine the tolerability of compound-10 by mice and generate preliminary data on efficacy of compound-10 in modulating the α-Catenin and PrKD1 expressions in inhibiting tumor progression. PrKD1, a novel serine threonine kinase, phosphorylates β-Catenin T120. In silico analysis, confirmed that T120 phosphorylation alters β- to α-Catenin binding. Forced expression of PrKD1 in prostate cancer cells increased β- and α-Catenin protein levels associated with reduced etoposide induced DNA damage. Downregulation of α-Catenin abrogates the PrKD1 mitigation of DNA damage. The in vitro results were corroborated in vivo using mouse prostate cancer patient derived xenograft model by inhibition of PrKD1 kinase activity with compound-10, a selective PrKD inhibitor, demonstrating decreased total β- and α-Catenin protein levels, and β-Catenin T120 phosphorylation. Alteration in DNA damage response pathways play major role in prostate cancer progression. The study identifies a novel mechanism of α-Catenin dependent DNA damage mitigation role for PrKD1 in prostate cancer.

Extracellular Vesicles Derived from Ligament Tissue Transport Interleukin‐17A to Mediate Ligament‐To‐Bone Crosstalk in Ankylosing Spondylitis

AbstractPathological new bone formation is a critical feature of the progression of ankylosing spondylitis (AS), and spine ankylosis is a distinctive feature of this condition. Ligaments are the primary regions of pathological new bone formation in AS. Here, it is demonstrated that ligament tissue‐derived extracellular vesicles (EVs) and their interleukin‐17A (IL‐17A) cargo mediate the communication between the tissue and other cells. The investigation revealed that IL‐17A in EVs can activate the JAK‐STAT3 pathway, thereby stimulating the expression of MMP14 in AS ligament. Overexpression of MMP14 can lead to changes in the cytoskeleton and mechanical signaling of mesenchymal stem cells and other cells. These alterations in cellular cytoskeleton and mechanical signaling at ligament sites in patients with AS or in stem cells treated with EVs can result in pathological new bone formation. Finally, inhibiting IL‐17A activity and EV endocytosis effectively controlled inflammation and pathological new bone formation. Overall, these data suggest that ligament‐derived EVs and the enclosed IL‐17A have a potential role in driving pathological new bone formation in AS, and targeting EVs may therefore emerge as a novel approach to delaying ectopic ossification in AS.

Plectin: Dual Participation in Tumor Progression.

The plectin gene can encode a cytoskeletal linking protein, plectin, known for its interaction with three critical components of the cellular cytoskeleton: intermediate filaments, microtubules, and actin filaments. In recent years, more and more studies have reported that plectin is closely related to tumorigenesis and development, exhibiting both tumor-suppressive and tumor-promoting functions. Here, we first introduce the molecular structure and function of plectin, and then we summarize the current understanding of the crucial role of plectin in cancer progression. Finally, we also discuss the possible reasons for the different roles of plectin expression in various types of cancer and highlight the double-edged sword role of plectin in tumor progression. The review aims to deepen the comprehensive understanding of plectin's role in cancer and further help to develop novel therapeutic strategies and drug targets.

CCKR signaling map, G-Protein bindings, hormonal regulation, and neural mechanisms may influence the osteogenic/cementogenic differentiation potential of hPDLSCs

ObjectivePeriodontal regeneration poses challenges due to the periodontium's complexity, relying on mesenchymal cells from the periodontal ligament (hPDLSCs) to regenerate hard tissues like bone and cementum. While some hPDLSCs have high regeneration potential (HOP-hPDLSCs), most are low potential (LOP-hPDLSCs). This study analyzed hPDLSCs from a single donor to minimize inter-individual variability and focus on key differences in differentiation potentials. DesignThis study used RNA-seq, genomic databases, and bioinformatics tools to explore signaling pathways (SPs), biological processes (BPs), and molecular functions (MFs) guiding HOP cells to mineralized matrix production. It also investigated limitations of LOP cells and strategies for enhancing their osteo/cementogenesis. ResultsIn basal conditions, HOP exhibited a multifunctional gene network with higher expression of genes related to osteo/cementogenesis, cell differentiation, immune modulation, stress response, and hormonal regulation. In contrast, LOP focused on steroid hormone biosynthesis and nucleic acid maintenance. During osteo/cementogenic induction, HOP showed strong modulation of genes related to angiogenesis, cell division, mesenchymal differentiation, and extracellular matrix production. LOP demonstrated neural synaptic-related processes and preserved cellular cytoskeleton integrity. CCKR map signaling and G-protein receptor bindings gained significance during osteo/cementogenesis in HOP-hPDLSCs. Both HOP and LOP shared common BPs related to gastrointestinal and reproductive system development. ConclusionThe osteo/cementogenic differentiation of HOP cells may be regulated by CCKR signaling, G-protein bindings, and specific hormonal regulation. LOP cells seem committed to neural mechanisms. This study sheds light on hPDLSCs' complex characteristics, offering a deeper understanding of their differentiation potential for future periodontal regeneration research and therapies.

