Cell structure and function最新文献

In macroautophagy, disk-shaped double-membrane structures called phagophores elongate to form cup-shaped structures, becoming autophagosomes upon closure. These autophagosomes then fuse with lysosomes to become autolysosomes and degrade engulfed material. Autophagosome formation is reported to involve other organelles, including the endoplasmic reticulum (ER) and mitochondria. Organelles are also taken up by autophagosomes as autophagy cargos. However, few studies have performed systematic spatiotemporal analysis of inter-organelle relationships during macroautophagy. Here, we investigated the organelles in contact with phagophores, autophagosomes, and autolysosomes by using three-dimensional correlative light and electron microscopy with array tomography in cells starved 30 min. As previously reported, all phagophores associate with the ER. The surface area of phagophores in contact with the ER decreases gradually as they mature into autophagosomes and autolysosomes. However, the ER still associates with 92% of autophagosomes and 79% of autolysosomes, suggesting that most autophagosomes remain on the ER after closure and even when they fuse with lysosomes. In addition, we found that phagophores form frequently near other autophagic structures, suggesting the presence of potential hot spots for autophagosome formation. We also analyzed the contents of phagophores and autophagosomes and found that the ER is the most frequently engulfed organelle (detected in 65% of total phagophores and autophagosomes). These quantitative three-dimensional ultrastructural data provide insights into autophagosome-organelle relationships during macroautophagy.Key words: 3D-CLEM, autophagosome, electron microscopy, endoplasmic reticulum, lysosome.

Phosphatase of regenerating liver (PRL) is frequently overexpressed in various malignant cancers and is known to be a driver of malignancy. Here, we demonstrated that PRL overexpression causes mitotic errors that accompany spindle misorientation and aneuploidy, which are intimately associated with cancer progression. Mechanistic analyses of this phenomenon revealed dysregulation of the energy sensor kinase, AMP-activated protein kinase (AMPK), in PRL-induced mitotic errors. Specifically, immunofluorescence analysis showed that levels of phosphorylated AMPK (P-AMPK), an activated form of AMPK, at the kinetochore were reduced by PRL expression. Moreover, artificial activation of AMPK using chemical activators, such as A769662 and AICAR, in PRL-expressing cells restored P-AMPK signals at the kinetochore and normalized spindle orientation. Collectively, these results indicate the crucial importance of the activation of kinetochore-localized AMPK in the normal progression of mitosis, which is specifically perturbed by PRL overexpression.Key words: cancer, AMPK, PRL, kinetochore, mitotic errors.

The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding α-catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of α-catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of α-catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that increased tension sensitivity of α-catenin affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive α-catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that the appropriate tension sensitivity of α-catenin is crucial for the coordinated round morphogenesis.Key words: α-catenin, vinculin, adherens junction, morphogenesis, mechanotransduction.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 spike protein in high yield. We found that a large proportion of pseudovirions produced with the conventional transient expression system lacked coronavirus spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus spike protein allowed the efficient production of progeny pseudoviruses decorated with spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.

The Rab27 effector granuphilin plays an indispensable role in stable docking of secretory granules to the plasma membrane by interacting with the complex of Munc18-1 and the fusion-incompetent, closed form of syntaxins-1~3. Although this process prevents spontaneous granule exocytosis, those docked granules actively fuse in parallel with other undocked granules after stimulation. Therefore, it is postulated that the closed form of syntaxins must be converted into the fusion-competent open form in a stimulus-dependent manner. Although Munc13 family proteins are generally thought to prime docked vesicles by facilitating conformational change in syntaxins, it is unknown which isoform acts in granuphilin-mediated, docked granule exocytosis. In the present study, we show that, although both Munc13a and Munc13b are expressed in mouse pancreatic islets and their beta-cell line MIN6, the silencing of Munc13b, but not that of Munc13a, severely affects glucose-induced insulin secretion. Furthermore, Munc13b accumulates on a subset of granules beneath the plasma membrane just prior to fusion during stimulation, whereas Munc13a is translocated to the plasma membrane where granules do not exist. When fluorescently labeled granuphilin was introduced to discriminate between molecularly docked granules and other undocked granules in living cells, Munc13b downregulation was observed to preferentially decrease the fusion of granuphilin-positive granules immobilized to the plasma membrane. These findings suggest that Munc13b promotes insulin exocytosis by clustering on molecularly docked granules in a stimulus-dependent manner.Key words: docking, insulin, live cell imaging, priming, TIRF microscopy.

Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA or self-DNA leaked from mitochondria/nuclei. In response to the emergence of such DNAs in the cytosol, STING relocates from the endoplasmic reticulum (ER) to the Golgi, and activates TANK-binding kinase 1 (TBK1), a cytosolic kinase essential for the activation of STING-dependent downstream signalling. To understand at which subcellular compartments TBK1 becomes associated with STING, we generated cells stably expressing fluorescent protein-tagged STING (mNeonGreen-STING) and TBK1 (TBK1-mScarletI). We found that after STING stimulation, TBK1 became associated with the trans-Golgi network (TGN), not the other parts of the Golgi. STING variants that constitutively induce the type I interferon response have been identified in patients with autoinflammatory diseases named "STING-associated vasculopathy with onset in infancy (SAVI)". Even in cells expressing these constitutively active STING variants, TBK1 was found to be associated with TGN, not the other parts of the Golgi. These results suggest that TGN acts as a specific platform where STING associates with and activates TBK1.Key words: the Golgi, membrane traffic, innate immunity, STING.

Ubiquitin-like 3 (UBL3) is a well-conserved ubiquitin-like protein (UBL) in eukaryotes and regulates the ubiquitin cascade, but the significant roles of UBL3 in cellular processes remained unknown. Recently, UBL3 was elucidated to be a post-translational modification factor that promotes protein sorting to small extracellular vesicles (sEVs). Proteins sorted into sEVs have been studied as etiologies of sEV-related diseases. Also, there have been attempts to construct drug delivery systems (DDSs) by loading proteins into sEVs. In this review, we introduce the new concept that UBL3 has a critical role in the protein-sorting system and compare structure conservation between UBL3 and other UBLs from an evolutionary perspective. We conclude with future perspectives for the utility of UBL3 in sEV-related diseases and DDS.Key words: UBL3, small extracellular vesicles, protein sorting, ubiquitin-like protein, post-translational modification.

IFN-γ secreted from immune cells exerts pleiotropic effects on tumor cells, including induction of immune checkpoint and antigen presentation, growth inhibition, and apoptosis induction. We combined a dual promoter system with an IFN-γ signaling responsive promoter to generate a reporter named the interferon sensing probe (ISP), which quantitates the response to IFN-γ by means of fluorescence and bioluminescence. The integration site effect of the transgene is compensated for by the PGK promoter-driven expression of a fluorescent protein. Among five potential IFN-γ-responsive elements, we found that the interferon γ-activated sequence (GAS) exhibited the best performance. When ISP-GAS was introduced into four cell lines and subjected to IFN-γ stimulation, dose-dependency was observed with an EC₅₀ ranging from 0.2 to 0.9 ng/mL, indicating that ISP-GAS can be generally used as a sensitive biosensor of IFN-γ response. In a syngeneic transplantation model, the ISP-GAS-expressing cancer cells exhibited bioluminescence and fluorescence signals in an IFN-γ receptor-dependent manner. Thus, ISP-GAS could be used to quantitatively monitor the IFN-γ response both in vitro and in vivo.Key words: in vivo imaging, tumor microenvironment, interferon-gamma, dual promoter system.

Among the inheritance of cellular components during cell division, deoxyribonucleic acid (DNA) and its condensate (chromosome) are conventionally visualized using chemical tag-labeled nucleotide analogs. However, associated mutagenesis with nucleotide analogs in the visualization of chromosomes is cause for concern. This study investigated the efficiency of using stable isotope labels in visualizing the replicating cultured human cell-chromosomes, in the absence of analog labels, at a high spatial resolution of 100 nm. The distinct carbon isotope ratio between sister chromatids reflected the semi-conservative replication of individual DNA strands through cell cycles and suggested the renewal of histone molecules in daughter chromosomes. Thus, this study provides a new, powerful approach to trace and visualize cellular components with stable isotope labeling.Key words: stable isotope, chromosome replication, semi-conservative replication, imaging, mass spectrometry.

XRN2 is a 5'-to-3' exoribonuclease that is predominantly localized in the nucleus. By degrading or trimming various classes of RNA, XRN2 contributes to essential processes in gene expression such as transcription termination and ribosome biogenesis. Despite limited substrate specificity in vitro, XRN2 targets a specific subset of RNA by interacting with other proteins in cells. Here we review the functions of proteins that have an evolutionarily conserved XRN2-binding domain, XTBD. These proteins modulate activity of XRN2 by stabilizing it, controlling its subcellular localization or recruiting it to specific RNA targets, and thereby impact on various cellular processes.Key words: RNA regulation, XRN2, XTBD, ribosome biogenesis, subcellular localization.