self-assemblingsas-6multimerisacorecentriole … · self-assemblingsas-6multimerisacorecentriole...

Self-assembling SAS-6 Multimer Is a Core CentrioleBuilding Block*□S

Received for publication, December 10, 2009, and in revised form, January 14, 2010 Published, JBC Papers in Press, January 18, 2010, DOI 10.1074/jbc.M109.092627

Jayachandran Gopalakrishnan‡, Paul Guichard§¶, Andrew H. Smith‡, Heinz Schwarz�, David A. Agard**,Sergio Marco§¶, and Tomer Avidor-Reiss‡1

From the ‡Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, the §Institut Curie, Centre deRecherche, and ¶INSERM, U759, Orsay F-91405, France, the �Max-Planck Institut fur Entwicklungsbiologie, Spemannstrasse 35,Tubingen D-72076, Germany, and the **Howard Hughes Medical Institute and Department of Biochemistry and Biophysics,University of California, San Francisco, California 94158

Centrioles are conserved microtubule-based organelleswith 9-fold symmetry that are essential for cilia and mitoticspindle formation. A conserved structure at the onset of cen-triole assembly is a “cartwheel” with 9-fold radial symmetryand a central tubule in its core. It remains unclear how thecartwheel is formed. The conserved centriole protein, SAS-6,is a cartwheel component that functions early in centrioleformation. Here, combining biochemistry and electronmicroscopy, we characterize SAS-6 and show that it self-as-sembles into stable tetramers, which serve as building blocksfor the central tubule. These results suggest that SAS-6 self-assembly may be an initial step in the formation of the cart-wheel that provides the 9-fold symmetry. Electron micros-copy of centrosomes identified 25-nm central tubules withrepeating subunits and show that SAS-6 concentrates at thecore of the cartwheel. Recombinant and native SAS-6 self-oligomerizes into tetramers with �6-nm subunits, and thesetetramers are components of the centrosome, suggesting thattetramers are the building blocks of the central tubule. This isfurther supported by the observation that elevated levels ofSAS-6 in Drosophila cells resulted in higher order structuresresembling central tubule morphology. Finally, in the pres-ence of embryonic extract, SAS-6 tetramers assembled intohigh density complexes, providing a starting point for theeventual in vitro reconstruction of centrioles.

Centrioles are conserved microtubule-based organelleswith 9-fold symmetry organized around a central tubule andspoke structure termed the “cartwheel.” This conserved9-fold symmetry is also shared with basal body and cilia(1–3). Such architectural similarity is indicative of the role ofthe centriole as a template for cilia formation. Cilia aremicrotubule-rich cellular organelles involved in signalingpathways and sensory functions in eukaryotic cells (4, 5).Centrioles also recruit a pericentriolar matrix to form cen-trosomes, major microtubule organizing centers requiredfor cell proliferation and differentiation (6, 7). In ciliated

cells, one of the centrioles inherited by the daughter cellmigrates to the plasma membrane after cell division to serveas a template for the cell cilium. However, when a centrioleduplicates, the new centriole does not use the existing cent-riole as a template (8, 9), raising the question of how the newcentriole establishes its 9-fold symmetry.Centriole biogenesis is a multistep process that includes

an intermediate known as the procentriole (10). The procen-triole has an internal cartwheel structure that is required forthe stability of the centriole and the 9-fold symmetry of cilia(11). The cartwheel has a central tubule from which ninespokes emanate, each attaching to centriolar microtubules(supplemental Fig. S1A). Based on the structure of the cart-wheel, it is expected that the central tubule would itself dis-play 9-fold symmetry (12) (supplemental Fig. S1A). It hasbeen proposed that the central tubule generates the 9-foldsymmetry of the centriole (13, 14). However, the composi-tion of the central tubule and the mechanisms underlying itsbiogenesis remain unknown.SAS-6 is a coiled-coil protein that is required for centriole

