Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization

Abstract

As the main microtubule-organizing centre in animal cells, the centrosome has a fundamental role in cell function. Surrounding the centrioles, the pericentriolar material (PCM) provides a dynamic platform for nucleating microtubules. Although the importance of the PCM is established, its amorphous electron-dense nature has made it refractory to structural investigation. By using SIM and STORM subdiffraction-resolution microscopies to visualize proteins critical for centrosome maturation, we demonstrate that the PCM is organized into two main structural domains: a layer juxtaposed to the centriole wall, and proteins extending farther away from the centriole organized in a matrix. Analysis of Pericentrin-like protein (PLP) reveals that its carboxy terminus is positioned at the centriole wall, it radiates outwards into the matrix and is organized in clusters having quasi-nine-fold symmetry. By RNA-mediated interference (RNAi), we show that PLP fibrils are required for interphase recruitment and proper mitotic assembly of the PCM matrix.

Figure 1. 3D SIM of proteins critical for centrosome maturation identifies two distinct structural domains within the PCM

a) Maximum intensity projections along the Z-axis of centrioles from S2 cells labeled with antibodies against centriolar proteins obtained either with widefield or 3D SIM microscopy. b) Summary schematic of 3D sub-volume iterative alignment strategy based on cross-correlation in real space used for alignment and analysis of experimental 3D volumes. See methods for a detailed description. c) 2D projections of the average aligned volumes. Sas-6 n=10 (antibodies anti-eGFP), Sas-4 n=88 (antibodies against a.a. 2–150), Asl n=21 (a.a. 958–972 of Asl), Plp n=82 (a.a. 1–381 of Plp-PB), Spd-2 n=19 (a.a. 375–695 of Asl), Cnn n=22 (a.a. 1–571 of Cnn), γTub n=33. d) Fluorescence intensities profiles from the center of the centriole image outward measured from radially averaged 2D projections of average volumes of centrosomal proteins. e) Radially averaged fluorescence intensities values obtained from individual centrosomal protein projected volumes were fit to an offset Gaussian function to calculate the center position and deviation of the distribution. f) Centrosomes isolated from Drosophila syncytial blastoderm embryos were allowed to regrow microtubules with rhodamine-Tubulin and stained with rabbit anti-Plp (a.a. 1–381 of Plp-PB) or mouse anti-γTub antibodies recognized with anti-mouse 488. Bottom, image gallery of centrosomes from Drosophila embryos stained with rabbit anti-Plp NTD and anti-rabbit Alexa 488. Scale bar 500 nm. f) High pressure frozen Drosophila syncytial blastoderm embryos embedded in resin were sectioned (75 nm) and stained with rabbit antibody anti-Plp NTD (a.a. 1–381) and anti-rabbit immunogold labeled secondary antibodies (10 nm beads). Scale 100 nm.

Figure 2. The molecular architecture of Pericentrin-like protein

a) Amino-acid map of Drosophila Plp predicted from the Flybase database. Sequence prediction of coiled-coil conformation was performed with the software Coils using a window of 28 residues. Amino-acid stretches were considered coiled-coil if predicted with a probability of ≥70%. b) Top: Wild type S2 cells co-stained with rabbit anti-Plp MD primary antibodies (a.a 1805–2137, anti-rabbit Alexa 488) and with guinea pig anti-Plp NTD primary antibodies (a.a. 1–381, anti-guinea pig Alexa 555). Bottom: S2 cells expressing eGFP-PACT co-stained with mouse anti-eGFP monoclonal antibody (anti-mouse Alexa 488) and with rabbit anti-Plp NTD primary antibodies (a.a. 1–381, anti-rabbit Alexa 555). DNA is labeled with DAPI stain. Scale bar 1µM. c) 2D projections of the average aligned volumes. Plp NTD=82; Plp MD n=29; eGFP-PACT n=9. d) Fluorescence intensity profiles from the center of the centriole image outward measured from radially averaged 2D projections of average volumes of centrosomal proteins. e) Radially averaged fluorescence intensities values obtained from individual centrosomal protein projected volumes were fit to an offset Gaussian to calculate the center position and deviation of the distribution. Scale bar 200 nm. f) STORM image of centrioles from S2 cells stained with rabbit anti-Plp MD antibody (a.a 1805–2137) and anti-rabbit secondary antibodies conjugated with Alexa 647/Alexa 405 dye pairs.

Figure 3. Plp fibrils associated with mother centrioles form a gap where daughter centriole assembles

a) Centrosome from wild type S2 cells co-stained with rabbit anti-Plp NTD antibodies (a.a. 1–381, anti-rabbit 555) together with mouse anti-Sas-4 antibodies (anti-mouse 488). Bottom panel. Centrosomes from S2 cells expressing eGFP-Sas-6 co-stained with mouse anti-GFP antibodies (anti-mouse 488). b) Volume rendering of G2 centriole from panel a) stained with mouse anti-Sas-4 and rabbit anti-Plp NTD shown from end-on and side views. c) Gap distance measurements obtained from 3D SIM volumes of a subpopulation of G2 centrosomes stained with rabbit Plp NTD antibody. d) Rendering of volumes averages of G1 and G2 centrosomes stained with anti-Plp NTD antibody. G2 centrosomes were divided into two separate populations for sub-class averaging of centrioles with an open gap or partially open gap. e) STORM image of centrioles from S2 cells stained with rabbit anti-Plp NTD antibody (a.a. 1–381, left) and anti-rabbit secondary antibodies conjugated with Alexa 647/Alexa 405 dye pairs. Given the cluster variability, we used a blinded study to quantify how often a missing cluster could be recognized (58%) in STORM images of mother centrioles stained with anti-Plp antibody from cells blocked in G2.

