Histochemistry and Cell Biology最新文献

The development of progressively sophisticated tools complemented by the integration of live cell imaging enhances our understanding of the four-dimensional (4D) nucleome, revealing elaborate molecular interactions and chromatin states. Yet, the dynamics of chromosomes in relation to nuclear organelles or to each other across cell cycle in living cells are underexplored. We have developed photoconvertible GFP H3-Dendra2 stably expressing in PC3M cells. The nuclear lamina and perinucleolar associated heterochromatin or diffuse chromosome regions were photoconverted through a single-point activation using a confocal microscope. The results demonstrated a dynamic nature for both types of chromosomes in the same cell cycle and across mitosis. While some chromosome domains were heritably associated with either nuclear lamina or nucleoli, others changed alliance to different nuclear organelles postmitotically. In addition, co-photoconverted chromosome domains often do not stay together within the same cell cycle and across mitosis, suggesting a transient nature of chromosome neighborhoods. Long-range spreading and movement of chromosomes were also observed. Interestingly, when cells were treated with a low concentration of actinomycin D that inhibits Pol I transcription through intercalating GC-rich DNA, chromosome movement was significantly blocked. Treatment with another Pol I inhibitor, metarrestin, which does not impact DNA, had little effect on the movement, suggesting that the DNA structure itself plays a role in chromosome dynamics. Furthermore, inhibition of Pol II transcription with α-amanitin also reduced the chromosome movement, demonstrating that Pol II, but not Pol I transcription, is important for chromosome dynamics in the nucleus.

Since Robert Feulgen first stained DNA in the cell, visualizing genome chromatin has been a central issue in cell biology to uncover how chromatin is organized and behaves in the cell. To approach this issue, we have developed single-molecule imaging of nucleosomes, a basic unit of chromatin, to unveil local nucleosome behavior in living cells. In this study, we investigated behaviors of nucleosomes with various histone H4 mutants in living HeLa cells to address the role of H4 tail acetylation, including H4K16Ac and others, which are generally associated with more transcriptionally active chromatin regions. We ectopically expressed wild-type (wt) or mutated H4s (H4K16 point; H4K5,8,12,16 quadruple; and H4 tail deletion) fused with HaloTag in HeLa cells. Cells that expressed wtH4-Halo, H4K16-Halo mutants, and multiple H4-Halo mutants had euchromatin-concentrated distribution. Consistently, the genomic regions of the wtH4-Halo nucleosomes corresponded to Hi-C contact domains (or topologically associating domains, TADs) with active chromatin marks (A-compartment). Utilizing single-nucleosome imaging, we found that none of the H4 deacetylation or acetylation mimicked H4 mutants altered the overall local nucleosome motion. This finding suggests that H4 mutant nucleosomes embedded in the condensed euchromatic domains with excess endogenous H4 nucleosomes cannot cause an observable change in the local motion. Interestingly, H4 with four lysine-to-arginine mutations displayed a substantial freely diffusing fraction in the nucleoplasm, whereas H4 with a truncated N-terminal tail was incorporated in heterochromatic regions as well as euchromatin. Our study indicates the power of single-nucleosome imaging to understand individual histone/nucleosome behavior reflecting chromatin environments in living cells.

The mystery of how human DNA is compactly packaged into a nucleus-a space a hundred thousand times smaller-while still allowing for the regulation of gene function, has long been one of the greatest enigmas in cell biology. This puzzle is gradually being solved, thanks in part to the advent of new technologies. Among these, innovative genome-labeling techniques combined with high-resolution imaging methods have been pivotal. These methods facilitate the visualization of DNA within intact nuclei and have significantly contributed to our current understanding of genome organization. This review will explore various labeling and imaging approaches that are revolutionizing our understanding of the three-dimensional organization of the genome, shedding light on the relationship between its structure and function.