biogenesis (15, 16). Overexpression of SAS-6 produces mul-tiple procentrioles (17). In Chlamydomonas and Tetrahy-mena, SAS-6 localizes to the center of the cartwheel (11, 18).Null mutants of SAS-6 in Chlamydomonas (11) and Dro-sophila (11, 19) lack the cartwheel. The SAS-6 null mutantsof Chlamydomonas and Drosophila are also defective inestablishing the 9-fold symmetry of centrioles. In Caenorh-abditis elegans, RNAi studies show that SAS-6 is required forthe formation of a centriole central tube. Direct evidencethat SAS-6 is required for central tube assembly in wormscomes from time resolved electron tomography of C. elegansembryos demonstrating that centriole formation begins withassembly of a central tube, a process that requires SAS-6(21). This study also implied that the tube could itself orga-nize 9-fold symmetry, but it was not clear if the process wasdriven by steric constraints or was patterned by intrinsiccues on the outer surface of the tube. It was proposed thatSAS-6 brings together nine pre-centriolar units called ena-tosomes, which then form a tube-like centriole precursor(22) and that SAS-6 is the repeating subunit of the centraltubule (12).To investigate the mechanism of SAS-6 function, we em-

ployed aDrosophilamodel that allows for biochemical isolation

* This work was supported in part by a grant from the bequest of the WilliamF. Milton and Stewart Trust Pilot Projects.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table 1, Figs. S1–S4, and Movies 1 and 2.

1 To whom correspondence should be addressed. E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 12, pp. 8759 –8770, March 19, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

MARCH 19, 2010 • VOLUME 285 • NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 8759

at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from

http://www.jbc.org/content/suppl/2010/01/18/M109.092627.DC1.htmlSupplemental Material can be found at:

http://www.jbc.org/cgi/content/full/M109.092627/DC1



http://www.jbc.org/

and electron microscopy (EM)2 imaging of centrioles and cart-wheels. In vertebrates the cartwheel is transient and isrestricted to the “procentriole” stage (23). In worms the cart-wheel is not apparent (21), and in protozoa it is restricted to theproximal end of basal bodies (18, 24).Drosophila centrioles arestructurally similarly to the procentrioles of higher organisms(7), and cartwheels are constitutive components of Drosophilacentrioles. EM studies of in situ centrioles in Drosophilaembryos reported that the cartwheel extends through the fulllength of the centriole (25).Here, we report that in Drosophila, the central tubule of the

cartwheel has repeating subunits and that SAS-6 localizes at thecentral tubule. Recombinant SAS-6 produced in Escherichiacoli or Pichia pastoris forms tetrameric structures that are frac-tionated at 7.4 S. Most of the native SAS-6 exists as 7.4 S struc-tures, which is themost common soluble form of SAS-6. NativeSAS-6 is also found as part of 50 S structures and centrosomes.Elevated levels of SAS-6 in Drosophila cells resulted in higherorder structures built from multiple central tubule-like struc-tures. Affinity purification of these high density SAS-6 com-plexes contained tetramers. Upon disassembly, these higherorder complexes resulted in tetramers. In vitro assembly ofthese tetramers could then be assembled into high density com-plexes in the presence of embryonic extract. Collectively thesedata suggest that the SAS-6 tetramers serve as building blocksof a higher order central tubule at the core of centriolearchitecture.

MATERIALS AND METHODS

Transgenic Constructs—The generation of ana1 and sas-6fused with GFP was described previously (28). The flies weregrown according to standard procedures and maintained at25 °C.Drosophila Embryo and S2 Cell Extract Preparation—Dro-

sophila embryo extract was prepared as described previously(26). Briefly, Drosophila embryos of 0–12 h or tissue-culturedS2 cells were homogenized in extract buffer containing 80 mM

