Parallel RNAi screens across different cell lines identify generic and cell type-specific regulators of actin organization and cell morphology

Abstract

Background: In recent years RNAi screening has proven a powerful tool for dissecting gene functions in animal cells in culture. However, to date, most RNAi screens have been performed in a single cell line, and results then extrapolated across cell types and systems.

Results: Here, to dissect generic and cell type-specific mechanisms underlying cell morphology, we have performed identical kinome RNAi screens in six different Drosophila cell lines, derived from two distinct tissues of origin. This analysis identified a core set of kinases required for normal cell morphology in all lines tested, together with a number of kinases with cell type-specific functions. Most significantly, the screen identified a role for minibrain (mnb/DYRK1A), a kinase associated with Down's syndrome, in the regulation of actin-based protrusions in CNS-derived cell lines. This cell type-specific requirement was not due to the peculiarities in the morphology of CNS-derived cells and could not be attributed to differences in mnb expression. Instead, it likely reflects differences in gene expression that constitute the cell type-specific functional context in which mnb/DYRK1A acts.

Conclusions: Using parallel RNAi screens and gene expression analyses across cell types we have identified generic and cell type-specific regulators of cell morphology, which include mnb/DYRK1A in the regulation of protrusion morphology in CNS-derived cell lines. This analysis reveals the importance of using different cell types to gain a thorough understanding of gene function across the genome and, in the case of kinases, the difficulties of using the differential gene expression to predict function.

Figure 1

The cell morphology and gene expression profiles of six Drosophila cell lines. (a) The three CNS-derived cell lines BG2-c2, BG3-c1 and BG3-c2 have a bipolar, spiky cell shape, whereas the three embryonic hemocyte-derived cell lines S2, S2R+ and Kc167 have a symmetrical morphology. Gene expression profiles for each cell line were normalized and hierarchical clustering was used to generate the dendrogram shown. This analysis reveals that cell lines from the same origin have closely related gene expression profiles (Table 1). (b) Kinase RNAi screens were carried out in all six cell lines. An RNAi library targeting 265 kinases and kinase regulatory subunits (Additional file 1) was combined with cells in 384-well plates and incubated for 5 days before fixing and staining to visualize F-actin, microtubules and DNA.

Figure 2

Parallel RNAi screens reveal cell line-specific phenotypes. (a) Different cell lines exhibited different hit rates in RNAi screens (Additional file 3). (b) Venn diagrams depict the segregation of screen hits between related cell lines. (c) A Venn diagram depicts the classification of hits into three distinct classes: those that are hits in both CNS and hemocyte cell lines; those that are hits in neuronal cell lines only; and those that are hits in hemocyte cell lines only. (d) Hierarchical clustering of hits across cell lines (depicted in the form of a tree) was used to give a more detailed picture of the three hit classes. Two hits of particular interest, CG7236 and minibrain (mnb), are highlighted. Note that the relationships defined by the functional analysis (depicted in the form of a tree at the top of figure) mirror the relationships defined by the microarray analysis (see Table 1 for the Pearson correlation coefficients in each case).

Figure 3

CG7236 and minibrain show cell line-specific phenotypes. (a) Silencing of the cdc2-related kinase CG7236 in S2R+ cells gives rise to large cells that frequently contain multiple nuclei or a single large nucleus, whereas silencing in BG3-c2 cells has no discernable phenotype. (b) Silencing of the DYRK family kinase minibrain in BG3-c2 cells causes an increase in peripheral actin and an increase in the number of protrusions per cell, whereas silencing in S2R+ cells has no phenotype. Also, the BG3-c2 cells forced to spread by plating on concanavalin A (ConA) exhibit large lamellipodia when in the presence of a non-targeting dsRNA, but not in the presence of mnb dsRNA. (c) Quantification of the mnb RNAi phenotype shows a significant twofold increase in the number of long finger-like protrusions around the cell body. (d) Q-PCR analysis reveals that CG7236 and minibrain are effectively silenced by RNAi reagents in both S2R+ and BG3-c2 cells. Error bars indicate the standard error of the mean.

Figure 4

Genes with cell line-specific phenotypes are not differentially expressed. Chart of the expression levels of CG7236, mnb and Pvr in S2R+ and BG3-c2 cells as measured by Q-PCR. Expression levels were established by taking the ratio of expression of each gene compared to the control ribosomal component rp49 in three independent experiments. The error bars represent the standard error in the mean across those experiments. Plus sign indicates RNAi treatments that resulted in an observed phenotype.