Protein-protein interactions and genome engineering : novel strategies to study cell polarity

Cell polarity is a fundamental property of cells. The identification of conserved polarity regulators that control polarity in a variety of distinct tissues raises a number of questions. How are the same components used and integrated in tissue-specific ways to give rise to the wide variety of polarized tissues? What are the downstream components through which the polarity program acts? We developed a tissue-specific affinity purification/mass spectrometry approach for C. elegans based on in vivo biotinylation of Avi-tagged proteins of interest by the bacterial biotin ligase BirA. Tissue-specific biotinylation is accomplished by expressing BirA from a tissue-specific promoter, while the Avi-tagged protein is expressed from its native regulatory sequences. Biotinylated bait proteins are subsequently purified and interacting proteins identified by mass spectrometry. We confirmed the tissue-specificity of our approach and applied it to several polarity proteins. Tissue-specific purification of DLG-1 and CDC-42 resulted in the identification of several known interaction partners, demonstrating that our approach can identify valid interactions from specific tissues. It is important to gain a detailed mechanistic understanding of the proteins that regulate polarity. The functioning of a protein can in large part be dictated by the protein interactions it engages in. Here, we expand the concept of Y2H-based interaction domain mapping to the genome-wide scale. We generated a human prey library by fragmenting an ORFeome collection with ultrasonication and demonstrated the quality of the library by screening it with polarity and cell division proteins. We identified several interactions previously described in literature as well as novel interactions, and validated 55% of all identified interactions by affinity purifications in cell culture. The best approach for studying the function of a protein in vivo is to precisely modify the genome to express a mutated protein that for example lacks interaction domains. We developed the CRISPR/Cas9 system for C. elegans to be able to specifically engineer its genome. By expressing a guide RNA the Cas9 endonuclease is targeted to a specific locus in the genome where it creates a double strand break. Imprecise repair of the break can yield mutations and we obtained mutants for all four genes tested. Several other groups independently adapted the CRISPR/Cas9 system for C. elegans, and we reviewed the different approaches taken. We used our CRISPR/Cas9 approach to gain a better understanding of the functioning of the Crumbs proteins in C. elegans. While the Crumbs protein is essential for epithelial polarity and viability in Drosophila, the two Crumbs homologs identified in C. elegans thus far do not appear to play a major role in polarity establishment. We identified a third Crumbs homolog in C. elegans, which localizes in a polarized pattern in several tissues. We developed a variation of our CRISPR/Cas9 approach to delete entire genes, and generated a triple Crumbs deletion mutant. Remarkably, animals lacking all three Crumbs homologs are viable. Overexpression of all three Crumbs homologs caused changes in the localization pattern of the polarity protein PAR-3, suggesting that the C. elegans Crumbs homologs play a non-essential role in polarity establishment.