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Life Science Alliance Aug 2024Dynamic rearrangements of the F-actin cytoskeleton are a hallmark of tumor metastasis. Thus, proteins that govern F-actin rearrangements are of major interest for...
Dynamic rearrangements of the F-actin cytoskeleton are a hallmark of tumor metastasis. Thus, proteins that govern F-actin rearrangements are of major interest for understanding metastasis and potential therapies. We hypothesized that the unique F-actin binding and bundling protein SWAP-70 contributes importantly to metastasis. Orthotopic, ectopic, and short-term tail vein injection mouse breast and lung cancer models revealed a strong positive dependence of lung and bone metastasis on SWAP-70. Breast cancer cell growth, migration, adhesion, and invasion assays revealed SWAP-70's key role in these metastasis-related cell features and the requirement for SWAP-70 to bind F-actin. Biophysical experiments showed that tumor cell stiffness and deformability are negatively modulated by SWAP-70. Together, we present a hitherto undescribed, unique F-actin modulator as an important contributor to tumor metastasis.
Topics: Animals; Actins; Mice; Humans; Female; Cell Line, Tumor; Neoplasm Metastasis; Breast Neoplasms; Lung Neoplasms; Microfilament Proteins; Cell Movement; Actin Cytoskeleton; Cell Proliferation; Cell Adhesion; Protein Binding
PubMed: 38760173
DOI: 10.26508/lsa.202302307 -
Parasite (Paris, France) 2024Eimeria tenella is an obligate intracellular parasite which causes great harm to the poultry breeding industry. Protein phosphorylation plays a vital role in host...
Quantitative phosphoproteomic analysis of chicken DF-1 cells infected with Eimeria tenella, using tandem mass tag (TMT) and parallel reaction monitoring (PRM) mass spectrometry.
Eimeria tenella is an obligate intracellular parasite which causes great harm to the poultry breeding industry. Protein phosphorylation plays a vital role in host cell-E. tenella interactions. However, no comprehensive phosphoproteomic analyses of host cells at various phases of E. tenella infection have been published. In this study, quantitative phosphoproteomic analysis of chicken embryo DF-1 fibroblasts that were uninfected (UI) or infected with E. tenella for 6 h (PI6, the early invasion phase) or 36 h (PI36, the trophozoite development phase) was conducted. A total of 10,122 phosphopeptides matched to 3,398 host cell phosphoproteins were identified and 13,437 phosphorylation sites were identified. Of these, 491, 1,253, and 275 differentially expressed phosphorylated proteins were identified in the PI6/UI, PI36/UI, and PI36/PI6 comparisons, respectively. KEGG pathway enrichment analysis showed that E. tenella modulated host cell processes through phosphorylation, including focal adhesion, regulation of the actin cytoskeleton, and FoxO signaling to support its early invasion phase, and modulating adherens junctions and the ErbB signaling pathway to favor its trophozoite development. These results enrich the data on the interaction between E. tenella and host cells and facilitate a better understanding of the molecular mechanisms underlying host-parasite relationships.
Topics: Animals; Eimeria tenella; Chickens; Proteomics; Phosphoproteins; Tandem Mass Spectrometry; Phosphorylation; Fibroblasts; Cell Line; Poultry Diseases; Host-Parasite Interactions; Coccidiosis; Chick Embryo; Signal Transduction
PubMed: 38759153
DOI: 10.1051/parasite/2024027 -
The Journal of Cell Biology Jun 2024There has long been conflicting evidence as to how bundled actin filaments, found in cellular structures such as filopodia, are disassembled. In this issue, Chikireddy...
There has long been conflicting evidence as to how bundled actin filaments, found in cellular structures such as filopodia, are disassembled. In this issue, Chikireddy et al. (https://doi.org/10.1083/jcb.202312106) provide a detailed in vitro analysis of the steps involved in fragmentation of fascin-bundled actin filaments and propose a novel mechanism for severing two-filament bundles.
Topics: Actin Cytoskeleton; Microfilament Proteins; Actins; Pseudopodia; Humans; Animals; Carrier Proteins
PubMed: 38748453
DOI: 10.1083/jcb.202403191 -
Zoological Research May 2024Proper regulation of synapse formation and elimination is critical for establishing mature neuronal circuits and maintaining brain function. Synaptic abnormalities, such...
Proper regulation of synapse formation and elimination is critical for establishing mature neuronal circuits and maintaining brain function. Synaptic abnormalities, such as defects in the density and morphology of postsynaptic dendritic spines, underlie the pathology of various neuropsychiatric disorders. Protocadherin 17 (PCDH17) is associated with major mood disorders, including bipolar disorder and depression. However, the molecular mechanisms by which PCDH17 regulates spine number, morphology, and behavior remain elusive. In this study, we found that PCDH17 functions at postsynaptic sites, restricting the number and size of dendritic spines in excitatory neurons. Selective overexpression of PCDH17 in the ventral hippocampal CA1 results in spine loss and anxiety- and depression-like behaviors in mice. Mechanistically, PCDH17 interacts with actin-relevant proteins and regulates actin filament (F-actin) organization. Specifically, PCDH17 binds to ROCK2, increasing its expression and subsequently enhancing the activity of downstream targets such as LIMK1 and the phosphorylation of cofilin serine-3 (Ser3). Inhibition of ROCK2 activity with belumosudil (KD025) ameliorates the defective F-actin organization and spine structure induced by PCDH17 overexpression, suggesting that ROCK2 mediates the effects of PCDH17 on F-actin content and spine development. Hence, these findings reveal a novel mechanism by which PCDH17 regulates synapse development and behavior, providing pathological insights into the neurobiological basis of mood disorders.