K-Pipes, pH 6.8, 1 mM MgCl2, 1 mM Na3EGTA, 14% sucrose,100 mM KCl, 1 mM phenylmethylsulfonyl fluoride and proteaseinhibitor mixture (Sigma) plus EDTA-free complete tablets(RocheApplied Science). A similar buffer with 500mMKCl wasused for homogenizing S2 cells expressing SAS-6-GFP-FLAGbefore affinity purification of SAS-6 complexes. The clearembryo extract was obtained by centrifuging the crude extractfor 20 min at 1500 � g at 4 °C and subsequently used to isolatecentrosomes and their substructures using sucrose gradientvelocity sedimentation.Sucrose Gradient Velocity Sedimentation—To create contin-

uous sucrose gradients of 15–60 or 5–40%, sucrose was dis-solved in a buffer containing 80 mM K-Pipes, pH 6.8, 1 mM

MgCl2, 1 mM Na3EGTA, 1 mM GTP, and 500 mM KCl. Thegradient was generated with a Biocomp gradient maker. Cen-trifugation was performed at 100,000 � g in a SW-40 rotor

(Beckman) for 13 h at 4 °C to reach equilibrium. Fractions werecollected beginning from the top andwere analyzed byWesternblot.Western Blot—Extracts of embryo and tissue cultured Dro-

sophila S2 cells were boiled in SDS-PAGE sample buffer andresolved in 8% acrylamide gel. 25-�l sucrose gradient fractionswere used for theWestern blotting analyses. The proteins weretransferred to a nitrocellulose membrane and incubated withprimary antibodies overnight at 4 °C followed by secondaryantibody at room temperature for 1 h. Chemiluminescent sub-strate (Pierce) was used to reveal peroxidase activity, and thesignal was detected using autoradiographic film or a Fugi LAS3000 image processor. Apparent molecular masses were deter-mined by comparing with molecular standards (ProSieve,Lonza).Antibodies—Rabbit anti-SAS-6 (supplemental Fig. S3C) was

produced by us using full-length Drosophila SAS-6 cDNAcloned into pet23b vector provided byDr.M. Bettencourt-Dias.The protein was affinity-purified using a nickel column andinjected into a rabbit.For the Western blot, antibodies were rabbit anti-SAS-6

(1:5000), chicken anti-SAS-6 (1:500) (19),mouse anti-�-tubulin(1:5000, Sigma), mouse anti-Misato (1:5000, Santa Cruz),mouse anti-V5 (1:10,000, Abcam), mouse anti-GFP (1:5000,Roche Applied Science), mouse anti-FLAG (1:1000), andmouse anti-Myc (1:200). The secondary antibodies (1:5000)used were conjugated with peroxidase (Vector labs).For immunofluorescence, antibodies were rat anti-�-tubulin

(1:200, Chemicon) and mouse anti-�-tubulin (1:200, Sigma).All fluorescent secondary antibodies were from JacksonImmunoResearch and used at 1:200: Cy5 goat anti-mouse andrhodamine donkey anti-rat. 4�,6-Diamidino-2-phenylindole(1 �g/ml; Sigma) was used to stain DNA.Immunofluorescence and Staining—Testis from transgenic

flies expressing SAS-6-GFP and ANA1-GFP were dissected insaline solution (0.7% NaCl) and fixed 5 min in formaldehyde(3.7% in PBS). The slides were then mounted in mountingmedia (Biomedia) and examined using a Leica TCS SP5 scan-ning confocal microscope. Images were processed using AdobePhotoshop.Drosophila S2 cells expressing SAS-6-GFP or SAS-6-GFP-

FLAG were established by co-transfecting p(UAST)-SAS-6-GFP or p(UAST)-SAS-6-GFP-FLAG with hygromycin-resist-ant plasmid. The cells were grown on concanavalin-coatedcoverslips for 1 h at 25 °C before they were fixed using formal-dehyde (3.7% in PBS) for 5 min. After washing with PBS, thecells were permeabilized with PBS, 0.1% Triton X-100 for 10min and blocked with PBS, 1% bovine serum albumin, 0.1%Triton X-100 for 45 min. Antibody staining was performed for1 h at room temperature followed by three washes with PBS.The slides were then mounted in mounting media (Biomedia)and examined using a scanning confocal fluorescence micro-scope as described above.Immunoelectron Microscopy of Isolated Centrosomes—Frac-

tions from sucrose gradient-containing centrosomeswere fixedin 3.7% paraformaldehyde plus 0.1% glutaraldehyde in 80 mM

K-Pipes, pH 6.8, 1 mM MgCl2, 1 mM Na3EGTA at 4 °C for 10min. The fixed centrosomes were spun onto previously glow-

2 The abbreviations used are: EM, electron microscopy; PCM, pericentriolarmaterial; �-TuRC, �-tubulin ring complex; �-TuSC, �-tubulin small com-plexes; GFP, green fluorescent protein; Pipes, 1,4-piperazinediethanesul-fonic acid; PBS, phosphate-buffered saline.

