Importin-α調控神經細胞之微管成核與形態發生

Abstract

微管(Microtubule)在神經系統中扮演著非常重要的角色，是一個在神經發育中主要的細胞骨架。TPX2是一個微管蛋白，已經被證實可以幫助微管的成核及在分裂的哺乳類細胞中協助紡錘絲的形成。我們實驗室最近最近發現TPX2在神經細胞中可以促進非中心體微管的成核，且TPX2的活性會受到Ran-importin訊息傳遞路徑的調控。本研究旨在了解importin-α如何調控TPX2的活性及如何影響神經形態發生。首先，我們檢視所有importin-α蛋白質在有絲分裂細胞與神經細胞中的表現及分佈。此外，我們在海馬迴神經細胞中大量表現了中樞神經系統中含量最高的三個importin-α異構物 (KPNA1, 4, 6)，並觀察到大量importin-α會影響神經形態發生。最後，我們觀察到KPNA1, 4, 6大量表現後會對神經末梢的微管成核現象產生影響，但對於微管的聚合速度與時間則沒有影響。我們的實驗結果顯示神經元細胞質中的importin會控制微管成核及神經形態發生。

Microtubule is the major cytoskeleton in the nervous system and plays crucial roles when neurons generate their polarized and elaborated morphology. TPX2 is a microtubule-associated protein which has been shown to promote microtubule nucleation and spindle formation in mitotic cells. Our lab has recently discovered an important role of TPX2 in post-mitotic neurons to promote acentrosomal microtubule nucleation. We also found that the activity of TPX2 is regulated by Ran-importin signaling pathway. The current study focuses on the function of importin-α isoforms in regulating TPX2 activity and neuronal morphogenesis. We first examined the expression level and subcellular localization of all importin-α isoforms in mitotic cells and post-mitotic neuron. Additionally, we overexpressed CNS-enriched importin-α isoforms (KPNA1, 4, 6) in dissociated mouse hippocampal neurons and observed the inhibitory effect of these importin-α isoforms on neuronal morphogenesis. Finally, we observed that KPNA1, 4, or 6 overexpression specially compromised microtubule nucleation at the neurite tip without affecting microtubule polymerization rate and persistence. Our data suggests the existence of an importin-regulated mechanism in the neuronal cytoplasm controlling microtubule nucleation and neuronal morphogenesis.