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. 2019 Mar 11;10(1):1151.
doi: 10.1038/s41467-019-08838-2.

Molecular Architecture of a Cylindrical Self-Assembly at Human Centrosomes

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Free PMC article

Molecular Architecture of a Cylindrical Self-Assembly at Human Centrosomes

Tae-Sung Kim et al. Nat Commun. .
Free PMC article

Abstract

The cell is constructed by higher-order structures and organelles through complex interactions among distinct structural constituents. The centrosome is a membraneless organelle composed of two microtubule-derived structures called centrioles and an amorphous mass of pericentriolar material. Super-resolution microscopic analyses in various organisms revealed that diverse pericentriolar material proteins are concentrically localized around a centriole in a highly organized manner. However, the molecular nature underlying these organizations remains unknown. Here we show that two human pericentriolar material scaffolds, Cep63 and Cep152, cooperatively generate a heterotetrameric α-helical bundle that functions in conjunction with its neighboring hydrophobic motifs to self-assemble into a higher-order cylindrical architecture capable of recruiting downstream components, including Plk4, a key regulator for centriole duplication. Mutations disrupting the self-assembly abrogate Plk4-mediated centriole duplication. Because pericentriolar material organization is evolutionarily conserved, this work may offer a paradigm for investigating the assembly and function of centrosomal scaffolds in various organisms.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Mutual dependency of Cep63 and Cep152 for interaction and subcellular localization. ac Immunoprecipitation (IP) and immunoblotting analyses using transfected HEK293 cells. FL, full-length; ∆P1b, Cep63 lacking residues 490–541; ∆M4e and ∆M4d, Cep152 lacking residues 1257–1272 and 1257–1295, respectively. Note that IP for b was carried out using a mixture of lysates from cells transfected separately with each of the indicated constructs and that the provision of HA-Cep63 greatly increased the level of coprecipitated HA-Cep152 (see also the Supplementary Fig. 1i legend). GFP-Cep152 M4f, which fails to bind to Cep63 (Supplementary Fig. 1b), serves as a control in c. CC, coiled-coil domains predicted by COILS server; numbers, relative signal intensities. Arrows indicate input signals. d Schematic diagram showing the regions for the heterotetrameric Cep63–Cep152 and homomeric Cep63–Cep63 or Cep152–Cep152 interactions. e Confocal images showing immunostained transfected HEK293 cells. Dotted boxes, areas of enlargement. Bar, 10 μm. Quantified data obtained from three independent experiments are provided in Supplementary Fig. 1l
Fig. 2
Fig. 2
Cooperative formation of a dynamic cylindrical self-assembly by Cep63 and Cep152. a, b Sedimentation velocity analyses with Cep63 P1•Cep152 M4d or its respective P1(4A)•M4d(5A) mutant complex, using the complexes shown in Supplementary Fig. 2a, left. A high-molecular weight (MW) species (arrow) with rapid sedimentation velocity (~4S) detected at higher concentrations may likely represent a dimer of the ~2.74S heterotetramer (i.e., heterooctamer). Weighted-average sedimentation coefficients obtained from the c(s) profiles are presented in b for the Cep63 P1•Cep152 M4d (red) and P1(4A)•M4d(5A) mutant (blue) complexes as a function of concentration. c Three-dimensional structured-illumination microscopy (3D-SIM (top) and surface rendering (bottom) of the in vitro self-assembly generated by the mCherry-Cep63 P1•mGFP-Cep152 M4d complex. Bar, 2 μm; bar for rendered image, 0.5 μm. df The dimension of the self-assemblies (d, e) and the diameter difference for mCherry-Cep63 P1 and mGFP-Cep152 M4d fluorescence (f) were determined from a total of 429 cylindrical assemblies obtained from three independent experiments. The Zeiss Zen software allowed to determine the inter-signal distance of up to 33nm/pixel (see Methods for details). A sharp drop in the number of small assemblies (<100 nm in diameter) in d is due to the resolution limit of SIM. The interpolated line of best fit is shown in e. Bars, s.d. g Summary of diameter differences between mGFP and mCherry fluorescence for the indicated cylindrical assemblies. Center line, median; cross, mean; box limits, upper and lower quartiles; whiskers, maximum or minimum of the data. **P < 0.01; ****P < 0.0001 (unpaired two-tailed t test). h, i SIM-total internal reflection fluorescence (TIRF) time lapse of the self-assembling process of the mCherry-Cep63 P1•mGFP-Cep152 M4d complex in vitro (h) and the diameters of the assemblies as a function of time (i). Data are representative of a total of eight independent time-lapse movies analyzed. Bar, 1 μm. j Fluorescent recovery after photobleaching (FRAP) analysis for the mCherry-Cep63 P1•mGFP-Cep152 M4d self-assemblies in vitro. Representative data from a total of 14 independent FRAP experiments are shown. Bar, 1 μm. mGFP monomeric green fluorescent protein
