Cell structure and function最新文献

Multiplex live imaging enables simultaneous visualization of multiple signaling pathways in living cells, offering real-time insights into complex cellular networks. This methodology is essential in research fields such as cancer biology, where signaling activities exhibit heterogeneity, feedback regulation, crosstalk, and dynamic changes during pathological progression and the acquisition of therapeutic resistance. While conventional biochemical assays advanced our understanding of signaling signatures through static or population-level analyses, they lack the temporal resolution required to capture dynamic events at single-cell resolution. Recent methodological innovations have expanded multiplex live imaging through several strategies. Spectral multiplexing exploits broadened fluorescent protein palettes and optimized biosensor combinations, sometimes coupled with intracellular multiplexing methods that distinguish signals by targeting fluorescence to subcellular compartments. Intercellular multiplexing distributes reporters across cell populations, and temporal multiplexing leverages optical switching to separate signals over time. Additional modalities such as fluorescence anisotropy, fluorescence lifetime, and Raman imaging provide orthogonal readouts. Furthermore, computational approaches reinforce multiplex strategies by improved spectral unmixing, often complemented by deep learning-based algorithms. Collectively, these advances enable simultaneous tracking of multiple signaling pathways within single cells, revealing how diverse inputs are integrated into cellular responses. Here we review current strategies for multiplex live imaging, especially highlighting its applications to cancer signaling networks. Progress in fluorescent biosensor development, imaging technologies, and computational analysis will further promote the exploration of dynamic cellular regulations in basic research and translational medicine.Key words: multiplex live imaging, fluorescent biosensors, signal dynamics, image analysis, cancer heterogeneity.

Nuclear morphology alters during development and disease. Nuclear size and shape are regulated through several mechanisms. In this study, details of phenotypes of temperature-sensitive mutants, which were isolated from Chinese hamster CHO-K1 cells to identify genes responsible for the maintenance of chromosome integrity, were characterized with particular focus on changes in their nuclear size and shape. DNA replication has been implicated in a mutant exhibiting elongation of the nucleus with an increase in its ellipticity during incubation at the nonpermissive temperature of 39°C. Incubation at this temperature also resulted in nuclear enlargement in other mutants accompanied by increased DNA damage and led to a remarkable increase in cells harboring an abnormal nucleus, particularly multiple nuclei or segmented nuclei. These findings may lead to the discovery of a novel mechanism that regulates nuclear size and shape. Identification of genes responsible for these defects is highly desirable.Key words: 53BP1, γH2AX, mammalian, replication, temperature-sensitive mutant.

Tubular-shaped recycling endosomes, known as tubular endosomes, are present in certain types of cells, including HeLa cells, and they regulate the recycling of clathrin-independent endocytosed (CIE) cargo proteins to the plasma membrane. Several key regulators of tubular endosomes, including Rab small GTPases and related proteins, have been identified thus far, but the entire process of tubular endosome formation is not yet fully understood. We previously showed that expression of a Golgi-related Rab-GTPase-activating protein TBC1D22B in HeLa cells caused their tubular structures to disappear, suggesting a possible link between tubular endosome formation and a certain Golgi function(s). However, nothing is known about the target Rab(s) of TBC1D22B in tubular endosome formation or about the functional relationship between tubular endosomes and the Golgi apparatus. Here, we performed comprehensive Rab-knockdown screening in combination with dominant-negative Rab expression and succeeded in identifying 12 Rabs as regulators of tubular endosome formation. One of them, Rab30, a Golgi-resident Rab, is a novel target of TBC1D22B and involved in both tubular endosome formation and CIE cargo trafficking. We also showed that a Rab30-BICD2-KIF5B axis is likely to be involved in tubular endosome formation. Our findings suggest the importance of Rab30-mediated post-Golgi trafficking in tubular endosome formation.Key words: Golgi, GTPase-activating protein (GAP), Rab30, siRNA screening, tubular endosome.

The Golgi stress response is a homeostatic mechanism that augments Golgi function when Golgi function becomes insufficient (Golgi stress). Glycosylation of the core proteins of proteoglycans is one of the important functions of the Golgi. If the production of core proteins is increased and the amount of glycosylation enzymes for proteoglycans becomes insufficient (PG-type Golgi stress), the proteoglycan pathway of the Golgi stress response is activated, resulting in the transcriptional induction of glycosylation enzymes, including NDST2, HS6ST1 and GLCE. The transcriptional induction of these glycosylation enzymes is regulated by the enhancer element, PGSE-A; however, transcription factors that induce transcription from PGSE-A have not yet been identified. We herein identified KLF2 and KLF4 as transcription factors that directly bind to PGSE-A, and found that overexpression of KLF2 and KLF4 augments transcriptional induction from PGSE-A during PG-type Golgi stress, whereas their dominant negative mutants suppress the transcriptional induction. Moreover, expression of KLF2 and KLF4 was up-regulated in response to PG-type Golgi stress. Transcriptional induction of human KLF4 gene is regulated by PGSE-A, while that of human KLF2 gene is mainly controlled by a novel enhancer called PGSE-C. These results suggest that KLF2 and KLF4 are important regulators of the proteoglycan pathway of the mammalian Golgi stress response.Key words: Golgi stress, proteoglycan, ER stress, organelle zone, organelle autoregulation, KLF2, KLF4, xyloside.

