A genetic view on the interior of the cell

Abstract

In chapter 1 of this thesis an overview of functional genetic approaches is given with an emphasis on the contribution of cDNA libraries on the identification of the regulators of sterol homeostasis, which is also the topic of chapter 5. Additionally, RNA-based approaches aimed at inactivating gene function, such as RNAi and CRISPR/CAS9-mediated genome editing, are described. Finally, the background and use of haploid genetics as a tool to link genes to phenotypes is described. In chapter 2 we describe the current state of knowledge regarding the function and enzymes of the tubulin detyrosination cycle. This cycle involves the enzymatic removal of the carboxyterminal tyrosine on a-tubulin on microtubules. This tubulin-specific post-translational modification is involved in the binding of microtubule-associated proteins and thereby contributes to the regulation of a wide variety of biological processes such as mitosis, cardiomyocyte contraction and the migration of vesicles over microtubules. Chapter 3 describes the development of a novel application of haploid genetics. This approach allows us to combine the power of haploid genetics to identify the regulators of intracellular phenotypes. To this end, mutagenized haploid cells are stained using antibodies recognizing the (intra-)cellular trait of interest. Subsequently, the mutants possessing the highest and lowest abundancy of the trait are isolated and mutations are determined. After showing the versatility of the technique using antibodies recognizing a suite of protein fates we perform an in-depth analysis of the Akt/PKB pathway. Chapter 4 describes the identification of vasohibins as the first tubulin detyrosinating enzymes. Previously, vasohibins were studied as secreted regulators of angiogenesis and no enzymatic function had been assigned to them. Vasohibin-1 as well as Vasohibin-2 possess tubulin detyrosinating activity and contribute to the endogenous levels of detyrosinated tubulin in the tested cell culture models. As predicted previously, microtubules, rather than a/b-tubulin dimers, are preferred targets for detyrosination. Surprisingly however, in cells lacking both vasohibins a remaining detyrosinating activity is observed, indicating the presence of yet to be identified detyrosinating enzymes. Chapter 5 reports the identification of the Golgi-resident protein C12orf49 as a regulator of sterol homeostasis. Various haploid genetic screens identify C12orf49 together with SREBP2 and accessory factors. Cells lacking C12orf49 show a poor induction of SREBP2 target genes as well as a defect in the localization of SCAP, a protein recycling between ER and Golgi-apparatus that aids to the activation of SREBP2 in the Golgi-apparatus. Although the exact molecular function of C12orf49 remains unclear, it is suggested that C12orf49 could contribute to the release of SCAP from the Golgi-apparatus to allow its recycling to the ER. The discussion of this thesis is divided in two chapters. Chapter 6 further describes the biological and technical aspects of the experimental chapters. Chapter 7 focusses on the application of the technique described in chapter 3 and how this relates to other techniques that lead to the formation of biological networks. In addition, this final chapter provides an outlook on the generation of a comprehensive wiring map of the human cell.