SAS-6 Tetramer Is a Core Centriole Building Block

8760 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 12 • MARCH 19, 2010

at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from



http://www.jbc.org/

discharged ACLAR coverslips (Ted Pella, Inc., Redding, CA)coated with polylysine (Sigma). Using 3% glutaraldehyde, thecentrosomes were post-fixed for 10 min followed by 1%osmium tetroxide and 0.5% potassium ferricyanide in 0.1 M

cacodylate buffer, pH 7.4, for 15 min at 4 °C. The specimenswere stained overnight with 1% aqueous uranyl acetate at 4 °Cfollowed by dehydration through graded cold alcohol series andbrought to room temperature with absolute alcohol. The sam-ples were embedded onto epon/araldite using standard proto-col and remounted for thin serial sections. Thin sections of 70nm were cut using ultra cut microtome, and the thin sectionswere collected on Formvar-coated copper grids. The specimenswere post-stained with 1% uranyl acetate in 50% methanol fol-lowed by aqueous lead citrate and viewed in a Tecnai G SpiritBioTWIN transmission electron microscope (FEI, Hillsboro,OR) operated at 80 kV. For immunolabeling of centrosomes,the fixation was modified. Centrosomes on the coverslips wereinitially blockedwith a buffer containing 2%bovine serumalbu-min and 0.1% coldwater fish skin gelatin inTris-buffered saline.The samples were labeled using chicken anti-SAS-6 or anti-GFP diluted to 1:20 in the same buffer and then with rabbitanti-chicken followed by protein A with 5-nm colloidal gold.Negative Stain Electron Microscopy and EM Tomogram—

The sucrose from the fractionswas removed by buffer exchangeusing PD-10 Superdex-25 columns (GE Healthcare) with abuffer containing 80 mM K-Pipes, pH 6.8, 1 mM MgCl2, 1 mM

Na3EGTA, and 500mMKCl. 3.5 �l of sample was placed onto aglow-discharged carbon-coated copper grid for 1 min. Theexcess sample was blotted using filter paper, and the grids werewashed with a drop of deionized water twice. The grids werethen stained with two drops of freshly prepared 0.75% uranylformate and were air-dried subsequent to blotting with filterpaper. The grids were observed in a Jeol 1200EX electronmicroscope operating at 80 kV. Image analysis was performedusing ImageJ (33) and Xmipp (34) packages.Tomogram reconstructions were performed by the EMCAT

package within the PRIISM software developed at University ofCalifornia, San Francisco. Final tomograms were filtered usinganisotropic diffusion filter (35) or Fourier low-pass filters forsmall structures up to 4 nm for tomograms with noise becausethe anisotropic diffusion does not preserve the individual pro-tein densities. Rotational spectra analysis was performed usingthe Xmipp (34) package by Fourier-Bessel decomposition(36). Tomogram segmentation and volume rendering was per-formed using ImageJ (33) and University of California, SanFrancisco chimera (37), respectively. Briefly, semiautomaticsegmentation was carried out by selecting three independentregions of interest corresponding to the central tubule, spokes,andmicrotubules on the different tomogram slices. Voxels out-side each independent regions of interest were set to 0 beforepseudo-color assignment on each region of interest based onvoxel intensity. Volumes from regions of interest and tomo-grams were combined to generate the final video stream.E. coli Strains, Constructs, and Protein Purification—Dro-