Tumor endothelial cells (TECs) play a key role in tumor growth and metastasis. They exhibit distinct phenotypic and functional features compared with normal endothelial cells (NECs). Although microRNAs (miRNAs) play diverse roles in tumor progression, the differences in the miRNA expression profiles between TECs and NECs and their role in the TEC phenotype remain unclear.In this study, we isolated NECs and TECs from non-tumor and tumor regions of human renal cancer tissues and conducted an miRNA array analysis. Of the 13 differentially expressed miRNAs, miR-199a-3p was expressed the highest in TECs. Overexpression of miR-199a-3p in NECs significantly enhanced their proliferation, migration, and invasion.Target gene analysis using four databases identified CD151 as a potential target of miR-199a-3p. CD151 expression was downregulated in TECs compared with NECs, and miR-199a-3p transfection decreased CD151 expression in NECs. CD151 knockdown by siRNA promotes cell proliferation, migration, and invasion, and reduces cell adhesion to the extracellular matrix. Moreover, miR-199a-3p overexpression and CD151 silencing upregulated MMP2 expression in TECs. Taken together, these results suggest that miR-199a-3p contributes to the proangiogenic phenotype in TECs by suppressing CD151 expression.Key words: miR-199a-3p, tumor endothelial cells, CD151, angiogenesis, cell migration and invasion.

Podocytes are terminally differentiated renal epithelial cells that play a crucial role in kidney filtration. Given this essential function, podocyte dysfunction results in kidney diseases known as podocytopathies. Previous studies have demonstrated that maintaining the activation-deactivation balance of mechanistic target of rapamycin complex 1 (mTORC1) is vital for podocyte function. Podocyte-specific knockout (KO) mouse models revealed that abnormal mTORC1 activation leads to severe podocytopathy. Therefore, elucidating the mechanism underlying mTORC1 activation in podocytes may contribute to the development of treatments for certain podocytopathies. In our previous study, we showed that macropinocytosis-large-scale endocytosis-is involved in the molecular mechanism of mTORC1 activation in podocytes. Growth factor (GF) stimulation induces circular dorsal ruffles (CDRs), which are large membrane protrusions on the dorsal surface of podocytes. CDRs serve as precursors to macropinocytosis, generating vesicles called macropinosomes, which transport extracellular nutrients to lysosomes, thereby activating mTORC1. These findings suggest that CDRs-derived macropinosomes modulate the mTORC1 pathway. In the present study, we investigated the molecular mechanism underlying macropinosome formation in podocytes, focusing on flotillin-1 (Flot1), a protein enriched in lipid microdomains. Imaging analysis revealed the localization of Flot1 at CDRs, and Flot1 depletion reduced macropinosome formation. Biochemical analysis further demonstrated impaired GF-stimulated mTORC1 activation in Flot1-KO cells, which exhibited slower growth than control cells. Notably, immuno-staining analysis showed that Flot1 is expressed specifically in podocytes but not in other renal cells. These findings indicate that Flot1 participates in the formation of CDRs-derived macropinosomes and contributes to macropinosome-dependent mTORC1 activation in podocytes.Key words: Flot1, circular dorsal ruffles, macropinocytosis, mTORC1, podocytes.

A primary cilium is a hair-like organelle that protrudes from the cell surface in many cell types. Growing evidence indicates that extracellular vesicles are released from primary cilia, and research is increasingly focused on defining the functions of these cilia-derived extracellular vesicles (EVs). EVs are known to modulate the behavior of various cancer cells, and structural and functional abnormalities in primary cilia have been reported in multiple cancer types. We previously demonstrated that PANC-1 cells, a human pancreatic ductal adenocarcinoma cell line, acquire enhanced primary cilia formation after surviving solitary culture conditions, and that their cilia contribute to tumor-like cell mass formation. Here, we explored part of the underlying mechanism of this phenotype by investigating the contribution of EVs released from the primary cilia of PANC-1 cells. PANC-1 clones generated by limiting dilution exhibited enhanced ciliogenesis and distinct ciliary morphologies compared with parental cells. These clones also released higher levels of cilia-derived EVs, including an expanded population of freely floating EVs within the culture environment. Biochemical analyses further showed that this increase was selective for primary cilia-derived EVs rather than reflecting a global rise in total EV production. Functionally, EV fractions enriched in cilia-derived EVs suppressed parental PANC-1 cell migration, altered cell morphology, and promoted cell aggregation, mimicking key behavioral traits of solitary condition-surviving PANC-1 clones. Together, these findings identify enhanced release of primary cilia-derived EVs as a distinct feature of PANC-1 cells adapted to solitary growth and suggest their potential involvement in the malignant and metastatic behaviors of pancreatic cancer.Key words: primary cilia, PDAC, extracellular vesicles, cell migration, cell aggregation.

