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

Liu T, Sims D, Baum B - Genome Biol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Laboratory of Molecular Cell Biology, UCL, London, UK. tao.liu@ucl.ac.uk

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.

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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.
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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.

Mentions: The C3 cluster identified a cyclin-dependent kinase, CG7236 [20], which elicited an RNAi phenotype only when targeted in hemocyte cell lines. Cyclin-dependent kinases are known to regulate cell cycle-dependent changes in cell organization together with a host of other processes, such as RNA Polymerase II activity [21]. In hemocyte cell lines RNAi-mediated silencing of CG7236 led to the accumulation of large cells with multiple or enlarged nuclei (Figure 3a), as verified using independent dsRNAs and confocal imaging (Figure 3a, bottom panels). This suggests a role for CG7236 in the regulation of the cell division cycle. However, RNAi-mediated silencing of CG7236 caused no detectable change in the appearance of neuronal cell lines such as BG3-c2 (Figure 3a), even though a quantitative PCR (Q-PCR) analysis revealed that CG7236 is both expressed and effectively silenced by RNAi in both S2R+ and BG3-c2 cells (Figure 3d). CG7236 has not been studied in detail before, but was previously identified as a cell cycle kinase in an RNAi screen in S2 cells [22], and as having a cytokinesis defect in RNAi screens in Drosophila hemocyte cell lines [14,23,24]. By analyzing its function across cell types, our analysis suggests that CG7236 differs from many other kinases involved in cell cycle control in performing a cell type-specific function.


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

Liu T, Sims D, Baum B - Genome Biol. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2690997&req=5

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.
Mentions: The C3 cluster identified a cyclin-dependent kinase, CG7236 [20], which elicited an RNAi phenotype only when targeted in hemocyte cell lines. Cyclin-dependent kinases are known to regulate cell cycle-dependent changes in cell organization together with a host of other processes, such as RNA Polymerase II activity [21]. In hemocyte cell lines RNAi-mediated silencing of CG7236 led to the accumulation of large cells with multiple or enlarged nuclei (Figure 3a), as verified using independent dsRNAs and confocal imaging (Figure 3a, bottom panels). This suggests a role for CG7236 in the regulation of the cell division cycle. However, RNAi-mediated silencing of CG7236 caused no detectable change in the appearance of neuronal cell lines such as BG3-c2 (Figure 3a), even though a quantitative PCR (Q-PCR) analysis revealed that CG7236 is both expressed and effectively silenced by RNAi in both S2R+ and BG3-c2 cells (Figure 3d). CG7236 has not been studied in detail before, but was previously identified as a cell cycle kinase in an RNAi screen in S2 cells [22], and as having a cytokinesis defect in RNAi screens in Drosophila hemocyte cell lines [14,23,24]. By analyzing its function across cell types, our analysis suggests that CG7236 differs from many other kinases involved in cell cycle control in performing a cell type-specific function.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Laboratory of Molecular Cell Biology, UCL, London, UK. tao.liu@ucl.ac.uk

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.

Show MeSH
Related in: MedlinePlus