探討細胞質NuMA在早期神經細胞型態發生中之功能

Abstract

NuMA是一個分子量為238-kDa的蛋白質，此蛋白在細胞間期主要位於在細胞核內而在進行細胞分裂時則位於紡錘體的兩極。NuMA的蛋白質結構主要可以分為N端球狀區域、中段呈現捲曲的螺旋結構及C端的球狀區域。在C端的區域中具有微管結合位(microtubule binding site)和核定位信號(NLS)，其中微管結合位可以跟微管作連結並且有穩定微管的功能，而核定位信號主要是六個胺基酸組成的序列其功能為促使NuMA能夠被運送至細胞核內作用。在有絲分裂細胞中，從先前的研究已經得知NuMA所扮演的功能是幫助微管的穩定及組織。有趣的是，NuMA不僅在有絲分裂細胞中會表現，在神經細胞中依然會表現，但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顯示，當初代的海馬迴神經細胞利用小髮夾RNA (shRNA)抑制NuMA表現後，神經細胞的神經突(neurite)呈現出較短的現象。而這個結果表示NuMA在神經細胞的型態發生(neurite morphogenesis)中具有功能存在。另外，在免疫螢光的染色圖中顯示出NuMA會表現在神經細胞中的細胞核及細胞質內。為了瞭解會影響神經型態的NuMA是細胞核內的功能還是在細胞質中所扮演的功能，因此我們建立三種不同結構的NuMA: 第一種是正常的人類NuMA且接有增強綠色螢光蛋白(EGFP)的結構(EGFP-NuMA)；第二種是將NLS序列移除的NuMA且接有EGFP (EGFP-NuMA-ΔNLS)；第三種則是將NLS序列突變成由甘胺酸(glycine)及丙胺酸(alanine)組成的序列的NuMA並接有EGFP (EGFP-NuMA-*NLS)。我們發現不具有NLS序列的NuMA無論在有絲分裂細胞或神經細胞中皆無法被運送至細胞核內。同時我們也發現，這些不具NLS序列的NuMA會位在神經細胞中的中心體(centrosome)上。另外令人值得注意的是，當我們大量表現這些不具有NLS的NuMA對於培養兩天(2DIV)的初代海馬迴神經細胞不會造成影響。因此，我們的結果顯示細胞質NuMA並不會對早期的神經細胞型態發生造成影響。

The nuclear mitotic apparatus (NuMA) protein is a 238-kDa protein that localizes to the nucleus during interphase and to spindle poles during mitosis. The structure of NuMA protein is composed of N-terminal globular domain, coiled-coil central region, and C-terminal globular tail domain. In the C-terminus, there is a microtubule binding site which can bind to microtubule to stabilize microtubule as well as a nuclear localization signal (NLS) which is a six amino acid stretch that facilitates its transportation into the nucleus. It has been shown that NuMA interacts with microtubules and regulates their stability and organization in mitotic cells. Interestingly, NuMA expresses not only in mitotic cells but also in neurons, but the function in neurons is still unclear. Recent studies from our laboratory showed that depleting NuMA by small hairpin RNA (shRNA) in primary hippocampal neurons resulted in reduced neurite length. This suggests that NuMA has additional non-mitotic functions on neurite morphogenesis. Immunofluorescence staining of NuMA indicated that it localized to both the nucleus and cytoplasm in neurons. To determine whether the nuclear NuMA or cytoplasmic NuMA affected neurite morphogenesis, we established 3 constructs: a full length NuMA fused to EGFP (NuMA-EGFP), a NLS deleted NuMA fused to EGFP (EGFP-NuMA-ΔNLS), and a NuMA with its NLS mutated to glycine and alanine fused to EGFP (EGFP-NuMA-*NLS). We discovered that NuMA without NLS (EGFP-NuMA-ΔNLS and EGFP-NuMA-*NLS) cannot be transported into the nucleus in either mitotic cells or in neurons. We also found that NuMA without NLS proteins localized to the centrosome in neurons. Interestingly, overexpressing NuMA without NLS did not affect neurite morphogenesis in 2DIV hippocampal neurons. Our data therefore suggest that the cytoplasmic NuMA does not affect early neuronal morphogenesis.