Singar1, a Novel RUN Domain-containing Protein, Suppresses Formation of Surplus Axons for Neuronal Polarity

Abstract

Although neuronal functions depend on their robust polarity, the mechanisms that ensure generation and maintenance of only a single axon remain poorly understood. Using highly sensitive two-dimensional electrophoresis-based proteomics, we identified here a novel protein, single axon-related (singar)1/KIAA0871/RPIPx/RUFY3, which contains a RUN domain and is predominantly expressed in the brain. Singar1 expression became up-regulated during polarization of cultured hippocampal neurons and remained at high levels thereafter. Singar1 was diffusely localized in hippocampal neurons and moderately accumulated in growth cones of minor processes and axons. Overexpression of singar1 did not affect normal neuronal polarization but suppressed the formation of surplus axons induced by excess levels of shootin1, a recently identified protein located upstream of phosphoinositide-3-kinase and involved in neuronal polarization. Conversely, reduction of the expression of singar1 and its splicing variant singar2 by RNA interference led to an increase in the population of neurons bearing surplus axons, in a phosphoinositide-3-kinase-dependent manner. Overexpression of singar2 did not suppress the formation of surplus axons induced by shootin1. We propose that singar1 ensures the robustness of neuronal polarity by suppressing formation of surplus axons. Although neuronal functions depend on their robust polarity, the mechanisms that ensure generation and maintenance of only a single axon remain poorly understood. Using highly sensitive two-dimensional electrophoresis-based proteomics, we identified here a novel protein, single axon-related (singar)1/KIAA0871/RPIPx/RUFY3, which contains a RUN domain and is predominantly expressed in the brain. Singar1 expression became up-regulated during polarization of cultured hippocampal neurons and remained at high levels thereafter. Singar1 was diffusely localized in hippocampal neurons and moderately accumulated in growth cones of minor processes and axons. Overexpression of singar1 did not affect normal neuronal polarization but suppressed the formation of surplus axons induced by excess levels of shootin1, a recently identified protein located upstream of phosphoinositide-3-kinase and involved in neuronal polarization. Conversely, reduction of the expression of singar1 and its splicing variant singar2 by RNA interference led to an increase in the population of neurons bearing surplus axons, in a phosphoinositide-3-kinase-dependent manner. Overexpression of singar2 did not suppress the formation of surplus axons induced by shootin1. We propose that singar1 ensures the robustness of neuronal polarity by suppressing formation of surplus axons. Most neurons develop polarity by forming a single long axon and multiple short dendrites (1Craig A.M. Banker G. Annu. Rev. Neurosci. 1994; 17: 267-310Crossref PubMed Scopus (651) Google Scholar, 2Winckler B. Mellman I. Neuron. 1999; 23: 637-640Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The ability of neurons to develop and maintain polarity is essential for their basic functions. The polarization processes have been extensively studied in cultured hippocampal neurons (1Craig A.M. Banker G. Annu. Rev. Neurosci. 1994; 17: 267-310Crossref PubMed Scopus (651) Google Scholar, 3Dotti C.G. Sullivan C.A. Banker G.A. J. Neurosci. 1988; 8: 1454-1468Crossref PubMed Google Scholar). These cells first form several minor processes, any of which appears capable of becoming either an axon or a dendrite, during the first 12-24 h after plating (stage 2). One of the neurites then rapidly elongates to acquire axonal characteristics (stage 3), whereas the others later become dendrites (stage 4). Hippocampal neurons must use a robust internal mechanism that ensures polarization as they regenerate a single axon and multiple dendrites even when polarity is altered by axonal amputation (4Dotti C.G. Banker G.A. Nature. 