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FASEB Journal : Official Publication of... Mar 2024Stem cells respond and remember mechanical cues from the microenvironment, which modulates their therapeutic effects. Chromatin organization and energy metabolism...
Stem cells respond and remember mechanical cues from the microenvironment, which modulates their therapeutic effects. Chromatin organization and energy metabolism regulate the stem cell fate induced by mechanical cues. However, the mechanism of mechanical memory is still unclear. This study aimed to investigate the effects of mechanical amplitude, frequency, duration, and stretch cycle on mechanical memory in mesenchymal stem cells. It showed that the amplitude was the dominant parameter to the persistence of cell alignment. F-actin, paxillin, and nuclear deformation are more prone to be remolded than cell alignment. Stretching induces transcriptional memory, resulting in greater transcription upon subsequent reloading. Cell metabolism displays mechanical memory with sustained mitochondrial fusion and increased ATP production. The mechanical memory of chromatin condensation is mediated by histone H3 lysine 27 trimethylation, leading to much higher smooth muscle differentiation efficiency. Interestingly, mechanical memory can be transmitted based on direct cell-cell interaction, and stretched cells can remodel the metabolic homeostasis of static cells. Our results provide insight into the underlying mechanism of mechanical memory and its potential benefits for stem cell therapy.
Topics: Chromatin; Stress, Mechanical; Cell Differentiation; Mesenchymal Stem Cells; Muscle, Smooth; Cell Proliferation
PubMed: 38482729
DOI: 10.1096/fj.202302178R -
Investigative Ophthalmology & Visual... Mar 2024To explore the role of substrate stiffness and the mechanism beneath corneal endothelial cells' (CECs') stemness maintenance and differentiation.
PURPOSE
To explore the role of substrate stiffness and the mechanism beneath corneal endothelial cells' (CECs') stemness maintenance and differentiation.
METHODS
CECs were divided into central zone (8 mm trephined boundary) and peripheral zone (8 mm trephined edge with attached limbal). Two zones were analyzed by hematoxylin-eosin staining and scanning electron microscopy for anatomic structure. The elastic modulus of Descemet's membrane (DM) was analyzed by atomic force microscopy. Compressed type I collagen gels with different stiffness were constructed as an in vitro model system to test the role of stiffness on phenotype using cultured rabbit CECs. Cell morphology, expression and intracellular distribution of Yes-associated protein (YAP), differentiation (ZO-1, Na+/K+-ATPase), stemness (FOXD3, CD34, Sox2, Oct3/4), and endothelial-mesenchymal transition (EnMT) markers were analyzed by immunofluorescence, quantitative RT-PCR, and Western blot.
RESULTS
The results showed that the peripheral area of rabbit and human DM is softer than the central area ex vivo. Using the biomimetic extracellular matrix collagen gels in vitro model, we then demonstrated that soft substrate weakens the differentiation and EnMT in the culture of CECs. It was further proved by the inhibitor experiment that soft substrate enhances stemness maintenance via inhibition of paxillin-YAP signaling, which was activated on a stiff substrate.
CONCLUSIONS
Our findings confirm that substrate stiffness modulates the stemness maintenance and differentiation of CECs and suggest a potential strategy for CEC-based corneal tissue engineering.
Topics: Humans; Animals; Rabbits; Paxillin; Endothelium, Corneal; Endothelial Cells; Cornea; Sodium-Potassium-Exchanging ATPase; Gels
PubMed: 38466286
DOI: 10.1167/iovs.65.3.15 -
The Journal of Cell Biology Apr 2024Focal adhesions (FAs) are transmembrane protein assemblies mediating cell-matrix connection. Although protein liquid-liquid phase separation (LLPS) has been tied to the...
Focal adhesions (FAs) are transmembrane protein assemblies mediating cell-matrix connection. Although protein liquid-liquid phase separation (LLPS) has been tied to the organization and dynamics of FAs, the underlying mechanisms remain unclear. Here, we experimentally tune the LLPS of PXN/Paxillin, an essential scaffold protein of FAs, by utilizing a light-inducible Cry2 system in different cell types. In addition to nucleating FA components, light-triggered PXN LLPS potently activates integrin signaling and subsequently accelerates cell spreading. In contrast to the homotypic interaction-driven LLPS of PXN in vitro, PXN condensates in cells are associated with the plasma membrane and modulated by actomyosin contraction and client proteins of FAs. Interestingly, non-specific weak intermolecular interactions synergize with specific molecular interactions to mediate the multicomponent condensation of PXN and are efficient in promoting FA assembly and integrin signaling. Thus, our data establish an active role of the PXN phase transition into a condensed membrane-associated compartment in promoting the assembly/maturation of FAs.
Topics: Humans; Actin Cytoskeleton; Focal Adhesions; Integrins; Paxillin; Phase Separation
PubMed: 38466167
DOI: 10.1083/jcb.202209027 -
ACS Applied Materials & Interfaces Mar 2024The design of implantable biomaterials involves precise tuning of surface features because the early cellular fate on such engineered surfaces is highly influenced by...
The design of implantable biomaterials involves precise tuning of surface features because the early cellular fate on such engineered surfaces is highly influenced by many physicochemical factors [roughness, hydrophilicity, reactive oxygen species (ROS) responsiveness, etc.]. Herein, to enhance soft tissue integration for successful implantation, Ti substrates decorated with uniform layers of nanoceria (Ce), called Ti@Ce, were optimally developed by a simple and cost-effective in situ immersion coating technique. The characterization of Ti@Ce shows a uniform Ce distribution with enhanced roughness (∼3-fold increase) and hydrophilicity (∼4-fold increase) and adopted ROS-scavenging capacity by nanoceria coating. When human gingival fibroblasts were seeded on Ti@Ce under oxidative stress conditions, Ti@Ce supported cellular adhesion, spreading, and survivability by its cellular ROS-scavenging capacity. Mechanistically, the unique nanocoating resulted in higher expression of amphiphysin (a nanotopology sensor), paxillin (a focal adhesion protein), and cell adhesive proteins (collagen-1 and fibronectin). Ti@Ce also led to global chromatin condensation by decreasing histone 3 acetylation as an early differentiation feature. Transcriptome analysis by RNA sequencing confirmed the chromatin remodeling, antiapoptosis, antioxidant, cell adhesion, and TGF-β signaling-related gene signatures in Ti@Ce. As key fibroblast transcription (co)factors, Ti@Ce promotes serum response factor and MRTF-α nucleus localization. Considering all of this, it is proposed that the surface engineering approach using Ce could improve the biological properties of Ti implants, supporting their functioning at soft tissue interfaces and utilization as a bioactive implant for clinical conditions such as peri-implantitis.
Topics: Humans; Reactive Oxygen Species; Titanium; Cells, Cultured; Surface Properties; Cell Adhesion; Fibroblasts; Cerium
PubMed: 38466038
DOI: 10.1021/acsami.4c02119 -
BioRxiv : the Preprint Server For... Feb 2024Transmembrane signalling receptors, such as integrins, organise as nanoclusters that are thought to provide several advantages including, increasing avidity, sensitivity...
Transmembrane signalling receptors, such as integrins, organise as nanoclusters that are thought to provide several advantages including, increasing avidity, sensitivity (increasing the signal-to-noise ratio) and robustness (signalling above a threshold rather than activation by a single receptor) of the signal compared to signalling by single receptors. Compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, if nanoclusters function as signalling hubs remains poorly understood. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at sub-diffraction limited resolution of ~100nm length scale within a focal adhesion to examine the dynamics of diverse focal adhesion proteins. We show that (i) subregions of focal adhesions are enriched in immobile population of integrin β3 organised as nanoclusters, which (ii) in turn serve to organise nanoclusters of associated key adhesome proteins- vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signalling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct dynamics dependent on function. (iv) long-lived nanoclusters function as signalling hubs- wherein phosphorylated FAK and paxillin formed stable nanoclusters in close proximity to immobile integrin nanoclusters which are disassembled in response to inactivation signal by phosphatase PTPN12 (v) signalling takes place in response to an external signal such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. Taken together, these results demonstrate that signalling downstream of transmembrane receptors is organised as hubs of signalling proteins (FAK, paxillin, vinculin) seeded by nanoclusters of the transmembrane receptor (integrin).