Topics: Animals; Mice; Actin Cytoskeleton; Cadherins; Dendritic Spines; Gene Expression Regulation; rho-Associated Kinases; Protocadherins
PubMed: 38747058
DOI: 10.24272/j.issn.2095-8137.2024.055 -
BioRxiv : the Preprint Server For... May 2024Asthma is a chronic inflammatory disease of the airways that involves crosstalk between myeloid-derived regulatory cells (MDRCs) and CD4+ T cells. Although small...
RATIONALE
Asthma is a chronic inflammatory disease of the airways that involves crosstalk between myeloid-derived regulatory cells (MDRCs) and CD4+ T cells. Although small extracellular vesicles (sEVs) are known to mediate cell-cell communication, the role of sEV signaling via mitochondria in perpetuating asthmatic airway inflammation is unknown.
OBJECTIVES
We investigated the effects of MDRC-derived exosomes on dysregulated T cell responses in asthmatics.
METHODS
Small extracellular vesicles isolated from bronchoalveolar lavage fluid or airway MDRCs of mild to moderate asthmatics or healthy controls were co-cultured with autologous peripheral and airway CD4+ T lymphocytes. sEV internalization, sEV-mediated transfer of mitochondria targeted GFP to T cells, sEV mitochondrial signaling, and subsequent activation, proliferation and polarization of CD4+ T lymphocytes to Th1, Th2 and Th17 subsets were assessed.
MEASUREMENTS AND MAIN RESULTS
Airway MDRC-derived sEVs from asthmatics mediated T cell receptor engagement and transfer of mitochondria that induced antigen-specific activation and polarization into Th17 and Th2 cells, drivers of chronic airway inflammation in asthma. CD4+ T cells internalized sEVs containing mitochondria predominantly by membrane fusion, and blocking mitochondrial oxidant signaling in MDRC-derived exosomes mitigated T cell activation. Reactive oxygen species-mediated signaling that elicited T cell activation in asthmatics was sEV-dependent. A Drp1-dependent mitochondrial fission in pro-inflammatory MDRCs promoted mitochondrial packaging within sEVs, which then co-localized with the polarized actin cytoskeleton and mitochondrial networks in the organized immune synapse of recipient T cells.
CONCLUSIONS
Our studies indicate a previously unrecognized role for mitochondrial fission and exosomal mitochondrial transfer in dysregulated T cell activation and Th cell differentiation in asthma which could constitute a novel therapeutic target.
PubMed: 38746361
DOI: 10.1101/2024.04.30.589227 -
BioRxiv : the Preprint Server For... May 2024The plasma membrane and the underlying skeleton form a protective barrier for eukaryotic cells. The molecules forming this complex composite material constantly...
The plasma membrane and the underlying skeleton form a protective barrier for eukaryotic cells. The molecules forming this complex composite material constantly rearrange under mechanical stress to confer this protective capacity. One of those molecules, spectrin, is ubiquitous in the membrane skeleton and primarily located proximal to the inner leaflet of the plasma membrane and engages in protein-lipid interactions via a set of membrane-anchoring domains. Spectrin is linked by short actin filaments and its conformation varies in different types of cells. In this work, we developed a generalized network model for the membrane skeleton integrated with myosin contractility and membrane mechanics to investigate the response of the spectrin meshwork to mechanical loading. We observed that the force generated by membrane bending is important to maintain a smooth skeletal structure. This suggests that the membrane is not just supported by the skeleton, but has an active contribution to the stability of the cell structure. We found that spectrin and myosin turnover are necessary for the transition between stress and rest states in the skeleton. Our model reveals that the actin-spectrin meshwork dynamics are balanced by the membrane forces with area constraint and volume restriction promoting the stability of the membrane skeleton. Furthermore, we showed that cell attachment to the substrate promotes shape stabilization. Thus, our proposed model gives insight into the shared mechanisms of the membrane skeleton associated with myosin and membrane that can be tested in different types of cells.
PubMed: 38746295
DOI: 10.1101/2024.04.29.591779 -
BioRxiv : the Preprint Server For... May 2024The actin filament (F-actin) bundling protein fascin-1 is highly enriched in many metastatic cancers. Fascin's contribution to metastasis have been ascribed to its...