Condensate interfacial forces reposition DNA loci and probe chromatin viscoelasticity

Biomolecular condensates assemble in living cells through phase separation and related phase transitions. An underappreciated feature of these dynamic molecular assemblies is that they form interfaces with other cellular structures, including membranes, cytoskeleton, DNA and RNA, and other membraneless compartments. These interfaces are expected to give rise to capillary forces, but there are few ways of quantifying and harnessing these forces in living cells. Here, we introduce viscoelastic chromatin tethering and organization (VECTOR), which uses light-inducible biomolecular condensates to generate capillary forces at targeted DNA loci. VECTOR can be utilized to programmably reposition genomic loci on a timescale of seconds to minutes, quantitatively revealing local heterogeneity in the viscoelastic material properties of chromatin. These synthetic condensates are built from components that naturally form liquid-like structures in living cells, highlighting the potential role for native condensates to generate forces and do work to reorganize the genome and impact chromatin architecture.

Hypoxic ischemic encephalopathy (HIE).

The morbidity and mortality of acute ischemic hypoxic encephalopathy in newborns have not been dramatically modified over the last 20 years. The purpose of this review is to describe the use of hyperbaric oxygenation therapy (HBOT) in the management of acute ischemic hypoxic encephalopathy in newborns. A review of the medical literature was conducted on the use of HBOT in the pathophysiology of this condition and its impact on outcomes of patients treated at an early stage. When HBOT is administered promptly, it can promote the survival of the penumbra, modulate the cytokine storm, modify inflammatory cascades, restore mitochondrial function, inhibit apoptosis, reinstate cellular communication and cytoskeleton function, reinstall the functioning of the kinase system, reduce cytotoxic and tissue edema, promote microcirculation, and provide an antioxidant effect. All these secondary mechanisms aid in saving, rescuing, and protecting the marginal tissue. When used promptly, HBOT is a non-invasive adjunct treatment that can preserve the marginal tissue affected by ischemia, hypoxia, meet the metabolic needs of the penumbra, reduce inflammatory cascades, prevent the extension of the damaged tissue, and modulate ischemia-reperfusion injury.

Orthogonal DNA Self-Assembly-Based Expansion Microscopy Platform for Amplified, Multiplexed Biomarker Imaging.

Expansion microscopy (ExM) facilitates nanoscale imaging under conventional microscopes, but it frequently encounters challenges such as fluorescence losses, low signal-to-noise ratio (SNR), and limited detection throughput. To address these issues, a method of orthogonal DNA self-assembly-based ExM (o-DAExM) platform is developed, which employs hybridization chain reaction instead of conventional fluorescence labeling units, showcasing signal amplification efficacy, enhancement of SNR, and expandable multiplexing capability at any stage of the ExM process. In this work, o-DAExM has been applied to compare with immunofluorescence-based ExM for cellular cytoskeleton imaging, and the resolved nanoscale spatial distributions of cytoskeleton show outstanding performance and reliability of o-DAExM. Furthermore, the study demonstrates the utility of o-DAExM in accurately revealing exosome heterogeneous information and multiplexed analysis of protein targets in single cells, which provides infinite possibilities in super-resolution imaging of cells and other samples. Therefore, o-DAExM offers a straightforward expansion and signal labeling method, highlighting future prospects to study nanoscale structures and functional networks in biological systems.

