[Different concentrations of adapalene induce differentiation and apoptosis of SH-SY5Y cells]. / 不同浓度阿达帕林诱导SH-SY5Y细胞的分化及凋亡.

OBJECTIVES: To investigate the effects of different concentrations of adapalene on the morphology and functions of neuroblastoma cell line SH-SY5Y, as well as its role in inducing cell differentiation and apoptosis. METHODS: SH-SY5Y cells were divided into control group, low concentration (0.1 µM and 1 µM) adapalene groups, and high concentration (10 µM) adapalene group. Time-lapse microscopy was used to observe the morphological changes of SH-SY5Y cells. Immunofluorescence staining was performed to detect the expression of neuronal specific marker ßIII-tubulin and mature neuronal marker neurofilament heavy polypeptide (NFH). Multi-electrode array was used to record the electrophysiological features of SH-SY5Y cells. Cell apoptosis was evaluated using a cell apoptosis detection kit. RESULTS: Low concentrations of adapalene promoted the formation of neurite outgrowth in SH-SY5Y cells, with the neurites interconnected to form a network. Spontaneous discharge activity was observed in SH-SY5Y cells treated with low concentrations of adapalene. Compared to the control group, the expression of ßIII-tubulin and NFH increased in the 1 µM adapalene group, while the level of cell apoptosis increased in the high concentration adapalene group (P<0.05). CONCLUSIONS: Low concentrations of adapalene can induce differentiation of SH-SY5Y cells into mature functional neurons, while high concentrations of adapalene can induce apoptosis in SH-SY5Y cells.

A transcription factor complex in Dictyostelium enables adaptive changes in macropinocytosis during the growth-to-development transition.

Macropinocytosis, an evolutionarily conserved endocytic pathway, mediates nonselective bulk uptake of extracellular fluid. It is the primary route for axenic Dictyostelium cells to obtain nutrients and has also emerged as a nutrient-scavenging pathway for mammalian cells. How cells adjust macropinocytic activity in various physiological or developmental contexts remains to be elucidated. We discovered that, in Dictyostelium cells, the transcription factors Hbx5 and MybG form a functional complex in the nucleus to maintain macropinocytic activity during the growth stage. In contrast, during starvation-induced multicellular development, the transcription factor complex undergoes nucleocytoplasmic shuttling in response to oscillatory cyclic adenosine 3',5'-monophosphate (cAMP) signals, which leads to increased cytoplasmic retention of the complex and progressive downregulation of macropinocytosis. Therefore, by coupling macropinocytosis-related gene expression to the cAMP oscillation system, which facilitates long-range cell-cell communication, the dynamic translocation of the Hbx5-MybG complex orchestrates a population-level adjustment of macropinocytic activity to adapt to changing environmental conditions.

Loss of Myo19 increases metastasis by enhancing microenvironmental ROS gradient and chemotaxis.

Tumor metastasis involves cells migrating directionally in response to external chemical signals. Reactive oxygen species (ROS) in the form of H2O2 has been demonstrated as a chemoattractant for neutrophils but its spatial characteristics in tumor microenvironment and potential role in tumor cell dissemination remain unknown. Here we investigate the spatial ROS distribution in 3D tumor spheroids and identify a ROS concentration gradient in spheroid periphery, which projects into a H2O2 gradient in tumor microenvironment. We further reveal the role of H2O2 gradient to induce chemotaxis of tumor cells by activating Src and subsequently inhibiting RhoA. Finally, we observe that the absence of mitochondria cristae remodeling proteins including the mitochondria-localized actin motor Myosin 19 (Myo19) enhances ROS gradient and promotes tumor dissemination. Myo19 downregulation is seen in many tumors, and Myo19 expression is negatively associated with tumor metastasis in vivo. Together, our study reveals the chemoattractant role of tumor microenvironmental ROS and implies the potential impact of mitochondria cristae disorganization on tumor invasion and metastasis.

mTORC1 Mediates Biphasic Mechano-Response to Orchestrate Adhesion-Dependent Cell Growth and Anoikis Resistance.

Cells constantly sense and respond to not only biochemical but also biomechanical changes in their microenvironment, demanding for dynamic metabolic adaptation. ECM stiffening is a hallmark of cancer aggressiveness, while survival under substrate detachment also associates with poor prognosis. Mechanisms underlying this, non-linear mechano-response of tumor cells may reveal potential double-hit targets for cancers. Here, an integrin-GSK3ß-FTO-mTOR axis is reported, that can integrate stiffness sensing to ensure both the growth advantage endowed by rigid substrate and cell death resistance under matrix detachment. It is demonstrated that substrate stiffening can activate mTORC1 and elevate mTOR level through integrins and GSK3ß-FTO mediated mRNA m6 A modification, promoting anabolic metabolism. Inhibition of this axis upon ECM detachment enhances autophagy, which in turn conveys resilience of tumor cells to anoikis, as it is demonstrated in human breast ductal carcinoma in situ (DCIS) and mice malignant ascites. Collectively, these results highlight the biphasic mechano-regulation of cellular metabolism, with implications in tumor growth under stiffened conditions such as fibrosis, as well as in anoikis-resistance during cancer metastasis.

