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1.
Nat Commun ; 15(1): 7956, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39261466

RESUMO

Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8+ T cell proliferation, but is required for sustained IL-2 dependent CD8+ effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8+ T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8+ T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8+ T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling.


Assuntos
Linfócitos T CD8-Positivos , Citoesqueleto , Interleucina-2 , Camundongos Endogâmicos C57BL , Fator de Resposta Sérica , Transativadores , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/metabolismo , Interleucina-2/genética , Animais , Transativadores/metabolismo , Transativadores/genética , Citoesqueleto/metabolismo , Camundongos , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/genética , Listeriose/microbiologia , Actinas/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Camundongos Knockout , Proliferação de Células , Ativação Linfocitária
2.
Commun Biol ; 7(1): 1146, 2024 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-39277687

RESUMO

Microtubules are essential for various cellular processes. The functional diversity of microtubules is attributed to the incorporation of various α- and ß-tubulin isotypes encoded by different genes. In this work, we investigated the functional role of ß4B-tubulin isotype (TUBB4B) in hearing and vision as mutations in TUBB4B are associated with sensorineural disease. Using a Tubb4b knockout mouse model, our findings demonstrate that TUBB4B is essential for hearing. Mice lacking TUBB4B are profoundly deaf due to defects in the inner and middle ear. Specifically, in the inner ear, the absence of TUBB4B lead to disorganized and reduced densities of microtubules in pillar cells, suggesting a critical role for TUBB4B in providing mechanical support for auditory transmission. In the middle ear, Tubb4b-/- mice exhibit motile cilia defects in epithelial cells, leading to the development of otitis media. However, Tubb4b deletion does not affect photoreceptor function or cause retinal degeneration. Intriguingly, ß6-tubulin levels increase in retinas lacking ß4B-tubulin isotype, suggesting a functional compensation mechanism. Our findings illustrate the essential roles of TUBB4B in hearing but not in vision in mice, highlighting the distinct functions of tubulin isotypes in different sensory systems.


Assuntos
Cílios , Camundongos Knockout , Tubulina (Proteína) , Animais , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Cílios/metabolismo , Camundongos , Citoesqueleto/metabolismo , Cóclea/metabolismo , Microtúbulos/metabolismo
3.
FASEB J ; 38(18): e70049, 2024 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-39275889

RESUMO

In this study, we have explored the role of the KATNB1 gene, a microtubule-severing protein, in the seminiferous epithelium of the rat testis. Our data have shown that KATNB1 expressed in rat brain, testes, and Sertoli cells. KATNB1 was found to co-localize with α-tubulin showing a unique stage-specific distribution across the seminiferous epithelium. Knockdown of KATNB1 by RNAi led to significant disruption of the tight junction (TJ) permeability barrier function in primary Sertoli cells cultured in vitro with an established functional TJ-barrier, as well as perturbations in the microtubule and actin cytoskeleton organization. The disruption in these cytoskeletal structures, in turn, led to improper distribution of TJ and basal ES proteins essential for maintaining the Sertoli TJ function. More importantly, overexpression of KATNB1 in the testis in vivo was found to block cadmium-induced blood-testis barrier (BTB) disruption and testis injury. KATNB1 exerted its promoting effects on BTB and spermatogenesis through corrective spatiotemporal expression of actin- and microtubule-based regulatory proteins by maintaining the proper organization of cytoskeletons in the testis, illustrating its plausible therapeutic implication. In summary, Katanin regulatory subunit B1 (KATNB1) plays a crucial role in BTB and spermatogenesis through its effects on the actin- and microtubule-based cytoskeletons in Sertoli cells and testis, providing important insights into male reproductive biology.


Assuntos
Barreira Hematotesticular , Katanina , Células de Sertoli , Animais , Masculino , Células de Sertoli/metabolismo , Ratos , Katanina/metabolismo , Katanina/genética , Barreira Hematotesticular/metabolismo , Citoesqueleto/metabolismo , Ratos Sprague-Dawley , Junções Íntimas/metabolismo , Espermatogênese/fisiologia , Células Cultivadas , Epitélio Seminífero/metabolismo , Testículo/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo
4.
Stem Cell Res Ther ; 15(1): 284, 2024 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-39243052

RESUMO

BACKGROUND: Periodontal ligament stem cells (PDLSCs) are important seed cells in tissue engineering and clinical applications. They are the priority receptor cells for sensing various mechanical stresses. Yes-associated protein (YAP) is a recognized mechanically sensitive transcription factor. However, the role of YAP in regulating the fate of PDLSCs under tension stress (TS) and its underlying mechanism is still unclear. METHODS: The effects of TS on the morphology and fate of PDLSCs were investigated using fluorescence staining, transmission electron microscopy, flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). Then qRT-PCR, western blotting, immunofluorescence staining and gene knockdown experiments were performed to investigate the expression and distribution of YAP and its correlation with PDLSCs proliferation. The effects of cytoskeleton dynamics on YAP nuclear translocation were subsequently explored by adding cytoskeleton inhibitors. The effect of cytoskeleton dynamics on the expression of the LINC complex was proved through qRT-PCR and western blotting. After destroying the LINC complex by adenovirus, the effects of the LINC complex on YAP nuclear translocation and PDLSCs proliferation were investigated. Mitochondria-related detections were then performed to explore the role of mitochondria in YAP nuclear translocation. Finally, the in vitro results were verified by constructing orthodontic tooth movement models in Sprague-Dawley rats. RESULTS: TS enhanced the polymerization and stretching of F-actin, which upregulated the expression of the LINC complex. This further strengthened the pull on the nuclear envelope, enlarged the nuclear pore, and facilitated YAP's nuclear entry, thus enhancing the expression of proliferation-related genes. In this process, mitochondria were transported to the periphery of the nucleus along the reconstructed microtubules. They generated ATP to aid YAP's nuclear translocation and drove F-actin polymerization to a certain degree. When the LINC complex was destroyed, the nuclear translocation of YAP was inhibited, which limited PDLSCs proliferation, impeded periodontal tissue remodeling, and hindered tooth movement. CONCLUSIONS: Our study confirmed that appropriate TS could promote PDLSCs proliferation and periodontal tissue remodeling through the mechanically driven F-actin/LINC complex/YAP axis, which could provide theoretical guidance for seed cell expansion and for promoting healthy and effective tooth movement in clinical practice.


Assuntos
Citoesqueleto , Membrana Nuclear , Ligamento Periodontal , Células-Tronco , Animais , Humanos , Masculino , Ratos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proliferação de Células , Células Cultivadas , Citoesqueleto/metabolismo , Membrana Nuclear/metabolismo , Ligamento Periodontal/metabolismo , Ligamento Periodontal/citologia , Células-Tronco/metabolismo , Células-Tronco/citologia , Estresse Mecânico , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Sinalização YAP/metabolismo
5.
Commun Biol ; 7(1): 1064, 2024 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-39215206

RESUMO

Biophysical cues have the ability to enhance cellular signaling response to Bone Morphogenetic Proteins, an essential growth factor during bone development and regeneration. Yet, therapeutic application of Bone Morphogenetic Protein 2 (BMP2) is restricted due to uncontrolled side effects. An understanding of the temporal characteristics of mechanically regulated signaling events and underlying mechanism is lacking. Using a 3D bioreactor system in combination with a soft macroporous biomaterial substrate, we mimic the in vivo environment that BMP2 is acting in. We show that the intensity and duration of BMP2 signaling increases with increasing loading frequency in synchrony with the number and size of focal adhesions. Long-term mechanical stimulation increases the expression of BMP receptor type 1B, specific integrin subtypes and integrin clustering. Together, this triggered a short-lived mechanical echo that enhanced BMP2 signaling even when BMP2 is administered directly after mechanical stimulation, but not when it is applied after a resting period of ≥30 min. Interfering with cytoskeletal remodeling hinders focal adhesion remodeling verifying its critical role in shifting cells into a state of high BMP2 responsiveness. The design of biomaterials that exploit this potential locally at the site of injury will help to overcome current limitations of clinical growth factor treatment.


