訊息蛋白SH2B1β透過與IRSp53及Eps8交互作用促進海馬迴神經細胞的絲狀偽足形成及樹突分支

SH2B1β enhances filopodium formation and dendritic branching of hippocampal neurons through interacting with IRSp53 and Eps8

陳建任(Chien-Jen Chen)

指導教授 ： 陳令儀

國立清華大學/生命科學院/分子醫學研究所/博士(2014年)

https://doi.org/10.6843/NTHU.2014.00182

摘要

SH2B1為一訊息蛋白可啓動PC12細胞、皮質及海馬迴神經細胞的神經軸生長(neurite outgrowth)。在本論文研究中，我們推測大量表現SH2B1β啓動海馬迴神經細胞的絲狀偽足(filopodium) 及樹突分支(dendritic branching)的形成。在神經軸起始時(neurite initiation)，絲狀偽足的形成是必需的，微小管(microtubule)會沿著絲狀偽足延伸出去，推測SH2B1β也許藉由調控肌動蛋白的重組(actin-remodeling)，進而促進神經軸起始及生長。為進一步了解SH2B1β如何影響神經軸形成，調控肌動蛋白重組的IRSp53及Eps8蛋白被研究是否與SH2B1β有協同作用。IRSp53為一具有不同功能區域(multi-domain)的蛋白，可調控肌動蛋白細胞骨架的相關蛋白並導致絲狀偽足形成。Eps8具有結合在肌動蛋白的正端(barbed-ends)活性而調控肌動蛋白的動態。藉由活化的Cdc42調控，促使IRSp53-Eps8複合體的形成，可導致肌動蛋白束(actin bundles)的形成，進而促進絲狀偽足的突起。我們發現SH2B1β透過proline-rich區域與IRSp53及Eps8結合。大量表現SH2B1β與IRSp53可增強絲狀偽足形成及神經軸起始。相反的，藉由shRNA抑制內源性的SH2B1或表現缺少proline-rich區域的SH2B1β突變型皆會抑制絲狀偽足及神經軸分支形成。除此之外，SH2B1β、IRSp53及Eps8共同表現在細胞膜上，利用Triton X-100分離出不溶的細胞骨架部份，三者表現量隨著神經細胞發育而增加。從這些結果推測藉由啓動肌動蛋白聚合(actin polymerization)，進而促使絲狀偽足形成、神經軸起始及分支，SH2B1β、IRSp53及Eps8彼此間的交互作用是必需的。

關鍵字

SH2B1β 訊息蛋白 ； 神經軸生長 ； 樹突分支 ； 絲狀偽足 ； IRSp53 蛋白 ； Eps8 蛋白

並列摘要

SH2B1 is an adapter protein that promotes neurite outgrowth of PC12 cells, cortical and hippocampal neurons. In this study, we provide evidence suggesting that overexpression of SH2B1β promotes filopodium formation and dendritic branching of hippocampal neurons. In neurite initiation, filopodium formation is required and microtubule extension into filopodia, suggesting that SH2B1β may regulate actin remodeling to enhance neurite initiation and outgrowth. To understand how SH2B1β may affect neurite formation, the collaborative efforts of SH2B1β with two actin-remodeling proteins, IRSp53 (Insulin receptor tyrosine kinase substrate p53) and Eps8 (Epidermal growth factor receptor kinase substrate 8) have been investigated. IRSp53 is a multi-domain protein that can regulate actin cytoskeleton-associated proteins and thus filopodium formation. Eps8 regulates actin dynamics through actin barbed-ends capping activity. The formation of IRSp53-Eps8 complex, regulated by active Cdc42, contributes to the formation of actin bundles, thus promoting filopodium protrusions. We found that the proline-rich domains of SH2B1 interact with IRSp53 and Eps8. Overexpressing SH2B1β and IRSp53 enhanced filopodium formation and neurite initiation. In contrast, shRNA for SH2B1 to silence endogenous SH2B1 or the deletion mutants of SH2B1β which lack the proline-rich domains that inhibit filopdium formation and neuronal branching. In addition, SH2B1, IRSp53 and Eps8 co-localize at the plasma membrane and their levels increase in the Triton X-100-insoluble fraction of developing neurons. These results suggest that the interaction among SH2B1, IRSp53 and Eps8 is required to promote actin polymerization and thus filopodium formation, neurite initiation and neuronal branching.

並列關鍵字

SH2B1β adapter protein ； neurite outgrowth ； dendritic branching ； filopodium ； IRSp53 ； Eps8

參考文獻

1. Thomson JA, et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282(5391):1145-1147.

2. Preynat-Seauve O, et al. (2009) Neural progenitors derived from human embryonic stem cells are targeted by allogeneic T and natural killer cells. Journal of cellular and molecular medicine 13(9B):3556-3569.

3. Mumaw JL, et al. (2010) Neural differentiation of human embryonic stem cells at the ultrastructural level. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada 16(1):80-90.

6. Arimura N & Kaibuchi K (2007) Neuronal polarity: from extracellular signals to intracellular mechanisms. Nature reviews. Neuroscience 8(3):194-205.

7. Papa M, Bundman MC, Greenberger V, & Segal M (1995) Morphological analysis of dendritic spine development in primary cultures of hippocampal neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience 15(1 Pt 1):1-11.