Forum 05/28/2009

Electron Cryo-Microscopy Study of the Microtubule Organizing Centre in Model Organisms

Sam Li – Department of Biochemistry & Biophysics, University of California San Francisco

The microtubule-organizing centre (MTOC) is an organelle in most eukaryotic cells where microtubules nucleate. It plays important roles in diverse cellular processes, including mitosis, meiosis, cell polarity, organelle positioning, and cellular trafficking. We have been using electron cryo-tomography to study the molecular assembly of the MTOC in several model organisms.  First, we have been studying the spindle pole body (SPB), the MTOC in budding yeast. Tomographic reconstructed SPB in different cell-cycle stages show that this is a highly dynamic complex embedded in the nuclear envelope. The SPB is composed of multiple layers in a well-ordered assembly. In particular, multiple copies of Spc42p, one of the major SPB components, arrange in a crystalline lattice, providing a template during initiation of the SPB assembly.  The nuclear microtubules attached to the SPB are obvious in the reconstruction. They are closed at the minus ends and open at the plus ends, indicating their characteristic dynamic instability. Meanwhile, we are also using electron cryo-tomography to study the basal bodies in Chlamydomonas reinhardtii which is homologous to the human centriole. Our results show the basal body is a rigid and well-ordered cylindrical structure formed by nine microtubule triplets. There is a distinct position where the microtubule triplets transition to doublets. The lumen of the basal body is filled with filamentous structures. Overall, our electron tomographic reconstructions have revealed the assembly the MTOCs, both the spindle pole body and the basal body, in unprecedented detail. We are currently striving to improve the electron tomography resolution so that the molecular architecture of these MTOCs may be realized in the near future.

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