MOTIVATION: The nuclear pore complex (NPC) is the only passageway for macromolecules between nucleus and cytoplasm, and an important reference standard in microscopy: it is massive and stereotypically arranged. The average architecture of NPC proteins has been resolved with pseudoatomic precision, however observed NPC heterogeneities evidence a high degree of divergence from this average. Single-molecule localization microscopy (SMLM) images NPCs at protein-level resolution, whereupon image analysis software studies NPC variability. However, the true picture of this variability is unknown. In quantitative image analysis experiments, it is thus difficult to distinguish intrinsically high SMLM noise from variability of the underlying structure. RESULTS: We introduce CIR4MICS ('ceramics', Configurable, Irregular Rings FOR MICroscopy Simulations), a pipeline that synthesizes ground truth datasets of structurally variable NPCs based on architectural models of the true NPC. Users can select one or more N- or C-terminally tagged NPC proteins, and simulate a wide range of geometric variations. We also represent the NPC as a spring-model such that arbitrary deforming forces, of user-defined magnitudes, simulate irregularly shaped variations. Further, we provide annotated reference datasets of simulated human NPCs, which facilitate a side-by-side comparison with real data. To demonstrate, we synthetically replicate a geometric analysis of real NPC radii and reveal that a range of simulated variability parameters can lead to observed results. Our simulator is therefore valuable to test the capabilities of image analysis methods, as well as to inform experimentalists about the requirements of hypothesis-driven imaging studies. AVAILABILITY AND IMPLEMENTATION: Code: https://github.com/uhlmanngroup/cir4mics. Simulated data: BioStudies S-BSST1058.

BACKGROUND: Loss-of-function phenotypes are widely used to infer gene function using the principle that similar phenotypes are indicative of similar functions. However, converting phenotypic to functional annotations requires careful interpretation of phenotypic descriptions and assessment of phenotypic similarity. Understanding how functions and phenotypes are linked will be crucial for the development of methods for the automatic conversion of gene loss-of-function phenotypes to gene functional annotations. RESULTS: We explored the relation between cellular phenotypes from RNAi-based screens in human cells and gene annotations of cellular functions as provided by the Gene Ontology (GO). Comparing different similarity measures, we found that information content-based measures of phenotypic similarity were the best at capturing gene functional similarity. However, phenotypic similarities did not map to the Gene Ontology organization of gene function but to functions defined as groups of GO terms with shared gene annotations. CONCLUSIONS: Our observations have implications for the use and interpretation of phenotypic similarities as a proxy for gene functions both in RNAi screen data analysis and curation and in the prediction of disease genes.

MOTIVATION: Most biological processes remain only partially characterized with many components still to be identified. Given that a whole genome can usually not be tested in a functional assay, identifying the genes most likely to be of interest is of critical importance to avoid wasting resources. RESULTS: Given a set of known functionally related genes and using a state-of-the-art approach to data integration and mining, our Functional Lists (FUN-L) method provides a ranked list of candidate genes for testing. Validation of predictions from FUN-L with independent RNAi screens confirms that FUN-L-produced lists are enriched in genes with the expected phenotypes. In this article, we describe a website front end to FUN-L. AVAILABILITY AND IMPLEMENTATION: The website is freely available to use at http://funl.org

BACKGROUND: The combination of time-lapse imaging of live cells with high-throughput perturbation assays is a powerful tool for genetics and cell biology. The Mitocheck project employed this technique to associate thousands of genes with transient biological phenotypes in cell division, cell death and migration. The original analysis of these data proceeded by assigning nuclear morphologies to cells at each time-point using automated image classification, followed by description of population frequencies and temporal distribution of cellular states through event-order maps. One of the choices made by that analysis was not to rely on temporal tracking of the individual cells, due to the relatively low image sampling frequency, and to focus on effects that could be discerned from population-level behaviour. RESULTS: Here, we present a variation of this approach that employs explicit modelling by dynamic differential equations of the cellular state populations. Model fitting to the time course data allowed reliable estimation of the penetrance and time of appearance of four types of disruption of the cell cycle: quiescence, mitotic arrest, polynucleation and cell death. Model parameters yielded estimates of the duration of the interphase and mitosis phases. We identified 2190 siRNAs that induced a disruption of the cell cycle at reproducible times, or increased the durations of the interphase or mitosis phases. CONCLUSIONS: We quantified the dynamic effects of the siRNAs and compiled them as a resource that can be used to characterize the role of their target genes in cell death, mitosis and cell cycle regulation. The described population-based modelling method might be applicable to other large-scale cell-based assays with temporal readout when only population-level measures are available.