1987; 330: 254-256Crossref PubMed Scopus (220) Google Scholar, 5Goslin K. Banker G. J. Cell Biol. 1989; 108: 1507-1516Crossref PubMed Scopus (336) Google Scholar). Recent studies have begun to reveal molecules involved in neuronal polarization (6Arimura N. Kaibuchi K. Neuron. 2005; 48: 881-884Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 7Jiang H. Rao Y. Nat. Neurosci. 2005; 8: 544-546Crossref PubMed Scopus (30) Google Scholar, 8Wiggin G.R. Fawcett J.P. Pawson T. Dev. Cell. 2005; 8: 803-816Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Localization, overexpression, and loss of function studies showed that spatially localized intracellular signals of a number of molecules, such as phosphoinositide-3-kinase (PI 3-kinase) 2The abbreviations used are: PI 3-kinase, phosphoinositide-3-kinase; 2-DE, two-dimensional electrophoresis; CMFDA, 5-chloromethylfluorescein diacetate; DIV, day in vitro; E, embryonic day; P, postnatal day; GST, glutathione S-transferase; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; MARK2, microtubule affinity-regulation kinase; RUN domain, RPIP8, UNC-14 and NESCA homology domain; RPIPx, Rap2 interacting protein X; scRNA, scramble RNA; siRNA, small interfering RNA; RNAi, RNA interference; singar, single axon-related; PTEN, phosphatase and tensin homolog; GFP, green fluorescent protein. (9Shi S.H. Jan L.Y. Jan Y.N. Cell. 2003; 112: 63-75Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar), phosphatidylinositol (3Dotti C.G. Sullivan C.A. Banker G.A. J. Neurosci. 1988; 8: 1454-1468Crossref PubMed Google Scholar, 4Dotti C.G. Banker G.A. Nature. 1987; 330: 254-256Crossref PubMed Scopus (220) Google Scholar, 5Goslin K. Banker G. J. Cell Biol. 1989; 108: 1507-1516Crossref PubMed Scopus (336) Google Scholar) triphosphate (10Menager C. Arimura N. Fukata Y. Kaibuchi K. J. Neurochem. 2004; 89: 109-118Crossref PubMed Scopus (183) Google Scholar), Akt (11Jiang H. Guo W. Liang X. Rao Y. Cell. 2005; 120: 123-135Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar), glycogen synthase kinase-3β (11Jiang H. Guo W. Liang X. Rao Y. Cell. 2005; 120: 123-135Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 12Yoshimura T. Kawano Y. Arimura N. Kawabata S. Kikuchi A. Kaibuchi K. 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Atkins C.M. Copenagle L. Banker G.A. J. Neurosci. 2006; 26: 9462-9470Crossref PubMed Scopus (132) Google Scholar), and DOCK7 (20Watabe-Uchida M. John K.A. Janas J.A. Newey S.E. Van Aelst L. Neuron. 2006; 51: 727-739Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) are involved in axon specification for neuronal polarity formation. As downstream events, these pathways are thought to regulate cytoskeletal networks of actin filaments (21Bradke F. Dotti C.G. Science. 1999; 283: 1931-1934Crossref PubMed Scopus (429) Google Scholar) and microtubules (20Watabe-Uchida M. John K.A. Janas J.A. Newey S.E. Van Aelst L. Neuron. 2006; 51: 727-739Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 22Fukata Y. Itoh T.J. Kimura T. Menager C. Nishimura T. Shiromizu T. Watanabe H. Inagaki N. Iwamatsu A. Hotani H. Kaibuchi K. Nat. Cell Biol. 2002; 4: 583-591Crossref PubMed Scopus (637) Google Scholar). In contrast to the progress in defining the mechanisms of axon specification, our knowledge of how neurons generate and maintain only a single axon is limited. Reduction in the expression or activities of glycogen synthase kinase-3β, PTEN, or microtubule affinity-regulation kinase (MARK)2 in hippocampal neurons leads to formation of multiple axons (11Jiang H. Guo W. Liang X. Rao Y. Cell. 2005; 120: 123-135Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 12Yoshimura T. Kawano Y. Arimura N. Kawabata S. Kikuchi A. Kaibuchi K. Cell. 2005; 120: 137-149Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar, 23Chen Y.M. Wang Q.J. Hu H.S. Yu P.C. Zhu J. Drewes G. Piwnica-Worms H. Luo Z.G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8534-8539Crossref PubMed Scopus (129) Google Scholar), suggesting that these molecules may be