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研究生： 洪紹景
論文名稱： 探討 IRSp53 和 SH2B1beta 在形成絲狀偽足中的角色
Role of IRSp53 and SH2B1beta in filopodium formation
指導教授： 陳令儀
口試委員: 陳令儀
徐瑞洲
黃兆祺
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2013
畢業學年度： 101
語文別： 英文
論文頁數： 51
中文關鍵詞： 絲狀偽足
外文關鍵詞： SH2B1
相關次數： 點閱：58  下載：0 
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    • 絲狀偽足 (Filopodia) 是一細長且富含肌動蛋白的細胞膜突起結構。絲狀偽足在許多生理過程中扮演重要的角色，包括病原偵測，細胞遷移，傷口癒合以及生長發育。在神經發育過程中，樹突及樹突小刺形成之前，必須先形成絲狀偽足。Insulin receptor substrate protein of 53 kDa (IRSp53) 在先前已被證實會參與調控絲狀偽足的形成。我們初步的結果指出，大量表現一個訊息連接蛋白 SH2B1β 能夠增進海馬迴神經細胞的生長。因此，本研究假設 IRSp53 和 SH2B1β 可能會共同調控細胞骨架的重組，進而促成絲狀偽足的形成。本研究中顯示，在人類胚胎腎細胞株 (293T cells) 中，大量表現 IRSp53 或 SH2B1β 皆能誘導絲狀偽足的形成。此外， SH2B1β 會與 IRSp53 結合，共同增進絲狀偽足的形成以及造成絲狀偽足的分支。另外，本研究也進一步發現，增進絲狀偽足的形成需要 IRSp53 的 IMD 和 SH3 domain 以及 SH2B1β 的 N 端 proline-rich domain，而其中造成絲狀偽足增加的機制為 IRSp53 會吸引SH2B1β 到細胞膜上。最後，本研究也證實， IRSp53 和 SH2B1β 會共同作用，增進海馬迴神經細胞的生長與分枝。總結來說，本研究首先發現 SH2B1β 會透過 IRSp53 複合體，來促進絲狀偽足的形成以及神經軸的生長。

    Filopodia are thin, actin-enriched structure of membrane protrusions. Filopodia play instrumental roles in pathogen detection, cell migration, wound healing, and during development. During neurogenesis, filopodium formation precedes the formation of dendrites and spines. The insulin receptor substrate protein of 53 kDa (IRSp53) has been shown to regulate the formation of filopodia. Our preliminary results suggest that overexpressing an adaptor protein SH2B1β enhances neurite outgrowth of hippocampal neurons. Thus, I hypothesize that IRSp53 and SH2B1β may act together to regulate actin cytoskeleton remodeling and thus filopodium formation. In this thesis, I show that overexpression of either IRSp53 or SH2B1β induces the filopodium formation of 293T cells. In addition, SH2B1β interacts with IRSp53 to enhance filopodium formation and highly branched filopodia. Furthermore, IMD and SH3 domains of IRSp53 as well as N-terminal proline-rich domains of SH2B1β are required for the enhancement of filopodium formation. One mechanism for this enhancement of filopodia is that IRSp53 recruits SH2B1β to the plasma membrane. Finally, I demonstrate that IRSp53 and SH2B1β synergistically enhance neurite branching of hippocampal neurons. Taken together, this study provides novel findings that SH2B1β interacts with IRSp53-containing complexes to promote filopodium formation and thus neurite outgrowth.

    Abstract I 摘要 II 誌謝 III Index V Abbreviations VII Introduction 1 Small GTPases Rho family members regulate actin structures 1 IRSp53 family 2 Domains of IRSp53 isoforms 2 IMD domain 4 Partial CRIB motif 5 Src homology 3 domain 5 WW and PDZ-binding motif 6 Role of IRSp53-containing complexes in the formation of filopodia 6 IRSp53 in the formation of dendritic spines 7 SH2B1β 7 Materials and Methods 11 Reagents 11 Plasmids 12 Cell lines and cultures 12 Primary culture of hippocampal neurons 12 Transient transfection and immunofluorescence microscopy 13 Immunoblotting and immunoprecipitation 14 Subcellular fractionation 14 Results 16 IMD domain of IRSp53 is critical for filopodium induction 16 SH2B1β promotes long cytoplasmic extensions to foster filopodium formation 17 A positive role of SH2B1β in IRSp53-mediated filopodia formation 17 IRSp53 interacts with SH2B1β 18 N-terminal proline-rich domains of SH2B1β are required to enhance IRSp53-induced filopodium initiation 19 The SH3 domain of IRSp53 promotes SH2B1β-enhanced elongation and branching of filopodia 20 IRSp53 may modulate the subcellular distribution of SH2B1β 21 SH2B1β promotes IRSp53-enhanced neurite outgrowth 22 Discussion 24 References 27 Figures 35 Figure 1. The IMD domain of IRSp53 induces filopodia of 293T cells. 36 Figure 2. SH2B1β promotes cytoplasmic extensions of 293T cells. 38 Figure 3. Role of IRSp53 and SH2B1β in the formation of filopodia. 40 Figure 4. IRSp53 interacts with SH2B1β. 42 Figure 5. Effect of expressing various truncation constructs of GFP-tagged SH2B1β and myc-IRSp53 in filopodium formation. 44 Figure 6. Effect of co-expression of GFP-SH2B1β and various truncation constructs of myc-tagged IRSp53 in 293T cells. 46 Figure 7. Cellular distribution of IRSp53 and SH2B1β in 293T cells. 48 Figure 8. Effect of the co-expression of IRSp53 and various SH2B1β constructs in hippocampal neurons. 50 Table 51 Table 1. Oligonucleotide primers used in this thesis 51

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