Abstract
We have used cryo Electron Tomography, proteomics and immunolabeling to study centrosomes isolated from the young lamb thymus, an efficient source of quiescent differentiated cells. We compared the proteome of thymocyte centrosomes to data published for KE37 cells, focusing on proteins associated with centriole disengagement and centrosome separation. The data obtained enhances our understanding of the protein system joining the centrioles, a system comprised of a branched network of fibers linked to an apparently amorphous density that was partially characterized here. A number of proteins were localized to the amorphous density by immunolabeling (C-NAP1, cohesin SMC1, condensin SMC4 and NCAPD2), yet not DNA. In conjuction, these data not only extend our understanding of centrosomes but they will help refine the model that focus on the protein system associated with the centriolar junction.
Highlights
The centrosome is the main microtubule-organizing center (MTOC) in higher animals[1], serving as a pole for the mitotic spindle and a hub to regulate mitosis[2]
The centrioles are bound together by a network of proteins, the degree of structure and organization of which is poorly understood[17]. These elements are surrounded by a semi-dense proteinaceous cloud called the pericentriolar material (PCM), made up of layers that vary in their protein composition[18,19,20]
The whole process of duplication requires two types of linkages between the centrioles[28,29,30,31,32,33,34,35]: one between two mature centrioles that is loose and extends over a distance, the association established in quiescent cells; and the other between a mature and an immature centriole that is tight and rigid, maintaining the orthogonal organization inherited from the centriole/procentriole pair
Summary
The centrosome is the main microtubule-organizing center (MTOC) in higher animals[1], serving as a pole for the mitotic spindle and a hub to regulate mitosis[2]. Commencing with the autoassembly of a transient SAS6 structure, shaped as a 9-fold symmetric cartwheel[25,26], the procentriole elongates[2] and each centrosome, formed by a mature centriole and a new immature centriole, subsequently migrates to the poles of the mitotic spindle[17]. The whole process of duplication requires two types of linkages between the centrioles[28,29,30,31,32,33,34,35]: one between two mature centrioles that is loose and extends over a distance, the association established in quiescent cells; and the other between a mature and an immature centriole that is tight and rigid, maintaining the orthogonal organization inherited from the centriole/procentriole pair. The fiber-forming proteins LRRC4539 and CEP6840–42 are thought to contribute to this system, attaching either directly to C-NAP1 (LRRC45) or through a centlein interface (CEP68)[41]
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