A cytological F1 RNAi screen for defects in Drosophila melanogaster female meiosis.

Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, we have screened the VALIUM22 collection of RNAi constructs that target germline-expressing genes in a vector optimized for germline expression by driving RNAi with GAL4 under control of a germline-specific promoter (nanos or mat-alpha4). This allowed us to test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we could identify defects in sterile females. After screening >1,450 lines of the collection for two different defects (chromosome congression and the hypoxic sequestration of Mps1-GFP to ooplasmic filaments), we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.

A Cytological F1 RNAi Screen for Defects in Drosophila melanogaster Female Meiosis.

Genetic screens for recessive alleles induce mutations, make the mutated chromosomes homozygous, and then assay those homozygotes for the phenotype of interest. When screening for genes required for female meiosis, the phenotype of interest has typically been nondisjunction from chromosome segregation errors. As this requires that mutant females be viable and fertile, any mutants that are lethal or sterile when homozygous cannot be recovered by this approach. To overcome these limitations, our lab has screened the VALIUM22 collection produced by the Harvard TRiP Project, which contains RNAi constructs targeting genes known to be expressed in the germline in a vector optimized for germline expression. By driving RNAi with GAL4 under control of a germline-specific promoter (nanos or mat-alpha4), we can test genes that would be lethal if knocked down in all cells, and by examining unfertilized metaphase-arrested mature oocytes, we can identify defects associated with genes whose knockdown results in sterility or causes other errors besides nondisjunction. We screened this collection to identify genes that disrupt either of two phenotypes when knocked down: the ability of meiotic chromosomes to congress to a single mass at the end of prometaphase, and the sequestration of Mps1-GFP to ooplasmic filaments in response to hypoxia. After screening >1450 lines of the collection, we obtained multiple hits for both phenotypes, identified novel meiotic phenotypes for genes that had been previously characterized in other processes, and identified the first phenotypes to be associated with several previously uncharacterized genes.

Alteration of Membrane Cholesterol Content Plays a Key Role in Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Channel Activity.

Altered cholesterol homeostasis in cystic fibrosis patients has been reported, although controversy remains. As a major membrane lipid component, cholesterol modulates the function of multiple ion channels by complicated mechanisms. However, whether cholesterol directly modulates cystic fibrosis transmembrane conductance regulator (CFTR) channel function remains unknown. To answer this question, we determined the effects of changing plasma membrane cholesterol levels on CFTR channel function utilizing polarized fischer rat thyroid (FRT) cells and primary human bronchial epithelial (HBE) cells. Treatment with methyl-ß-cyclodextrin (MßCD) significantly reduced total cholesterol content in FRT cells, which significantly decreased forskolin (FSK)-mediated activation of both wildtype (WT-) and P67L-CFTR. This effect was also seen in HBE cells expressing WT-CFTR. Cholesterol modification by cholesterol oxidase and cholesterol esterase also distinctly affected activation of CFTR by FSK. In addition, alteration of cholesterol increased the potency of VX-770, a clinically used potentiator of CFTR, when both WT- and P67L-CFTR channels were activated at low FSK concentrations; this likely reflects the apparent shift in the sensitivity of WT-CFTR to FSK after alteration of membrane cholesterol. These results demonstrate that changes in the plasma membrane cholesterol level significantly modulate CFTR channel function and consequently may affect sensitivity to clinical therapeutics in CF patients.