Many important processes in cell biology require the association of distinct cellular components. It is not yet clear whether such associations can be accomplished by diffusion or would require active motility, and elucidating the role of diffusion in cellular assembly processes is a current challenge for physical cell biology. Here we investigate the potential for association of centrin-containing foci or granules with mother centrioles during the process of centriole assembly, as a model system for investigating the role of diffusion in organelle-scale assembly.
Centrioles are cylindrical microtubule-based structures that form the core of the centrosome, the main microtubule organizing center of the cell (Debec et al., 2010
). The apparent duplication of centrioles, in which new centrioles form adjacent to pre-existing ones (Dippell, 1968
; Kuriyama and Borisy, 1981
), has long been one of the most fascinating processes in cell biology. How can one structure give rise to a copy of itself? How much information does the mother centriole propagate to the daughter? It was once thought that the role of the mother might be obligatory in forming new centrioles, however numerous examples were discovered in which centrioles form de novo (e.g. Mizukami and Gall, 1966
). Centrioles can even form de novo in cells that normally undergo centriole duplication provided the mother centrioles are removed (Marshall et al., 2001
; Khodjakov et al., 2002
; Uetake et al., 2007
), suggesting that cells have two pathways for centriole assembly: a “templated” pathway catalyzed by the mother centriole, and a de novo pathway which is normally inhibited by the presence of the mother centriole (Loncarek and Khodjakov, 2009
One important molecule in centriole assembly is the EF hand protein centrin (Salisbury et al., 2002
; Koblenz et al., 2003
; Stemm-Wolf et al., 2005
; Pearson et al., 2009
). In many cell types, centrin is present in the form of cytoplasmic foci that have been variously termed granules, satellites, nucleus associated foci, or pre-centrioles (Baron et al., 1991
; La Terra et al., 2005
; Prosser et al., 2009
; Collins et al., 2010
). These terms are likely to encompass several distinct entities whose common feature is that they contain centrin. In no case is the precise function of these centrin-containing foci clearly understood.
Khodjakov and co-workers (La Terra et al, 2005
) found that vertebrate cells undergoing de novo centriole assembly contain multiple centrin foci, some of which appear to directly develop into centrioles. La Terra et al named these foci “precentrioles” and proposed that they might represent inherently unstable centriole precursor forms, which are then stabilized by the mother centriole after being captured at a defined docking site on the mother surface. If a mother centriole is missing, then one or more of the pre-centrioles may spontaneously develop into a mature centriole. This ingenious model represents a radical departure from the usual view that mothers actively nucleate formation of the new centriole by recruiting individual protein building blocks, and instead implies that the mother centriole provides a stabilizing function for partially formed precursors. Khodjakov has termed the original nucleation-based model and the new capture-based model “birth” and “adoption”, respectively. Although the adoption model was first proposed based on initial observation of pre-centrioles in cells undergoing de novo assembly, pre-centrioles have also been observed in cells undergoing normal centriole duplication (La Terra et al., 2005
) suggesting that they might indeed be a common precursor for centriole assembly in both the de novo and templated pathways. Such an adoption model apparently conflicts with evidence that individual centriole precursor proteins such as SAS-6 and Cep135 assemble step-wise on the mother centriole in response to Plk4 activity (Kleylein-Sohn et al., 2007
), but since centrin probably plays a role downstream of SAS-6 incorporation, the possibility that centrin foci represent a partially assembled centriole module remains open. Centrin-containing foci have been shown to contain centriole and centrosome proteins including gamma tubulin, PCM-1, and Cep135 (Prosser et a., 2009
; Collins et al., 2010
), further suggesting they could play a role in centriole assembly.
In order for these centrin foci or granules to play a direct role in building a daughter centriole, they would have to be able to reach the mother in a reasonable time frame before the next cell division. The simplest way to do this would be via diffusion, but one could also imagine a role for directed motility.
Here, we measured the motion of centrin-containing foci in human U2OS cells and found that it is indeed apparently diffusive in nature, with the foci undergoing a random walk through the cell. We determined the effective diffusion constant and used this to estimate the time scale for diffusion to capture of these foci by a docking site on mother centrioles. Our calculations indicate that there is not sufficient time for the apparent diffusive motion to allow capture by the mothers, suggesting that either the centrin foci do not directly incorporate into the growing daughter centriole, or that an additional active, directed motion, not observed in our images, is required to bring the centrin foci to the mother.