Enhanced RHO-ROCK signaling is associated with CRELD2 production and fibroblast recruitment in cutaneous squamous cell carcinoma.

A key characteristic of cancer cells is their ability to induce changes in their microenvironment that render it permissive to tumor growth, invasion and metastasis. Indeed, these changes are required for tumor progression. Consequently, the tumor microenvironment is emerging as a key source of new targets against cancer, with novel therapies aimed at reversing tumor-promoting changes, reinstating a tumor-hostile microenvironment and suppressing disease progression. RHO-ROCK signaling, and consequent tension within the cellular actomyosin cytoskeleton, regulates a paracrine signaling cascade that establishes a tumor-promoting microenvironment. Here, we show that consistent with our observations in breast cancer, enhanced ROCK activity and consequent production of CRELD2 is associated with the recruitment and tumor-promoting polarization of cancer-associated fibroblasts in cutaneous squamous cell carcinoma. Our observations provide support for the notion that the role of RHO-ROCK signaling in establishing a tumor-promoting microenvironment may be conserved across patients and potentially also different cancer types.

Microtubule polymerization induced by iridium‐fullerene photosensitizers for cancer immunotherapy via dual‐reactive oxygen species regulation strategy

AbstractMicrotubules (MTs) are key players in cell division, migration, and signaling, and they are regarded as important targets for cancer treatment. In this work, two fullerene (C60)‐functionalized Ir(III) complexes (Ir‐C601 and Ir‐C602) are rationally designed as dual reactive oxygen species (ROS) regulators and MT‐targeted Type I/II photosensitizers. In the dark, Ir‐C601 and Ir‐C602 serve as ROS scavengers to eliminate O2•− and •OH, consequently reducing the dark cytotoxicity and reversing dysfunctional T cells. Due to the efficiently populated C60‐localized intraligand triplet state, Ir‐C601 and Ir‐C602 can be excited by green light (525 nm) to produce O2•− and •OONO− (Type I) and 1O2 (Type II) to overcome tumor hypoxia. Moreover, Ir‐C601 is also able to photooxidize tubulin, consequently interfering with the cellular cytoskeleton structures, inducing immunogenic cell death and inhibiting cell proliferation and migration. Finally, Ir‐C601 exhibits promising photo‐immunotherapeutic effects both in vitro and in vivo. In all, we report here the first MT stabilizing photosensitizer performing through Type I/II photodynamic therapy pathways, which provides insights into the rational design of new photo‐immunotherapeutic agents targeting specific biomolecules.

The dishevelled associated activator of morphogenesis protein 2 (Daam2) regulates neural tube closure.

The Wnt signaling pathway is highly conserved in metazoans and regulates a large array of cellular processes including motility, polarity and fate determination, and stem cell homeostasis. Modulation of the actin cytoskeleton via the non-canonical Wnt pathway regulate cell polarity and cell migration that are required for proper vertebrate gastrulation and subsequent neurulation. However, the mechanism(s) of how the non-canonical pathway mediates actin cytoskeleton modulation is not fully understood. Herein, we characterize the role of the Formin-homology protein; dishevelled associated activator of morphogenesis 2 (Daam2) protein in the Wnt signaling pathway. Co-immunoprecipitation assays confirm the binding of Daam2 to dishevelled2 (Dvl2) as well as the domains within these proteins required for interaction; additionally, the interaction between Daam2 and Dvl2 was Wnt-regulated. Sub-cellular localization studies reveal Daam2 is cytoplasmic and regulates the cellular actin cytoskeleton by modulating actin filament formation. During Xenopus development, a knockdown or loss of Daam2 specifically produces neural tube closure defects indicative of a role in non-canonical signaling. Additionally, our studies did not identify any role for Daam2 in canonical Wnt signaling in mammalian culture cells or the Xenopus embryo. Our studies together identify Daam2 as a component of the non-canonical Wnt pathway and Daam2 is a regulator of neural tube morphogenesis during vertebrate development.

