Kiger A A, Baum B, Jones S, Jones M R, Coulson A, Echeverri C, Perrimon N
Department of Genetics, Harvard Medical School, Howard Hughes Medical Institute, Boston, MA 02115, USA.
J Biol. 2003;2(4):27. doi: 10.1186/1475-4924-2-27. Epub 2003 Oct 1.
The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies.
We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA.
Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.
后生动物细胞形状的多样性受动态细胞骨架网络的影响。随着RNA干扰（RNAi）技术的出现，现在有可能系统地筛选控制特定细胞生物学过程的基因，包括那些产生不同形态所需的基因。
我们将果蝇细胞培养中现有的RNAi技术应用于高通量筛选，以便对细胞形态发生进行全面的遗传学剖析。为了鉴定负责两种形态不同的细胞系特征形状的基因，我们在每个细胞系中用一组靶向994个预测的细胞形状调节因子的双链RNA（dsRNA）进行了RNAi筛选。使用自动荧光显微镜观察肌动蛋白丝、微管和DNA，我们检测到160个基因的形态学表型，其中三分之一以前在体内未被表征。具有相似表型的基因对应于控制细胞骨架组织和细胞形状的已知信号通路成分，这使我们对以前未被表征的基因提出了相似的功能。此外，我们能够通过共RNAi筛选发现作用于特定信号通路的基因，以鉴定由Pten dsRNA诱导的细胞形状变化的dsRNA抑制因子。
使用RNAi，我们鉴定了影响两种不同细胞类型中细胞骨架组织和形态的基因。一些基因在两种细胞类型中表现出相似的RNAi表型，而另一些基因似乎具有细胞类型特异性功能，部分反映了用于产生圆形或扁平细胞形态的不同机制。