Metastasis of cancer cells to the brain leads to a poor prognosis in patients with cancer. The brain environment is characterized by cell types, extracellular matrices (ECMs), and mechanical properties that differ from those of the primary tumors. A previous study using human melanoma cells (WM266.4 cells) and its highly brain-metastatic subline cells (WM266.4-BrM3 cells) revealed that WM266.4-BrM3 cells showed enhanced proliferation in brain tissues after cardiac injection in mice compared with WM266.4 cells. However, the effects of mechanical properties such as ECM stiffness on growth and gene expression in WM266.4-BrM3 cells remain to be clarified. In this study, we cultured these cells on ECMs of different stiffnesses. On a soft ECM, WM266.4-BrM3 cells showed significantly higher proliferation and lower expression of early growth response 1 (EGR1) and TP53 than WM266.4 cells. In contrast, on a stiff ECM, the proliferation and EGR1 expression of WM266.4 and WM266.4-BrM3 cells were not significantly different. Additionally, EGR1 knockdown by siRNA transfection in WM266.4 cells results in promoted cell proliferation and downregulated TP53 on a soft ECM. These results suggest that brain metastatic WM266.4 cells decrease EGR1 expression, thereby promoting cell proliferation via TP53 downregulation on a soft ECM.Key words: EGR1, ECM stiffness, metastasis, cancer, growth.

Mei-P26, a member of the TRIM-NHL family, plays a pivotal role in Drosophila germline development, including female meiosis. However, its role in male meiosis remains unclear. We observed abnormal spermatid cysts comprising 16 cells in mei-P26 mutant testes, which resulted from spermatid differentiation in the absence of meiosis. The same phenotype was observed in the cysts derived from spermatocytes subjected to mei-P26 knockdown. No cysts undergoing meiosis were observed, and cyclin-dependent kinase 1 (Cdk1) was not activated in the knockdown spermatocytes. However, these phenotypes are unlikely to result from altered phosphorylation of Cdk1, which is necessary for its activation. Instead, aberrant subcellular localization of Cyclin B (CycB) was observed. In wild-type, CycB first migrates into the nucleus of spermatocytes at earlier stages, is then exported from the nucleus, and re-enters the nucleus just before meiosis. By contrast, in mei-P26^mfs1 spermatocytes and mei-P26 knockdown cells, CycB remained accumulated in the nucleus before meiosis. Another M-phase Cyclin, Cyclin A also showed nuclear accumulation in mei-P26 knockdown spermatocytes. Interactions between CycB and the nuclear export factors Emb and Nup62 remained unchanged. Although mammalian PLK1 modifies nuclear export signal of CycB in mitosis, a Drosophila orthologue, Polo, is unlikely to be involved in this meiotic phenotype. The loss of mei-P26 resulted in continuous activation of the meiotic checkpoint, which retains M-phase cyclins within the nucleus until multiple conditions necessary for meiosis are satisfied, thereby preventing Cdk1 from full activation. Our findings will be useful for understanding the role of Mei-P26 in other developmental processes.Key words: Meiosis, Spermatogenesis, Drosophila, mei-P26, checkpoint, Cyclins, Cdk1.

In higher eukaryotic cells, genomic DNA is packaged into dynamic chromatin domains whose physical behavior is coupled to DNA transactions such as transcription and DNA repair. Although chromatin organization is altered in cancer, how oncogenic signals modulate chromatin dynamics over time remains unclear. To address this issue, we established a doxycycline-inducible carcinogenesis model in hTERT-immortalized human RPE-1 cells expressing HPV16 E6/E7, MYC(T58A), and KRAS(G12V) (EMR cells) and investigated chromatin behavior during oncogene-driven transformation. Upon induction, EMR cells displayed accelerated proliferation, loss of contact inhibition, anchorage-independent growth in soft agar, and tumor formation in nude mice. Using time-resolved single-nucleosome imaging to track local chromatin dynamics over days to weeks of oncogene induction, we found that local nucleosome motion was unchanged at 1-3 days, significantly increased at 5-7 days, and returned to parental levels by 4 weeks, despite sustained oncogene expression and stable malignant growth. To explore the basis of this transient increase, we quantified DNA damage, histone marks, and transcription. γH2AX foci were elevated in EMR cells, but ATM/ATR inhibition had only minor effects on local chromatin motion, indicating that the DNA damage response is not the principal driver. By contrast, H3/H4 acetylation and nascent RNA synthesis were upregulated specifically during the early window of enhanced dynamics, whereas the heterochromatin mark H3K9me3 decreased, consistent with transient chromatin loosening associated with increased transcription. These findings reveal a biphasic change in local chromatin dynamics during human oncogene-driven transformation and provide a physical and temporal framework for understanding how oncogenic pathways reorganize chromatin.Key words: cancer, oncogenesis, single-nucleosome imaging, chromatin dynamics.