3D reconstruction of cellular microtubule networks using time- resolved live cell imaging and serial electron tomography

Microtubules are part of the cell cytoskeleton and play crucial roles in cellular processes like intracellular transport and cell division. During cell division, the centrosome, found in most eukaryotes and also know as the microtubule organizing center, duplicates and the mitotic spindle is organized by microtubules which are nucleated and polymerized from tubulin at the two centrosomes. This process can be followed by light microscopy in live cells using fluorescently labelled tubulin. Due their small diameter (~25 nm) and high density at the centrosome, microtubules cannot be resolved by light microscopy and no precise quantitative analysis of tubulin polymerized in microtubules, on the ultrastructure of the network of tubes and their dependance on cell division stage, cell size, cell type can be obtained. To model the 3D organization of centrosomal microtubules in C. elegans embryo mitosis, we have chosen to use electron tomography on serial300nm-thick, sections, followed by segmentation of microtubules in the serial tomography stacks. In the perspective of doing a largescale statistical analysis of multiple samples, we have developed software in the ZIBAmira (ZIB, Germany)/Amira (FEI, The Netherlands) framework for automated tracing of microtubules centerlines in electron tomograms based on template matching [1]. Then, we have developed a method for automated stitching of microtubules across serial sections, using the previously segmented network of lines to register consecutive sections [2]. Finally we have applied this image processing workflow – from serial electron tomography to complete microtubule network reconstructions to cells from C. elegans embryo after live cell imaging and cell division staging.