研究神經元中細胞質Ran對於非中心體微管聚核之影響

Abstract

功能性神經系統取決於神經元發育過程中神經元之間的正確連接。已經顯示微管細胞骨架對於神經元發育是重要的。並且已經表明，非中心體微管組織中心在調節神經元微管中起重要作用。然而，這種非中心體微管組織中心的身份和本地化仍然難以捉摸。基於我們以前的研究，我們發現Ran GTP酶在調節微管成核中發揮作用。在這裡，我們嘗試通過過度表達組成型活性RanQ69L或神經元中的顯性負RanT24N來檢查細胞質Ran對acentrosomal微管成核的作用。首先，我們發現RanGTP集中在神經突末端。第二，我們發現RanGTP在神經突尖端的分佈以及沿軸突軸的微管密度可能受到過突變冉突變體的影響。此外，我們發現神經突末端的acentrosomal微管形成的頻率受到Ran突變體的影響。為了改善神經元形態的定量，我們建立了低密度神經元培養。最後，我們發現過度表達神經元中的冉突變體可能會影響神經突伸長。這些結果表明細胞質Ran在調節神經元中的非中心體微管成核和形態發生中起作用。

A functional nervous system depends on the correct connection between neurons during neuronal development. It has been shown that the microtubule cytoskeleton is important for neuronal development. And it has been shown that acentrosomal microtubule organizing center (MTOC) plays an important role in regulating the neuronal microtubules. However, the identity and localization of this acentrosomal MTOC remain elusive. Based on our previous research, we found that Ran GTPase plays a role in regulating microtubule nucleation. Here we attempt to examine the role of cytoplasmic Ran on acentrosomal microtubule nucleation by overexpressing a constitutively active RanQ69L or a dominant negative RanT24N in neurons. First, we found that RanGTP is concentrated at the tip of neurites. Second, we found that the distribution of RanGTP at the tip of neurites as well as the microtubule density along the neurite shaft can be affected by overexpressing the Ran mutants. Furthermore, we discovered that the frequency of acentrosomal microtubule formation at the tip of neurites is influenced by Ran mutants. To improve the quantification of neuronal morphology, we established the low-density neuron culture. Finally, we found that overexpressing the Ran mutants in neurons can affect neurite elongation. These results indicated that cytoplasmic Ran plays a role in regulating acentrosomal microtubule nucleation and morphogenesis in neurons.