Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk lo ) expressing 25% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild-type and Btk ؊/؊ mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk lo phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2-containing inositol phosphatase suppressed the Btk lo phenotype in vitro but not in vivo, whereas CD19 and the p85␣ form of phosphoinositide 3-kinase behaved as Btk lo enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85␣ haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C ␤, Fyn, CD22, G␣q, or G␣11 had no detectable effect on the function of Btk lo B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.In vivo genetic analysis is a powerful approach to define the relative contribution of signaling interactions to physiological processes. Small genomes, ease of mutagenesis, and rapid generation time have made Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae the major models for genetic dissection of eukaryotic signaling pathways. Although there are some examples of large-scale genetic screens in mice (1-3), time, space, and financial resources generally limit mammalian genetic studies to targeted mutations in known genes or the characterization of naturally occurring mutations.In a commonly used type of genetic screen, organisms carrying a defined mutation affecting the process of interest are further mutagenized and screened for second site mutations, or modifiers, that enhance (increase) or suppress (reduce) the severity of the parental phenotype (4, 5). The initial mutation often renders the system responsive to subtle changes in modifiers that would not be penetrant on an otherwise wild-type background. This sensitivity often permits a dominant screen, decreasing the time and resources required to identify new mutations.Complete deficiency of many signaling molecules results in loss of the cell type of interest or pleiotropic or lethal phenotypes, making the effects of additional m...

Heat shock proteins represent the major elements of the cellular stress response that protects cells from diseases caused by protein misfolding. Small-molecule amplifiers of heat shock proteins have shown promising results in several animal models, demonstrating the potential importance of such compounds for therapeutics. The expression of many heat shock proteins is controlled by HSF1, which forms stress granules in the nucleus when transcriptionally activated. Activation of the cellular stress also correlates with the translocation of HSP70 into nucleoli. The authors have developed an image-based, multiparametric assay to simultaneously monitor the effects of compounds on HSF1/HSP70 stress granule formation in heat-shocked Hela cells. Highcontent screening of the compound library was performed with a Z′ of 0.62, demonstrating a highly robust assay for large-scale screening. The resulting hits showed prolonged amplification of HSP70 induction in heat-stressed cells but no effects in cells without stress. Treatment of cells with selected hits exhibited significant cytoprotection from both oxygen glucose deprivation and rotenone-induced stresses. Thus, high-content screening of HSF1/HSP70 amplifiers provides a practical opportunity for clinical therapeutics targeting protein misfolding diseases. (Journal of Biomolecular Screening 2008:953-959)