The dynamics of DNA in the cell nucleus plays a role in cellular processes and fates but the interplay of DNA mobility with the hierarchical levels of DNA organization is still underexplored. Here, we made use of DNA replication to directly label genomic DNA in an unbiased genome-wide manner. This was followed by live-cell time-lapse microscopy of the labeled DNA combining imaging at different resolutions levels simultaneously and allowing one to trace DNA motion across organization levels within the same cells. Quantification of the labeled DNA segments at different microscopic resolution levels revealed sizes comparable to the ones reported for DNA loops using 3D super-resolution microscopy, topologically associated domains (TAD) using 3D widefield microscopy, and also entire chromosomes. By employing advanced chromatin tracking and image registration, we discovered that DNA exhibited higher mobility at the individual loop level compared to the TAD level and even less at the chromosome level. Additionally, our findings indicate that chromatin movement, regardless of the resolution, slowed down during the S phase of the cell cycle compared to the G1/G2 phases. Furthermore, we found that a fraction of DNA loops and TADs exhibited directed movement with the majority depicting constrained movement. Our data also indicated spatial mobility differences with DNA loops and TADs at the nuclear periphery and the nuclear interior exhibiting lower velocity and radius of gyration than the intermediate locations. On the basis of these insights, we propose that there is a link between DNA mobility and its organizational structure including spatial distribution, which impacts cellular processes.

Key reproductive events such as fertilization and early embryonic development occur in the lumen of the oviduct. Since investigating these processes in vivo is both technically challenging and ethically sensitive, cell culture models have been established to reproduce the oviductal microenvironment. Compartmentalized culture systems, particularly air-liquid interface cultures (ALI; cells access the culture medium only from the basolateral cell side), result in highly differentiated oviduct epithelial cell cultures. The oxygen (O₂) tension within the oviduct is 4-10% across species, and its reduced O₂ content is presumed to be important for early reproductive processes. However, cell culture models of the oviduct are typically cultivated without O₂ regulation and therefore at about 18% O₂. To investigate the impact of O₂ levels on oviduct epithelium functions in vitro, we cultured porcine oviduct epithelial cells (POEC) at the ALI using both physiological (5%) and supraphysiological (18%) O₂ levels and two different media regimes. Epithelium architecture, barrier function, secretion of oviduct fluid surrogate (OFS), and marker gene expression were comparatively assessed. Under all culture conditions, ALI-POEC formed polarized, ciliated monolayers with appropriate barrier function. Exposure to 18% O₂ accelerated epithelial differentiation and significantly increased the apical OFS volume and total protein content. Expression of oviduct genes and the abundance of OVGP1 (oviduct-specific glycoprotein 1) in the OFS were influenced by both O₂ tension and medium choice. In conclusion, oviduct epithelial cells can adapt to a supraphysiological O₂ environment. This adaptation, however, may alter their capability to replicate in vivo tissue characteristics.

The aim of this study is to determine the influence of the mitochondrial open-reading-frame of the twelve S rRNA-c (MOTS-c) peptide on pancreatic cell physiology. Moreover, in this study, we examined the changes in MOTS-c secretion and expression under different conditions. Our experiments were conducted using laboratory cell line cultures, specifically the INS-1E and αTC-1 cell lines, which represent β and α pancreatic cells, respectively. As the pancreas is an endocrine organ, we also tested its hormone regulation capabilities. Furthermore, we assessed the secretion of MOTS-c after incubating the cells with glucose and free fatty acids. Additionally, we examined key cell culture parameters such as cell viability, proliferation, and apoptosis. The results obtained from this study show that MOTS-c has a significant impact on the physiology of pancreatic cells. Specifically, it lowers insulin secretion and expression in INS-1E cells and enhances glucagon secretion and expression in αTC-1 cells. Furthermore, MOTS-c affects cell viability and apoptosis. Interestingly, insulin and glucagon affect the MOTS-c secretion as well as glucose and free fatty acids. These experiments clearly show that MOTS-c is an important regulator of pancreatic metabolism, and there are numerous properties of MOTS-c yet to be discovered.