Fig. 3
Fig. 3
Structural basis for forming the Cep63•Cep152 complex and its biological function. a Transmission electron micrograph showing the Cep63(220–541)•Cep152(1140–1295) complex after negative staining. Bar, 30 nm. b Size-exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis showing a stable heterotetrameric Cep63(220–541)•Cep152(1140–1295) complex. Bovine serum albumin (BSA) serves as a control. c Crystal structure of the Cep63 P1b5•Cep152 M4e15 complex forming an antiparallel four-helix bundle. The N and C termini of each protein are indicated. d Pairwise M4e15–M4e15 interactions (top) and P1b5 (A and C chains) interactions with M4e15 (B and D chains) (middle and bottom). The two P1b5 chains do not directly interact with each other. Side chains involved in the interaction are shown in the same color (i.e., green for M4e15 and red for Plb5) as the helices to which they belong. Side chains colored in yellow and gray indicate the residues mutated to 6A and 8A in Cep152 and Cep63, respectively. eg Immunoprecipitation (IP) and immunoblotting analyses using transfected HEK293 cells. The schematic diagram illustrates the region where marked mutations are introduced. Gray boxes, hydrophobic motifs; numbers, relative signal intensities. Note that the 6A mutations greatly impair not only the central M4e–M4e interaction but also the P1–M4e interaction (g). hk Analysis of U2OS cells stably expressing the indicated constructs after silencing for control luciferase (siGL), Cep63 (siCep63), or Cep152 (siCep152). The resulting cells were immunoblotted and immunostained (see Supplementary Fig. 3i–n). Relative fluorescence intensities of centrosome-associated Cep63 or Cep152 were quantified from three independent experiments (≥60 cells per experiment) (h, j). The number of centrosomal Sas6 dot signals per cell (classified as 0, 1, 2, and ≥ 3) was quantified from three independent experiments (≥200 cells per experiment) (i, k). Error bars, s.d.; **P < 0.01; ***P < 0.001, ****P < 0.0001 (unpaired two-tailed t test)
Fig. 4
Fig. 4
Hydrophobic motifs promote the localization and function of Cep63 and Cep152. a Schematic diagram illustrating the hydrophobic motifs (gray boxes) present in Cep63 P1b and Cep152 M4e. b Immunoprecipitation (IP) and immunoblotting analyses using transfected HEK293 cells. Numbers, relative signal intensities. cf Analysis of U2OS cells stably expressing the indicated constructs after silencing for luciferase (siGL), Cep63 (siCep63), or Cep152 (siCep152). The resulting cells were immunoblotted and immunostained (see Supplementary Fig. 4d–i). Relative fluorescence intensities of centrosome-associated Cep63 or Cep152 were quantified from three independent experiments (≥70 cells per experiment) (c, e). The number of the centrosomal Sas6 per cell was quantified from three independent experiments (≥200 cells per experiment) (d, f). Error bars, s.d.; **P < 0.01; ***P < 0.001, ****P <0.0001 (unpaired two-tailed t test)
Fig. 5
Fig. 5
Recruitment of Plk4 and Sas6 by the Cep63-Cep152 self-assembly. a Three-dimensional structured-illumination microscopy (3D-SIM) analysis showing the ability of Cep63 P1•Cep152 N70-M4d self-assemblies to recruit Plk4 in an N70 (a minimal Plk4-binding motif)-dependent manner in vitro (see Methods for details). Surface rendering (right) is generated from the assemblies marked by dotted boxes. Bar, 1 μm; bar for rendered image, 0.5 μm. b 3D-SIM analysis of transfected U2OS cells showing the ability of Cep63•Cep152 N217-M6 self-assemblies to recruit Plk4 and Sas6 (see details in Methods). Surface rendering (right) from the assembly marked in Stack 24 (dotted box). Bar, 5 μm; bar for rendered image, 0.5 μm. c Model illustrating how Cep63 and Cep152 interact to form a heterotetrameric α-helical bundle and self-assemble into a higher-order cylindrical architecture at the proximal end of centrioles. The process of forming the four-helix bundle is explained in Supplementary Fig. 4c. We propose that the four-helix bundle functions in concert with the adjacent hydrophobic moiety to achieve the radial arrangement of the Cep63•Cep152 complex in the Cep152 N terminus-outward fashion (see Discussion for details). The resulting self-assembly appears to be a highly dynamic architecture, constantly exchanging its components with those in the surroundings

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