Abstract PO5-19-12: Innovative Trial Design: ARTIDIS Nanomechanical Generated Measurements for Early Breast Lesions (ANGEL) - Multicentre Study

Abstract Background: The diagnosis of breast cancer based on histopathology of core needle biopsies and the delineation into breast cancer subtypes to drive clinical treatment decisions can be time consuming and cause patient (and clinician) anxiety. Alternative, near patient, approaches to conventional diagnostic pathways would be desirable to drive diagnostic and therapeutic planning. This study harnesses the novel concept of evaluating diagnostic cores for nanomechanical features at the time of core biopsy. Clinical manifestations of disease are often accompanied by biomechanical alterations at sub-/cellular, extracellular, tissue and organ level. Mechanical properties of cancer cells and their microenvironment play a critical role in cancer invasion, progression, and immune cell infiltration. Ultrastructural (sub-) cellular alterations (nanoscale level), including the remodulation and alteration of cellular cytoskeleton dynamics, contribute to altered mechanical state of cancer cells, and lead to increased deformability and motility of soft cancer cells to overcome stromal barrier, and spread into the adjacent tissues and distant sites in the body. This results in a direct mechanistic correlation between nanomechanical properties and clinical outcome in cancer tissues. The ARTIDIS Nanomechanical Generated measurements for Early Lesions (ANGEL) Study will prospectively evaluate the efficacy of the ARTIDIS system as an aid to diagnosis, as well as subtyping breast cancer and predicting clinical outcomes based on tissue nanomechanics, particularly in the context of neoadjuvant therapy (NAT). Methods: The study seeks to validate the predictive power of the nanomechanical signature, a unique tissue biomarker reflecting the tissue phenotype, by comparing it to standard histological assessment. A projected cohort of 2,705 patients will be enrolled, from diverse ethnic and socioeconomic backgrounds, with benign or malignant breast conditions undergoing diagnostic core biopsy. A single core biopsy (gauge agnostic) is placed in a cassette and subjected to thousands of mechanical measurements at the “nano” scale over a period of 1-3 hours. The ARTIDIS technology builds upon the Atomic Force Microscopy platform to non-disruptively detect up to 17 different nanomechanical parameters in soft tissues with a subcellular resolution. The same core biopsy can then undergo fixation and conventional histopathological analyses for comparison with the final pathology. The primary aim is to confirm the potential of the ARTIDIS system in diagnosis (benign vs malignant) and secondarily address whether the nanomechanical signature can differentiate subtypes of breast cancer, associate with clinical outcome, and guide treatment decisions. Particularly, we will investigate ARTIDIS potential as a predictor of treatment response for patients receiving NAT. Results: The multicenter ANGEL study is a prospective investigation that builds upon previous clinical data from the NANO study at the University Hospital Basel (Switzerland) and is designed to demonstrate the power of using a nanomechanical signature to evaluate breast biopsies within a routine clinical setting in a multicenter setting. The data will be used to develop ARTIDIS as an aid to diagnosis, as well as NEO Match test applications to predict and guide response to NAT. Conclusion: This study presents the first of its kind as a direct prospective, multi-center based clinical translation of the Physics of Cancer findings into clinical settings. This innovative trial design allows the prospective development of ARTIDIS as an aid to diagnosis and NAT outcome prediction and therapy guidance applications. Citation Format: Karla Sepulveda, Ashley Roark, Sagar Dhamne, Chandandeep Nagi, Ivan Marin, Iqbal Tabish, Susanne G. Hilsenbeck Hilsenbeck, Sara Nizzero, Mariam Gachechiladze, Tobias A Appenzeller, Marko Loparic, Marija Plodinec, Alastair Thompson. Innovative Trial Design: ARTIDIS Nanomechanical Generated Measurements for Early Breast Lesions (ANGEL) - Multicentre Study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-19-12.

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.

Abstract 3373: Protein kinase D1 alleviate etoposide induced DNA damage through up regulation of alpha-catenin