Assuntos
Proteína Morfogenética Óssea 2 , Citoesqueleto , Adesões Focais , Transdução de Sinais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/genética , Adesões Focais/metabolismo , Humanos , Citoesqueleto/metabolismo , Mecanotransdução Celular , Animais
6.
Int J Mol Sci ; 25(16)2024 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-39201657

RESUMO

Recently, magnetic fields (MFs) have received major attention due to their potential therapeutic applications and biological effects. This review provides a comprehensive analysis of the cellular and molecular impacts of MFs, with a focus on both in vitro and in vivo studies. We investigate the mechanisms by which MFs influence cell behavior, including modifications in gene expression, protein synthesis, and cellular signaling pathways. The interaction of MFs with cellular components such as ion channels, membranes, and the cytoskeleton is analyzed, along with their effects on cellular processes like proliferation, differentiation, and apoptosis. Molecular insights are offered into how MFs modulate oxidative stress and inflammatory responses, which are pivotal in various pathological conditions. Furthermore, we explore the therapeutic potential of MFs in regenerative medicine, cancer treatment, and neurodegenerative diseases. By synthesizing current findings, this article aims to elucidate the complex bioeffects of MFs, thereby facilitating their optimized application in medical and biotechnological fields.


Assuntos
Campos Magnéticos , Humanos , Animais , Estresse Oxidativo , Transdução de Sinais , Apoptose , Diferenciação Celular , Proliferação de Células , Citoesqueleto/metabolismo
7.
Cell Mol Life Sci ; 81(1): 354, 2024 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-39158743

RESUMO

Mature neurons have stable dendritic architecture, which is essential for the nervous system to operate correctly. The ability to undergo structural plasticity, required to support adaptive processes like memory formation, is still present in mature neurons. It is unclear what molecular and cellular processes control this delicate balance between dendritic structural plasticity and stabilization. Failures in the preservation of optimal dendrite structure due to atrophy or maladaptive plasticity result in abnormal connectivity and are associated with various neurological diseases. Vascular endothelial growth factor D (VEGFD) is critical for the maintenance of mature dendritic trees. Here, we describe how VEGFD affects the neuronal cytoskeleton and demonstrate that VEGFD exerts its effects on dendrite stabilization by influencing the actin cortex and reducing microtubule dynamics. Further, we found that during synaptic activity-induced structural plasticity VEGFD is downregulated. Our findings revealed that VEGFD, acting on its cognate receptor VEGFR3, opposes structural changes by negatively regulating dendrite growth in cultured hippocampal neurons and in vivo in the adult mouse hippocampus with consequences on memory formation. A phosphoproteomic screening identified several regulatory proteins of the cytoskeleton modulated by VEGFD. Among the actin cortex-associated proteins, we found that VEGFD induces dephosphorylation of ezrin at tyrosine 478 via activation of the striatal-enriched protein tyrosine phosphatase (STEP). Activity-triggered structural plasticity of dendrites was impaired by expression of a phospho-deficient mutant ezrin in vitro and in vivo. Thus, VEGFD governs the equilibrium between stabilization and plasticity of dendrites by acting as a molecular brake of structural remodeling.


Assuntos
Dendritos , Hipocampo , Plasticidade Neuronal , Transdução de Sinais , Animais , Dendritos/metabolismo , Camundongos , Hipocampo/metabolismo , Hipocampo/citologia , Camundongos Endogâmicos C57BL , Células Cultivadas , Citoesqueleto/metabolismo , Masculino , Neurônios/metabolismo , Neurônios/citologia , Actinas/metabolismo , Fosforilação , Microtúbulos/metabolismo
8.
Cells ; 13(16)2024 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-39195282

RESUMO

Human cancers express altered levels of actin-binding cytoskeletal filamin A (FLNA) protein. FLNA in mammals consists of an actin-binding domain at its N-terminus that is followed by 24 immunoglobulin-like repeat modules interrupted by two hinge regions between repeats 15-16 and 23-24. Cleavage of these hinge regions produces a naturally occurring C-terminal 90 kDa fragment of FLNA (FLNACT) that physically interacts with multiple proteins with diverse functions. This cleavage leads to actin cytoskeleton remodeling, which in turn contributes to cellular signaling, nucleocytoplasmic shuttling of transcriptional factors and nuclear receptors, and regulation of their transcriptional activities that are important for initiation and progression of cancers. Therefore, recent studies have proposed blocking FLNA cleavage as a means of cancer therapy. Here, we update how FLNA cleavage has been targeted by different approaches and their potential implications for future treatment of human cancers.


Assuntos
Filaminas , Neoplasias , Filaminas/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Citoesqueleto/metabolismo , Citoesqueleto de Actina/metabolismo
9.
Front Immunol ; 15: 1411957, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39114656

RESUMO

Introduction: CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Methods: Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. Results: We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Discussion: Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.