sophila SAS-6 cDNAwas cloned into pDONR 221 (Invitrogen)and then recombined into the pet23b vector previously modi-fied for the gateway system (Invitrogen). The cDNAwas taggedwith V5 and His6 at its C terminus (a kind gift from Betten-

court-Dias). The construct was transformed into E. coli strainBL21 (Stratagene) and grown in LB medium to absorbance600�0.60 nm. The protein expression was induced by 1 mM

isopropyl 1-thio-�-D-galactopyranoside. The cells were pel-leted and disrupted by lysozyme treatment followed by sonica-tion. The lysate was centrifuged at 30,000 � g for 30 min tosediment the membranes and aggregates. The supernatant-containing soluble form of SAS-6 fusion protein was boundwith a nickel-nitrilotriacetic acid column, and the boundmate-rial was elutedwith 250mM imidazole. The purified proteinwasfractionated using Superdex-200 column (GEHealthcare). Thestandard markers were obtained from Bio-Rad.P. pastoris Strains, Constructs, and Protein Purification—

P. pastoris strain GS115 (Invitrogen) was used for heterologousexpression of SAS-6. YPDS and Zeocin agar plates that con-tained 1% (w/v) yeast extract, 2% (w/v) Tryptone, 2% (v/v) dex-trose, 1 M sorbitol, and 100 mg/ml Zeocin were used for trans-formation. The expression trial and 1 liter of culture weregrown in BMGY (1% (w/v) yeast extract, 2% (w/v) Tryptone,100 mM potassium phosphate, pH 6.0, 1.34% (v/v) yeast nitro-gen base, 4 � 10�5 % (v/v) biotin, and 1% glycerol). TOP 10 F0E. coli (Invitrogen) was used for the formation of the construct.Drosophila SAS-6 coding sequence from (AT29216) was

inserted into pPICZB between sites EcoRI andNotI to generateSAS-6-Myc-His6. One microgram of SacI-linearized plasmidwas transformed into P. pastorisGS115 cells by electroporation(Bio-Radmicropulser) in a 0.2-cm cuvette. Onemilliliter of 1 M

sorbitol was added, and the electroporated cells were incubatedfor 1 h at 30 °C. Then 1ml of YPDwas added to the cell solutionand incubated with shaking for 1 h. The resulting solution wasspread onto YPDS � Zeocin plates and incubated at 30 °C for 3days. The yeast colonieswere picked aseptically and grownwith1 ml of YPD (yeast extract/peptone/dextrose) tubes containing1 mg/liter selective marker Zeocin. Each colony was PCRscreened for the presence of the insert and then screened forprotein expression. For the expression trial themediumBMMYwas used, and the culture was induced with 0.5% methanolevery 24 h. The protein expression was monitored every 8 huntil the optimum level was reached at 72 h. For preparativecultures the pre-grown nursery cultures from minimal mediawere transferred to 6-liter flasks for the protein expression. Thecultures were microscopically checked for any contamination.The yeast cells were spun down at 5000 rpm, washed with

distilled H2O, and resuspended with lysis buffer that contained6 mM Na2HPO4, 4 mM NaH2PO4, 100 mM NaCl, 2 mM EGTA,100 mM Na3VO4, and 0.1% TritonX1100 and rapidly frozenusing liquid nitrogen. The frozen cells were mechanically dis-rupted using a domestic mixer under frozen conditions or ballmill (Retsch). The disrupted cells were verified under lightmicroscope. Protease inhibitors and 40mM �-mercaptoethanolwere added, and the cell extract wasmixedwell at 4 °C and thensubjected to centrifugation at 30,000� g. The clear supernatantwas used for affinity purification using nickel-nitrilotriaceticacid resins. The binding buffer contained 50 mM imidazole toprevent nonspecific binding, and the final elution was carriedout using 250–500 mM imidazole. When required the proteinsample was concentrated using Millipore spin columns.



