Time-Resolved Quantification of Centrosomes by Automated Image Analysis Suggests Limiting Component to Set Centrosome Size in C. Elegans Embryos

The centrosome is a dynamic organelle found in all animal cells that serves as a microtubule organizing center during cell division. Most of the centrosome components have been identified by genetic screens over the last decade, but little is known about how these components interact with each other to form a functional centrosome. Towards a better understanding of the molecular organization of the centrosome, we investigated the mechanism that regulates the size of the centrosome in the early C. elegans embryo. For this, we monitored fluorescently labeled centrosomes in living embryos and developed a suite of image analysis algorithms to quantify the centrosomes in the resulting 3D time-lapse images. In particular, we developed a novel algorithm involving a two-stage linking process for tracking entrosomes, which is a multi-object tracking task. This fully automated analysis pipeline enabled us to acquire time-resolved data of centrosome growth in a large number of embryos and could detect subtle phenotypes that were missed by previous assays based on manual image analysis. In a first set of experiments, we quantified centrosome size over development in wild-type embryos and made three essential observations. First, centrosome volume scales proportionately with cell volume. Second, beginning at the 4-cell stage, when cells are small, centrosome size plateaus during the cell cycle. Third, the total centrosome volume the embryo gives rise to in any one cell stage is approximately constant. Based on our observations, we propose a ‘limiting component’ model in which centrosome size is limited by the amounts of maternally derived centrosome components. In a second set of experiments, we tested our hypothesis by varying cell size, centrosome number and microtubule-mediated pulling forces. We then manipulated the amounts of several centrosomal proteins and found that the conserved centriolar and pericentriolar material protein SPD-2 is one such component that determines centrosome size

Towards digital representation of Drosophila embryogenesis

Animal development can be described as a complex, threedimensional cellular system that changes dramatically across time as a consequence of cell proliferation, differentiation and movement. Using Drosophila embryogenesis as a model we are developing molecular, imaging and image analysis techniques to record an entire developmental system at cellular resolution. We image Drosophila embryos expressing fluorescent markers in toto using Single Plane Illumination Microscopy (SPIM). SPIM offers the unique ability to image large living biological specimens in their entirety by acquiring image stacks from multiple angles while also providing high temporal resolution necessary for following dynamic developmental processes. We have developed an image analysis pipeline that efficiently processes long-term time-lapse multi-view SPIM data by aligning the different angles with high precision for a single time point and propagating the alignment parameters throughout the time series. The registered views are fused using an approach that evaluates the image content in each view