Assuntos
Linfócitos T CD8-Positivos , Citosol , Ativação Linfocitária , Mitocôndrias , Biossíntese de Proteínas , Receptores de Antígenos de Linfócitos T , Humanos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Ativação Linfocitária/imunologia , Citosol/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/imunologia , Citoesqueleto/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Ribossomos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
10.
Int J Mol Sci ; 25(15)2024 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-39125632

RESUMO

The behavior and presence of actin-regulating proteins are characteristic of various clinical diseases. Changes in these proteins significantly impact the cytoskeletal and regenerative processes underlying pathological changes. Pituitary adenylate cyclase-activating polypeptide (PACAP), a cytoprotective neuropeptide abundant in the nervous system and endocrine organs, plays a key role in neuron differentiation and migration by influencing actin. This study aims to elucidate the role of PACAP as an actin-regulating polypeptide, its effect on actin filament formation, and the underlying regulatory mechanisms. We examined PACAP27, PACAP38, and PACAP6-38, measuring their binding to actin monomers via fluorescence spectroscopy and steady-state anisotropy. Functional polymerization tests were used to track changes in fluorescent intensity over time. Unlike PACAP27, PACAP38 and PACAP6-38 significantly reduced the fluorescence emission of Alexa488-labeled actin monomers and increased their anisotropy, showing nearly identical dissociation equilibrium constants. PACAP27 showed weak binding to globular actin (G-actin), while PACAP38 and PACAP6-38 exhibited robust interactions. PACAP27 did not affect actin polymerization, but PACAP38 and PACAP6-38 accelerated actin incorporation kinetics. Fluorescence quenching experiments confirmed structural changes upon PACAP binding; however, all studied PACAP fragments exhibited the same effect. Our findings indicate that PACAP38 and PACAP6-38 strongly bind to G-actin and significantly influence actin polymerization. Further studies are needed to fully understand the biological significance of these interactions.


Assuntos
Actinas , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Espectrometria de Fluorescência , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Actinas/metabolismo , Actinas/química , Animais , Espectrometria de Fluorescência/métodos , Citoesqueleto/metabolismo , Ligação Proteica , Citoesqueleto de Actina/metabolismo , Humanos , Cinética
11.
J Cell Sci ; 137(16)2024 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-39206824

RESUMO

Intermediate filaments (IFs) comprise a large family of versatile cytoskeletal proteins, divided into six subtypes with tissue-specific expression patterns. IFs have a wide repertoire of cellular functions, including providing structural support to cells, as well as active roles in mechanical support and signaling pathways. Consequently, defects in IFs are associated with more than 100 diseases. In this Cell Science at a Glance article, we discuss the established classes of IFs and their general features, their functions beyond structural support, and recent advances in the field. We also highlight their involvement in disease and potential use as clinical markers of pathological conditions. Finally, we provide our view on current knowledge gaps and the future directions of the IF field.


Assuntos
Filamentos Intermediários , Filamentos Intermediários/metabolismo , Humanos , Animais , Proteínas de Filamentos Intermediários/metabolismo , Proteínas de Filamentos Intermediários/genética , Transdução de Sinais , Citoesqueleto/metabolismo
12.
PLoS Genet ; 20(8): e1011219, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39173071

RESUMO

Protein tyrosine phosphatases non-receptor type (PTPNs) have been studied extensively in the context of the adaptive immune system; however, their roles beyond immunoregulation are less well explored. Here we identify novel functions for the conserved C. elegans phosphatase PTPN-22, establishing its role in nematode molting, cell adhesion, and cytoskeletal regulation. Through a non-biased genetic screen, we found that loss of PTPN-22 phosphatase activity suppressed molting defects caused by loss-of-function mutations in the conserved NIMA-related kinases NEKL-2 (human NEK8/NEK9) and NEKL-3 (human NEK6/NEK7), which act at the interface of membrane trafficking and actin regulation. To better understand the functions of PTPN-22, we carried out proximity labeling studies to identify candidate interactors of PTPN-22 during development. Through this approach we identified the CDC42 guanine-nucleotide exchange factor DNBP-1 (human DNMBP) as an in vivo partner of PTPN-22. Consistent with this interaction, loss of DNBP-1 also suppressed nekl-associated molting defects. Genetic analysis, co-localization studies, and proximity labeling revealed roles for PTPN-22 in several epidermal adhesion complexes, including C. elegans hemidesmosomes, suggesting that PTPN-22 plays a broad role in maintaining the structural integrity of tissues. Localization and proximity labeling also implicated PTPN-22 in functions connected to nucleocytoplasmic transport and mRNA regulation, particularly within the germline, as nearly one-third of proteins identified by PTPN-22 proximity labeling are known P granule components. Collectively, these studies highlight the utility of combined genetic and proteomic approaches for identifying novel gene functions.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Caenorhabditis elegans/genética , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Humanos , Muda/genética , Adesão Celular/genética , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Regulação da Expressão Gênica no Desenvolvimento , Citoesqueleto/metabolismo , Citoesqueleto/genética , Mutação com Perda de Função
14.
Proc Natl Acad Sci U S A ; 121(33): e2401816121, 2024 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-39106306

RESUMO

Many cytoskeletal networks consist of individual filaments that are organized into elaborate higher-order structures. While it is appreciated that the size and architecture of these networks are critical for their biological functions, much of the work investigating control over their assembly has focused on mechanisms that regulate the turnover of individual filaments through size-dependent feedback. Here, we propose a very different, feedback-independent mechanism to explain how yeast cells control the length of their actin cables. Our findings, supported by quantitative cell imaging and mathematical modeling, indicate that actin cable length control is an emergent property that arises from the cross-linked and bundled organization of the filaments within the cable. Using this model, we further dissect the mechanisms that allow cables to grow longer in larger cells and propose that cell length-dependent tuning of formin activity allows cells to scale cable length with cell length. This mechanism is a significant departure from prior models of cytoskeletal filament length control and presents a different paradigm to consider how cells control the size, shape, and dynamics of higher-order cytoskeletal structures.


Assuntos
Citoesqueleto , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Citoesqueleto/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Nat Commun ; 15(1): 7023, 2024 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-39174529

RESUMO

Neutrophil infiltration and subsequent extracellular trap formation (NETosis) is a contributing factor in sterile inflammation. Furthermore, neutrophil extracellular traps (NETs) are prothrombotic, as they provide a scaffold for platelets and red blood cells to attach to. In circulation, neutrophils are constantly exposed to hemodynamic forces such as shear stress, which in turn regulates many of their biological functions such as crawling and NETosis. However, the mechanisms that mediate mechanotransduction in neutrophils are not fully understood. In this study, we demonstrate that shear stress induces NETosis, dependent on the shear stress level, and increases the sensitivity of neutrophils to NETosis-inducing agents such as adenosine triphosphate and lipopolysaccharides. Furthermore, shear stress increases intracellular calcium levels in neutrophils and this process is mediated by the mechanosensitive ion channel Piezo1. Activation of Piezo1 in response to shear stress mediates calpain activity and cytoskeleton remodeling, which consequently induces NETosis. Thus, activation of Piezo1 in response to shear stress leads to a stepwise sequence of cellular events that mediates NETosis and thereby places neutrophils at the centre of localized inflammation and prothrombotic effects.


Assuntos
Cálcio , Armadilhas Extracelulares , Canais Iônicos , Mecanotransdução Celular , Neutrófilos , Estresse Mecânico , Neutrófilos/metabolismo , Canais Iônicos/metabolismo , Canais Iônicos/genética , Humanos , Armadilhas Extracelulares/metabolismo , Cálcio/metabolismo , Trifosfato de Adenosina/metabolismo , Calpaína/metabolismo , Lipopolissacarídeos/farmacologia , Citoesqueleto/metabolismo , Infiltração de Neutrófilos , Inflamação/metabolismo
16.
Int Immunopharmacol ; 141: 112950, 2024 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-39159563