Regulation of mitochondrial structure by the actin cytoskeleton.

Mitochondria are the powerhouse of the cell and play important roles in multiple cellular processes including cell metabolism, proliferation, and programmed cell death. Mitochondria are double-membrane organelles with the inner membrane folding inward to form cristae. Mitochondria networks undergo dynamic fission and fusion. Deregulation of mitochondrial structure has been linked to perturbed mitochondrial membrane potential and disrupted metabolism, as evidenced in tumorigenesis, neurodegenerative diseases, etc. Actin and its motors-myosins have long been known to generate mechanical forces and participate in short-distance cargo transport. Accumulating knowledge from biochemistry and live cell/electron microscope imaging has demonstrated the role of actin filaments in pre-constricting the mitochondria during fission. Recent studies have suggested the involvement of myosins in cristae maintenance and mitochondria quality control. Here, we review current findings and discuss future directions in the emerging fields of cytoskeletal regulation in cristae formation, mitochondrial dynamics, intracellular transport, and mitocytosis, with focus on the actin cytoskeleton and its motor proteins.

An Identification Method for Irregular Components Related to Terminal Blocks in Equipment Cabinet of Power Substation.

Despite the continuous advancement of intelligent power substations, the terminal block components within equipment cabinet inspection work still often require loads of personnel. The repetitive documentary works not only lack efficiency but are also susceptible to inaccuracies introduced by substation personnel. To resolve the problem of lengthy, time-consuming inspections, a terminal block component detection and identification method is presented in this paper. The identification method is a multi-stage system that incorporates a streamlined version of You Only Look Once version 7 (YOLOv7), a fusion of YOLOv7 and differential binarization (DB), and the utilization of PaddleOCR. Firstly, the YOLOv7 Area-Oriented (YOLOv7-AO) model is developed to precisely locate the complete region of terminal blocks within substation scene images. The compact area extraction model rapidly cuts out the valid proportion of the input image. Furthermore, the DB segmentation head is integrated into the YOLOv7 model to effectively handle the densely arranged, irregularly shaped block components. To detect all the components within a target electrical cabinet of substation equipment, the YOLOv7 model with a differential binarization attention head (YOLOv7-DBAH) is proposed, integrating spatial and channel attention mechanisms. Finally, a general OCR algorithm is applied to the cropped-out instances after image distortion to match and record the component's identity information. The experimental results show that the YOLOv7-AO model reaches high detection accuracy with good portability, gaining 4.45 times faster running speed. Moreover, the terminal block component detection results show that the YOLOv7-DBAH model achieves the highest evaluation metrics, increasing the F1-score from 0.83 to 0.89 and boosting the precision to over 0.91. The proposed method achieves the goal of terminal block component identification and can be applied in practical situations.

Reciprocal regulation of actin filaments and cellular metabolism.

For cells to adhere, migrate and proliferate, remodeling of the actin cytoskeleton is required. This process consumes a large amount of ATP while having an intimate connection with cellular metabolism. Signaling pathways that regulate energy homeostasis can also affect actin dynamics, whereas a variety of actin binding proteins directly or indirectly interact with the anabolic and catabolic regulators in cells. Here, we discuss the inter-regulation between actin filaments and cellular metabolism, reviewing recent discoveries on key metabolic enzymes that respond to actin remodeling as well as historical findings on metabolic stress-induced cytoskeletal reorganization. We also address emerging techniques that would benefit the study of cytoskeletal dynamics and cellular metabolism in high spatial-temporal resolution.

Transcriptome Analysis Reveals the Anti-Tumor Mechanism of Eucalyptol Treatment on Neuroblastoma Cell Line SH-SY5Y.

