This article explores why elevated pericentrin in trisomy 21 negatively impacts primary cilia. It is found that elevated pericentrin produces more pericentrosomal puncta that associate with and increase microtubules. Elevated pericentrin and microtubules mislocalize centriolar satellites in a pericentrosomal crowd.

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.

Trisomy 21, the genetic cause of Down syndrome, disrupts primary cilia formation and function, in part through elevated Pericentrin, a centrosome protein encoded on chromosome 21. Yet how trisomy 21 and elevated Pericentrin disrupt cilia-related molecules and pathways, and the in vivo phenotypic relevance remain unclear. Utilizing ciliogenesis time course experiments combined with light microscopy and electron tomography, we reveal that chromosome 21 polyploidy elevates Pericentrin and microtubules away from the centrosome that corral MyosinVA and EHD1, delaying ciliary membrane delivery and mother centriole uncapping essential for ciliogenesis. If given enough time, trisomy 21 cells eventually ciliate, but these ciliated cells demonstrate persistent trafficking defects that reduce transition zone protein localization and decrease sonic hedgehog signaling in direct anticorrelation with Pericentrin levels. Consistent with cultured trisomy 21 cells, a mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers at P4. Our work reveals that elevated Pericentrin from trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding mechanism results in signaling deficiencies consistent with the neurological phenotypes found in individuals with Down syndrome.

The primary cilium is a critical sensory organelle that is built of axonemal microtubules ensheathed by a ciliary membrane. In polarized epithelial cells, primary cilia reside on the apical surface and must extend these microtubules directly into the extracellular space and remain a stable structure. However, the factors regulating cross-talk between ciliation and cell polarization, as well as, axonemal microtubule growth and stabilization in polarized epithelia are not fully understood. In this study, we find TTLL12, a previously uncharacterized member of the Tubulin Tyrosine Ligase-Like (TTLL) family, localizes to the base of primary cilia and is required for cilia formation in polarized renal epithelial cells. We also show that TTLL12 directly binds to the tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications (PTMs). While all other TTLLs catalyze the addition of glutamate or glycine to microtubule C-terminal tails, TTLL12 uniquely affects tubulin PTMs by promoting both microtubule lysine acetylation and arginine methylation. Together, this work identifies a novel microtubule regulator and provides insight into the requirements for apical extracellular axoneme formation.

Trisomy 21, the cause of Down syndrome, causes a 0.5-fold protein increase of the chromosome 21-resident gene Pericentrin (PCNT) and reduces primary cilia formation and signaling. Here we investigate the mechanisms by which PCNT imbalances disrupt cilia. Using isogenic RPE-1 cells with increased chromosome 21 dosage, we find PCNT protein accumulates around the centrosome as a pericentrosomal cluster of enlarged cytoplasmic puncta that localize along and at MT ends. Cytoplasmic PCNT puncta impact the intracellular MT trafficking network required for primary cilia, as the PCNT puncta sequester cargo peripheral to centrosomes in what we call pericentrosomal crowding. The centriolar satellite proteins, PCM1, CEP131 and CEP290, important for ciliogenesis, accumulate at sites of enlarged PCNT puncta in trisomy 21 cells. Reducing PCNT when chromosome 21 ploidy is elevated is sufficient to decrease PCNT puncta, reestablish a normal density of MTs around the centrosome, restore ciliogenesis to wild type levels and decrease pericentrosomal crowding. A transient reduction in MTs also decreases pericentrosomal crowding and partially rescues ciliogenesis in trisomy 21 cells, indicating that increased PCNT leads to defects in the microtubule network deleterious to normal centriolar satellite distribution. We propose that chromosome 21 aneuploidy disrupts MT-dependent intracellular trafficking required for primary cilia.