STED nanoscopy of the centrosome linker reveals a CEP68-organized, periodic rootletin network anchored to a C-Nap1 ring at centrioles

Abstract

The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is ∼35 to 40 nm, leading to an estimated minimal rootletin length of ∼110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator.

Keywords: CEP68; STED superresolution; centrosome; centrosome linker; rootletin.

Fig. 1.

Rootletin assembles into a web-like network with a repeat unit of 75 nm. (A) STED superresolution image of the rootletin linker (root-C1) of RPE-1 cells in combination with polyglutamylated tubulin (GT335, Top) or γ-tubulin (Bottom). (B) Single rootletin filaments from four different cells (red dotted lines 75 nm apart are a guide to the eye). Although the majority of rootletin spots are separated by 75 nm, a few locations show slightly different distances (red arrows). (C) Histogram of fitted periodicity. The Gauss fit (red line) gave a periodicity of 75 nm (red dotted line; SD, 2 nm). The data from three samples [47 cells; 112 line profiles with a total length of 79.48 μm (∼1,060 periods)] were taken that could be fitted by using an automated procedure (SI Appendix, Fig. S2), with the length of the filament taken to weight. The maximum single fiber length is 2.5 μm. (D) Rootletin filaments are often thicker close to centrioles and taper off toward the tip (white arrows, Top). In some cases, these tapered filaments appear to be one fiber; in other cases, multiple fibers come together and align their periodicity (white arrows, Bottom). (E) Color-coded 3D rootletin (root-C2) network analyzed by 3D-STORM. The upper image shows the rootletin network (Movie S7). The lower image shows a single rootletin fiber (Movie S8). (All scale bars, 500 nm.) The STED images are Wiener deconvolved; the raw data are shown in SI Appendix, Fig. S11.

Fig. 2.

Cep68 binds to rootletin filaments in 75-nm intervals. (A) STED images of CEP68 together with C-Nap1 (Top) or γ-tubulin (Bottom). (B) Single CEP68 filaments from the cell in A and three other cells (red dotted lines 75 nm apart are a guide to the eye). Although the majority of the CEP68 spots are separated by 75 nm, a few locations show slightly different distances (red arrows). (C) Histogram of fitted periodicity. CEP68 shows a regular 75 ± 3 nm repeat organization [three samples, plus one data point from a fourth sample; 38 cells; 142 line profiles; total length, 37.54 μm (∼984 periods); maximum single fiber length, 1.34 μm]. (D) CEP68 filaments show thick to thin filaments (white arrow, Top), and even separate filaments synchronize their phase (white arrows, Bottom). (E) Rootletin (red) and CEP68 (green) colocalize along the same fiber [Left, STED; Right, intensity line profile and the filament (no. 1 or 2) it is taken from]. (F) Color-coded 3D CEP68 network of the cell shown in Movie S9 analyzed by 3D-STORM. (All scale bars, 500 nm.) The STED images are Wiener deconvolved; the raw data are shown in SI Appendix, Fig. S11.

Fig. 3.

Rootletin N-terminus and C-terminus localization along the fiber. (A) Raw-data STED image of the N- and C-terminus staining by root-N (red) and root-C2 (green) antibodies, respectively. Through confocal microscopy, PCNT (blue) is also imaged. (Scale bar, 500 nm.) (B) A deconvolved (Wiener) filament of the cell in A is shown, from which a line profile is drawn from centriole outward, as illustrated by the marked yellow area. (Scale bar, 500 nm.) (C) The line profiles show that both root-N and root-C2 give a 75-nm periodic signal along the fiber. The shift in phase of root-C2 compared with root-N (drawn from the centriole outward) is 43 nm, as indicated by the red and green lines at the peaks. (D) Histogram of the shift of the root-C2 signal compared with the root-N signal (26 cells; 48 line profiles with a total length of 43.6 μm). The root-C2 signal is, on average, shifted by 40 ± 7 nm compared with the root-N signal (as measured from the centriole outward). Blue data are from root-N to the kk114L dye and root-C2 to the star580 dye, and the purple data are from the reversed staining. (E) The results indicate an overlap between individual rootletin molecules, as drawn. More examples in other directions and with reversed staining are given in SI Appendix, Fig. S4, and the raw and Wiener deconvolved images are shown in SI Appendix, Fig. S11.

Fig. 4.