at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from


http://www.jbc.org/

RESULTS

SAS-6 Is a Component of the Centriole Central Tubule—Togain structural insights into the centriole central tubule, webiochemically isolated centrosomes from Drosophila embryoextract and processed them for thin-section EM as previouslydescribed (26). Our thin-section EM of chemically fixed iso-latedDrosophila centrosomes showed that the central tubule is25–30 nm in outer diameter (n� 15) (supplemental Fig. S1B) asdemonstrated previously (25). This is similar in size to the cen-tral tubules of most of the model organisms (supplementalTable 1)with the exception of the hexapodAcerentomonmicro-rhinus, where the diameter of the central tubule varied accord-ing to the centriole diameter (27). Analysis of cryo-EM tomo-grams of centrosomes suggested that the central tubule is anextended structure along the axis of the centriole (supplemen-tal Movies 1 and 2). This supported that the central tubule is apolymer of repeating subunits as was previously proposed (sup-plemental Fig. S1) (17).We then examined whether SAS-6 is a component of the

central tubule in Drosophila. Work in human cells has shownthat SAS-6 localizes to the proximal end of centrioles (17). SAS-6-GFP localization in the elongated centrioles of Drosophilaspermatocytes showed that SAS-6 is restricted to the proximalend (Fig. 1A). It was previously reported that SAS-6 is presentthroughout the full length of spermatocyte centrioles whenSAS-6 gene products were expressed using a strong promoter(19).We then performed ultrastructural immunolocalization of

SAS-6 using isolated Drosophila centrosomes. The centro-somes were isolated from extracts of transgenic Drosophilaembryos expressing SAS-6-GFP. For immunostaining, we usedGFP and SAS-6 antibodies separately. Additionally, to distin-guish the pericentriolar material (PCM) portion of the centro-some from the centriole core, we immuno-stained the centro-somes for Asl, a PCM protein (28) (Fig. 1D). Analysis ofimmunogold labeling of cross-sectioned centrioles showed thatin Drosophila, SAS-6 is specifically localized to the centraltubule at the center of the centriole (Fig. 1, Bi and Ci). In longi-tudinally sectioned centrioles, preferential SAS-6 labeling wasfound at the center part of the centriole (Fig. 1, B, ii–iv, and C,ii-iv). Similar results were previously reported inChlamydomo-nas (11) and Tetrahymena (18, 20). These studies indicate thatSAS-6 has an evolutionarily conserved function as a componentof the cartwheel and central tubule.Recombinant SAS-6 Produced in E. coli and P. pastoris Self-

oligomerizes to Form Tetramers—To check if SAS-6 can formsubunits similar to those found in the centriole central tubule,we produced and characterized recombinant SAS-6 in E. coliand in P. pastoris, expression systems that do not have centri-oles. In E. coli, the SAS-6-V5-His fusion protein was expressedas a�55-kDa protein foundmostly in inclusion bodies (supple-mental Fig. S2Ai). However, affinity purification from large vol-ume cultures allowed us to obtain a detectable amount of solu-ble protein (supplemental Fig. S2Aii). First, we analyzed itsoligomerization potential using sucrose gradient sedimenta-tion and size exclusion chromatography. In a 5–40% linear gra-dient, we found an intense signal centered at 7.4 S (Fig. 2A). In

size exclusion chromatography, the majority of SAS-6 waseluted in a manner consistent with a complex with a molecularweight slightly higher than 158 kDa, approximately the sizeof a SAS-6 tetramer and also at the boundary of the exclusionvolume (supplemental Fig. S2B). These data are consistentwith SAS-6 possessing homophilic binding properties form-ing multimers.We further tested if recombinant SAS-6 produced from

E. coli could interact with native SAS-6. We mixed SAS-6-V5-His with fly embryonic extract containing SAS-6-GFP andemployed nickel affinity resin to recover the SAS-6-V5-Hisalong with its binding partners. Elution of the resin followed byWestern analysis showed that recombinant SAS-6-V5-His spe-cifically binds SAS-6-GFP but not Misato, a non-relevant flymitochondrial protein (Fig. 2B).We then examined the purified 7.4 S structures using nega-

