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HAP1 helps to regulate actin-based transport of insulin-containing granules in pancreatic β cells

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Abstract

Huntingtin-associated protein 1 (HAP1) is enriched in neurons and binds to polyglutamine-expanded huntingtin. It consists of two alternatively spliced isoforms, HAP1A and HAP1B, which differ only in their short C-terminal sequences. Both HAP1A and HAP1B have been also detected in pancreatic β cells, where the loss of HAP1 impairs glucose-stimulated insulin secretion. Here, we use time-lapse laser scanning confocal microscopy to provide direct evidence that HAP1A, but not HAP1B, co-localizes and co-migrates with insulin-containing vesicles and actin-based myosin Va motor protein in the INS-1 pancreatic β cell line. Knocking down HAP1 expression using small interfering RNA significantly inhibited actin-based transport of insulin vesicles following glucose stimulation. Co-immunoprecipitation experiments demonstrated interaction between HAP1A, myosin Va, and phogrin, a transmembrane protein in insulin-containing vesicles. Stimulating INS-1 cells with glucose increased the association of HAP1A with myosin Va, while silencing HAP1 expression reduced the association of myosin Va with phogrin after glucose stimulation, without affecting levels of myosin Va or actin. Our results provide real-time evidence in living cells that HAP1 may help regulate transport of insulin-containing secretory granules along cortical actin filaments. This also raises the possibility that HAP1 may play an important role in actin-based secretory vesicle trafficking in neurons.

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Acknowledgments

This work was supported by a grant from the National Natural Science Foundation of China (No. 31171155) to HL. We are grateful to Professor Guy Rutter of Imperial College London for the kind gift of phogrin-EGFP plasmid.

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The authors report no conflicts of interest.

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Authors and Affiliations

  1. Division of Histology and Embryology, Department of Anatomy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China

    Zhiyong Wang, Ting Peng, Hongnian Wu, Jun He & He Li

  2. Department of Anatomy, Xinxiang Medical University, Xinxiang, 453003, People’s Republic of China

    Zhiyong Wang

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  1. Zhiyong Wang
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  2. Ting Peng
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  3. Hongnian Wu
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  4. Jun He
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Correspondence to He Li.

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418_2015_1311_MOESM3_ESM.tif

Fig. S1 Partial co-localization of myosin Va/actin and HAP1/actin. Immunofluorescence double labeling with antibodies against myosin Va (red) or HAP1(both HAP1A and HAP1B) (red) and actin (green) in INS-1 cells. Confocal microscopy was used to capture serial sections at 1-μm intervals of (A) myosin Va/actin and (B) HAP1/actin. Myosin Va and HAP1 partially co-localized with actin (arrows). (TIFF 985 kb)

418_2015_1311_MOESM4_ESM.tif

Fig. S2. Diffuse localization of HAP1B in the cytoplasm. INS-1 cultured in 6-well plates were transfected with 0.5, 1.0 or 2.0 μg per well of plasmid encoding EGFP-HAP1B (green) or pJred-HAP1B (red). Confocal microscopy showed a homogeneous cytoplasmic distribution of HAP1B. (TIFF 848 kb)

418_2015_1311_MOESM5_ESM.tif

Fig. S3 Dephosphorylated HAP1A binds more myosin Va than wild-type HAP1A. (A) INS-1 cells were transfected with plasmids expressing EGFP fusions of wild-type HAP1A (EGFP-HAP1A) or HAP1A(T598A) mutant (EGFP-T598A), and the ability of these fusion proteins to co-precipitate endogenous myosin Va was investigated. Immunoprecipitates were probed by Western blotting using a rabbit anti-GFP antibody. EGFP-T598A bound more myosin Va than wild-type HAP1A. (B) Densitometric analysis of the ratio of endogenous myosin Va to EGFP-HAP1A or EGFP-T598A from 3 experiments. (TIFF 304 kb)

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Wang, Z., Peng, T., Wu, H. et al. HAP1 helps to regulate actin-based transport of insulin-containing granules in pancreatic β cells. Histochem Cell Biol 144, 39–48 (2015). https://doi.org/10.1007/s00418-015-1311-9

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