involved in inhibiting the formation of surplus axons. Recently, we identified the protein shootin1, which is located upstream of PI 3-kinase and involved in neuronal polarization (24Toriyama M. Shimada T. Kim K.B. Mitsuba M. Nomura E. Katsuta K. Sakumura Y. Roepstorff P. Inagaki N. J. Cell Biol. 2006; 175: 147-157Crossref PubMed Scopus (107) Google Scholar). During polarization, shootin1 accumulated in a single neurite while being depleted in its sibling neurites, through competitive transport to multiple neurites. This in turn induced formation of a single axon, leading us to propose that the depletion of shootin1 from the sibling neurites of the nascent axon by competition prevents the formation of surplus axons (24Toriyama M. Shimada T. Kim K.B. Mitsuba M. Nomura E. Katsuta K. Sakumura Y. Roepstorff P. Inagaki N. J. Cell Biol. 2006; 175: 147-157Crossref PubMed Scopus (107) Google Scholar). We describe here a novel brain-specific protein, named single axon-related (singar)1. Singar1 became up-regulated during polarization of cultured hippocampal neurons and remained at high levels thereafter. Reduction of singar1 expression by RNAi led to an increase in the population of neurons bearing surplus axons, as has been reported for glycogen synthase kinase-3β, PTEN, and MARK2. Furthermore, overexpression of singar1 suppressed the formation of surplus axons induced by excess amounts of shootin1, without affecting normal polarization processes. These data suggest that singar1 suppresses the formation of surplus axons, thereby contributing to the robustness of neuronal polarity. Cultures and Metabolic Labeling—Hippocampal neurons prepared from E18 rat embryos were seeded on coverslips coated with poly-d-lysine (Sigma) and laminin (Iwaki) and cultured in Neurobasal medium (Invitrogen) supplemented with B-27 supplement (Invitrogen) and 1 without as N. Chihara K. Arimura N. Menager C. Kawano Y. Matsuo N. Nishimura T. Amano M. Kaibuchi K. Nat. Neurosci. 2001; 4: 781-782Crossref PubMed Scopus (466) Google Scholar). two-dimensional and neurons were with the medium of the normal levels of and in and for we used Neurobasal medium without and cells were cultured in medium supplemented with and by was as reported T. I. Katsuta K. Nomura E. M. Inagaki N. 2002; PubMed Scopus Google Scholar), a a N. Katsuta K. Curr. 2004; Google Scholar). 2-DE, neurons were as and protein by were by protein protein from rat were by the and by The protein to the were then from and as E. Katsuta K. T. M. T. Inagaki N. J. 2004; PubMed Scopus Google Scholar). laser desorption/ionization mass was a with were the and for of of rat singar1 and singar2 were by from a rat from rat an with the and The were then the of with a by J. H. K. J. 108: PubMed Scopus Google Scholar), and These were to generate the to and glutathione at the We did not use and singar1 was expressed in coli as a protein by with at for h and on a after which was from singar1 by was the singar1 and and and were as N. Chihara K. Arimura N. Menager C. Kawano Y. Matsuo N. Nishimura T. Amano M. Kaibuchi K. Nat. Neurosci. 2001; 4: 781-782Crossref PubMed Scopus (466) Google Scholar). and were used at and was used at 5-chloromethylfluorescein neurons were with for of neurons were a fluorescent with a or an and was a of the and were used at and was used at and were with or RNA by the N. Chihara K. Arimura N. Menager C. Kawano Y. Matsuo N. Nishimura T. Amano M. Kaibuchi K. Nat. Neurosci. 