Formation of a single new centriole from a pre-existing centriole is strictly controlled to maintain correct centrosome number and spindle polarity in cells. However, the mechanisms that govern this process are incompletely understood. Here, using several human cell lines, immunofluorescence and structured illumination microscopy methods, and ubiquitination assays, we show that the E3 ubiquitin ligase F-box and WD repeat domain–containing 7 (FBXW7), a subunit of the SCF ubiquitin ligase, down-regulates spindle assembly 6 homolog (HsSAS-6), a key protein required for procentriole cartwheel assembly, and thereby regulates centriole duplication. We found that FBXW7 abrogation stabilizes HsSAS-6 and increases its recruitment to the mother centriole at multiple sites, leading to supernumerary centrioles. Ultrastructural analyses revealed that FBXW7 is broadly localized on the mother centriole and that its presence is reduced at the site where the HsSAS-6–containing procentriole is formed. This observation suggested that FBXW7 restricts procentriole assembly to a specific site to generate a single new centriole. In contrast, during HsSAS-6 overexpression, FBXW7 strongly associated with HsSAS-6 at the centriole. We also found that SCFFBXW7 interacts with HsSAS-6 and targets it for ubiquitin-mediated degradation. Further, we identified putative phosphodegron sites in HsSAS-6, whose substitutions rendered it insensitive to FBXW7-mediated degradation and control of centriole number. In summary, SCFFBXW7 targets HsSAS-6 for degradation and thereby controls centriole biogenesis by restraining HsSAS-6 recruitment to the mother centriole, a molecular mechanism that controls supernumerary centrioles/centrosomes and the maintenance of bipolar spindles.
Carrier-free delivery of therapeutically relevant small molecules and functional oligonucleotides is extremely challenging and is one of the major hurdles in cancer therapy. Herein, we report a non-covalent approach for...
There is huge demand for developing guests that bind β‐CD and can conjugate multiple cargos for cellular delivery. We synthesized trioxaadamantane derivatives, which can conjugate up to three cargos per guest. 1H NMR titration and isothermal titration calorimetry revealed these guests form 1 : 1 inclusion complexes with β‐CD with association constants in the order of 103 M−1. Co‐crystallization of β‐CD with guests yielded crystals of their 1 : 1 inclusion complexes as determined by single‐crystal X‐ray diffraction. In all cases, trioxaadamantane core is buried within the hydrophobic cavity of β‐CD and three hydroxyl groups are exposed outside. We established biocompatibility using representative candidate G4 and its inclusion complex with β‐CD (β‐CD⊂G4), by MTT assay using HeLa cells. We incubated HeLa cells with rhodamine‐conjugated G4 and established cellular cargo delivery using confocal laser scanning microscopy (CLSM) and fluorescence‐activated cell sorting (FACS) analysis. For functional assay, we incubated HeLa cells with β‐CD‐inclusion complexes of G4‐derived prodrugs G6 and G7, containing one and three units of the antitumor drug (S)‐(+)‐camptothecin, respectively. Cells incubated with β‐CD⊂G7 displayed the highest internalization and uniform distribution of camptothecin. β‐CD⊂G7 showed higher cytotoxicity than G7, camptothecin, G6 and β‐CD⊂G6, affirming the efficiency of adamantoid derivatives in high‐density loading and cargo delivery.
There is huge demand for developing guests that bind β‐CD and can conjugate multiple cargos for cellular delivery. We synthesized trioxaadamantane derivatives, which can conjugate up to three cargos per guest. 1H NMR titration and isothermal titration calorimetry revealed these guests form 1 : 1 inclusion complexes with β‐CD with association constants in the order of 103 M−1. Co‐crystallization of β‐CD with guests yielded crystals of their 1 : 1 inclusion complexes as determined by single‐crystal X‐ray diffraction. In all cases, trioxaadamantane core is buried within the hydrophobic cavity of β‐CD and three hydroxyl groups are exposed outside. We established biocompatibility using representative candidate G4 and its inclusion complex with β‐CD (β‐CD⊂G4), by MTT assay using HeLa cells. We incubated HeLa cells with rhodamine‐conjugated G4 and established cellular cargo delivery using confocal laser scanning microscopy (CLSM) and fluorescence‐activated cell sorting (FACS) analysis. For functional assay, we incubated HeLa cells with β‐CD‐inclusion complexes of G4‐derived prodrugs G6 and G7, containing one and three units of the antitumor drug (S)‐(+)‐camptothecin, respectively. Cells incubated with β‐CD⊂G7 displayed the highest internalization and uniform distribution of camptothecin. β‐CD⊂G7 showed higher cytotoxicity than G7, camptothecin, G6 and β‐CD⊂G6, affirming the efficiency of adamantoid derivatives in high‐density loading and cargo delivery.
This work examines the roles played by wall thickness in determining the plasmonic properties of gold-silver (Ag-Au) nanocages. Ag-Au cages with different wall thicknesses, but the same void or outer...
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