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Cell and Tissue Research

, Volume 366, Issue 1, pp 75–87 | Cite as

Identification of a shootin1 isoform expressed in peripheral tissues

  • Yasuna Higashiguchi
  • Kazuhiro Katsuta
  • Takunori Minegishi
  • Shigenobu Yonemura
  • Akihiro Urasaki
  • Naoyuki Inagaki
Regular Article

Abstract

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.

Keywords

Shootin1b Clutch molecule Epithelial cell Dendritic cell 

Notes

Acknowledgments

EpH4 cells were the kind gift of Dr. E. Reichman. We thank Drs. Sadao Shiosaka and Michinori Toriyama for reviewing the manuscript.

Compliance with ethical standards

Funding

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.

Supplementary material

441_2016_2415_MOESM1_ESM.tif (1.7 mb)
Fig. S1 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)
441_2016_2415_MOESM2_ESM.tif (1.4 mb)
Fig. S2 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)
441_2016_2415_MOESM3_ESM.tif (9.2 mb)
Fig. S3 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 (ad), 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)
441_2016_2415_MOESM4_ESM.tif (1.2 mb)
Fig. S4 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)

References

  1. Brieher WM, Yap AS (2013) Cadherin junctions and their cytoskeleton(s). Curr Opin Cell Biol 25:39–46CrossRefPubMedGoogle Scholar
  2. Craig EM, Stricker J, Gardel M, Mogilner A (2015) Model for adhesion clutch explains biphasic relationship between actin flow and traction at the cell leading edge. Phys Biol 12:035002CrossRefPubMedPubMedCentralGoogle Scholar
  3. El Sayegh TY, Arora PD, Laschinger CA, Lee W, Morrison C, Overall CM, Kapus A, McCulloch CA (2004) Cortactin associates with N-cadherin adhesions and mediates intercellular adhesion strengthening in fibroblasts. J Cell Sci 117:5117–5131CrossRefPubMedGoogle Scholar
  4. Forscher P, Smith SJ (1988) Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J Cell Biol 107:1505–1516CrossRefPubMedGoogle Scholar
  5. Garcia M, Leduc C, Lagardere M, Argento A, Sibarita JB, Thoumine O (2015) Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones. Proc Natl Acad Sci U S A 112:6997–7002CrossRefPubMedPubMedCentralGoogle Scholar
  6. Helwani FM, Kovacs EM, Paterson AD, Verma S, Ali RG, Fanning AS, Weed SA, Yap AS (2004) Cortactin is necessary for E-cadherin-mediated contact formation and actin reorganization. J Cell Biol 164:899–910CrossRefPubMedPubMedCentralGoogle Scholar
  7. Inagaki N, Yamatodani A, Ando-Yamamoto M, Tohyama M, Watanabe T, Wada H (1988) Organization of histaminergic fibers in the rat brain. J Comp Neurol 273:283–300CrossRefPubMedGoogle Scholar
  8. Inagaki N, Goto H, Ogawara M, Nishi Y, Ando S, Inagaki M (1997) Spatial patterns of Ca2+ signals define intracellular distribution of a signaling by Ca2+/Calmodulin-dependent protein kinase II. J Biol Chem 272:25195–25199CrossRefPubMedGoogle Scholar
  9. Inagaki N, Toriyama M, Sakumura Y (2011) Systems biology of symmetry breaking during neuronal polarity formation. Dev Neurobiol 71:584–593CrossRefPubMedGoogle Scholar
  10. Kamiguchi H, Hlavin ML, Yamasaki M, Lemmon V (1998) Adhesion molecules and inherited diseases of the human nervous system. Annu Rev Neurosci 21:97–125CrossRefPubMedGoogle Scholar
  11. Katoh K, Hammar K, Smith PJ, Oldenbourg R (1999) Birefringence imaging directly reveals architectural dynamics of filamentous actin in living growth cones. Mol Biol Cell 10:197–210CrossRefPubMedPubMedCentralGoogle Scholar