RESUMO

Multiple myeloma (MM) is an incurable plasma cell malignancy that has prompted investigations into new potential therapeutic avenues. Epigallocatechin-3-gallate (EGCG), a major component of green tea, confers antioxidant, anti-inflammatory, and anti-tumor properties. Previous studies have shown that EGCG inhibits proliferation and induces apoptosis of multiple myeloma cells, however its underlying molecular mechanisms are largely unknown. In this study, we accordingly sought to examine the therapeutic effects and underlying mechanisms of EGCG on MM. Initially, using CCK8 (Cell Counting Kit-8) assays and Annexin V-FITC/PI staining, we demonstrated that EGCG dose-dependently reduced cell viability and induced apoptosis in the MM cell lines MM.1S and RPMI 8226. Subsequently, mRNA sequencing of EGCG-treated MM.1S cells revealed a significant upregulation of genes associated with endoplasmic reticulum stress (ERS), including P-eIF2α (phosphorylation-eukaryotic translation initiation factor 2 alpha), ATF4 (activating transcription factor 4), CHOP (C/EBP homologous protein, DDIT3), and PUMA (p53 upregulated modulator of apoptosis, BBC3), which were confirmed at the protein level by western blotting. Furthermore, treatment with the eIF2α inhibitor ISRIB reduced the rates of EGCG-induced apoptosis and promoted increases in the protein expression of all four ER stress-related molecules in MM cells. Additionally, mRNA-seq data revealed a downregulation of α-Tubulin 1b (TUBA1B) expression in EGCG-treated MM cells, which was confirmed by western blotting and immunofluorescence analyses. Moreover, we utilized a mouse model to show that EGCG inhibited myeloma tumor growth, which was inhibited by ISRIB. In summary, the findings of this novel study indicated that EGCG promotes apoptosis of MM cells, both via activation of the ER stress pathway and disruption of cytoskeletal integrity. These findings highlight the multi-faceted anti-tumor effects of EGCG and its potential clinical application in MM treatment.


Assuntos
Apoptose , Catequina , Estresse do Retículo Endoplasmático , Mieloma Múltiplo , Catequina/análogos & derivados , Catequina/farmacologia , Catequina/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Humanos , Linhagem Celular Tumoral , Camundongos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Fator de Transcrição CHOP/metabolismo , Fator de Transcrição CHOP/genética , Sobrevivência Celular/efeitos dos fármacos
17.
Matrix Biol ; 133: 77-85, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39147247

RESUMO

To form blood vessels, endothelial cells rearrange their cytoskeleton, generate traction stresses, migrate, and proliferate, all of which require energy. Despite these energetic costs, stiffening of the extracellular matrix promotes tumor angiogenesis and increases cell contractility. However, the interplay between extracellular matrix, cell contractility, and cellular energetics remains mechanistically unclear. Here, we utilized polyacrylamide substrates with various stiffnesses, a real-time biosensor of ATP, and traction force microscopy to show that endothelial cells exhibit increasing traction forces and energy usage trend as substrate stiffness increases. Inhibition of cytoskeleton reorganization via ROCK inhibition resulted in decreased cellular energy efficiency, and an opposite trend was found when cells were treated with manganese to promote integrin affinity. Altogether, our data reveal a link between matrix stiffness, cell contractility, and cell energetics, suggesting that endothelial cells on stiffer substrates can better convert intracellular energy into cellular traction forces. Given the critical role of cellular metabolism in cell function, our study also suggests that not only energy production but also the efficiency of its use plays a vital role in regulating cell behaviors and may help explain how increased matrix stiffness promotes angiogenesis.


Assuntos
Resinas Acrílicas , Citoesqueleto , Células Endoteliais , Matriz Extracelular , Matriz Extracelular/metabolismo , Humanos , Resinas Acrílicas/química , Células Endoteliais/metabolismo , Citoesqueleto/metabolismo , Metabolismo Energético , Trifosfato de Adenosina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/antagonistas & inibidores , Integrinas/metabolismo , Integrinas/genética , Manganês/metabolismo , Movimento Celular , Adesão Celular
18.
Aging (Albany NY) ; 16(16): 11776-11795, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39197170

