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Quantification of the plant endoplasmic reticulum

By Abdnacer Bouchekhima


One of the challenges of quantitative approaches to biological sciences is the lack of understanding of the interplay between form and function. Each cell is full of complex-shaped objects, which moreover change their form over time. To address this issue, we exploit recent advances in confocal microscopy, by using data collected from a series of optical sections taken at short regular intervals along the optical axis to reconstruct the Endoplasmic Reticulum (ER) in 3D, obtain its skeleton, then associate to each of its edges key geometric and dynamic characteristics obtained from the original filled in ER specimen. These properties include the total length, surface area, and volume of the ER specimen, as well as the length surface area, and volume of each of its branches. In a view to benefit from the well established graph theory algorithms, we abstract the obtained skeleton by a mathematical entity that is a graph. We achieve this by replacing the inner points in each edge in the skeleton by the line segment connecting its end points. We then attach to this graph the ER geometric properties as weights, allowing therefore a more precise quantitative characterisation, by thinning the filled in ER to its essential features. The graph plays a major role in this study and is the final and most abstract quantification of the ER. One of its advantages is that it serves as a geometric invariant, both in static and dynamic samples. Moreover, graph theoretic features, such as the number of vertices and their degrees, and the number of edges and their lengths are robust against different kinds of small perturbations. We propose a methodology to associate parameters such as surface areas and volumes to its individual edges and monitor their variations with time. One of the main contributions of this thesis is the use of the skeleton of the ER to analyse the trajectories of moving junctions using confocal digital videos. We report that the ER could be modeled by a network of connected cylinders (0.87μm±0.36 in diameter) with a majority of 3-way junctions. The average length, surface area and volume of an ER branch are found to be 2.78±2.04μm, 7.53±5.59μm2 and 1.81±1.86μm3 respectively. Using the analysis of variance technique we found that there are no significant differences in four different locations across the cell at 0.05 significance level. The apparent movement of the junctions in the plant ER consists of different types, namely: (a) the extension and shrinkage of tubules, and (b) the closing and opening of loops. The average velocity of a junction is found to be 0.25μm/sec±0.23 and lies in the range 0 to 1.7μm/sec which matches the reported actin filament range

Topics: QH301
OAI identifier: oai:wrap.warwick.ac.uk:2742

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