Eucalyptol (1.8-cineole), an active component in traditional Chinese medicine Artemisia argyi for moxibustion. Previous studies have shown that eucalyptol has anti-tumor effects on leukemia and colon cancer. Nonetheless, the effect and mechanism of eucalyptol on neuroblastoma remains unclear. In the present study, we intended to reveal the effect and mechanism of eucalyptol treatment on the neuroblastoma cell line SH-SY5Y through transcriptome analysis. In the group treated with eucalyptol, 566 brain genes were up-regulated, while 757 genes were down-regulated. GO function analysis showed that positive regulation of cell cycle was down-regulated in biological processes. Meanwhile, cancer-related pathways were identified in KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis, including pathways in cancer, PI3K-Akt signaling pathway, cAMP signaling pathway, TGF-beta signaling pathway, Hippo signaling pathway, p53 signaling pathway, and additional pathways. Furthermore, we found a key gene, such as MYC, by constructing a network of cancer related pathways with differentially expressed genes and transcription factor analysis. In conclusion, our research indicates that MYC might play a central role in the anit-tumor mechanisms of eucalyptol.

YAP Activation in Promoting Negative Durotaxis and Acral Melanoma Progression.

Directed cell migration towards a softer environment is called negative durotaxis. The mechanism and pathological relevance of negative durotaxis in tumor progression still requires in-depth investigation. Here, we report that YAP promotes the negative durotaxis of melanoma. We uncovered that the RhoA-myosin II pathway may underlie the YAP enhanced negative durotaxis of melanoma cells. Acral melanoma is the most common subtype of melanoma in non-Caucasians and tends to develop in a stress-bearing area. We report that acral melanoma patients exhibit YAP amplification and increased YAP activity. We detected a decreasing stiffness gradient from the tumor to the surrounding area in the acral melanoma microenvironment. We further identified that this stiffness gradient could facilitate the negative durotaxis of melanoma cells. Our study advanced the understanding of mechanical force and YAP in acral melanoma and we proposed negative durotaxis as a new mechanism for melanoma dissemination.

Mechanical instability generated by Myosin 19 contributes to mitochondria cristae architecture and OXPHOS.

The folded mitochondria inner membrane-cristae is the structural foundation for oxidative phosphorylation (OXPHOS) and energy production. By mechanically simulating mitochondria morphogenesis, we speculate that efficient sculpting of the cristae is organelle non-autonomous. It has long been inferred that folding requires buckling in living systems. However, the tethering force for cristae formation and regulation has not been identified. Combining electron tomography, proteomics strategies, super resolution live cell imaging and mathematical modeling, we reveal that the mitochondria localized actin motor-myosin 19 (Myo19) is critical for maintaining cristae structure, by associating with the SAM-MICOS super complex. We discover that depletion of Myo19 or disruption of its motor activity leads to altered mitochondria membrane potential and decreased OXPHOS. We propose that Myo19 may act as a mechanical tether for effective ridging of the mitochondria cristae, thus sustaining the energy homeostasis essential for various cellular functions.

Cloning, expression, and function of ferritins in the tick Haemaphysalis flava.

The full-length cDNA of two ferritins of Haemaphysalis flava were cloned after which recombinant Hf-FER1 and Hf-FER2 were expressed and their function was analyzed. In addition, RNA interference (RNAi) based on the injection of Hf-fer1 or Hf-fer2 dsRNA into fully engorged female ticks was performed. The cDNA encoding Hf-FER1 is 834 bp in length. It contains an iron-responsive element in the 5' untranslated region and encodes 174 amino acid residues. The full-length cDNA of Hf-FER2 contains 696 bp and encodes 199 amino acids, including a putative signal peptide sequence. Hf-FER1 and Hf-FER2 both have the ferroxidase iron center and the ferrihydrite nucleation center. The evolutionary relationship of Hf-FER1 and Hf-FER2 was established, and the predicted quaternary structures were assembled as typical spherical shells composed of 24 subunits which was demonstrated by nature PAGE. Real-time PCR showed that Hf-fer1 and Hf-fer2 were expressed in all developmental stages, with the highest expression in fully engorged females. The expression of Hf-fer1 and Hf-fer2 were relatively high in unfed larvae. Hf-fer1 was expressed in all tissues and was especially abundant in the salivary glands of fully engorged females. In contrast, the highest levels of Hf-fer2 were found in the midgut of fully engorged females, and no expression was found in the salivary glands of this life stage. Both recombinant Hf-FER1 and Hf-FER2 had iron-binding capabilities. Silencing of both Hf-fer1 and Hf-fer2 affected fecundity. Compared to the control, the percentage of ticks that laid eggs in the Hf-fer1 and Hf-fer2 RNAi groups was 73.3% and 66.7%, respectively. The silenced ticks that laid eggs had lower egg weight to body weight ratios, and the eggs had abnormal morphologies. The hatchability of eggs with normal morphology in the Hf-fer1 and Hf-fer2 silenced groups was 47.8% and 22.8%, respectively, which was significantly different from the control group (P < 0.005). These findings indicate that Hf-FER1 and Hf-FER2 play important roles in the iron storage of H. flava.

