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

Visualization and Analysis of 3D Microscopic Images

In a wide range of biological studies, it is highly desirable to visualize and analyze three-dimensional (3D) microscopic images. In this primer, we first introduce several major methods for visualizing typical 3D images and related multi-scale, multi-time-point, multi-color data sets. Then, we discuss three key categories of image analysis tasks, namely segmentation, registration, and annotation. We demonstrate how to pipeline these visualization and analysis modules using examples of profiling the single-cell gene-expression of C. elegans and constructing a map of stereotyped neurite tracts in a fruit fly brain

Aerospace medicine and biology: A continuing bibliography with indexes (supplement 335)

This bibliography lists 143 reports, articles and other documents introduced into the NASA Scientific and Technical Information System during March, 1990. Subject coverage includes: aerospace medicine and psychology, life support systems and controlled environments, safety equipment, exobiology and extraterrestrial life, and flight crew behavior and performance

Tracking-by-Assignment as a Probabilistic Graphical Model with Applications in Developmental Biology

This thesis presents a novel approach for tracking a varying number of divisible objects with similar appearance in the presence of a non-negligible number of false positive detections (more than 10%). It is applied to the reconstruction of cell lineages in developing zebrafish and fruit fly embryos from 3d time-lapse record- ings. The model takes the form of a chain graph—a mixed directed-undirected probabilistic graphical model—and a tracking is obtained simultaneously over all time slices from the maximum a-posteriori configuration. The tracking model is used as the second step in a two-step pipeline to produce digital embryos—maps of cell nuclei in an embryo and their ancestral fate; the first step being the segmentation of the fluorescently-stained cell nuclei in light sheet microscopy images. The pipeline is implemented as a software with an intuitive graphical user interface. It is the first freely available program of its kind and makes the presented methods accessible to a broad audience of users from the life sciences

Spindle organization in three dimensions

During cell division, chromosome segregation takes place on bipolar, microtubulebased spindles. Here, C. elegans is used to analyze spindle organization under both mitotic and meiotic conditions. First, the role of SAS-4 in organizing centrosome structure was analyzed. Partial depletion of SAS-4 in early embryos results in structurally defective centrioles. The study of this protein sheds light on the poorly understood role of the centrioles in dictating centrosome size. Second, the ultrastructure of wild-type mitotic spindle components was analyzed by electron tomography. This 3-D analysis reveals morphologically distinct microtubule end morphologies in the mitotic spindle pole. These results have structural implications for models of microtubule interactions with centrosomes Third, spindle assembly was studied in female meiosis. Specifically, the role of the microtubule severing complex katanin in spindle organization was analyzed. Electron tomography reveals fragmentation of spindle microtubules and suggests a novel katanin-dependent mechanism of meiotic spindle assembly. In this model, relatively long microtubules seen near the meiotic chromatin are converted into numerous short fragments, thus increasing the total number of polymers in an acentrosomal environment. Taken together, these results provide novel insights into the three-dimensional organization of microtubules during spindle assembly.Die Segregation der Chromosomen während der Zellteilung wird duch bipolare, von Microtubuli-aufgebauten Spindlen gewährleistet. In der vorliegenden Arbeit wird C. elegans zur Analyse der Spindelorganisation unter mitotischen und meiotischen Bedingungen herangezogen. Erstens wird die Rolle von SAS-4 in der Organisation von Zentrosomen untersucht. Die partielle Depletierung von SAS-4 in frühen Embryonen führt zu strukturell defekten Zentriolen und wirft somit Licht auf die wenig verstandene Rolle der Zentriolen in der Bestimmung der Zentrosomengröße. Zweitens wird die Ultrastruktur der mitotischen Spindelkomponenten im Wildtyp durch Elektronentomographie untersucht. Diese 3-D-Analyse zeigt, dass im mitotischen Spindlepol unterschiedliche Morphologien der Mikrotubulienden zu finden sind. Diese Ergebnisse haben strukturelle Implikationen für Modelle der Mikrotubuli-Zentrosomen-Interaktionen. Drittens wird der Aufbau der Spindel in der weiblichen Meiose, speziell die Rolle des Mikrotubuli-schneidenden Kataninkomplexes in der Spindelorganisation, untersucht. Die Elektronentomographie zeigt hier eine Fragmentierung der Spindelmikrotubuli. Basierend auf diesem Ergebnis wird ein neues Katanin-abhängiges Modell der Formierung der Meiosespindel entwickelt, in dem relativ lange Microtubuli in Nähe des meiotischen Chromatins in zahlreiche kurze Mikrotubuli “zerschnitten” werden. Dies erhöht die Anzahl der verfügbaren Polymere in dieser azentrosomalen Situation. Zusammenfassend bringen diese Ergebnisse neue Einsichten in die räumliche Organisation der Mikrotubuli während des Spindelaufbaus