Basal body duplication in Chlamydomonas
Eileen T. O'Toole1 and Susan K. Dutcher2
1) Boulder Laboratory for 3-D Electron Microscopy of Cells, Department of Molecular, Cellular, and Developmental Biology University of Colorado, Boulder, CO 80309-0347
2) Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110
Basal body/centriole duplication is a key event in eukaryotic cells and they are needed for a variety of cellular events. We used Chlamydomonas to investigate the pathway of basal body duplication. Cells were synchronized by light/dark cycles and prepared for electron tomography using high pressure freezing and freeze substitution. During interphase, Chlamydomonas has two full-length basal bodies and two probasal bodies. In preprophase, the probasal bodies elongate to a mean length of ~378 nm, which is shorter than the mature basal bodies. The first duplication intermediate is detected at this stage and consists of a nine-spoked pinwheel structure that is positioned orthogonally at the face of triplets 7 and 8 of the mature basal body. At prophase, the basal bodies separate and migrate as pairs to opposite sides of the nucleus. Rootlet microtubules (MT) remain associated with the basal bodies and there is a noticeable increase in the number of cytoplasmic MTs. Atmetaphase, the nascent basal bodies consist of a ring of 9 singlet MTs that are at right angles to triplet 8 of the mother basal bodies. Surprisingly, the nascent basal body microtubules are much longer (with a mean length of ~210 nm) than the probasal bodies at interphase. The pinwheel structure was also significantly elongated in the forming probasal bodies. The length of the probasal bodies at anaphase and telophase/cytokinesis shortens (~173 nm) and doublet and triplet MT blades are detected at the distal ends of the daughter basal bodies. The formation of the doublet and triplet microtubules at the distal end suggests a new model for the phenotype of the bld2-5 mutant that is defective at the proximal but not at the distal end of the basal body. Unexpectedly, the reconstruction of the nucleus and the basal body complex suggests that the basal bodies do not appear to serve as the centrosome for the mitotic spindle, but instead remains closely associated with the plasma membrane.
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