PubMed: 38464288
DOI: 10.1101/2024.02.25.581925 -
Frontiers in Cell and Developmental... 2024Prostate cancer (PCa) is a leading male malignancy worldwide, often progressing to bone metastasis, with limited curative options. Extracellular vesicles (EVs) have...
Prostate cancer (PCa) is a leading male malignancy worldwide, often progressing to bone metastasis, with limited curative options. Extracellular vesicles (EVs) have emerged as key players in cancer communication and metastasis, promoting the formation of supportive microenvironments in distant sites. Our previous studies have highlighted the role of PCa EVs in modulating osteoblasts and facilitating tumor progression. However, the early pre-metastatic changes induced by PCa EVs within the bone microenvironment remain poorly understood. To investigate the early effects of repeated exposure to PCa EVs , mimicking EVs being shed from the primary tumor, PCa EVs isolated from cell line PC3MLuc2a were fluorescently labelled and repeatedly administered via tail vein injection to adult CD1 NuNu male mice for a period of 4 weeks. imagining, histological analysis and gene expression profiling were performed to assess the impact of PCa EVs on the bone microenvironment. We demonstrate for the first time that PCa EVs home to both bone and lymph nodes following repeated exposures. Furthermore, the accumulation of EVs within the bone leads to distinct molecular changes indicative of disrupted bone homeostasis (e.g., changes to signaling pathways such as Paxillin = 0.0163, Estrogen Receptor = 0.0271, RHOA = 0.0287, Ribonucleotide reductase = 0.0307 and ERK/MAPK = 0.0299). Changes in key regulators of these pathways were confirmed on human osteoblasts. In addition, our data compares the known gene signature of osteocytes and demonstrates a high proportion of overlap (52.2%), suggesting a potential role for this cell type in response to PCa EV exposure. No changes in bone histology or immunohistochemistry were detected, indicating that PCa EV mediated changes were induced at the molecular level. This study provides novel insights into the alterations induced by PCa EVs on the bone microenvironment. The observed molecular changes indicate changes in key pathways and suggest a role for osteocytes in these EV mediated early changes to bone. Further research to understand these early events may aid in the development of targeted interventions to disrupt the metastatic cascade in PCa.
PubMed: 38455075
DOI: 10.3389/fcell.2024.1354606 -
Role of steroid hormones in the maintenance of focal adhesions in bovine oviductal epithelial cells.Domestic Animal Endocrinology Jul 2024The oviduct, the organ of the female reproductive system where fertilization and early embryonic development occur, provides an optimal environment for the final...
The oviduct, the organ of the female reproductive system where fertilization and early embryonic development occur, provides an optimal environment for the final maturation of oocytes, storage, and sperm capacitation and transport of gametes and embryos. During the estrous cycle, the oviduct is affected by ovarian sex hormones, resulting in changes aimed at maintaining an appropriate microenvironment. Normal cell migration is tightly regulated, its role being essential for the development and maintenance of organ and tissue functions as well as for regeneration following injury. Due to their involvement in focal contact formations, focal adhesion kinase (PTK2) and paxillin (PXN) are key proteins in the study of cell migration and adhesion. The objective of this work was to compare the expression of PTK2 and PXN in oviductal cells along the estrous cycle and to determine if their expression is regulated by the presence of 17-β estradiol (E) and/or progesterone (P). No transcripts of PTK2 or of PXN were detected in cells corresponding to the luteal phase. Additionally, hormonal stimulation experiments on bovine oviductal cell cultures (BOECs) were carried out, where P inhibited the expression of both genes. Migration assays demonstrated that P reduced BOECs migration capacity. P treatment also reduced cell adhesion, while E increased the number of adhered cells. In conclusion, the presence of E and P regulates the expression of genes involved in the formation of focal contacts and modifies the migration and adhesion of BOECs. Understanding the effect of steroid hormones on BOECs is critical to grasp the impact of steroid control on oviductal function and its contribution to establishing successful pregnancies.
Topics: Animals; Female; Cattle; Estradiol; Focal Adhesions; Progesterone; Epithelial Cells; Fallopian Tubes; Paxillin; Cell Movement; Estrous Cycle; Cells, Cultured; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation; Oviducts
PubMed: 38433026
DOI: 10.1016/j.domaniend.2024.106839 -
British Journal of Cancer May 2024FOXL2 is a transcription factor expressed in ovarian granulosa cells. A somatic variant of FOXL2 (c.402 C > G, p.Cys134Trp) is the hallmark of adult-type granulosa...
BACKGROUND
FOXL2 is a transcription factor expressed in ovarian granulosa cells. A somatic variant of FOXL2 (c.402 C > G, p.Cys134Trp) is the hallmark of adult-type granulosa cell tumours.
METHODS
We generated KGN cell clones either heterozygous for this variant (MUT) or homozygous for the wild-type (WT) allele by CRISPR/Cas9 editing. They underwent RNA-Seq and bioinformatics analyses to uncover pathways impacted by deregulated genes. Cell morphology and migration were studied.
RESULTS
The differentially expressed genes (DEGs) between WT/MUT and WT/WT KGN cells (DEGs-), pointed to several dysregulated pathways, like TGF-beta pathway, cell adhesion and migration. Consistently, WT/MUT cells were rounder than WT/WT cells and displayed a different distribution of stress fibres and paxillin staining. A comparison of the DEGs- with those found when FOXL2 was knocked down (KD) in WT/WT KGN cells showed that most DEGs- cells were not so in the KD experiment, supporting a gain-of-function (GOF) scenario. MUT-FOXL2 also displayed a stronger interaction with SMAD3.
CONCLUSIONS
Our work, aiming at better understanding the GOF scenario, shows that the dysregulated genes and pathways are consistent with this idea. Besides, we propose that GOF might result from an enhanced interaction with SMAD3 that could underlie an ectopic capacity of mutated FOXL2 to bind SMAD4.
Topics: Forkhead Box Protein L2; Humans; Female; Granulosa Cell Tumor; Cell Line, Tumor; Cell Movement; Ovarian Neoplasms; Smad3 Protein; CRISPR-Cas Systems; Gene Expression Regulation, Neoplastic
PubMed: 38429437
DOI: 10.1038/s41416-024-02613-x -
Zhonghua Zhong Liu Za Zhi [Chinese... Feb 2024To investigate the role and the mechanism of Ras-associated binding protein23 (RAB23) in the migration and invasion of esophageal squamous cell carcinoma (ESCC) cells....