tive-stain EM after removing the sucrose by buffer exchange.Importantly, the recombinant SAS-6 generated objects with adiameter of �12 nm with four �6-nm stain excluding regionsarranged in 4-fold symmetry (Fig. 2C). The four stain-excludingregions had identical densities, suggesting that these structuresare tetramers made of SAS-6 monomers. Analysis of the purityof the samples by silver staining detected SAS-6 as the majorcomponent (Fig. 2D).We conducted additional experiments in P. pastoris, an

eukaryotic expression system that may have better proteinexpression and folding properties. Using Myc antibody, wedetected the affinity-purified SAS-6-Myc-His fusion protein asa monomer at �55 kDa in reducing SDS-PAGE (supplementalFig. S2C). However, in the absence of the reducing agent�-mercaptoethanol, we detected oligomerized SAS-6 as asmear at highermolecular weight as well as a distinct signal thatcorresponds to a size of �150 kDa (Fig. 2E). In size exclusionchromatography, we detected a SAS-6 peak between 670 and158 kDa, including a peak at the boundary of the exclusionvolume (supplemental Fig. S2D). SAS-6 expressed from P. pas-toris also bound in a homophilic manner with endogenousSAS-6 from embryonic extract (Fig. 2F).

Like the recombinant SAS-6 produced from E. coli, the den-sity sedimentation of affinity-purified recombinant SAS-6 fromP. pastoris identified a 7.4 S peak. However, we also observedSAS-6-positive fractions around 50 S and higher (Fig. 2G). Neg-ative-stain EM analysis of pooled high density fractions identi-fied SAS-6 tetramers (Fig. 2,Hi andHii) similar to those formedby recombinant SAS-6 produced in E. coli and also agglomer-ates of tetramers (Fig. 2Hiii), some of which exhibited a curvedassembly. One question that remains is how free tetramerscan be present in the high density fractions. One possibilityis that the higher order structures disassociated into stabletetrameric building blocks during the sucrose removal andbuffer exchange process that preceded negative staining offractions. In an attempt to increase the number of particles,we concentrated the SAS-6-containing samples and foundthat SAS-6 was lost during the course of concentration (sup-plemental Fig. S2F), possibly due to its tendency to aggregateat high concentrations.Native SAS-6 Exists as Tetrameric Structures—To test if the

SAS-6 tetramers are functionally relevant, we performed a bio-



at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from


















http://www.jbc.org/

chemical characterization of native SAS-6. A major limitationof using biochemical methods to study centrioles is that mostcells have only two centrioles. To get enough starting material,we harvested large amounts of Drosophila embryos expressingSAS-6-GFP from Drosophila population cages. We fraction-

ated Drosophila embryonic extracts by velocity sedimentation.In a 5–40% linear gradient with 100 mM KCl, a wide distribu-tion of SAS-6-positive fractions was detected. Importantly,under this condition most of SAS-6 was distributed startingaround 7.4 S, where tetramers are fractionated (Fig. 3A). This

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 spermatocytecentrioles. The entire length of the spermatocyte centriole is marked by ANA1-tdTomato, a pan centriole marker (28). B and C, thin-section EM of isolatedDrosophila centrosomes immunogold-labeled with antibodies to GFP (B) and SAS-6 are shown. C, red arrows and circles highlight the immunogoldlabeling at the central tubule (Bi and Ci) and the preferential immunogold-labeling at the center of the longitudinally sectioned centrioles where thecentral 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 thecentrosome (red arrows). Most of them are located outside the yellow circle.



at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from


http://www.jbc.org/



at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from


http://www.jbc.org/

finding suggests that themost stablesoluble form of SAS-6 in cells is tet-ramers, and therefore, the tetramerscould serve as intermediate buildingblocks in assembling the higherorder central tubule structures.In an attempt to enrich core cen-

triolar structures, we increased thesalt concentration from 100 to 500mM, a condition that has previouslybeen used to strip away PCM pro-tein complexes (29). As expected,under these conditions the �-tubu-lin ring complex (�-TuRC) collapsesinto small complexes (�-TuSC) (Fig.3B), and the total protein in the 50 Sfraction was reduced from 213 to130 �g/ml. In addition, SAS-6 wasdetected as a distinct peak with asedimentation coefficient of �50 S(Fig. 3B and supplemental Fig. S3A).As the low density fractions are