RESUMO

Crucial for skin homeostasis, synthesis and degradation of extracellular matrix components are orchestrated by dermal fibroblasts. During aging, alterations of component expression, such as collagens and enzymes, lead to reduction of the mechanical cutaneous tension and defects of skin wound healing. The aim of this study was to better understand the molecular alterations underwent by fibroblasts during aging by comparing secretomic and proteomic signatures of fibroblasts from young (<35years) and aged (>55years) skin donors, in quiescence or TGF-stimulated conditions, using HLPC/MS. The comparison of the secretome from young and aged fibroblasts revealed that 16 proteins in resting condition, and 11 proteins after a 24h-lasting TGF-ß1-treatment, were expressed in significant different ways between the two cell groups (fold change>2, p-value <0.05), with a 77% decrease in the number of secreted proteins in aged cells. Proteome comparison between young and aged fibroblasts identified a significant change of 63 proteins in resting condition, and 73 proteins in TGF-ß1-stimulated condition, with a 67% increase in the number of proteins in aged fibroblasts. The majority of the differentially-expressed molecules belongs to the cytoskeleton-associated proteins and aging was characterized by an increase in Coronin 1C (CORO1C), and Filamin B (FLNB) expression in fibroblasts together with a decrease in Cofilin (CFL1), and Actin alpha cardiac muscle 1 (ACTC1) detection in aged cells, these proteins being involved in actin-filament polymerization and sharing co-activity in cell motility. Our present data reinforce knowledge about an age-related alteration in the synthesis of major proteins linked to the migratory and contractile functions of dermal human fibroblasts.


Assuntos
Envelhecimento , Citoesqueleto , Fibroblastos , Proteômica , Humanos , Fibroblastos/metabolismo , Citoesqueleto/metabolismo , Adulto , Pessoa de Meia-Idade , Envelhecimento/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Idoso , Pele/metabolismo , Pele/citologia , Proteoma/metabolismo , Células Cultivadas , Masculino , Secretoma/metabolismo , Feminino , Derme/citologia , Derme/metabolismo
19.
Acta Biomater ; 186: 300-315, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-39127326

RESUMO

Recently, a variety of microenvironmental biophysical stimuli have been proved to play a crucial role in regulating cell functions. Among them, morpho-physical cues, like curvature, are emerging as key regulators of cellular behavior. Changes in substrate curvature have been shown to impact the arrangement of Focal Adhesions (FAs), influencing the direction and intensity of cytoskeleton generated forces and resulting in an overall alteration of cell mechanical identity. In their native environment, cells encounter varying degrees of substrate curvature, and in specific organs, they are exposed to dynamic changes of curvature due to periodic tissue deformation. However, the mechanism by which cells perceive substrate curvature remains poorly understood. To this aim, a micro-pneumatic device was designed and implemented. This device enables the controlled application of substrate curvature, both statically and dynamically. Employing a combined experimental and simulative approach, human adipose-derived stem cells were exposed to controlled curvature intensity and frequency. During this exposure, measurements were taken on FAs extension and orientation, cytoskeleton organization and cellular/nuclear alignment. The data clearly indicated a significant influence of the substrate curvature on cell adhesion processes. These findings contribute to a better understanding of the mechanisms through which cells perceive and respond to substrate curvature signals. STATEMENT OF SIGNIFICANCE: This work is our contribution to the comprehension of substrate curvature's function as a crucial regulator of cell adhesion at the scale of focal adhesions and cell mechanical identity. In recent years, a large body of knowledge is continuously growing providing comprehension of the role of various microenvironmental biophysical stimuli in regulating cell functions. Nevertheless, little is known about the role of substrate curvature, in particular, when cells are exposed to this stimulus in a dynamic manner. To address the role of substrate curvature on cellular behavior, a micro-pneumatic device was designed and implemented. This device enables the controlled application of substrate curvature, both statically and dynamically. The experiment data made it abundantly evident that the substrate curvature had a major impact on the mechanisms involved in cell adhesion.


Assuntos
Adesão Celular , Adesões Focais , Humanos , Adesões Focais/metabolismo , Citoesqueleto/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Tecido Adiposo/citologia
20.
Curr Opin Cell Biol ; 90: 102408, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39121805

RESUMO

Biologists have the capability to edit a genome at the nanometer scale and then observe whether or not the edit affects the structure of a developing organ or organism at the centimeter scale. Our understanding of the underlying mechanisms driving this emergent phenomenon from a multiscale perspective remains incomplete. This review focuses predominantly on recent experimental developments in uncovering the mechanical interplay between the chromatin and cell scale since mechanics plays a major role in determining nuclear, cellular, and tissue structure. Here, we discuss the generation and transmission of forces through the cytoskeleton, affecting chromatin diffusivity and organization. Decoding such pieces of these multiscale connections lays the groundwork for solving the genotype-to-phenotype puzzle in biology.


Assuntos
Cromatina , Citoesqueleto , Cromatina/metabolismo , Cromatina/química , Humanos , Animais , Citoesqueleto/metabolismo , Montagem e Desmontagem da Cromatina
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