2001; 4: 781-782Crossref PubMed Scopus (466) Google Scholar), or (Invitrogen) or after for of the of small interfering RNA on of neurons was plating of the of on neuronal after polarization, neurons were by the on day in the neurons were with or RNA neurons in Neurobasal medium were with complex for they were by and in and on h after the the medium was by the cells were with by the The rat singar1 and singar2 to in the of singar1 in the of The scramble RNA the as the but to any The were from cells were with the expression and in h after cells were with and in the 1 1 on for the were then at for at The were with of and were with protein of with the the were in the and to CMFDA, and were from and was from of PI 3-kinase was by from a rat as the for the neurons with long neurite and as long as the neurites were as neurons cells not neurons with multiple was by the of Singar1 by a of and Hippocampal involved in neuronal polarity we of cultured rat hippocampal neurons a N. Katsuta K. Curr. 2004; Google Scholar). We protein on and that were up-regulated during the and (24Toriyama M. Shimada T. Kim K.B. Mitsuba M. Nomura E. Katsuta K. Sakumura Y. Roepstorff P. Inagaki N. J. Cell Biol. 2006; 175: 147-157Crossref PubMed Scopus (107) Google Scholar). and mass of of located at a mass of and in identified to rat and of protein in and and of and of and that contains RUN and domain I. J. B. J.P. Sci. 2001; 26: Full Text Full Text PDF PubMed Scopus Google Scholar) and Recently, the of its RUN domain was reported M. T. K. T. T. M. Watanabe S. A. Y. T. M. S. J. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). We with reported its as and homology to RUN such as RPIP8, and NESCA identified and of and in and In a splicing variant of was identified in rat and and singar2 and Singar1 and in the and during an singar1 to its and intracellular The at and in of rat The to the of whereas the to that of singar2 suggesting that the splicing are expressed in the brain. of with led to of the and increase in the of the whereas of cells singar1 with the phosphatase the of the we that the to the form of The were not in suggesting that singar1 and singar2 are predominantly expressed in the brain. with the data for the protein the of singar1 expression during the from to in cultured hippocampal neurons and remained high expression of singar1 in rat was on embryonic day postnatal day and in the expression were for singar2 and singar1 in that the expression of singar2 in hippocampal neurons to a at and of singar1 was in the cultured neurons and and Singar1 and in of and we the of in cultured hippocampal showed a of in and neurons In showed a in growth cones of minor processes of growth cones showed growth In accumulated in growth cones of axons in to of minor processes and of axonal growth cones showed growth whereas of minor growth cones showed growth In accumulated in growth cones of axons of axonal growth cones showed growth was localized in dendrites but did not We used a green CMFDA, as an internal to of (24Toriyama M. Shimada T. Kim K.B. Mitsuba M. Nomura E. Katsuta K. Sakumura Y. Roepstorff P. Inagaki N. J. Cell Biol. 2006; 175: 147-157Crossref PubMed Scopus (107) Google Scholar). The of in growth cones of minor processes and axons were growth and growth that in In growth was localized in and As the singar1 and we the of singar1 and singar2 and expressed in hippocampal of and were to that of the and Overexpression of Singar1 the of by of without the functions of singar1 and singar2 in neuronal polarization, we singar1 and singar2 in hippocampal neurons the were with and the of neurons was at h in of neurons neurons and of neurons neurons as in the of neurons with neurons of were from neurons with and We cultured the neurons with and We did not in either the or the number of axons and dendrites these neurons and we that overexpression of singar1 or singar2 not affect normal polarization processes. Recently, we identified the protein shootin1, which is located at an upstream in polarity signals PI 3-kinase (24Toriyama M. Shimada T. Kim K.B. Mitsuba M. Nomura E. Katsuta K. Sakumura Y. Roepstorff P. Inagaki N. J. Cell Biol. 2006; 175: 147-157Crossref PubMed Scopus (107) Google Scholar). Overexpression of shootin1 in hippocampal neurons leads to formation of surplus axons. of singar1 led to a reduction in the of neurons with surplus axons induced by shootin1 overexpression neurons when with neurons and the overexpression of singar2 did not suppress the formation of surplus axons induced by shootin1 neurons showed that overexpression of singar1 did not suppress the