Self-assembling SAS-6 multimer is a core centriole building block - PubMed (original) (raw)
Self-assembling SAS-6 multimer is a core centriole building block
Jayachandran Gopalakrishnan et al. J Biol Chem. 2010.
Abstract
Centrioles are conserved microtubule-based organelles with 9-fold symmetry that are essential for cilia and mitotic spindle formation. A conserved structure at the onset of centriole assembly is a "cartwheel" with 9-fold radial symmetry and a central tubule in its core. It remains unclear how the cartwheel is formed. The conserved centriole protein, SAS-6, is a cartwheel component that functions early in centriole formation. Here, combining biochemistry and electron microscopy, we characterize SAS-6 and show that it self-assembles into stable tetramers, which serve as building blocks for the central tubule. These results suggest that SAS-6 self-assembly may be an initial step in the formation of the cartwheel that provides the 9-fold symmetry. Electron microscopy of centrosomes identified 25-nm central tubules with repeating subunits and show that SAS-6 concentrates at the core of the cartwheel. Recombinant and native SAS-6 self-oligomerizes into tetramers with approximately 6-nm subunits, and these tetramers are components of the centrosome, suggesting that tetramers are the building blocks of the central tubule. This is further supported by the observation that elevated levels of SAS-6 in Drosophila cells resulted in higher order structures resembling central tubule morphology. Finally, in the presence of embryonic extract, SAS-6 tetramers assembled into high density complexes, providing a starting point for the eventual in vitro reconstruction of centrioles.
Figures
FIGURE 1.
SAS-6 is a component of the centriole central tubule. A, SAS-6-GFP localizes to centrioles and to the proximal end of elongated spermatocyte centrioles. The entire length of the spermatocyte centriole is marked by ANA1-tdTomato, a pan centriole marker (28). B and C, thin-section EM of isolated Drosophila centrosomes immunogold-labeled with antibodies to GFP (B) and SAS-6 are shown. C, red arrows and circles highlight the immunogold labeling at the central tubule (Bi and Ci) and the preferential immunogold-labeling at the center of the longitudinally sectioned centrioles where the central tubule is found (B, ii and iii, and C, ii and iii). Biv and Civ quantify the labeling pattern observed in longitudinally sectioned centrioles (B, ii and iii, and C, ii and iii). B–D, dashed yellow lines define the centriole core from PCM portion. D, Asl immunogold particles label the PCM portion of the centrosome (red arrows). Most of them are located outside the yellow circle.
FIGURE 2.
Recombinant SAS-6 expressed in E. coli (A–D) and P. pastoris (E–H) produces tetrameric structures. A, purified recombinant SAS-6 forms 7.4 S structures that are fractionated in a 5–40% linear sucrose gradient and probed with V5 antibody. A distinct peak of signal was detected only at around 7.4 S. Sedimentation coefficient markers were run on an identical gradient. B, recombinant SAS-6 from E. coli binds to SAS-6-GFP from fly embryo extract as assessed by a pulldown assay of recombinant SAS-6-V5-His using nickel affinity resins. The complex was eluted using 500 m
m
imidazole and immunoblotted for the presence of SAS-GFP. HI, heat-inactivated. C, negative stain EM of 7.4 S fractions shows single particles that have a diameter of ∼12 nm and contain four identical stain excluding regions with diameters of ∼6 nm. D, shown is a silver stain of protein sample concentrated 10-fold by trichloroacetic acid precipitation (i) and parallel Western analysis using rabbit anti-SAS-6 antibody (ii). The asterisk (*) marks a barely visible protein signal, possible evidence of contamination. E, Western blot of purified SAS-6-Myc-His from P. pastoris identifies oligomers of SAS-6 (O1 and O2) in non-reducing conditions and a monomer (M) in the presence of β-mercaptoethanol. F, recombinant SAS-6 from P. pastoris binds to SAS-6-GFP from Drosophila embryo extract. G, affinity-purified SAS-6 run on a 15–60% linear sucrose gradient forms 7.4 S and denser (marked by dashed line rectangle) structures. Sedimentation coefficient markers were run on an identical gradient. H, negative-stain EM of pooled dense fractions finds SAS-6 tetramers (dashed squares) that are shown in a wide field (i) and as single particles (ii) and subunit-rich agglomerates of tetramers (iii), some of which curve. iv, distribution of particles were observed in a field of 0.45 μm (n = 20).
FIGURE 3.
Native SAS-6 forms 7.4 S and 50 S structures. A, SAS-6-GFP Drosophila embryo extract was fractionated through a 5–40% linear sucrose gradient with 100 m
m
KCl. Fractions were subjected to SDS-PAGE and immunoblotted for the presence of SAS-6-GFP and Misato. Note that SAS-6 was detected as an intense peak starting only at around 7.4 S where tetramers are fractionated, whereas the loading control Misato was detected right from first fractions where monomers are expected to fractionate indicating undetectable or no free monomeric SAS-6. B, SAS-6-GFP Drosophila embryo extract fractionated through a 15–60% linear sucrose gradient with 500 m
m
KCl is shown. Fractions were subjected to SDS-PAGE and immunoblotted for the presence of SAS-6-GFP and γ-tubulin. The fractions containing γ-TuSC and centrosomes are indicated as is the expected region of γ-TuRC. Note that at 500 m
m
KCl, SAS-6-containing centrosomal substructures were stripped, only a 50 S core structure (marked by a square) remained, and γ-TuRC collapsed into γ-TuSC. C, negative-stain EM of SAS-6-GFP-enriched 50 S fraction (i) detected structures measuring ∼12 nm with four ∼6-nm stain-excluding regions (ii). D, centrosomes containing SAS-6-GFP were pretreated with 0.5 or 1.5
m
KCl for 2 h at 4 °C and subjected to velocity sedimentation in the presence of 500 m
m
KCl. No SAS-6-GFP was detected at 7.4 S when the centrosomes were not disrupted with high salt and fractionated at the bottom of the gradient (i), whereas in the presence of 1.5
m
KCl, SAS-6-GFP was detected at 7.4 S, where the tetrameric molecular sizes are fractionated (ii).