Abstract Interaction between E-cadherin and β-catenin at cell-cell adherens junctions is pivotal in cell adhesion, and dysregulation of these genes is associated with disease progression in various solid tumors, notably in prostate cancer. α-catenin acts as a physical linker between the cadherin/β-catenin complex and actin cytoskeleton, through its mechanosensory properties has potential to modify interactions between cadherins and cytoskeleton and results in sensitivity of cells to DNA damage and toxicity. Membranous β-catenin interacts with the cellular cytoskeleton through threonine 120 (T120) phosphorylation and its association with α-catenin. We want to investigate the role of protein Kinase D1 (PrKD1) in DNA damage response, using various prostate cancer cell lines; PrKD1-silenced cells (LNCaP sh-PrKD1) and overexpression of PrKD1 (C4-2 PrKD1). PrKD1 silence and overexpress cells were treated with PrKD1 inhibitor and Etoposide to observe the effect of DNA damage repair altering various genes in prostate cancer cells. Silencing α-Catenin increased p-γH2AX (ser-139) expression in C4-2 PrKD1 cells. Conversely silenced PrKD1 (LNCaP sh-PrKD1) cells represented decreased α-Catenin expression, and increased p-γH2AX (ser-139). Overexpression of PrKD1 in prostate cancer cells increased α-catenin levels and conferred resistance to etoposide-induced DNA damage. Silencing PrKD1 compromised DNA damage protective effect. Furthermore, we elucidate the role of Protein kinase D1 (PrKD1), a unique serine-threonine kinase that phosphorylates β-catenin at T120, in this interaction. We treated C4-2 and C4-2 PrKD1 cells with PrKD1 inhibitor and immuno-precipitated β-Catenin complex represented decrease in β-Catenin (T-120) and α-Catenin than untreated C4-2 PrKD1 cells. We observed crosstalk between α-catenin and PrKD1 in DNA damage repair. In-silico analysis affirmed that the loss of T120 phosphorylation disrupts β- and α-catenin interaction. PrKD1 inhibitor decreased cell viability in dose response fashion, and also decrease the p-γH2AX (ser-139) in C4-2 PrKD1 cells than C4-2 cells. In vitro findings were corroborated in a mouse prostate cancer PDX xenograft model by inhibiting PrKD1 kinase activity, resulting in reduced β-catenin T120 phosphorylation and α-catenin levels. In vivo experiments with PrKD1 inhibitor represented tolerability in mice and its efficacy in modulating α-catenin and PrKD1 expression to inhibit tumor progression. Study for the first time provides a mechanistic basis of the protective role of PrKD1 against DNA damage. Citation Format: Sanjeev Shukla, Samuel Serrano, Mohammed Al-Toubat, Teruko Osumi, K.C. Balaji. Protein kinase D1 alleviate etoposide induced DNA damage through up regulation of alpha-catenin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3373.

Balancing competing effects of tissue growth and cytoskeletal regulation during Drosophila wing disc development

How a developing organ robustly coordinates the cellular mechanics and growth to reach a final size and shape remains poorly understood. Through iterations between experiments and model simulations that include a mechanistic description of interkinetic nuclear migration, we show that the local curvature, height, and nuclear positioning of cells in the Drosophila wing imaginal disc are defined by the concurrent patterning of actomyosin contractility, cell-ECM adhesion, ECM stiffness, and interfacial membrane tension. We show that increasing cell proliferation via different growth-promoting pathways results in two distinct phenotypes. Triggering proliferation through insulin signaling increases basal curvature, but an increase in growth through Dpp signaling and Myc causes tissue flattening. These distinct phenotypic outcomes arise from differences in how each growth pathway regulates the cellular cytoskeleton, including contractility and cell-ECM adhesion. The coupled regulation of proliferation and cytoskeletal regulators is a general strategy to meet the multiple context-dependent criteria defining tissue morphogenesis.

An Overview on Nanotechnology and Cancer Diagnosis and Therapy

Nanotechnology is a rapidly increasing area of research and recent advancements have demonstrated its promise in medicinal applications. One such application is in cancer diagnosis and treatment. These led to the creation of innovative, sophisticated applications that diagnose cancer cells, transport medications to target tissue, record treatment outcomes and observe intracellular alterations to assist stop precancerous cells from turning malignant.They assist in the investigation of factors such as cellular mechanics, morphology and cytoskeleton, which have proven difficult to attain with traditional technologies.

Avian Influenza A Viruses Modulate the Cellular Cytoskeleton during Infection of Mammalian Hosts.

