Abstract
In animal cells, the centrosome is the main microtubule-organizing centre where microtubules are nucleated and anchored. The centriole subdistal appendages (SDAs) are the key structures that anchor microtubules in interphase cells, but the composition and assembly mechanisms of SDAs are not well understood. Here, we reveal that centrosome-binding proteins, coiled-coil domain containing (CCDC) 120 and CCDC68 are two novel SDA components required for hierarchical SDA assembly in human cells. CCDC120 is anchored to SDAs by ODF2 and recruits CEP170 and Ninein to the centrosome through different coiled-coil domains at its N terminus. CCDC68 is a CEP170-interacting protein that competes with CCDC120 in recruiting CEP170 to SDAs. Furthermore, CCDC120 and CCDC68 are required for centrosome microtubule anchoring. Our findings elucidate the molecular basis for centriole SDA hierarchical assembly and microtubule anchoring in human interphase cells.
Highlights
In animal cells, the centrosome is the main microtubule-organizing centre where microtubules are nucleated and anchored
To characterize Coiled-coil domain containing 120 (CCDC120), we generated rabbit and mouse polyclonal antibodies against its N terminus (1–200 amino acids, Supplementary Fig. 1a). Both antibodies recognized a band at B85 kDa by immunoblotting in U2OS cells, which was of similar size as that recognized by a commercial anti-CCDC120 antibody, and nearly abolished after transfection with short interfering RNA targeting CCDC120 (Supplementary Fig. 1b,c), a nonspecific band at B60 kDa was detected by the rabbit polyclonal antibody (Supplementary Fig. 1b)
In this study we identified two new subdistal appendages (SDAs) components, CCDC120 and Coiled-coil domain containing 68 (CCDC68), which cooperate with known SDA components ODF2, human Ninein (hNinein) and CEP170, and are required for hierarchical SDA assembly and microtubule anchoring in interphase cells (Fig. 8)
Summary
The centrosome is the main microtubule-organizing centre where microtubules are nucleated and anchored. Recent studies have shown that ODF2 controls DA and SDA assembly through different domains[21] Depleting these SDA proteins disturbs microtubule anchorage to the centrosomes in interphase cells[11,12,13,14,15]. Other proteins, including those comprising the dynein/dynactin complex (containing p50/dynamitin, p150Glued and p24), EB1, Kif3a and trichoplein (TCHP), which localize near SDAs or the subdistal ends of centrioles, function in anchoring microtubules to mother centrioles[22,23,24,25,26,27]. We identify CCDC120 and CCDC68 as SDA components and propose a hierarchical assembly model of SDAs by uncovering their roles in cooperating with known SDA components such as ODF2, Ninein and CEP170, as well as in microtubule anchoring in interphase cells
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