  12. Katsuno H, Toriyama M, Hosokawa Y, Mizuno K, Ikeda K, Sakumura Y, Inagaki N (2015) Actin migration driven by directional assembly and disassembly of membrane-anchored actin filaments. Cell Rep 12:648–660CrossRefPubMedGoogle Scholar
  13. Kubo Y, Baba K, Toriyama M, Minegishi T, Sugiura T, Kozawa S, Ikeda K, Inagaki N (2015) Shootin1-cortactin interaction mediates signal-force transduction for axon outgrowth. J Cell Biol 210:663–676CrossRefPubMedPubMedCentralGoogle Scholar
  14. Le Clainche C, Carlier MF (2008) Regulation of actin assembly associated with protrusion and adhesion in cell migration. Physiol Rev 88:489–513CrossRefPubMedGoogle Scholar
  15. Leenen PJ, Radosevic K, Voerman JS, Salomon B, van Rooijen N, Klatzmann D, van Ewijk W (1998) Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity, expression of macrophage markers, and subpopulation turnover. J Immunol 160:2166–2173PubMedGoogle Scholar
  16. MacGrath SM, Koleske AJ (2012) Cortactin in cell migration and cancer at a glance. J Cell Sci 125:1621–1626CrossRefPubMedPubMedCentralGoogle Scholar
  17. Mallavarapu A, Mitchison T (1999) Regulated actin cytoskeleton assembly at filopodium tips controls their extension and retraction. J Cell Biol 146:1097–1106CrossRefPubMedPubMedCentralGoogle Scholar
  18. McIlroy D, Troadec C, Grassi F, Samri A, Barrou B, Autran B, Debre P, Feuillard J, Hosmalin A (2001) Investigation of human spleen dendritic cell phenotype and distribution reveals evidence of in vivo activation in a subset of organ donors. Blood 97:3470–3477CrossRefPubMedGoogle Scholar
  19. Metlay JP, Witmer-Pack MD, Agger R, Crowley MT, Lawless D, Steinman RM (1990) The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies. J Exp Med 171:1753–1771CrossRefPubMedGoogle Scholar
  20. Mitchison T, Kirschner M (1988) Cytoskeletal dynamics and nerve growth. Neuron 1:761–772CrossRefPubMedGoogle Scholar
  21. Pollard TD, Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112:453–465CrossRefPubMedGoogle Scholar
  22. Preis M, Gardner TB, Gordon SR, Pipas JM, Mackenzie TA, Klein EE, Longnecker DC, Gutmann EJ, Sempere LF, Korc M (2011) MicroRNA-10b expression correlates with response to neoadjuvant therapy and survival in pancreatic ductal adenocarcinoma. Clin Cancer Res 17:5812–5821CrossRefPubMedPubMedCentralGoogle Scholar
  23. Ramsey VG, Doherty JM, Chen CC, Stappenbeck TS, Konieczny SF, Mills JC (2007) The maturation of mucus-secreting gastric epithelial progenitors into digestive-enzyme secreting zymogenic cells requires Mist1. Development 134:211–222CrossRefPubMedGoogle Scholar
  24. Reichmann E, Ball R, Groner B, Friis RR (1989) New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally. J Cell Biol 108:1127–1138CrossRefPubMedGoogle Scholar
  25. Ren G, Helwani FM, Verma S, McLachlan RW, Weed SA, Yap AS (2009) Cortactin is a functional target of E-cadherin-activated Src family kinases in MCF7 epithelial monolayers. J Biol Chem 284:18913–18922CrossRefPubMedPubMedCentralGoogle Scholar
  26. Sapir T, Levy T, Sakakibara A, Rabinkov A, Miyata T, Reiner O (2013) Shootin1 acts in concert with KIF20B to promote polarization of migrating neurons. J Neurosci 33:11932–11948CrossRefPubMedGoogle Scholar
  27. Shimada T, Toriyama M, Uemura K, Kamiguchi H, Sugiura T, Watanabe N, Inagaki N (2008) Shootin1 interacts with actin retrograde flow and L1-CAM to promote axon outgrowth. J Cell Biol 181:817–829CrossRefPubMedPubMedCentralGoogle Scholar
  28. Suter DM, Forscher P (2000) Substrate-cytoskeletal coupling as a mechanism for the regulation of growth cone motility and guidance. J Neurobiol 44:97–113CrossRefPubMedGoogle Scholar