Figure 4. Plp is associated exclusively with mother centrioles until metaphase

a) S2 cells expressing eGFP-Sas-6 stained with mouse anti-GFP antibodies (anti-mouse 488) and with rabbit anti-Plp NTD (a.a. 1–381, anti-rabbit 555). DNA is labeled with DAPI stain. Scale bar 1µM. White arrows point at the lack of or partial recruitment of Plp on daughter centrioles at metaphase. Grey arrows on the telophase panels show complete recruitment of Plp on daughter centrioles. b) Wild type S2 cells stained with mouse anti-Sas-4 antibodies (anti-mouse 488) and rabbit anti-Plp NTD antibodies (a.a. 1–381, anti-rabbit 555). DNA is labeled with DAPI stain. Scale bar 1µM. White arrows point at the lack of or partial recruitment of Plp on daughter centrioles at metaphase. Notice the green arrow on the metaphase panel showing the separation of Sas-4 stained centrioles compared to G2 cells. Grey arrows on the telophase panels show complete recruitment of Plp on daughter centrioles. c) Quantification of the number of Sas-4, Sas-6 clusters or Plp rings per centrosomes per S2 cells. (G1 n=26, G2=29, Telophase =10). d) S2 cells expressing eGFP-Sas-6 co-stained with guinea pig anti-Plp NTD antibodies (a.a. 1–381, anti-guinea pig 405) and rabbit anti-GFP antibodies (anti-rabbit 488) and mouse anti γTubulin (anti-mouse 555). Scale bar 1µM.

Figure 5. Plp is required for the initial recruitment and proper 3D assembly of the PCM distal layer

a) Left. Plp genomic region obtained from the Flybase database (5’ and 3’UTR regions not displayed). The position of the sequences on Plp exons used for dsRNA production is annotated on the sequence. Right, western blot on extracts obtained from S2 cells treated for control or Plp dsRNA probed with rabbit anti-Plp NTD antibody. b) S2 cells treated with control dsRNA or dsRNA specific for Plp exon 10 were stained with either anti-Cnn antibodies (anti-mouse 488) or mouse anti-γTubulin antibodies or rabbit anti-Spd2 antibodies (anti-rabbit or mouse 555). Widefield images were collected with a 60X objective and deconvolved with a non-linear positivity-constrained iterative algorithm. DNA was labeled with DAPI stain. Scale bar 10µM c) Mitotic S2 cells treated with dsRNA specific for Plp exon 10 were stained with guinea pig anti-Plp NTD (anti-guinea pig 555 and rabbit anti-Cnn 1–571 (anti-rabbit 488). DNA was labeled with DAPI stain. SIM Images were acquired in the camera linear range of response and are displayed with identical intensity settings. Scale bar 1µM. d) Volume rendering of control and Plp RNAi S2 cells shown in panel b. Scale bar 500 nm. e) Volumes obtained from stained S2 cells as in panel b were quantified for the volume occupied and their surface area with Chimera software (control n=16, Plp Rnai n=18). f) Volumes were approximated to a spherical object to measure Vol^2/Area^3.

Figure 6. The molecular architecture of Kendrin/Pericentrin is similar to that of Plp

a) Linear map of the amino-acid sequence of human Kendrin predicted from the UCSC database. The antibodies on the cartoon show the location of Plp protein sequence used for immunization. Sequence prediction of coiled-coil conformation was performed with the software Coils using a window of 28 residues. Amino-acid stretches were considered coiled-coil if predicted with a probability of ≥70%. b) Human RPE cells were labeled with antibodies against Kendrin NTD (a.a. 744–909, anti-rabbit 555) and Kendrin CTD (a.a. 3197–3336, anti-rabbit 555) with mouse acetylated-Tubulin or mouse anti γTubulin antibodies (anti-mouse 488). Nuclei were labeled with DAPI stain. The white arrow points at the diffused population of Kendrin in interphase. Scale bar 1 µm. c) 2D projections of the average aligned volumes (Kendrin NTD n=16, Kendrin CTD n=15). d) Fluorescence intensities profiles from the center of the centriole image outward measured from radially averaged 2D projections of average volumes of centrosomal proteins. e) Radially averaged fluorescence intensities values obtained from individual centrosomal protein projected volumes were fitted to an offset mirrored Gaussian function to calculate the center position of the distribution. e) HeLa cells expressing eGFP-PACT were co-stained with antibodies against Kendrin NTD (a.a. 744–909, anti-rabbit 555) and anti-GFP (anti-mouse 488). Scale bar 500 nm.

Figure 7. Pericentrin-like protein forms elongated fibrils that extend radially from the centriole wall to support the 3D organization of the PCM

During centrosome maturation, the PCM is organized in two distinct structural domains: a layer juxtaposed to the centriole wall, and proteins extending further away from the centriole organized in a matrix. In this proximal PCM domain, we found elongated Plp fibrils that are anchored with the PACT domain to the centriole wall and with their N-terminus extending outwards. Plp is exclusively associated with mother centrioles until metaphase and form a gap where daughter centriole assembles. During centrosome maturation, Plp facilitates the proper 3D assembly of the PCM distal layer by organizing a shell of Cnn molecules that is in place around the wall of mother centrioles from the interphase cell cycle stage.