The actin filament (F-actin) bundling protein fascin-1 is highly enriched in many metastatic cancers. Fascin's contribution to metastasis have been ascribed to its enhancement of cell migration and invasion. However, mouse genetic studies clearly point to functions also in tumorigenesis, yet without mechanistic underpinnings. Here, we show that fascin expression promotes the formation of a non-canonical signaling complex that enables anchorage-independent proliferation. This complex shares similarities to focal adhesions and we refer to them as pseudo-adhesion signaling scaffolds (PASS). PASS are enriched with tyrosine phosphorylated proteins and require fascin's F-actin-bundling activity for its assembly. PASS serve as hubs for the Rac1/PAK/JNK proliferation signaling axis, driven by PASS-associated Rac-specific GEFs. Experimental disruption of either fascin or RacGEF function abrogates sustained proliferation of aggressive cancers and . These results add a new molecular element to the growing arsenal of metabolic and oncogenic signaling programs regulated by the cytoskeleton architecture.
PubMed: 38746129
DOI: 10.1101/2024.05.04.592404 -
Scientific Reports May 2024MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal...
MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-β (PDGFR-β)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-β. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-β. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p.
Topics: Animals; MicroRNAs; Fibrosis; Mice; Mice, Knockout; Receptor, Platelet-Derived Growth Factor beta; Kidney; Mesenchymal Stem Cells; Ribonuclease III; Signal Transduction; Kidney Diseases; DEAD-box RNA Helicases; Male
PubMed: 38745066
DOI: 10.1038/s41598-024-61560-y -
Materials Today. Bio Jun 2024The mechanical environment of vascular endothelial cells (ECs) encompasses a wide range of curvatures due to variations in blood vessel diameters. Integrins, key...
The mechanical environment of vascular endothelial cells (ECs) encompasses a wide range of curvatures due to variations in blood vessel diameters. Integrins, key mediators of cell-matrix interactions, establish connections between the extracellular matrix and the actin cytoskeleton, influencing diverse cellular behaviors. In this study, we explored the impact of spatial confinement on human umbilical vein ECs (HUVECs) cultured within three-dimensional hydrogel microgrooves of varying curvatures and the underlying role of integrins in mediating cellular responses. Employing maskless lithography, we successfully fabricated precise and wall curvatures-controlled hydrogel microgrooves, conferring spatial constraints on the cells. Our investigations revealed substantial alterations in HUVEC behavior within the hydrogel microgrooves with varying sidewall curvatures, marked by reduced cell size, enhanced orientation, and increased apoptosis. Interestingly, microgroove curvature emerged as a crucial factor influencing cell orientation and apoptosis, with rectangular microgrooves eliciting distinct changes in cell orientation, while ring-form microgrooves exhibited higher apoptosis rates. The side-wall effect in the 20 μm region near the microgroove wall had the greatest influence on cell orientation and apoptosis. HUVECs within the microgrooves exhibited elevated integrin expression, and inhibition of αV-integrin by cilengitide significantly curtailed cell apoptosis without affecting proliferation. Additionally, integrin-mediated cell traction force closely correlated with the spatial confinement effect. Cilengitide not only reduced integrin and focal adhesion expression but also attenuated cell traction force and cytoskeletal actin filament alignment. Overall, our findings elucidate the spatial confinement of ECs in hydrogel microgrooves and underscores the pivotal role of integrins, particularly αV-integrin, in mediating cell traction force and apoptosis within this microenvironment.
PubMed: 38736613
DOI: 10.1016/j.mtbio.2024.101074 -
Cancer Cell International May 2024Forkhead box E1 (FOXE1), also known as thyroid transcription factor 2 (TTF-2), belongs to a large family of forkhead transcription factors. It plays important roles in...
Forkhead box E1 (FOXE1), also known as thyroid transcription factor 2 (TTF-2), belongs to a large family of forkhead transcription factors. It plays important roles in embryogenesis, cell growth, and differentiation. Cancer-specific FOXE1 hypermethylation events have been identified in several cancers. However, the expression and function of FOXE1 in the tumorigenesis of colorectal cancer remain still unknown. In this study, we examined FOXE1 expression and methylation in normal colon mucosa, colorectal cancer (CRC) cell lines, and primary tumors by immunohistochemistry, semi-quantitative RT-PCR, methylation-specific PCR, and bisulfite genomic sequencing. We found that FOXE1 was frequently methylated and silenced in CRC cell lines and was downregulated in CRC tissues compared with paired adjacent non-tumor tissues. Meanwhile, low FOXE1 expression was significantly correlated with lymph node metastasis and advanced TNM stages, indicating its potential as a tumor marker. Subsequently, we established colon cancer cell lines with stable FOXE1 expression to observe the biological effect on colorectal cancer, including cell growth, migration, actin cytoskeleton, and growth of human colorectal xenografts in nude mice. Ectopic expression of FOXE1 could suppress tumor cell growth and migration and affect the organization of the actin cytoskeleton together with suppressing tumorigenicity in vivo. FOXE1 methylation was frequently seen in association with a complete absence of or downregulated gene expression, and FOXE1 plays a suppressive role in the development and progression of colorectal cancer.
PubMed: 38734646
DOI: 10.1186/s12935-024-03352-y