Aberrant glycosylation is an important factor in facilitating tumor progression and therapeutic resistance. In this study, using Wisteria floribunda agglutinin (WFA), we examined the expression of WFA-binding glycans (WFAG) in cholangiocarcinoma (CCA). The results showed that WFAG was highly detected in precancerous and cancerous lesions of human CCA tissues, although it was rarely detected in normal bile ducts. The positive signal of WFAG in the cancerous lesion accounted for 96.2% (50/52) of the cases. Overexpression of WFAG was significantly associated with lymph node and distant metastasis (P < 0.05). The study using the CCA hamster model showed that WFAG is elevated in preneoplastic and neoplastic bile ducts as early as 1 month after being infected with liver fluke and exposed to N-nitrosodimethylamine. Functional analysis was performed to reveal the role of WFAG in CCA. The CCA cell lines KKU-213A and KKU-213B were treated with WFA, followed by migration assay. Our data suggested that WFAG facilitates the migration of CCA cells via the activation of the Akt and ERK signaling pathways. In conclusion, we have demonstrated the association of WFAG with carcinogenesis and metastasis of CCA, suggesting its potential as a target for the treatment of the disease.

Breast cancer treatment encompasses various therapeutic modalities, including surgery, radiotherapy, and chemotherapy. Breast-conserving surgery has been an integral part of breast cancer management. However, radiotherapy, an important component of breast cancer management, can lead to complications, particularly fibrosis, affecting reconstructive surgery outcomes. We conducted an in vivo study using 48 female Wistar Albino rats, employing segmental mastectomy and radiotherapy to simulate post-mastectomy conditions. The rats were divided into six groups: control, mastectomy, mastectomy + radiotherapy, mastectomy + platelet-rich plasma (PRP) + radiotherapy, mastectomy + infliximab + radiotherapy, and mastectomy + infliximab + PRP + radiotherapy. Edema, hyperemia, inflammation, and fibrosis were assessed as indicators of tissue response. Histopathological analysis revealed that mastectomy + infliximab and mastectomy + infliximab + PRP groups showed significant reductions in fibrosis compared to other groups. Edema, hyperemia, and inflammation were also less severe in these groups compared to the control group. Radiotherapy-induced fibrosis is a major concern in breast reconstruction. Our study suggests that local PRP application and systemic infliximab administration, either alone or in combination, could mitigate the adverse effects of radiotherapy. This approach has the potential to improve reconstructive outcomes in patients undergoing or having the possibility to undergo radiotherapy. This is the first study showing the effectiveness of infliximab and PRP combination on wound healing. The provided experimental rat model might offer guidance for further research. This study provides insights into optimizing outcomes in reconstructive breast surgery, paving the way for further research and clinical studies.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Respecting the age of the patients and the tumor aggressiveness, investigation of the molecular mechanisms of RMS tumorigenesis is directed toward the identification of novel therapeutic targets. To contribute to a better understanding of the molecular pathology of RMS, we investigated ankyrin repeat domain 1 (ANKRD1), designated as a potential marker for differential diagnostics. In this study, we used three RMS cell lines (SJRH30, RD, and HS-729) to assess its expression profile, intracellular localization, and turnover. They express wild-type ANKRD1, as judged by the sequencing of the open reading frame. Each cell line expressed a different amount of ANKRD1 protein, although the transcript level was similar. According to western blot analysis, ANKRD1 protein was expressed at detectable levels in the SJRH30 and RD cells (SJRH30 > RD), but not in the HS-729, even after immunoprecipitation. Immunocytochemistry revealed nuclear and cytoplasmic localization of ANKRD1 in all examined cell lines. Moreover, the punctate pattern of ANKRD1 staining in the nuclei of RD and HS-729 cells overlapped with coilin, indicating its association with Cajal bodies. We have shown that RMS cells are not able to overexpress ANKRD1 protein, which can be attributed to its proteasomal degradation. The unsuccessful attempt to overexpress ANKRD1 in RMS cells indicates the possibility that its overexpression may have detrimental effects for RMS cells and opens a window for further research into its role in RMS pathogenesis and for potential therapeutic targeting.