  29. Syu LJ, El-Zaatari M, Eaton KA, Liu Z, Tetarbe M, Keeley TM, Pero J, Ferris J, Wilbert D, Kaatz A, Zheng X, Qiao X, Grachtchouk M, Gumucio DL, Merchant JL, Samuelson LC, Dlugosz AA (2012) Transgenic expression of interferon-γ in mouse stomach leads to inflammation, metaplasia, and dysplasia. Am J Pathol 181:2114–2125CrossRefPubMedPubMedCentralGoogle Scholar
  30. Thievessen I, Thompson PM, Berlemont S, Plevock KM, Plotnikov SV, Zemljic-Harpf A, Ross RS, Davidson MW, Danuser G, Campbell SL, Waterman CM (2013) Vinculin-actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth. J Cell Biol 202:163–177CrossRefPubMedPubMedCentralGoogle Scholar
  31. Toriyama M, Shimada T, Kim KB, Mitsuba M, Nomura E, Katsuta K, Sakumura Y, Roepstorff P, Inagaki N (2006) Shootin1: a protein involved in the organization of an asymmetric signal for neuronal polarization. J Cell Biol 175:147–157CrossRefPubMedPubMedCentralGoogle Scholar
  32. Toriyama M, Sakumura Y, Shimada T, Ishii S, Inagaki N (2010) A diffusion-based neurite length-sensing mechanism involved in neuronal symmetry breaking. Mol Syst Biol 6:394CrossRefPubMedPubMedCentralGoogle Scholar
  33. Toriyama M, Kozawa S, Sakumura Y, Inagaki N (2013) Conversion of a signal into forces for axon outgrowth through Pak1-mediated shootin1 phosphorylation. Curr Biol 23:529–534CrossRefPubMedGoogle Scholar
  34. Troy TC, Arabzadeh A, Yerlikaya S, Turksen K (2007) Claudin immunolocalization in neonatal mouse epithelial tissues. Cell Tissue Res 330:381–388CrossRefPubMedGoogle Scholar
  35. Truffi M, Dubreuil V, Liang X, Vacaresse N, Nigon F, Han SP, Yap AS, Gomez GA, Sap J (2014) RPTPalpha controls epithelial adherens junctions, linking E-cadherin engagement to c-Src-mediated phosphorylation of cortactin. J Cell Sci 127:2420–2432CrossRefPubMedGoogle Scholar
  36. Wang YL (1985) Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J Cell Biol 101:597–602CrossRefPubMedGoogle Scholar
  37. Weed SA, Parsons JT (2001) Cortactin: coupling membrane dynamics to cortical actin assembly. Oncogene 20:6418–6434CrossRefPubMedGoogle Scholar
  38. Wen YA, Liu D, Zhou QY, Huang SF, Luo P, Xiang Y, Sun S, Luo D, Dong YF, Zhang LP (2011) Biliary intervention aggravates cholestatic liver injury, and induces hepatic inflammation, proliferation and fibrogenesis in BDL mice. Exp Toxicol Pathol 63:277–284CrossRefPubMedGoogle Scholar
  39. Yadav N, Kanjirakkuzhiyil S, Kumar S, Jain M, Halder A, Saxena R, Mukhopadhyay A (2009) The therapeuic effect of bone marrow-drived liver cells in the phenotypic correction of murine hemophilia A. Blood 114:4552–4562CrossRefPubMedGoogle Scholar
  40. Yonemura S (2011) Cadherin-actin interactions at adherens junctions. Curr Opin Cell Biol 23:515–522CrossRefPubMedGoogle Scholar

Further Reading

  1. Ergin V, Erdogan M, Menevse A (2015) Regulation of shootin1 gene expression involves NGF-induced alternative splicing during neuronal differentiation of PC12 cells. Sci Rep 5:17931Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Yasuna Higashiguchi
    • 1
  • Kazuhiro Katsuta
    • 1
  • Takunori Minegishi
    • 1
  • Shigenobu Yonemura
    • 2
  • Akihiro Urasaki
    • 1
  • Naoyuki Inagaki
    • 1
  1. 1.Laboratory of Systems Neurobiology and Medicine, Graduate School of Biological SciencesNara Institute of Science and TechnologyIkomaJapan
  2. 2.Department of Cell Biology, Institute of Biomedical SciencesTokushima University Graduate SchoolTokushimaJapan

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