04-Nov-2022
Seminar Akira Noga (Ishikawa group, PSI)
Cryo-electron tomography identified novel filamentous structures composed of Bld10p/Cep135 that determine nine-fold symmetry of centriole
November 4th 10.00
Abstract:
Centrioles have a unique structure composed of nine triplet microtubules arranged rotationally. This nine-fold symmetrical structure functions as a core of cilia growth (called basal body) and a center of microtubule network for cell division (centriole), and is highly conserved among eukaryotes. Functional analysis revealed that SAS-6, a conserved centriolar protein, constitutes the cartwheel and that structure is essential to establish the nine-fold symmetry of the centriole. However, in bld12 mutant, a null mutant of SAS-6, 70% of centrioles still have nine triplet microtubules. Moreover, an engineered SAS-6 that in vitro self-assembles into a cartwheel-like structure with six-fold symmetry, when transformed into Chlamydomonas cell, produces centrioles with mostly nine triplet microtubules in vivo (Hilbert, Noga, et al., NCB 2016). These results indicate another SAS-6-independent mechanism to determine nine-fold symmetry. To elucidate the molecular entity of the SAS-6-independent mechanism we carried out the functional and structural analysis of Bld10p (homolog of mammalian Cep135) (Noga, et al., EMBO J., 2022). Immuno-electron microscopy analysis showed that Bld10p is localized linearly between pinheads, bulge structures connecting the cartwheel spokes and triplet microtubules. Cryo-electron tomography analysis identified novel filamentous structures that crosslink between adjacent pinheads. The positions of these structures coincide with localization of our immuno-gold label to Bld10p. These results suggest that Bld10p constitutes novel filamentous structures. Since these filamentous structures have fluctuation, we directly observed these structures by exploiting Volta Phase Plate. We observed centriole structures in strains expressing truncated Bld10p in bld10bld12, a double mutant that lacks both Bld10p and SAS-6. In these strains, the numbers of triplets are reduced and shorter crosslink structures were observed. These results indicate that Bld10p determines the nine-fold symmetrical structure by constituting the crosslinks in a manner independent of the cartwheel. In addition to the filamentous structures composed of Bld10p, we are analyzing the other structures in centrioles and cilia by cryo-electron tomography. I will show these preliminary results.