Influenza is one of the most prevalent causes of death worldwide. Influenza A viruses (IAVs) naturally infect various avian and mammalian hosts, causing seasonal epidemics and periodic pandemics with high morbidity and mortality. The recent SARS-CoV-2 pandemic showed how an animal virus strain could unpredictably acquire the ability to infect humans with high infection transmissibility. Importantly, highly pathogenic avian influenza A viruses (AIVs) may cause human infections with exceptionally high mortality. Because these latter infections pose a pandemic potential, analyzing the ecology and evolution features of host expansion helps to identify new broad-range therapeutic strategies. Although IAVs are the prototypic example of molecular strategies that capitalize on their coding potential, the outcome of infection depends strictly on the complex interactions between viral and host cell factors. Most of the studies have focused on the influenza virus, while the contribution of host factors remains largely unknown. Therefore, a comprehensive understanding of mammals' host response to AIV infection is crucial. This review sheds light on the involvement of the cellular cytoskeleton during the highly pathogenic AIV infection of mammalian hosts, allowing a better understanding of its modulatory role, which may be relevant to therapeutic interventions for fatal disease prevention and pandemic management.

Coupled single-cell and bulk RNA-seq analysis reveals the engulfment role of endothelial cells in atherosclerosis

The clearance of apoptotic cell debris, containing professional phagocytosis and non-professional phagocytosis, is essential for maintaining the homeostasis of healthy tissues. Here, we discovered that endothelial cells could engulf apoptotic cell debris in atherosclerotic plaque. Single-cell RNA sequencing (RNA-seq) has revealed a unique endothelial cell subpopulation in atherosclerosis, which was strongly associated with vascular injury-related pathways. Moreover, integrated analysis of three vascular injury-related RNA-seq datasets showed that the expression of scavenger receptor class B type 1 (SR-B1) was up-regulated and specifically enriched in the phagocytosis pathway under vascular injury circumstances. Single-cell RNA-seq and bulk RNA-seq indicate that SR-B1 was highly expressed in a unique endothelial cell subpopulation of mouse aorta and strongly associated with the reorganization of cellular adherent junctions and cytoskeleton which were necessary for phagocytosis. Furthermore, SR-B1 was strongly required for endothelial cells to engulf apoptotic cell debris in atherosclerotic plaque of both mouse and human aorta. Overall, this study demonstrated that apoptotic cell debris could be engulfed by endothelial cells through SR-B1 and associated with the reorganization of cellular adherent junctions and cytoskeleton.

The interplay between Wnt signaling pathways and microtubule dynamics.

Wnt signaling pathways represent an evolutionarily highly conserved, intricate network of molecular interactions that regulates various aspects of cellular behavior, including embryonic development and tissue homeostasis. Wnt signaling pathways share the β-catenin-dependent (canonical) and the multiple β-catenin-independent (non-canonical) pathways. These pathways collectively orchestrate a wide range of cellular processes through distinct mechanisms of action. Both the β-catenin-dependent and β-catenin-independent pathways are closely intertwined with microtubule dynamics, underscoring the complex crosstalk between Wnt signaling and the cellular cytoskeleton. This interplay involves several mechanisms, including how the components of Wnt signaling can influence the stability, organization, and distribution of microtubules. The modulation of microtubule dynamics by Wnt signaling plays a crucial role in coordinating cellular behaviors and responses to external signals. In this comprehensive review, we discussed the current understanding of how Wnt signaling and microtubule dynamics intersect in various aspects of cellular behavior. This study provides insights into our understanding of these crucial cellular processes.

A polarized cell system amenable to subcellular resolution imaging of influenza virus infection.

The life cycle of influenza A viruses (IAV), and notably intracellular trafficking of the viral genome, depends on multiple interactions with the cellular cytoskeleton and endomembrane system. A limitation of the conventional cellular models used for mechanistic study and subcellular imaging of IAV infection is that they are cultured in two dimensions (2D) under non-polarizing conditions, and therefore they do not recapitulate the intracellular organization of the polarized respiratory epithelial cells naturally targeted by IAVs. To overcome this limitation, we developed an IAV-infection assay in a 3D cell culture system which allows imaging along the baso-lateral axis of polarized cells, with subcellular resolution. Here we describe a protocol to grow polarized monolayers of Caco2-TC7 cells on static Cytodex-3 microcarrier beads, infect them with IAV, and subsequently perform immunostaining and confocal imaging, or electron microscopy, on polarized IAV-infected cells. This method can be extended to other pathogens that infect human polarized epithelial cells.