TLR4 signalling via Piezo1 engages and enhances the macrophage mediated host response during bacterial infection.

TLR4 signaling plays key roles in the innate immune response to microbial infection. Innate immune cells encounter different mechanical cues in both health and disease to adapt their behaviors. However, the impact of mechanical sensing signals on TLR4 signal-mediated innate immune response remains unclear. Here we show that TLR4 signalling augments macrophage bactericidal activity through the mechanical sensor Piezo1. Bacterial infection or LPS stimulation triggers assembly of the complex of Piezo1 and TLR4 to remodel F-actin organization and augment phagocytosis, mitochondrion-phagosomal ROS production and bacterial clearance and genetic deficiency of Piezo1 results in abrogation of these responses. Mechanistically, LPS stimulates TLR4 to induce Piezo1-mediated calcium influx and consequently activates CaMKII-Mst1/2-Rac axis for pathogen ingestion and killing. Inhibition of CaMKII or knockout of either Mst1/2 or Rac1 results in reduced macrophage bactericidal activity, phenocopying the Piezo1 deficiency. Thus, we conclude that TLR4 drives the innate immune response via Piezo1 providing critical insight for understanding macrophage mechanophysiology and the host response.

Proteome-scale analysis of phase-separated proteins in immunofluorescence images.

Phase separation is an important mechanism that mediates the spatial distribution of proteins in different cellular compartments. While phase-separated proteins share certain sequence characteristics, including intrinsically disordered regions (IDRs) and prion-like domains, such characteristics are insufficient for making accurate predictions; thus, a proteome-wide understanding of phase separation is currently lacking. Here, we define phase-separated proteomes based on the systematic analysis of immunofluorescence images of 12 073 proteins in the Human Protein Atlas. The analysis of these proteins reveals that phase-separated candidate proteins exhibit higher IDR contents, higher mean net charge and lower hydropathy and prefer to bind to RNA. Kinases and transcription factors are also enriched among these candidate proteins. Strikingly, both phase-separated kinases and phase-separated transcription factors display significantly reduced substrate specificity. Our work provides the first global view of the phase-separated proteome and suggests that the spatial proximity resulting from phase separation reduces the requirement for motif specificity and expands the repertoire of substrates. The source code and data are available at https://github.com/cheneyyu/deepphase.

LIMD1 phase separation contributes to cellular mechanics and durotaxis by regulating focal adhesion dynamics in response to force.

Cells sense and respond to extracellular mechanical cues through cell-matrix adhesions. Interestingly, the maturation of focal adhesions (FAs) is reciprocally force dependent. How biomechanical cues dictate the status of cell motility and how FAs spatial temporally coordinate force sensing and self-organization remain enigmatic. Here, we identify that LIMD1, a member of the LIM domain scaffolding proteins, undergoes force-sensitive condensation at the FAs. We also unveil that the multivalent interactions of LIMD1 intrinsically disordered region (IDR) and the LIM domains concertedly drive this phase transition under the regulation of phosphorylation. Intriguingly, formation of condensed LIMD1 protein compartments is sufficient to specifically enrich and localize late FA proteins. We further discover that LIMD1 regulates cell spreading, maintains FA dynamics and cellular contractility, and is critical for durotaxis-the ability of cells to crawl along gradients of substrate stiffness. Our results suggest a model that recruitment of LIMD1 to the FAs, via mechanical force triggered inter-molecular interaction, serves as a phase separation hub to assemble and organize matured FAs, thus allowing for efficient mechano-transduction and cell migration.

Eight-lncRNA signature of cervical cancer were identified by integrating DNA methylation, copy number variation and transcriptome data.

BACKGROUND: Copy number variation (CNV) suggests genetic changes in malignant tumors. Abnormal expressions of long non-coding RNAs (lncRNAs) resulted from genomic and epigenetic abnormalities play a driving role in tumorigenesis of cervical cancer. However, the role of lncRNAs-related CNV in cervical cancer remained largely unclear. METHODS: The data of messenger RNAs (mRNAs), DNA methylation, and DNA copy number were collected from 292 cervical cancer specimens. The prognosis-related subtypes of cervical cancer were determined by multi-omics integration analysis, and protein-coding genes (PCGs) and lncRNAs with subtype-specific expressions were identified. The CNV pattern of the subtype-specific lncRNAs was analyzed to identify the subtype-specific lncRNAs. A prognostic risk model based on lncRNAs was established by least absolute shrinkage and selection operator (LASSO). RESULTS: Multi-omics integration analysis identified three molecular subtypes incorporating 617 differentially expressed lncRNAs and 1395 differentially expressed PCGs. The 617 lncRNAs were found to intersect with disease-related lncRNAs. Functional enrichment showed that 617 lncRNAs were mainly involved in tumor metabolism, immunity and other pathways, such as p53 and cAMP signaling pathways, which are closely related to the development of cervical cancer. Finally, according to CNV pattern consistent with differential expression analysis, we established a lncRNAs-based signature consisted of 8 lncRNAs, namely, RUSC1-AS1, LINC01990, LINC01411, LINC02099, H19, LINC00452, ADPGK-AS1, C1QTNF1-AS1. The interaction of the 8 lncRNAs showed a significantly poor prognosis of cervical cancer patients, which has also been verified in an independent dataset. CONCLUSION: Our study expanded the network of CNVs and improved the understanding on the regulatory network of lncRNAs in cervical cancer, providing novel biomarkers for the prognosis management of cervical cancer patients.