expression of shootin1 thereby the that the reduction in the of neurons with surplus axons is by a in the of shootin1. Reduction of by RNAi to an in the of the of in neuronal polarization, we expression an of hippocampal neurons with an plating in a in the levels of singar1 and singar2 when with after with the of in hippocampal neurons by the was of neurons with the led to a reduction in when with the after with the neurons neurons when with of the neurons or axons that were by and but were for a on In only neurons of neurons with axons. We the and number of the neurites. by axonal were dendrites The of the of neurites in the neurons bearing surplus axons was that in neurons neurite number in the neurons with surplus axons was to that of neurons reduction of expression appears to surplus axons without the number of neurites. In reduction of expression the formation of surplus axons induced by shootin1 overexpression neurons when with of the neurons with shootin1 and multiple axons. In neurons of neurons with shootin1 and of the of neurons with suppressed expression of the of neurons with multiple axons and the of neurons bearing or axons. of neurons with and neurons with siRNA, was in were identified by axon and of neurons with multiple axons. of neurons with and neurons with siRNA, was S.E. C and neurite and neurite number of neurons and neurons with multiple axons of neurons with and neurons with siRNA, was S.E. E, of RNAi on the formation of surplus axons induced by overexpression of shootin1. with and or and were on and of neurons for with and neurons for with siRNA, was in were identified by S.E. We expression after hippocampal neurons polarity. were cultured to (stage 3), after which they were with the or scRNA, and their was on neurons when with of the neurons multiple axons that were by and not but were for not In neurons of neurons with axons. with the data of singar1 and overexpression, these suggest that an in suppressing the formation of axons for neuronal polarity. Singar1 to PI 3-kinase and by the PI 3-kinase is reported to axons through the PI 3-kinase (24Toriyama M. Shimada T. Kim K.B. Mitsuba M. Nomura E. Katsuta K. Sakumura Y. Roepstorff P. Inagaki N. J. Cell Biol. 2006; 175: 147-157Crossref PubMed Scopus (107) Google Scholar), and multiple axon formation was suppressed by singar1 overexpression and and by RNAi We singar1 with the PI 3-kinase Singar1 was with the or of PI 3-kinase in singar1 was of the and was and Singar1 was with the and that with PI We the formation of surplus axons induced by reduction of levels on PI kinase of PI 3-kinase by led to a reduction in the of neurons with multiple axons induced by RNAi Overexpression of PI 3-kinase is reported to formation of multiple axons as in the of shootin1 overexpression (24Toriyama M. Shimada T. Kim K.B. Mitsuba M. Nomura E. Katsuta K. Sakumura Y. Roepstorff P. Inagaki N. J. Cell Biol. 2006; 175: 147-157Crossref PubMed Scopus (107) Google Scholar, T. Arimura N. Kawano Y. Kawabata S. Wang S. Kaibuchi K. 2006; PubMed Scopus Google Scholar). in contrast to the of shootin1 overexpression RNAi did not affect the formation of surplus axons induced by the PI 3-kinase These that formation of surplus axons induced by the reduction of is on PI 3-kinase but surplus axon formation induced by the PI 3-kinase is not by Using highly sensitive proteomics, we have identified here a novel protein, which is predominantly expressed in the brain. Singar1 expression became up-regulated during polarization of cultured hippocampal neurons and remained at high levels thereafter. Overexpression of singar1 suppressed the formation of surplus axons induced by excess levels of shootin1. Conversely, of by RNAi led to an increase in the population of neurons bearing surplus axons. Reduction of expression the formation of surplus axons induced by shootin1 the levels of singar1 and and we not for singar1 or only singar1 showed the to suppress the formation of surplus axons induced by shootin1, we that the of RNAi from the reduction