以高內含分析技術探討P19神經分化細胞模型中kinesin動力蛋白之功能

Abstract

神經元的型態發生是一個非常重要的過程，最終可以形成互相連結的神經網絡。神經中微管(microtubule)是最主要的細胞骨架並在神經生成中扮演重要角色。而在神經元發育的過程中，微管動力蛋白(microtubule-based proteins)像是kinesin和dynein皆扮演重要角色:調控神經突的開始及延長、樹突及軸突的生成並可讓神經突產生分枝。目前已知微管動力蛋白對於細胞內物質的運輸非常重要，特別是在神經細胞中，為了有效將物質運送至長距離的樹突和軸突，更需要微管動力蛋白來進行胞內運輸。在本研究中，我們在神經分化的過程中用shRNA系統性地抑制所有的kinesin家族微管動力蛋白，並觀察這些kinesin動力蛋白對神經元型態的影響。我們以P19細胞株作為模型，通過表現proneuronal gene Ascl1可以有效率的讓P19細胞分化成神經細胞。在本研究中，我們利用高內含顯微影像技術進行自動影像擷取和分析，藉此一窺所有kinesin動力蛋白於神經突生成所扮演的角色。

Neuronal morphogenesis is an important developmental process that ultimately leads to the interconnected neuronal network. Microtubule is one of the major cytoskeletons in neurons and it plays essential roles during this process. During neuronal morphogenesis, microtubule-based motors like kinesin and dynein play crucial roles in regulating neurite initiation, neurite elongation, axon-dendrite specification, and neurite branching. It is known that microtubule-based motors is the power behind intracellular transport, which is essential for neurons in which cellular components need to be transported over long distance into axons and dendrites. In this study, we used shRNAs to target each and every kinesin motor during neuronal differentiation and examined their effect on the morphology of the neuron. We used mouse embryonal carcinoma (P19) cells as our model system. P19 cells can be efficiently differentiated into neurons by transient expression of the proneural gene Ascl1. High-content assay was used to acquire and analyze the large volume of image data generated in this study. Our study provided the first comprehensive view of the function of all kinesin motors during neuronal morphogenesis.