1. Cell Biology

Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis

  1. Kunli Zhao
  2. Kohichi Matsunaga
  3. Kouichi Mizuno
  4. Hao Wang
  5. Katsuhide Okunishi
  6. Tetsuro Izumi  Is a corresponding author
  1. Gunma University, Japan
https://doi.org/10.7554/eLife.82821
Share this article
Cite this article
  1. Kunli Zhao
  2. Kohichi Matsunaga
  3. Kouichi Mizuno
  4. Hao Wang
  5. Katsuhide Okunishi
  6. Tetsuro Izumi
(2023)
Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis
eLife 12:e82821.
https://doi.org/10.7554/eLife.82821
  2. Download BibTeX
  3. Download .RIS

Abstract

The Rab27 effectors are known to play versatile roles in regulated exocytosis. In pancreatic beta cells, exophilin-8 anchors granules in the peripheral actin cortex, whereas granuphilin and melanophilin mediate granule fusion with and without stable docking to the plasma membrane, respectively. However, it is unknown whether these coexisting effectors function in parallel or in sequence to support the whole insulin secretory process. Here, we investigate their functional relationships by comparing the exocytic phenotypes in mouse beta cells simultaneously lacking two effectors with those lacking just one of them. Analyses of prefusion profiles by total internal reflection fluorescence microscopy suggest that melanophilin exclusively functions downstream of exophilin-8 to mobilize granules for fusion from the actin network to the plasma membrane after stimulation. The two effectors are physically linked via the exocyst complex. Downregulation of the exocyst component affects granule exocytosis only in the presence of exophilin-8. The exocyst and exophilin-8 also promote fusion of granules residing beneath the plasma membrane prior to stimulation, although they differentially act on freely diffusible granules and those stably docked to the plasma membrane by granuphilin, respectively. The present study is the first to diagram the multiple intracellular pathways of granule exocytosis and the functional hierarchy among different Rab27 effectors within the same cell.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Kunli Zhao

    Department of Molecular Medicine, Gunma University, Maebashi, Japan
    Competing interests
    The authors declare that no competing interests exist.

  2. Kohichi Matsunaga

    Department of Molecular Medicine, Gunma University, Maebashi, Japan
    Competing interests
    The authors declare that no competing interests exist.

  3. Kouichi Mizuno

    Department of Molecular Medicine, Gunma University, Maebashi, Japan
    Competing interests
    The authors declare that no competing interests exist.

  4. Hao Wang

    Department of Molecular Medicine, Gunma University, Maebashi, Japan
    Competing interests
    The authors declare that no competing interests exist.

  5. Katsuhide Okunishi

    Department of Molecular Medicine, Gunma University, Maebashi, Japan
    Competing interests
    The authors declare that no competing interests exist.

  6. Tetsuro Izumi

    Department of Molecular Medicine, Gunma University, Maebashi, Japan
    For correspondence
    tizumi@gunma-u.ac.jp
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0974-7384

Funding

Japan Society for the Promotion of Science (JP19H03449)

  • Tetsuro Izumi

Japan Society for the Promotion of Science (JP20K06535)

  • Kouichi Mizuno

Japan Society for the Promotion of Science (JP20K15742)

  • Hao Wang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Jonathan S Bogan, Yale School of Medicine, United States

Ethics

Animal experimentation: Animal experiments were performed according to the rules and regulations of the Animal Care and Experimental Committees of Gunma University (permit number: 22-010; Maebashi, Japan).

Version history

  1. Received: August 18, 2022
  2. Preprint posted: August 23, 2022 (view preprint)
  3. Accepted: February 21, 2023
  4. Accepted Manuscript published: February 21, 2023 (version 1)
  5. Version of Record published: March 6, 2023 (version 2)

Copyright

© 2023, Zhao et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,139
    views
  • 195
    downloads
  • 5
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Kunli Zhao
  2. Kohichi Matsunaga
  3. Kouichi Mizuno
  4. Hao Wang
  5. Katsuhide Okunishi
  6. Tetsuro Izumi
(2023)
Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis
eLife 12:e82821.
https://doi.org/10.7554/eLife.82821

Share this article

https://doi.org/10.7554/eLife.82821

Categories and tags

Research organism

Further reading

    1. Cancer Biology
    2. Cell Biology

    A syngeneic spontaneous zebrafish model of tp53-deficient, EGFRvIII, and PI3KCAH1047R-driven glioblastoma reveals inhibitory roles for inflammation during tumor initiation and relapse in vivo

    Alex Weiss, Cassandra D'Amata ... Madeline N Hayes
    Research Article

    High-throughput vertebrate animal model systems for the study of patient-specific biology and new therapeutic approaches for aggressive brain tumors are currently lacking, and new approaches are urgently needed. Therefore, to build a patient-relevant in vivo model of human glioblastoma, we expressed common oncogenic variants including activated human EGFRvIII and PI3KCAH1047R under the control of the radial glial-specific promoter her4.1 in syngeneic tp53 loss-of-function mutant zebrafish. Robust tumor formation was observed prior to 45 days of life, and tumors had a gene expression signature similar to human glioblastoma of the mesenchymal subtype, with a strong inflammatory component. Within early stage tumor lesions, and in an in vivo and endogenous tumor microenvironment, we visualized infiltration of phagocytic cells, as well as internalization of tumor cells by mpeg1.1:EGFP+ microglia/macrophages, suggesting negative regulatory pressure by pro-inflammatory cell types on tumor growth at early stages of glioblastoma initiation. Furthermore, CRISPR/Cas9-mediated gene targeting of master inflammatory transcription factors irf7 or irf8 led to increased tumor formation in the primary context, while suppression of phagocyte activity led to enhanced tumor cell engraftment following transplantation into otherwise immune-competent zebrafish hosts. Altogether, we developed a genetically relevant model of aggressive human glioblastoma and harnessed the unique advantages of zebrafish including live imaging, high-throughput genetic and chemical manipulations to highlight important tumor-suppressive roles for the innate immune system on glioblastoma initiation, with important future opportunities for therapeutic discovery and optimizations.

    1. Cancer Biology
    2. Cell Biology

    Cancer: Modeling glioblastoma

    Ian Lorimer
    Insight

    Establishing a zebrafish model of a deadly type of brain tumor highlights the role of the immune system in the early stages of the disease.

    1. Cell Biology
    2. Neuroscience

    Presynaptic Rac1 in the hippocampus selectively regulates working memory

    Jaebin Kim, Edwin Bustamante ... Scott H Soderling
    Research Article

    One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear. Here, we show that spatial working memory in mice is selectively impaired following the expression of a genetically encoded Rac1 inhibitor at presynaptic terminals, while longer-term cognitive processes are affected by Rac1 inhibition at postsynaptic sites. To investigate the regulatory mechanisms of this presynaptic process, we leveraged new advances in mass spectrometry to identify the proteomic and post-translational landscape of presynaptic Rac1 signaling. We identified serine/threonine kinases and phosphorylated cytoskeletal signaling and synaptic vesicle proteins enriched with active Rac1. The phosphorylated sites in these proteins are at positions likely to have regulatory effects on synaptic vesicles. Consistent with this, we also report changes in the distribution and morphology of synaptic vesicles and in postsynaptic ultrastructure following presynaptic Rac1 inhibition. Overall, this study reveals a previously unrecognized presynaptic role of Rac1 signaling in cognitive processes and provides insights into its potential regulatory mechanisms.