Dynamical modelling of phenotypes in a genome-wide RNAi live-cell imaging assay.

International audienceBACKGROUND: 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-levelbehaviour. 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

Shapes of cell signaling

Cell signaling is a complex process organized in time and space. Signal transduction is constantly modulated by cell-intrinsic and cell-extrinsic input cues and the resulting phenotypic responses such as morphological can feed back into the system. This provides cells with a responsive, accurate, and rugged system to deal with changes in the surroundings or the genome. Whilst signaling networks (dynamic transient protein–protein interactions modulated by post-translational modifications in response to input cues) have been researched for decades, further analysis of their spatial organization is critical for both basic and disease biology and will benefit from recent advances in computational modeling and image analysis using deep/machine learning and in microscopy and imaging. Furthermore, mathematical modeling with reaction-diffusion approaches on time-varying geometries complements the investigations, allowing to conceptualize the organizational principles of signaling and information transduction in the four dimensions of time and space.Peer Reviewe

Advances in quantitative microscopy

Microscopy allows us to peer into the complex deeply shrouded world that the cells of our body grow and thrive in. With the emergence of automated digital microscopes and software for anlysing and processing the large numbers of image that they produce; quantitative microscopy approaches are now allowing us to answer ever larger and more complex biological questions. In this thesis I explore two trends. Firstly, that of using quantitative microscopy for performing unbiased screens, the advances made here include developing strategies to handle imaging data captured from physiological models, and unsupervised analysis screening data to derive unbiased biological insights. Secondly, I develop software for analysing live cell imaging data, that can now be captured at greater rates than ever before and use this to help answer key questions covering the biology of how cells make the decision to arrest or proliferate in response to DNA damage. Together this thesis represents a view of the current state of the art in high-throughput quantitative microscopy and details where the field is heading as machine learning approaches become ever more sophisticated.Open Acces

Cohesion failure and Mitosis: From Molecular Mechanisms to Organismal Consequences

"Mitosis is a dynamic culmination of the cell cycle, resulting in generation of two daughter cells from one mother. In order for this to happen, the cell must package its DNA into chromosomes and divide it equally amongst progeny. To ensure this process happens accurately, the cell glues identical chromosomes together so it can segregate them in symmetrical fashion during anaphase. The glue holding chromosomes together is a molecule called cohesin, which encompasses replicated DNA fibers via topological entrapment. The aim of this thesis was to study the immediate mitotic response to premature cohesion loss, as well as the long term consequences of such perturbed mitosis for the cell and the whole organism.(...)

Functional dissection of a gene expression oscillator in C. elegans

Gene expression oscillations control diverse biological processes. One such example of gene expression oscillations, are those found for thousands of genes during C. elegans larval development. However, it remains unclear whether and how gene expression oscillations regulate development processes in C. elegans. In this work, I aimed to study the molecular architecture and the system properties of the C. elegans oscillator to provide insight into potential developmental functions and reveal features that are unique, as well as those that are shared among oscillators. Here, performing temporally highly resolved mRNA-sequencing across all larval stages (L1-L4) of C. elegans development, we identified 3,739 genes, whose transcripts revealed high-amplitude oscillations (>2-fold from peak to trough), peaking once every larval stage with stable amplitudes, but variable periods. Oscillations appeared tightly coupled to the molts, but were absent from freshly hatched larvae, developmentally arrested dauer larvae and adults. Quantitative characterization of transitions between oscillatory and stable states of the oscillator showed that the stable states are similar to a particular phase of the oscillator, which coincided with molt exit. Given that these transitions are sensitive to food, we postulate that feeding might impact the state of the oscillator. These features appear rather unique, and hence a better understanding may help to reveal general principles of gene expression oscillators. Our RNAPII ChIP-seq revealed rhythmic occupancy of RNAPII at the promoters of oscillating genes, suggesting that mRNA transcript oscillations arise from rhythmic transcription. Given that oscillations are coupled to the repetitive molts and that the molecular mechanisms that regulate molting are unknown, we aimed to find transcription factors important for molting and oscillations. Hence, we screened 92 transcription factors that oscillate on the mRNA level for their role in molting and identified grh-1, myrf1, blmp-1, bed-3, nhr-23, nhr-25 and ztf-6. We showed that oscillatory activity of GRH-1 is required for timely completion of the molt, to prevent cuticle rupturing, and for oscillatory expression of structural components of the cuticle and ‘ECM regulators’, among others, including grh-1 itself. Hence, we propose GRH-1 as a putative component of the (sub-)oscillator that regulates molting. We showed that loss of BLMP-1 increased the duration of molts, affected cuticle integrity, and changed the oscillatory dynamics of a subset of genes in diverse ways. We postulate that BLMP-1 acts as factor that couples gene expression oscillations, and potentially sub-oscillators or repetitive developmental processes. In conclusion, this work provides insight into the function of the oscillator, and its system properties. Moreover, we identified relevant factors, which we propose as a starting point to unravel the molecular wiring of the C. elegans oscillator and its functional relevance

Neurofly 2008 abstracts : the 12th European Drosophila neurobiology conference 6-10 September 2008 Wuerzburg, Germany

This volume consists of a collection of conference abstracts

Lyapunov exponents and phase diagrams reveal multi-factorial control over TRAIL-induced apoptosis

Kinetic modeling, phase diagrams analysis, and quantitative single-cell experiments are combined to investigate how multiple factors, including the XIAP:caspase-3 ratio and ligand concentration, regulate receptor-mediated apoptosis

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin