利用螢光共振能量轉移技術觀察TPX2和importin-α在活體神經細胞中之交互作用

Abstract

TPX2是微管相關蛋白質，調控細胞分裂紡錘絲之形成。TPX2藉由兩段核定位序列跟importin-α結合形成蛋白質複合體。在我們先前的研究中，發現TPX2會幫助微管成核作用(microtubule nucleation)以及影響神經型態發生。再者，我們先前研究也發現TPX2的活性是藉由Ran GTPase所調控。目前已知在進行有絲分裂的細胞中，活化態的Ran (GTP-bound Ran)在染色體附近有較高濃度，這造成TPX2的活性不會被importin-α抑制。然而，至今仍無研究顯示在已分化之神經細胞中importin-α如何與TPX2交互作用以及控制TPX2的活性。在此，我們選用螢光共振能量轉移技術來觀察TPX2和importin-α蛋白質複合體的分布。我們分別使用已發表分子內及分子間的螢光共振能量轉移試劑來驗證我們的影像及分析系統。首先，我們偵測在進行有絲分裂細胞中TPX2與importin-α的交互作用。我們的結果顯示在細胞間期時，TPX2跟importin-α交互作用發生最強的位置分布在細胞核附近；而在有絲分裂時期，則在紡錘絲上有較強的交互作用。接著，我們在已分化之神經細胞中檢驗TPX2跟importin-α交互作用的位置；然而，因為外源性表現的FRET分子蛋白質表現量變化較大，以至於我們無法確定在神經細胞中TPX2與importin-α的交互作用位置。

TPX2 is a microtubule-associated protein that regulates the mitotic spindle formation in the mammalian cells. TPX2 binds to importin-α and form a protein complex via its bipartite nuclear localization sequence (NLS). We previous discovered that TPX2 promotes microtubule nucleation and affects neuronal morphogenesis. Moreover, we found that the activity of TPX2 is regulated by the small GTPase Ran. It is known that a high concentration of active Ran (GTP-bound Ran) around the chromosome prevents importin-α from binding to and inhibiting TPX2 during mitosis. However, where importin-α interacts with TPX2 to regulate its activity in post-mitotic neurons remains elusive. Here we examine the spatial distribution of TPX2-importin-α interaction using fluorescence resonance energy transfer (FRET). Well-documented intramolecular and intermolecular FRET sensors were used to validate our imaging and analysis system. We first examined the interaction sites of TPX2-importin-α in mitotic cells. TPX2 and importin-α have the highest interaction around the nuclear periphery in interphase and on the mitotic spindle during mitosis. We then examined the interaction sites of TPX2-importin-α in post-mitotic neurons. However, due to the variable protein level of exogenously expressed FRET molecules, we were unable to consistently determine the location of TPX2-importin-α interaction in primary neurons.