Shootin1 is a brain-specific cytoplasmic protein involved in neuronal polarity formation and axon outgrowth. It accumulates at the leading edge of axonal growth cones, where it mediates the mechanical coupling between F-actin retrograde flow and cell adhesions as a clutch molecule, thereby producing force for axon outgrowth. In this study, we report a novel splicing isoform of shootin1 which is expressed not only in the brain but also in peripheral tissues. We have renamed the brain-specific shootin1 as shootin1a and termed the novel isoform as shootin1b. Immunoblot and immunohistochemical analyses with a shootin1b-specific antibody revealed that shootin1b is distributed in various mouse tissues including the lung, liver, stomach, intestines, spleen, pancreas, kidney and skin. Interestingly, shootin1b immunoreactivity was widely detected in epithelial cells that constitute simple and stratified epithelia; in some cells, it colocalized with E-cadherin and cortactin at cell–cell contact sites. Shootin1b also localized in dendritic cells in the spleen. These results suggest that shootin1b may function in various peripheral tissues including epithelial cells.
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EpH4 cells were the kind gift of Dr. E. Reichman. We thank Drs. Sadao Shiosaka and Michinori Toriyama for reviewing the manuscript.
This research was supported in part by JSPS Grant-in-Aid for Scientific Research on Innovative Areas (25102010), JSPS KAKENHI (26290007), Osaka Medical Research Foundation for Incurable Diseases and Mitsubishi Foundation.
After the submission of this paper, a report by Ergin et al. (2015) was published. This paper describes shootin1b expression in a PC12 cell line.
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Two splice variants and isoforms of the mouse shootin1 gene (a). The structure of the shootin1 locus and transcripts. (i) The structure of the mouse shootin1 genomic locus. (ii) Transcripts in the shootin1 locus. The shootin1 gene has two splice variants, a 3767-nt mRNA (NM_175172.4) encoding a protein of 456 amino acids and a 4081-nt mRNA (NM_001114312.1) encoding a protein of 631 amino acids. Gray and white boxes indicate exons and untranslated regions, respectively. Positions of stop codons (TAA and TGA) are shown. b Alignment of shootin1 isoforms. The amino acid sequences were obtained from the following sources: shootin1a (ABK56021.1) and shootin1b (NP_001107784.1). (TIF 1787 kb)
Immunoblot analyses of shootin1b in adult mouse tissues and E18.5 mouse tissues with anti-shootin1b antibody. Whole scans of the immunoblotted membranes of Fig. 1d are shown. (TIF 1417 kb)
Control staining of the skin and forestomach without anti-shootin1b antibody. Two serial sections of the adult (a) and E18.5 (b and e) mouse skin, and the adult (c) and E18.5 (d) forestomach were immunostained: one section stained with anti-shootin1b antibody (shootin1b) and the other stained without the antibody (negative control). They were also co-stained with DAPI (a–d), anti-E-cadherin antibody (e) and anti-cortactin antibody (e) (red). CL cornified layer; KL keratinized layer. The data in (e) represent control data of those in Fig. 9. Bars 50 μm. (TIF 9471 kb)
RT-PCR analysis of shootin1b mRNA in the skin. RNA was extracted from the cornified layer and whole skin of P5 mouse. PCR was carried out by using shootin1b-specific primers (left) and β-actin-specific primers (right) as a positive control. The PCR products were electrophoresed on a 2 % agarose gel. The expected 233-bp shootin1b and 165-bp β-actin PCR products were detected both in the cornified layer (lane 1) and whole skin (lane 2). M, 1 kb DNA ladder marker. (TIF 1239 kb)
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Higashiguchi, Y., Katsuta, K., Minegishi, T. et al. Identification of a shootin1 isoform expressed in peripheral tissues. Cell Tissue Res 366, 75–87 (2016). https://doi.org/10.1007/s00441-016-2415-9
- Clutch molecule
- Epithelial cell
- Dendritic cell