探討NuMA在體外培養之早期神經細胞的分布及功能

Abstract

神經突 (Neurite) 生長對於建立複雜的神經系統是必要的過程，微管的生長幫助神經的細胞形成並維持他們特殊的型態。NuMA是一個分子量238-kDa的蛋白質，會透過其C端球狀區域的微管結合位與微管結合。在有絲分裂的細胞中，NuMA透過與微管結合扮演著維持和聚集紡錘極 (spindle poles) 的重要角色。然而，NuMA不僅在有絲分裂的細胞中表現，在不會進行細胞分裂的神經細胞中也有NuMA的存在，但目前對於NuMA在神經細胞中所扮演的功能至今尚未知。從我們實驗室先前的研究中發現，在初代海馬迴神經細胞中抑制NuMA的表現，會導致神經細胞的神經突發生縮短的現象。此外，在神經細胞中大量表現不具有核定位序列(NLS)的NuMA，發現它們會聚集在中心體 (centrosome) 上。因此，我們想要確定這些分布在細胞質的NuMA是否會影響神經細胞的型態發生。首先我們發現在神經細胞中，內生型NuMA主要分布在細胞核中，但也可以觀察到較微弱的訊號在細胞質裡，尤其是在較成熟的神經細胞中，細胞質的NuMA訊號更為明顯。然而，我們並沒有看到NuMA分布在中心體上。此外，在我們的研究結果顯示，分布在細胞質的NuMA並不影響早期神經細胞的型態發生。最後，我們成功建立了低密度培養神經的方法，幫助我們更容易地定量神經細胞型態。

Neurite elongation is an essential process for building complex nervous system. Microtubules are particularly important for neurite elongation that allows them to acquire and maintain their specialized morphology. NuMA (Nuclear Mitotic Apparatus) is a 238 kDa protein that binds to microtubules via its carboxyl terminal region. In mitotic cells, it plays an essential role in mitotic spindle assembly and maintenance. It has been shown that NuMA not only expresses in mitotic cells but also in post-mitotic neurons. However, the function of NuMA in neurons remains unknown. Recently studies from our laboratory showed that NuMA depletion causes the reduction of neurite length. Using NuMA constructs without NLS (Nuclear Localization Signal), we found that cytoplasm-localized NuMA concentrated at the centrosome in young neurons. To determine whether cytoplasm NuMA is involve in neuronal morphogenesis, we first examined the localization of endogenous NuMA in neurons. We found that NuMA localized primarily in the nucleus and to a lesser extent in the cytoplasm. The cytoplasmic localization of NuMA was especially obvious in more mature neurons. However, we did not observe endogenous NuMA localization at the centrosome. Additionally, our results suggested that the cytoplasm-localized NuMA does not affect early neuronal morphogenesis. Finally, we successfully established a low-density neuronal culture to allow easy neuronal morphology quantification.