To investigate the role and the mechanism of Ras-associated binding protein23 (RAB23) in the migration and invasion of esophageal squamous cell carcinoma (ESCC) cells. RAB23 mRNA levels were measured in 16 pairs of ESCC and adjacent normal tissues via real-time polymerase chain reactions. RAB23 mRNA levels in the ESCC and adjacent normal tissues of dataset GSE20347 deposited in the Gene Expression Omnibus (GEO) database were also analyzed. Immunohistochemistry (IHC) was used to detect the RAB23 protein expressions in 106 pairs of ESCC and adjacent normal tissues, as well as in the lymph glands and primary tumor tissues of 33 patients with positive lymph nodes and 10 patients with negative lymph nodes. Endogenous RAB23 expression was transiently depleted using siRNAs (si-NC, si-RAB23-1, and si-RAB23-9) or stably reduced using shRNAs (sh-NC and sh-RAB23) in ESCC KYSE30 and KYSE150 cells, and the knockdown efficiency was tested using Western blot assays. Cell counting kit-8 assays and mouse xenograft models were used to test the proliferation of ESCC cells Transwell assays and tail vein-pulmonary metastasis models in immunocompromised mice were used to examine the migration and invasion of ESCC cells. Cell adhesion assays were used to test the adhesion of ESCC cells. RNA-seq assays were used to analyze how RAB23 knockdown influenced the expression profile of ESCC cells and the implicated signal pathways were confirmed using Western blot assays. The RAB23 mRNA expression in 16 cases of ESCC tissues was 0.009 7±0.008 9, which was markedly higher than that in adjacent normal tissues (0.003 2±0.003 7, =0.006). GEO analysis on RAB23 expressions in ESCC and adjacent normal tissues showed that the RAB23 mRNA level in ESCC tissues (4.30±0.25) was remarkably increased compared with their normal counterparts (4.10±0.17, 0.037). Among the 106 pairs of ESCC and tumor-adjacent normal tissues, 51 cases exhibited low expression of RAB23 and 55 cases showed high expression of RAB23, whereas in the paired tumor-adjacent normal tissues 82 cases were stained weakly and 24 strongly for RAB23 protein. These results indicated that RAB23 expression was markedly increased in ESCC tissues (<0.001). Additionally, only 1 out of 33 primary ESCC tissues with positive lymph nodes showed low RAB23 protein expression. On the other hand, 7 samples of primary ESCC tissues with negative lymph nodes were stained strongly for RAB23 while its level in the other 3 samples was weak. These results showed that RAB23 expression was remarkably increased in primary ESCC tissues with positive lymph nodes compared with those with negative lymph nodes (=0.024). Further tests showed that 32 out of 33 positive lymph nodes were stained strongly for RAB23, whereas no negative lymph nodes (=10) exhibited high expression of RAB23 (<0.001). Both transient and stable knockdown of endogenous RAB23 expression failed to cause detectable changes in the proliferation of KYSE30 cells and , but attenuated the migration and invasion of KYSE30 cells as well as the invasion of KYSE150 cells. RAB23 knockdown was found to significantly decrease the number of adhesive KYSE30 cells in the sh-RAB23 group (313.75±89.34) compared with control cells in the sh-NC group (1 030.75±134.29, <0.001). RAB23 knockdown was also found to significantly decrease the number of adhesive KYSE150 cells in the sh-RAB23 group (710.5±31.74) compared with the number of control cells in the sh-NC group (1 005.75±61.09, <0.001). RNA-seq assays demonstrated that RAB23 knockdown using two siRNAs targeting RAB23 mRNA markedly impaired focal adhesion-related signal pathways, and decreased the levels of phosphorylated FAK (p-FAK) and phosphorylated paxillin (p-paxillin) in KYSE30 and KYSE150 cells. Significantly increased RAB23 in ESCC tissues positively correlates with lymph node metastasis. Depleted RAB23 expression attenuates focal adhesion-related signal pathways, thus impairing the invasion, metastasis, and adhesion of ESCC cells.
Topics: Humans; Animals; Mice; Esophageal Squamous Cell Carcinoma; Esophageal Neoplasms; Paxillin; Carrier Proteins; ras Proteins; Cell Line, Tumor; Cell Movement; Neoplasm Invasiveness; Cell Proliferation; RNA, Small Interfering; RNA, Messenger; Gene Expression Regulation, Neoplastic; rab GTP-Binding Proteins
PubMed: 38418184
DOI: 10.3760/cma.j.cn112152-20231026-00258 -
BioRxiv : the Preprint Server For... Feb 2024Single-molecule super-resolution imaging is instrumental for investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric...
Single-molecule super-resolution imaging is instrumental for investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise, 3D nanoscale studies of a wide range of cellular structures. Here we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi-and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D single-molecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin - a protein located in the focal adhesion complex - and actin in human osteosarcoma cells.
PubMed: 38405960
DOI: 10.1101/2024.02.08.579549