Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis

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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.

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

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Kunli Zhao

Department of Molecular Medicine, Gunma University, Maebashi, Japan

Competing interests
The authors declare that no competing interests exist.
Kohichi Matsunaga

Department of Molecular Medicine, Gunma University, Maebashi, Japan

Competing interests
The authors declare that no competing interests exist.
Kouichi Mizuno

Department of Molecular Medicine, Gunma University, Maebashi, Japan

Competing interests
The authors declare that no competing interests exist.
Hao Wang

Department of Molecular Medicine, Gunma University, Maebashi, Japan

Competing interests
The authors declare that no competing interests exist.
Katsuhide Okunishi

Department of Molecular Medicine, Gunma University, Maebashi, Japan

Competing interests
The authors declare that no competing interests exist.
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.

"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.

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).

Copyright

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.