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Microtubule-dependent formation of the stigmoid body as a cytoplasmic inclusion distinct from pathological aggresomes

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Abstract

The stigmoid body (STB) is a neurocytoplasmic inclusion containing huntingtin-associated protein 1 (HAP1), an interactor of huntingtin, and its formation is induced by transfection of HAP1-cDNA into cultured cells. Although STB is believed to play a protective role in polyglutamine diseases, including Huntington’s disease and spinal and bulbar muscular atrophy, by sequestering the causative proteins, huntingtin and androgen receptor, respectively, its physiological function and formation remain poorly understood. Therefore, STB is occasionally confused with another cytoplasmic inclusion observed in polyglutamine diseases, the aggresome. Here we examined the subcellular dynamics of STB and compared it immunohistochemically and cytochemically with the aggresome in the rat brain and COS-7 or HeLa cells transfected with HAP1 and/or polyglutamine disease-associated genes. In time-lapse image analysis of HAP1-transfected cells, the HAP1-induced STB is formed from multiple fusions of small HAP1 inclusions characterized by vigorous cytoplasmic movement. In HAP1-transfected cells treated with a microtubule-depolymerizing drug, although the formation of small HAP1 inclusions was not affected, their fusion was critically inhibited. Immunohistochemistry and cytochemistry revealed the absence of association between STB and aggresomal markers, such as ubiquitin/proteasome, intermediate filaments, and the centrosome. Taken together, we concluded that STB is formed by a two-step process comprising microtubule-independent formation of small HAP1 inclusions and microtubule-dependent fusion of these inclusions, and that STB is distinct from pathological aggresomes.

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Acknowledgments

We cordially thank Dr. Keith R. Yamamoto (School of Medicine, University of California, San Francisco, CA, USA) for providing HA-ARN127(Q65) plasmid, and Drs. Warren J. Strittmatter and James R. Burke (Duke University Medical Center, Durham, NC, USA) for the Q80-GFP plasmid. We are grateful to Dr. Makoto Inui (Yamaguchi University) for his advice on the preparation of stable transformant. We also thank Dr. Changjiu Zhao, Dr. Kumiko Yoshida, Mr. Chikahisa Matsuo, Mr. Jun Oba, and Mrs. Yumiko Matsuzaki for their technical assistance and administrative help. We would also like to acknowledge the DNA Core facility of the Center for Gene Research, Yamaguchi University, supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and the technical expertise of the Institute for Biomedical Research and Education, Yamaguchi University Science Research Center. This study was supported by Grant-in-Aids for Young Scientists (B) (No. 17700333) and for Scientific Research on Priority Areas (No.19040020) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and Grant-in-Aids for Scientific Research (C) (No.17500231) and for Young Scientists (Start-up) (No. 19800027) from the Japan Society for the Promotion of Science (JSPS).

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  1. Division of Neuroanatomy, Department of Neuroscience, Yamaguchi University School of Medicine, Minami-Kogushi 1-1-1, Ube, Yamaguchi, 755-8505, Japan

    Ryutaro Fujinaga, Yukio Takeshita, Kanako Uozumi, Akie Yanai, Kazuhiro Yoshioka, Keiji Kokubu & Koh Shinoda

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  1. Ryutaro Fujinaga
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  2. Yukio Takeshita
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  3. Kanako Uozumi
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  4. Akie Yanai
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  6. Keiji Kokubu
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  7. Koh Shinoda
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Correspondence to Koh Shinoda.

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Fujinaga, R., Takeshita, Y., Uozumi, K. et al. Microtubule-dependent formation of the stigmoid body as a cytoplasmic inclusion distinct from pathological aggresomes. Histochem Cell Biol 132, 305–318 (2009). https://doi.org/10.1007/s00418-009-0618-9

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