of not singar2 was in rat and but not in The functions of singar2 in rat and hippocampal neurons remain the suggest that singar1 an in suppressing formation of surplus axons. Recent studies have reported that reduction in the expression or activities of glycogen synthase kinase-3β, PTEN, or in hippocampal neurons leads to formation of multiple axons (11Jiang H. Guo W. Liang X. Rao Y. Cell. 2005; 120: 123-135Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 12Yoshimura T. Kawano Y. Arimura N. Kawabata S. Kikuchi A. Kaibuchi K. Cell. 2005; 120: 137-149Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar, 23Chen Y.M. Wang Q.J. Hu H.S. Yu P.C. Zhu J. Drewes G. Piwnica-Worms H. Luo Z.G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8534-8539Crossref PubMed Scopus (129) Google Scholar). Although these are to that of RNAi, their overexpression Overexpression of and glycogen synthase kinase-3β (11Jiang H. Guo W. Liang X. Rao Y. Cell. 2005; 120: 123-135Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 12Yoshimura T. Kawano Y. Arimura N. Kawabata S. Kikuchi A. Kaibuchi K. Cell. 2005; 120: 137-149Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar), (11Jiang H. Guo W. Liang X. Rao Y. Cell. 2005; 120: 123-135Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar), and Y.M. Wang Q.J. Hu H.S. Yu P.C. Zhu J. Drewes G. Piwnica-Worms H. Luo Z.G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8534-8539Crossref PubMed Scopus (129) Google Scholar) formation and of axons during polarization processes. the singar1 overexpression did not affect the formation or of axons during normal polarization processes but the formation of surplus axons induced by our suggest that singar1 may ensure the robustness of neuronal polarity by suppressing formation of surplus axons. As in small of neurons with surplus axons are reported in the H. P. J. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar). to be singar1 suppresses the formation of surplus axons in the mechanisms by which singar1 the formation of surplus axons are Singar1 overexpression the formation of surplus axons induced by shootin1 overexpression, whereas singar1 RNAi During polarization, shootin1 in a single whereas being depleted in its sibling neurites (24Toriyama M. Shimada T. Kim K.B. Mitsuba M. Nomura E. Katsuta K. Sakumura Y. Roepstorff P. Inagaki N. J. Cell Biol. 2006; 175: 147-157Crossref PubMed Scopus (107) Google Scholar). This in turn of PI 3-kinase (24Toriyama M. Shimada T. Kim K.B. Mitsuba M. Nomura E. Katsuta K. Sakumura Y. Roepstorff P. Inagaki N. J. Cell Biol. 2006; 175: 147-157Crossref PubMed Scopus (107) Google Scholar), which is essential for neuronal polarization (6Arimura N. Kaibuchi K. Neuron. 2005; 48: 881-884Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, S.H. Jan L.Y. Jan Y.N. Cell. 2003; 112: 63-75Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar). the shootin1 overexpression its in the growth cones of minor processes, which in turn in of PI 3-kinase and formation of surplus axons (24Toriyama M. Shimada T. Kim K.B. Mitsuba M. Nomura E. Katsuta K. Sakumura Y. Roepstorff P. Inagaki N. J. Cell Biol. 2006; 175: 147-157Crossref PubMed Scopus (107) Google Scholar). In the of a of singar1 was in minor processes and In reduction of expression by RNAi led to an increase in the population of neurons bearing surplus axons in a PI manner. singar1 in minor processes and dendrites may the formation of surplus axons by upstream of PI shootin1 and singar1 may to the maintenance of a single axon in In singar1 accumulated in the axon not be involved in suppressing axon formation. the a form of the that the functions of 1 may be altered by protein In the has identified a novel RUN protein suggest that singar1 may ensure the robustness of neuronal polarity by suppressing formation of surplus axons without affecting normal neuronal polarization. The in functions of singar1 and the of how singar1 the formation of surplus axons remain for We and the of and for Itoh for for the and for the and for