The Golgi microtubules regulate single cell durotaxis.

Current understandings on cell motility and directionality rely heavily on accumulated investigations of the adhesion-actin cytoskeleton-actomyosin contractility cycles, while microtubules have been understudied in this context. Durotaxis, the ability of cells to migrate up gradients of substrate stiffness, plays a critical part in development and disease. Here, we identify the pivotal role of Golgi microtubules in durotactic migration of single cells. Using high-throughput analysis of microtubule plus ends/focal adhesion interactions, we uncover that these non-centrosomal microtubules actively impart leading edge focal adhesion (FA) dynamics. Furthermore, we designed a new system where islands of higher stiffness were patterned within RGD peptide coated polyacrylamide gels. We revealed that the positioning of the Golgi apparatus is responsive to external mechanical cues and that the Golgi-nucleus axis aligns with the stiffness gradient in durotaxis. Together, our work unveils the cytoskeletal underpinning for single cell durotaxis. We propose a model in which the Golgi-nucleus axis serves both as a compass and as a steering wheel for durotactic migration, dictating cell directionality through the interaction between non-centrosomal microtubules and the FA dynamics.

Loss of 4.1N in epithelial ovarian cancer results in EMT and matrix-detached cell death resistance.

Epithelial ovarian cancer (EOC) is one of the leading causes of death from gynecologic cancers and peritoneal dissemination is the major cause of death in patients with EOC. Although the loss of 4.1N is associated with increased risk of malignancy, its association with EOC remains unclear. To explore the underlying mechanism of the loss of 4.1N in constitutive activation of epithelial-mesenchymal transition (EMT) and matrix-detached cell death resistance, we investigated samples from 268 formalin-fixed EOC tissues and performed various in vitro and in vivo assays. We report that the loss of 4.1N correlated with progress in clinical stage, as well as poor survival in EOC patients. The loss of 4.1N induces EMT in adherent EOC cells and its expression inhibits anoikis resistance and EMT by directly binding and accelerating the degradation of 14-3-3 in suspension EOC cells. Furthermore, the loss of 4.1N could increase the rate of entosis, which aggravates cell death resistance in suspension EOC cells. Moreover, xenograft tumors in nude mice also show that the loss of 4.1N can aggravate peritoneal dissemination of EOC cells. Single-agent and combination therapy with a ROCK inhibitor and a 14-3-3 antagonist can reduce tumor spread to varying degrees. Our results not only define the vital role of 4.1N loss in inducing EMT, anoikis resistance, and entosis-induced cell death resistance in EOC, but also suggest that individual or combined application of 4.1N, 14-3-3 antagonists, and entosis inhibitors may be a promising therapeutic approach for the treatment of EOC.

ER-mitochondria contacts promote mtDNA nucleoids active transportation via mitochondrial dynamic tubulation.

A human cell contains hundreds to thousands of mitochondrial DNA (mtDNA) packaged into nucleoids. Currently, the segregation and allocation of nucleoids are thought to be passively determined by mitochondrial fusion and division. Here we provide evidence, using live-cell super-resolution imaging, that nucleoids can be actively transported via KIF5B-driven mitochondrial dynamic tubulation (MDT) activities that predominantly occur at the ER-mitochondria contact sites (EMCS). We further demonstrate that a mitochondrial inner membrane protein complex MICOS links nucleoids to Miro1, a KIF5B receptor on mitochondria, at the EMCS. We show that such active transportation is a mechanism essential for the proper distribution of nucleoids in the peripheral zone of the cell. Together, our work identifies an active transportation mechanism of nucleoids, with EMCS serving as a key platform for the interplay of nucleoids, MICOS, Miro1, and KIF5B to coordinate nucleoids segregation and transportation.