Cell Structure and Function
Online ISSN : 1347-3700
Print ISSN : 0386-7196
ISSN-L : 0386-7196
Volume 47, Issue 1
Displaying 1-4 of 4 articles from this issue
  • Yusuke Takanashi, Tomoaki Kahyo, Sae Kamamoto, Hengsen Zhang, Bin Chen ...
    Article type: Mini-review and Review
    2022 Volume 47 Issue 1 Pages 1-18
    Published: 2022
    Released on J-STAGE: February 23, 2022

    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

  • Haruka Kemmoku, Yoshihiko Kuchitsu, Kojiro Mukai, Tomohiko Taguchi
    Article type: Short Communication
    2022 Volume 47 Issue 1 Pages 19-30
    Published: 2022
    Released on J-STAGE: March 08, 2022
    Advance online publication: February 05, 2022
    Supplementary material

    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

  • Kouichi Mizuno, Tetsuro Izumi
    Article type: Full Article
    2022 Volume 47 Issue 1 Pages 31-41
    Published: 2022
    Released on J-STAGE: May 21, 2022
    Advance online publication: April 06, 2022
    Supplementary material

    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

  • Yoichiro Fujioka, Sayaka Kashiwagi, Aiko Yoshida, Aya O. Satoh, Mari F ...
    Article type: Full Article
    2022 Volume 47 Issue 1 Pages 43-53
    Published: 2022
    Released on J-STAGE: June 25, 2022
    Advance online publication: April 28, 2022
    Supplementary material

    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.

    Key words: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein