Cell Structure and Function

Phosphatidylserine (PS) is a constituent of the cell membrane, being especially abundant in the cytoplasmic leaflet, and plays important roles in a number of cellular functions, including the formation of cell polarity and intracellular vesicle transport. Several studies in mammalian cells have suggested the role of PS in retrograde membrane traffic through endosomes, but in yeast, where PS is localized primarily at the plasma membrane (PM), the role in intracellular organelles remains unclear. Additionally, it is reported that polarized endocytic site formation is defective in PS-depleted yeast cells, but the role in the endocytic machinery has not been well understood...

The claudin family of membrane proteins is responsible for the backbone structure and function of tight junctions (TJs), which regulate the paracellular permeability of epithelia. It is thought that each claudin subtype has its own unique function and the combination of expressed subtypes determines the permeability property of each epithelium. However, many issues remain unsolved in regard to claudin functions, including the detailed functional differences between claudin subtypes and the effect of the combinations of specific claudin subtypes on the structure and function of TJs...

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...

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...

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...

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...

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...

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)...

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...

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 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 monomeric GTPase Rab27 regulates exocytosis of a broad range of vesicles in multicellular organisms. Several effectors bind GTP-bound Rab27a and/or Rab27b on secretory vesicles to execute a series of exocytic steps, such as vesicle maturation, movement along microtubules, anchoring within the peripheral F-actin network, and tethering to the plasma membrane, via interactions with specific proteins and membrane lipids in a local milieu. Although Rab27 effectors generally promote exocytosis, they can also temporarily restrict it when they are involved in the rate-limiting step...

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...

Glycosylphosphatidylinositol (GPI)-anchored proteins are post-transcriptionally modified with GPI and anchored to the plasma membrane. GPI is attached to nascent proteins in the endoplasmic reticulum by the GPI transamidase complex, which consists of PIGT, PIGK, GPAA1, PIGU, and PIGS. Of these, PIGK is a catalytic subunit that is unstable without PIGT. This study investigated the pathway by which unassembled PIGK not incorporated into the complex is degraded. We showed that unassembled PIGK was degraded via the proteasome-dependent pathway and that Hrd1 (also known as SYVN1), a ubiquitin ligase involved in the endoplasmic reticulum-associated degradation pathway, was responsible for degradation of unassembled PIGK...

Most organisms have multiple α- and β-tubulin isotypes that likely contribute to the diversity of microtubule (MT) functions. To understand the functional differences of tubulin isotypes in Caenorhabditis elegans, which has nine α-tubulin isotypes and six β-tubulin isotypes, we systematically constructed null mutants and GFP-fusion strains for all tubulin isotypes with the CRISPR/Cas9 system and analyzed their expression patterns and levels in adult hermaphrodites. Four isotypes-α-tubulins TBA-1 and TBA-2 and β-tubulins TBB-1 and TBB-2-were expressed in virtually all tissues, with a distinct tissue-specific spectrum...

Dysfunction of the endoplasmic reticulum (ER), so-called ER stress, is accompanied with accumulation of unfolded proteins in the ER. Eukaryotic cells commonly have an ER-located transmembrane protein, Ire1, which triggers cellular protective events against ER stress. In animal cells, PERK and ATF6 also initiate the ER-stress response. As a common strategy to control the activity of these ER-stress sensors, an ER-resident molecular chaperone, BiP, serves as their negative regulator, and dissociates from them in response to ER stress...

Stable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biology technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5'UTR of the endogenous gene loci. With this 5'UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high efficiencies ranging from 50 to 80% in antibiotic selected cells...

The development of hearing in mammals requires the formation and maturation of a highly organized and specialized epithelium known as the organ of Corti. This epithelium contains two types of cells, the sensory cells, which are the true receptors of auditory information, and the surrounding supporting cells, which are composed of a highly developed cytoskeleton essential to the architecture of the mature organ of Corti. The supporting cells are the only mammalian cells reported to contain the unusual 15-protofilament microtubules...

The somatic haploidy is unstable in diplontic animals, but cellular processes determining haploid stability remain elusive. Here, we found that inhibition of mevalonate pathway by pitavastatin, a widely used cholesterol-lowering drug, drastically destabilized the haploid state in HAP1 cells. Interestingly, cholesterol supplementation did not restore haploid stability in pitavastatin-treated cells, and cholesterol inhibitor U18666A did not phenocopy haploid destabilization. These results ruled out the involvement of cholesterol in haploid stability...

The smallest arenavirus gene product, Z protein, plays critical roles in the virus life cycle. Z is the major driving force of budding and particle production because of a unique property that defines self-assembly. In addition to the roles in budding, Z also participates in the suppression of type I interferon production to evade host antiviral immunity. Therefore, Z and its assembled form are an attractive drug target for arenaviral hemorrhagic fever, such as Lassa fever. Here, we developed a biosensor that enabled the evaluation of the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV), Z assembly using the principle of Förster resonance energy transfer (FRET)...