Single-cell heterogeneity in cell populations arises from a combination of intrinsic and extrinsic factors. This heterogeneity has been measured for gene transcription, phosphorylation, cell morphology and drug perturbations, and used to explain various aspects of cellular physiology. In all cases, however, the causes of heterogeneity were not studied. Here we analyse, for the first time, the heterogeneous patterns of related cellular activities, namely virus infection, endocytosis and membrane lipid composition in adherent human cells. We reveal correlations with specific cellular states that are defined by the population context of a cell, and we derive probabilistic models that can explain and predict most cellular heterogeneity of these activities, solely on the basis of each cell's population context. We find that accounting for population-determined heterogeneity is essential for interpreting differences between the activity levels of cell populations. Finally, we reveal that synergy between two molecular components, focal adhesion kinase and the sphingolipid GM1, enhances the population-determined pattern of simian virus 40 (SV40) infection. Our findings provide an explanation for the origin of heterogeneity patterns of cellular activities in adherent cell populations.

A two-step, automated, high-throughput RNAi silencing screen was used to identify host cell factors required during vaccinia virus infection. Validation and analysis of clustered hits revealed previously unknown processes during virus entry, including a mechanism for genome uncoating. Viral core proteins were found to be already ubiquitinated during virus assembly. After entering the cytosol of an uninfected cell, the viral DNA was released from the core through the activity of the cell's proteasomes. Next, a Cullin3-based ubiquitin ligase mediated a further round of ubiquitination and proteasome action. This was needed in order to initiate viral DNA replication. The results accentuate the value of large-scale RNAi screens in providing directions for detailed cell biological investigation of complex pathways. The list of cell functions required during poxvirus infection will, moreover, provide a resource for future virus-host cell interaction studies and for the discovery of antivirals.

A large set of high-content RNAi screens investigating mammalian virus infection and multiple cellular activities is analysed to reveal the impact of population context on phenotypic variability and to identify indirect RNAi effects.

A genome wide RNAi screen identifies 72 host cell genes affecting S. Typhimurium entry, including actin regulators and COPI. This study implicates COPI-dependent cholesterol and sphingolipid localization as a common mechanism of infection by bacterial and viral pathogens.

Source code, user manual and SaveObjectSegmentation CellProfiler module available for download at www.cellclassifier.ethz.ch under the GPL license (implemented in Matlab).

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