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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.

Basal bodies (BBs) are microtubule-based organelles that template 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 ofTetrahymena thermophilato characterize Poc5 BB functions.TetrahymenaPoc5 (TtPoc5) uniquely incorporates into assembling BBs and is then removed from mature BBs prior to ciliogenesis. Complete genomic knockout ofTtPOC5leads to a significantly increased production of BBs yet a markedly reduced ciliary density, both of which are rescued by reintroduction of TtPoc5. A secondTetrahymena POC5-like gene,SFR1, is similarly implicated in modulating BB production. WhenTtPOC5andSFR1are co-deleted, cell viability is compromised, and levels of BB overproduction are 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 betweenhPOC5mutations and impaired cilia.SUMMARY STATEMENTLoss ofTetrahymena thermophilaPoc5 reveals an important role for this centrin-binding protein in basal body maturation, which also impacts basal body production and ciliogenesis.

Hepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifically bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modified with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core. Au102pMBA44 (Au102) was an ideal conjugate given its small 2.5 nm size and detectability in cryoEM. Au102 was bound directly to nanocapsid surface cysteines via direct ligand exchange. In addition, Au102 was functionalized with a maleimide linker (Au102_C6MI) for maleimide-thiol conjugation to nanocapsid cysteines. The AuNC-bound nanocapsid constructs were conjugated in various conditions. We found Au102_C6MI to bind nanocapsid more efficiently, while Au102 remained more soluble over time. Nanocapsids conjugated to Au102_C6MI were imaged in cryoEM for single particle reconstruction to localize AuNC position on the nanocapsid surface. We resolved five unique high intensity volumes that formed a ring-shaped density at the 5-fold symmetry center. This finding was further supported by independent rigid modeling.