Summary Centriole duplication is coordinated such that a single round of duplication occurs during each cell cycle. Disruption of this synchrony causes defects including supernumerary centrosomes in cancer and perturbed ciliary signaling [1–5]. To preserve the normal number of centrioles, the level, localization, and post-translational modification of centriole proteins is regulated so that when centriole protein expression and/or activity is increased, centrioles self-assemble. Assembly is initiated by the formation of the cartwheel structure that comprises the base of centrioles [6–11]. SAS-6 constitutes the cartwheel and SAS-6 levels remain low until centriole assembly is initiated at S-phase onset [3, 12, 13]. Cep135 physically links to SAS-6 near the site of microtubule nucleation and binds to CPAP for triplet microtubule formation [13, 14]. We identify two distinct protein isoforms of Cep135 that antagonize each other to modulate centriole duplication: full length Cep135 (Cep135full) promotes new assembly while a short isoform, Cep135mini, represses it. Cep135mini represses centriole duplication by limiting the centriolar localization of Cep135full binding proteins (SAS-6 and CPAP) and the pericentriolar localization of γ-tubulin. The Cep135 isoforms exhibit distinct and complementary centrosomal localization during the cell cycle. Cep135mini protein decreases from centrosomes upon anaphase onset. We suggest that the decrease in Cep135mini from centrosomes promotes centriole assembly. The repression of centriole duplication by a splice isoform of a protein that normally promotes it serves as a novel mechanism to limit centriole duplication.

The centrosome, composed of two centrioles surrounded by pericentriolar material, is the cell’s central microtubule-organizing center. Centrosome duplication is coupled with the cell cycle such that centrosomes duplicate once in S phase. Loss of such coupling produces supernumerary centrosomes, a condition called centrosome amplification (CA). CA promotes cell invasion and chromosome instability, two hallmarks of cancer. We examined the contribution of centriole overduplication to CA and the consequences for genomic stability in breast cancer cells. CEP135, a centriole assembly protein, is dysregulated in some breast cancers. We previously identified a short isoform of CEP135, CEP135mini, that represses centriole duplication. Here, we show that the relative level of full-length CEP135 (CEP135full) to CEP135mini (the CEP135full:mini ratio) is increased in breast cancer cell lines with high CA. Inducing expression of CEP135full in breast cancer cells increases the frequency of CA, multipolar spindles, anaphase-lagging chromosomes, and micronuclei. Conversely, inducing expression of CEP135mini reduces centrosome number. The differential expression of the CEP135 isoforms in vivo is generated by alternative polyadenylation. Directed genetic mutations near the CEP135mini alternative polyadenylation signal reduces the CEP135full:mini ratio and decreases CA. We conclude that dysregulation of CEP135 isoforms promotes centriole overduplication and contributes to chromosome segregation errors in breast cancer cells.

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.

Microtubules (MTs) perform 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. The PCM proteins, including -tubulin and Pericentrin, promote MT nucleation and organization. Centrosomes in cancer cells are commonly numerically amplified. However, the question of how amplification of centrosomes alters the MT organization capacity is not well-studied. We developed a quantitative image-processing and machine learning-aided approach for the 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 analyzed the spatial distribution of PCM proteins, the growing ends of MTs and the total MT density in breast cancer cells. We find that breast cancer cells with supernumerary centrosomes not only have increased PCM protein but also exhibit expansion in PCM size. Moreover, centrosome amplified cells have a greater MT density and more growing MT ends near centrosomes than unamplified cells. The semi-automated approach developed here enables facile, unbiased and quantitative measurements of centrosome aberrations. We show that these aberrations increase MT nucleation and promote changes to MT density and the spatial distribution of MTs around amplified centrosomes.