FIGURE 4.
SAS-6 overexpression in Drosophila cells induces higher order ectopic complexes containing 25-nm ring-like substructures. A, fractionation of Drosophila cells overexpressing SAS-6-GFP at 100 m
m
KCl identifies a SAS-6-induced ectopic structure (marked by a rectangle) that co-fractionates with a γ-tubulin peak (marked by a rectangle) (i). A measurement of the intensity of the protein signals in each fraction (graphs beneath the Western blots) shows discernible peaks for SAS-6 and γ-tubulin. Unlike native SAS-6 structures, the SAS-6-induced ectopic structure is denser than 50 S (compare with Fig. 3_B_). ii, in control un-transfected cells the γ-TuSC and γ-TuRC are intact, and there was no γ-tubulin enrichment in the fractions corresponding to the SAS-6-induced ectopic structure. iii, at 500 m
m
, SAS-6-induced structures are stable, and the fractions do not contain γ-tubulin. B, negative-stain EM of the ∼50 S fraction (square 1 in Fig. 4_Aiii_) showing structures measuring ∼12 nm with four ∼6-nm stain-excluding regions (4 ± 1 particles in a field of 0.45 μm, n = 5). C, negative-stain EM images of SAS-6-rich fraction (square 2 in Fig. 4_Aiii_) reveal large ectopic structures (13 ± 1 observed in a field of 0.45 μm, n = 5) (i) that contain identical rings ∼25 nm in diameter arranged one after the other (arrows in ii and iii). Circles in iii highlight the individual rings, and arrows in iii point to individual subunits.
FIGURE 5.
7.4 S SAS-6 structures are stable components of higher order structures. A, velocity sedimentation of FLAG tag affinity-purified low density (i) and high density (ii) SAS-6 complexes is shown. Both complexes fractionate around 7.4 S, indicating that the 7.4 S structures are stable structural intermediates that remain intact after the disassembly of higher order structures. B, negative-stain EM of low density SAS-6 complexes fractionated at 7.4 S (boxed fraction in Ai) identifies tetrameric objects (iii), tetrameric objects with appendages (iv), and subunit-rich curved structures (v). B, i and ii, show the particles in a wide field. C, negative-stain EM of high density SAS-6 complexes fractionated at 7.4 S (boxed fraction in Aii) also identifies tetrameric objects (ii) and curved structures (iii). Ci shows the particles in a wide field. D, shown is distribution of particles in a 0.45-μm field (n = 20) after sedimentation of low (7.4 S) and high density (50 S) SAS-6 complexes. E, shown are a silver stain (i) and Western blot of the boxed fraction from Ai that was used for EM analysis. The blot was probed with anti-FLAG (ii) and anti-SAS-6 (iii). FLAG, fraction containing affinity-purified SAS-6 complex; Control, affinity-purified cell extracts obtained from cells stably expressing SAS-6-GFP. Note that anti-FLAG (ii) recognizes SAS-GFP-FLAG, whereas anti-SAS-6 (iii) recognizes both the fusion protein and the endogenous SAS-6. F, shown are a silver stain (i) and Western blot of the boxed fraction from Aii that was used for negative-stain EM analysis. The blot was probed with anti-FLAG (ii) and anti-SAS-6 (iii).
FIGURE 6.
In vitro assembly of higher order structures from 7.4 S SAS-6 structures. A, the 7.4 S recombinant SAS-6 from E. coli (Fig. 2_A_) and SAS-6-GFP from low density embryonic extract (fractions around 7.4 S from Fig. 3_B_) form an 11.3 S complex. B, in the presence of embryonic extract, purified 7.4 S SAS-6-GFP-FLAG from Drosophila cells (A) assembled into higher order structures that are fractionated at around 30 S. Such a shift in density is not observed for Misato or when the SAS-6 from E. coli or Drosophila cells was incubated with the embryonic extract buffer. An equivalent amount of purified SAS-6 from E. coli and Drosophila cells (6 μg/ml) was mixed with (3 mg/ml) of low density embryonic extract. The mixture was incubated for 2 h at 4 °C before being subjected to velocity sedimentation in a 15–60% gradient.
References
- Azimzadeh J., Bornens M. (2004) in Centrosomes in Development and Disease ( Nigg E. A. ed.) pp93–116, Wiley-VCH, Weinheim, Germany
- Rosenbaum J. (2002) Curr. Biol. 12, R125. - PubMed
- Scholey J. M., Anderson K. V. ( 2006) Cell 125, 439– 442 - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources