ABSTRACT Microtubules (MTs) promote important cellular functions including migration, intracellular trafficking, and chromosome segregation. The centrosome, comprised of two centrioles surrounded by the pericentriolar material (PCM), is the cell's central MT-organizing center. Centrosomes in cancer cells are commonly numerically amplified. However, the question of how the amplification of centrosomes alters MT organization capacity is not well studied. We developed a quantitative image-processing and machine learning-aided approach for the semi-automated analysis of MT organization. We designed a convolutional neural network-based approach for detecting centrosomes, and an automated pipeline for analyzing MT organization around centrosomes, encapsulated in a semi-automatic graphical tool. Using this tool, we find that breast cancer cells with supernumerary centrosomes not only have more PCM protein per centrosome, which gradually increases with increasing centriole numbers, but also exhibit expansion in PCM size. Furthermore, cells with amplified centrosomes have more growing MT ends, higher MT density and altered spatial distribution of MTs around amplified centrosomes. Thus, the semi-automated approach developed here enables rapid and quantitative analyses revealing important facets of centrosomal aberrations.

ABSTRACT Basal bodies (BBs) are microtubule-based organelles that act as a template for and stabilize cilia at the cell surface. Centrins ubiquitously associate with BBs and function in BB assembly, maturation and stability. Human POC5 (hPOC5) is a highly conserved centrin-binding protein that binds centrins through Sfi1p-like repeats and is required for building full-length, mature centrioles. Here, we use the BB-rich cytoskeleton of Tetrahymena thermophila to characterize Poc5 BB functions. Tetrahymena Poc5 (TtPoc5) uniquely incorporates into assembling BBs and is then removed from mature BBs prior to ciliogenesis. Complete genomic knockout of TtPOC5 leads to a significantly increased production of BBs, yet a markedly reduced ciliary density, both of which are rescued by reintroduction of TtPoc5. A second Tetrahymena POC5 -like gene, SFR1 , is similarly implicated in modulating BB production. When TtPOC5 and SFR1 are co-deleted, cell viability is compromised and BB overproduction is exacerbated. Overproduced BBs display defective transition zone formation and a diminished capacity for ciliogenesis. This study uncovers a requirement for Poc5 in building mature BBs, providing a possible functional link between hPOC5 mutations and impaired cilia. This article has an associated First Person interview with the first author of the paper .

Summary Directed fluid flow, which is achieved by the coordinated beating of motile cilia, is required for processes as diverse as cellular swimming, developmental patterning and mucus clearance. Cilia are nucleated, anchored and aligned at the plasma membrane by basal bodies, which are cylindrical microtubule-based structures with ninefold radial symmetry. In the unicellular ciliate Tetrahymena thermophila , two centrin family members associated with the basal body are important for both basal body organization and stabilization. We have identified a family of 13 proteins in Tetrahymena that contain centrin-binding repeats related to those identified in the Saccharomyces cerevisiae Sfi1 protein. We have named these proteins Sfr1–Sfr13 (for Sfi1-repeat). Nine of the Sfr proteins localize in unique polarized patterns surrounding the basal body, suggesting non-identical roles in basal body organization and association with basal body accessory structures. Furthermore, the Sfr proteins are found in distinct basal body populations in Tetrahymena cells, indicating that they are responsive to particular developmental programs. A complete genetic deletion of one of the family members, Sfr13, causes unstable basal bodies and defects in daughter basal body separation from the mother, phenotypes also observed with centrin disruption. It is likely that the other Sfr family members are involved in distinct centrin functions, providing specificity to the tasks that centrins perform at basal bodies.