C-Nap1 forms a ring at centrioles and functions as rootletin filament organizer. (A) Three examples of dual-color STED analysis of C-Nap1 [monoclonal anti-C-Nap1 mouse antibody (34)] labeled with kk114L and of CEP68 (polyclonal anti-rabbit antibody) labeled with star600 (Top) and star580 (Middle and Bottom). (Top) For optimal imaging of the C-Nap1 ring, C-Nap1 was imaged first, followed by CEP68, in each scanned line. (Middle and Bottom) The imaging order in Top was reversed to increase the resolution of the CEP68. The C-Nap1 ring is illustrated in the Middle and Bottom Insets. (B) Dual-color STED analysis of CEP68 and rootletin shows ringlike colocalization of both proteins. (Scale bars, 500 nm.) The STED images are Wiener deconvolved; the raw data are shown in SI Appendix, Fig. S11.

Fig. 5.

STED analysis of centriolar rootletin/CEP68 structures. PCNT in confocal imaging was used as a marker for centrioles. (A) Rings and often a single filament of rootletin and residual CEP68 at PCNT-marked centrosomes in response to siRNA CEP68 depletion. The NSC siRNA control is shown as comparison. (B) Mild overexpression of CROCC (rootletin) in combination with siRNA depletion of CEP68 or C-Nap1 in the absence of Dox. siRNA depletion of CEP68 in CROCC (rootletin)-overexpressing cells exposes a strong centriole rootletin/CEP68 ring. C-Nap1 is essential for centriolar rootletin/CEP68 ring assembly, even when CROCC (rootletin) is overexpressed. A single or very few elongated rootletin/CEP68 filaments that originate at centrosomes were observed in siC-Nap1-depleted cells. (Scale bars, 500 nm.) More examples are shown in SI Appendix, Fig. S8C. The STED images are Wiener deconvolved; the raw data are shown in SI Appendix, Fig. S11.

Fig. 6.

CEP68 plays a role in rootletin filament bundling. (A) In the RPE-1 TetON rootletin-HA cell line, mild overexpression of CROCC (rootletin) promotes assembly of thin and tick rootletin filaments. Only thick filaments were associated with CEP68 (arrowheads). Thin filaments (asterisks) were mostly devoid of CEP68. Low and high images reflect different contrast and brightness settings of the same image. Note that thin filaments were detected only with the higher sensitivity of the high setting. The line scan (Right) indicates colocalization of CEP68 and rootletin (stained with root-C2) on thick filaments. (B) Analysis of rootletin filaments of cells from A for CEP68 and rootletin by confocal microscopy and dual-color STED microscopy. The smaller images at the Right and Bottom show enlargements of thin and thick rootletin filaments, respectively. The STED images are Wiener deconvolved; the raw data are shown in SI Appendix, Fig. S11. (C) siRNA depletion of CEP68 in RPE-1 TetON rootletin-HA cells decreases the number of cells with cytosolic rootletin thick filaments. Instead, thin rootletin filaments encircle the nuclear envelope. These thin filaments were more visible with the high setting. They were resolved as single rootletin filaments by STED microscopy (SI Appendix, Fig. S9B). (D) Quantification of C: n = 3; n ≥ 20; mean and SD are shown. (A and C scale bars, 5 µm; B scale bars, 500 nm.)

Fig. 7.

The C terminus of CEP68 containing the spectrin repeat mediates binding to centrosomes. (A) The C terminus of CEP68 contains a spectrin repeat. Sequence alignment of CEP68 with Nesprin1 identified a spectrin repeat in the CEP68 C terminus (amino acids 628 to 728). (B) The C-terminal CEP68 fragment (amino acids 618 to 757) localizes to the centrosome, as does CEP68. Shown are stable RPE-1 TetON CEP68-HA cells with Dox (+Dox) or without (−Dox). Root-C1 antibody was used to stain rootletin. γ-Tubulin was used as marker for centrosomes. The smaller images to the Right of each cell are enlargements of the centrosomal area and indicate CEP68-618–757aa-HA recruitment to centrosomes upon Dox induction (+Dox). CEP68-HA was used as a positive control. In contrast, the N-terminal CEP68 fragment (amino acids 1 to 298) shows no defined localization (SI Appendix, Fig. S10A). (C) The rootletin fragment R3 recruits CEP68. Cells with expression of the HA-tagged CROCC (rootletin) constructs in stable RPE-1 TetON cell lines. Antibodies against HA, CEP68, and γ-tubulin were used. DNA was stained with DAPI. Low (Left) and high (Center) images reflect different contrast and brightness settings of the same CEP68 image. (Right) Enlargement(s) of the boxed area in the image to the left. CEP68 localization in cells expressing R2 is shown in SI Appendix, Fig. S10C. (D) Quantification of C: n = 2; n = 20; mean and SD are shown. (B and C scale bars, 5 µm.)

Fig. 8.

Model. (A) Rootletin filament assembly with and without CEP68. (B) Assembly hierarchy of centrosome linker C-Nap1, CEP68, and rootletin at centrosomes. See text for details.