Endocytosis is an essential cellular process consisting of the internalization of extracellular cargo and its transport towards different intracellular destinations. Multiple endocytic routes are tailored for the internalization and trafficking of different types of cargo and multiple endocytic organelles provide specialized biochemical environments where different molecular events take place. Membrane receptors and cargo molecules are internalized by both Clathrin-dependent and –independent endocytosis into early endosomes. From here two main endocytic routes are followed: 1) the recycling route, mainly followed by membrane receptor and other molecules like Transferrin, brings the cargo back to the plasma membrane and 2) the degradative route, followed by molecules like Epidermal Growth Factor (EGF) and Lipoprotein particles (LDL), leads the cargo to degradation into late endosomes/lysosomes. In addition to the basic function of intracellular cargo transport, the endocytic system fulfils many other cellular and developmental functions such as transmission of proliferative and survival signals and defence against pathogens. In order for cells to properly perform their various and numerous functions in organs and tissues, the activity of the endocytic system needs to be coordinated between cells and, within individual cells, integrated with other cellular functions. Even though molecules orchestrating the endocytic sorting and transport of different types of cargo have long been investigated, our understanding of the molecular machinery underlying endocytosis and its coordination into the cellular systems remains fragmentary. The work presented in this thesis aimed at understanding how this high-order regulation and integration is achieved. This requires not only a comprehensive analysis of molecular constituents of the endocytic system but also an understanding of the general design principles underlying its function. To this end, in collaboration with several members of the Zerial group and with the HT-Technology Development Studio (TDS) at MPI-CBG, I developed a new strategy to accurately profile the activity of human genes with respect to Transferrin (Tfn) and Epidermal Growth Factor (EGF) endocytosis by combining genome-wide RNAi with several siRNA/esiRNA per gene, automated high-resolution confocal microscopy, quantitative multi-parametric image analysis and high-performance computing. This provided a rich and complex genomic dataset that was subsequently subjected to analysis with a combination of tools such as a multi-parametric correlation of oligo profiles, phenotypic clustering and pathways analysis, and a Bayesian network reconstruction of key endocytic features. Altogether, the genomic endeavour and the subsequent analyses provided a number of important results: first, they revealed a much higher extent of off-target effects from RNAi and provided novel tools to infer the specific effects of genes loss of function; second, they identified a large number of novel molecules exerting a regulatory role on the endocytic system, including uncharacterized genes and genes implicated in human diseases; third, they uncovered the regulatory activity of signalling pathways such as Wnt, Integrin, TGF-β, and Notch, and found new genes regulating the sorting of cargo to a specialized subset of early endosomes that function as intracellular signalling platforms; and fourth, a systems analysis by Bayesian networks revealed that the cell specifically regulates the number, size, concentration of cargo and intracellular position of endosomes, thus uncovering novel properties of the endocytic system. In conclusion, the work presented here not only provided a dataset extremely rich of information whose potential has just begun to be uncovered but also shows how genomic datasets can be used to reveal design principles governing the functioning of biological processes.

Endocytosis is an essential cellular process consisting of the internalization of extracellular cargo and its transport towards different intracellular destinations. Multiple endocytic routes are tailored for the internalization and trafficking of different types of cargo and multiple endocytic organelles provide specialized biochemical environments where different molecular events take place. Membrane receptors and cargo molecules are internalized by both Clathrin-dependent and –independent endocytosis into early endosomes. From here two main endocytic routes are followed: 1) the recycling route, mainly followed by membrane receptor and other molecules like Transferrin, brings the cargo back to the plasma membrane and 2) the degradative route, followed by molecules like Epidermal Growth Factor (EGF) and Lipoprotein particles (LDL), leads the cargo to degradation into late endosomes/lysosomes. In addition to the basic function of intracellular cargo transport, the endocytic system fulfils many other cellular and developmental functions such as transmission of proliferative and survival signals and defence against pathogens. In order for cells to properly perform their various and numerous functions in organs and tissues, the activity of the endocytic system needs to be coordinated between cells and, within individual cells, integrated with other cellular functions. Even though molecules orchestrating the endocytic sorting and transport of different types of cargo have long been investigated, our understanding of the molecular machinery underlying endocytosis and its coordination into the cellular systems remains fragmentary. The work presented in this thesis aimed at understanding how this high-order regulation and integration is achieved. This requires not only a comprehensive analysis of molecular constituents of the endocytic system but also an understanding of the general design principles underlying its function. To this end, in collaboration with several members of the Zerial group and with the HT-Technology Development Studio (TDS) at MPI-CBG, I developed a new strategy to accurately profile the activity of human genes with respect to Transferrin (Tfn) and Epidermal Growth Factor (EGF) endocytosis by combining genome-wide RNAi with several siRNA/esiRNA per gene, automated high-resolution confocal microscopy, quantitative multi-parametric image analysis and high-performance computing. This provided a rich and complex genomic dataset that was subsequently subjected to analysis with a combination of tools such as a multi-parametric correlation of oligo profiles, phenotypic clustering and pathways analysis, and a Bayesian network reconstruction of key endocytic features. Altogether, the genomic endeavour and the subsequent analyses provided a number of important results: first, they revealed a much higher extent of off-target effects from RNAi and provided novel tools to infer the specific effects of genes loss of function; second, they identified a large number of novel molecules exerting a regulatory role on the endocytic system, including uncharacterized genes and genes implicated in human diseases; third, they uncovered the regulatory activity of signalling pathways such as Wnt, Integrin, TGF-β, and Notch, and found new genes regulating the sorting of cargo to a specialized subset of early endosomes that function as intracellular signalling platforms; and fourth, a systems analysis by Bayesian networks revealed that the cell specifically regulates the number, size, concentration of cargo and intracellular position of endosomes, thus uncovering novel properties of the endocytic system. In conclusion, the work presented here not only provided a dataset extremely rich of information whose potential has just begun to be uncovered but also shows how genomic datasets can be used to reveal design principles governing the functioning of biological processes.