以高含量顯微影像分析技術篩選對神經型態有影響之微管動力蛋白

Abstract

神經系統的功能有賴於神經元間神經突(neurite)的相互連結。為了正確的連接神經突，神經元生成和分化的種種步驟都必須被嚴格監控。包括神經突的起始、延長、極化、分枝及成熟的步驟，這一連串的步驟又合稱為神經突生成(neuritogenesis)。在神經生成的過程中，每個階段細胞型態的變化仰賴著其內部細胞骨架例如微管(microtubule)與微絲(actin)的互相合作，其中微管是在神經突中最主要的細胞骨架，並且在神經突向外生長時提供必要的助力，而與微管相關的微管蛋白(microtubule associated proteins)也能幫助增加微管的穩定性，在許多微管蛋白中，有一項分類為微管動力蛋白(microtubule-based motors)，它們能在極性的微管陣列上行有特定方向的移動，這些動力蛋白可以分成kinesin與dynein兩大家族，並在神經細胞內細胞本體與神經突末端之間提供了重要的物質聯絡管道。在人類或是老鼠身上，kinesin家族已經發現了45種基因並分為14大類；另一方面，cytoplasmic dynein通常會與dynactin形成複合物。在本研究中，我們計畫用shRNA系統性地抑制個別且所有的微管動力蛋白，以研究神經突生成中每個微管動力蛋白的功能。我們將以P19細胞株作為模型，在通過proneural gene將P19細胞分化為神經的同時， shRNA也會進入細胞並抑制各個微管動力蛋白。本研究將可初次一窺所有微管動力蛋白於神經突生成所扮演之角色。

A functional nervous system depends on the complex architecture of neuron networks. These neurons are morphologically characterized by its long and thin protrusions called neurites. Undifferentiated cells must extend and polarize their axons and dendrites to the targets in a process called neuritogenesis. Each process including initiation, elongation, polarization, branching, and maturation of neurites must be properly regulated. The morphological change of neuritogenesis involves coordinated changes between the actin cytoskeleton and the microtubule network. The microtubule is the major cytoskeleton in the axons and dendrites and it plays crucial roles during neuritogenesis. Bundled microtubules form a continuous array within neurites and act as compression resistant struts to support neurite elongation. Because of elongated morphology of neurons, mRNAs or proteins necessary for the neurites must be transported down these axons and dendrites after synthesis in the cell body. So the intracellular transport system is particularly important for establishing a functional neuron network. These microtubule-based motors can be divided into two superfamilies, the kinesin superfamily (KIF) and the dynein superfamily, both of them hydrolyze ATP to move along microtubule in a specific direction. In mammals such as human and mouse, the total number of Kif genes is 45 and has been classified into 14 classes. On the other hand, cytoplasmic dynein usually form a complex with an associated protein complex called dynactin. In this study, we propose to examine the function of all microtubule-based motors in neuritogenesis by systematically suppressing each and every motor using shRNA. As our model, mouse P19 embryonal carcinoma cells can be differentiated into neurons by transient expression of proneural gene Ascl1 (previously Mash1). We introduce Ascl1 and microtubule-based motor targeting shRNA into P19 cells simultaneously. This study provides the first comprehensive view of the function of all microtubule-based motors in neuritogenesis.