rich in various proteins, we initiallypursued a negative stain EM analy-sis of the 50 S SAS-6 rich fractionsand identified a mixture of struc-tures as well as enrichment with tet-ramers (Fig. 3Cii), suggesting thatSAS-6 tetramers are components ofthe 50 S structure.To test if SAS-6 tetramers are

part of the centrosome, we dis-rupted centrosomes at high saltconditions and analyzed usingvelocity sedimentation. Treatingcentrosomes with 0.5 M KCl did notaffect SAS-6 distribution and wasfractionated in the high densityfractions at the bottom of thesucrose gradient (Fig. 3Di). How-ever, when centrosomes are treatedwith 1.5 M KCl, SAS-6 was foundaround 7.4 S where tetramers arefractionated, and only a smallamount of SAS-6 was still detectedin the centrosome fraction (Fig.3Dii). Therefore, the observed SAS-6

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 Sstructures 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 assessedby a pulldown assay of recombinant SAS-6-V5-His using nickel affinity resins. The complex was eluted using 500 mM imidazole and immunoblotted for thepresence 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 fouridentical 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 thepresence of �-mercaptoethanol. F, recombinant SAS-6 from P. pastoris binds to SAS-6-GFP from Drosophila embryo extract. G, affinity-purified SAS-6 run on a15– 60% linear sucrose gradient forms 7.4 S and denser (marked by dashed line rectangle) structures. Sedimentation coefficient markers were run on an identicalgradient. 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) andsubunit-rich agglomerates of tetramers (iii), some of which curve. iv, distribution of particles were observed in a field of 0.45 �m (n � 20).

FIGURE 3. Native SAS-6 forms 7.4 S and 50 S structures. A, SAS-6-GFP Drosophila embryo extract was frac-tionated through a 5– 40% linear sucrose gradient with 100 mM KCl. Fractions were subjected to SDS-PAGE andimmunoblotted for the presence of SAS-6-GFP and Misato. Note that SAS-6 was detected as an intense peakstarting only at around 7.4 S where tetramers are fractionated, whereas the loading control Misato wasdetected right from first fractions where monomers are expected to fractionate indicating undetectable or nofree monomeric SAS-6. B, SAS-6-GFP Drosophila embryo extract fractionated through a 15– 60% linear sucrosegradient with 500 mM KCl is shown. Fractions were subjected to SDS-PAGE and immunoblotted for the pres-ence of SAS-6-GFP and �-tubulin. The fractions containing �-TuSC and centrosomes are indicated as is theexpected region of �-TuRC. Note that at 500 mM KCl, SAS-6-containing centrosomal substructures werestripped, only a 50 S core structure (marked by a square) remained, and �-TuRC collapsed into �-TuSC. C, negative-stain EM of SAS-6-GFP-enriched 50 S fraction (i) detected structures measuring �12 nm with four �6-nmstain-excluding regions (ii). D, centrosomes containing SAS-6-GFP were pretreated with 0.5 or 1.5 M KCl for 2 hat 4 °C and subjected to velocity sedimentation in the presence of 500 mM KCl. No SAS-6-GFP was detected at7.4 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 sizesare fractionated (ii).



at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from



http://www.jbc.org/

at 7.4 S is detached from the centrioles, suggesting that SAS-6in the centriole central tubule is composed of tetramers.Elevated Levels of SAS-6 in Drosophila Cells Induce Microtu-

bule Organizing Centers That Contain Central Tubule-likeStructures—A previous report showed that overexpression ofSAS-6 in Drosophila embryos induces large structures thatfunction as microtubule organizing centers (19). These struc-tures appeared to be hollow tubes of 2.2 times larger in diameterthan endogenous centrioles. This led us to speculate that SAS-6overexpression inDrosophila cells would lead to ectopic SAS-6structures stable enough to be fractionated and analyzed byEM. We generated Drosophila S2 cells stably overexpressingSAS-6-GFP. These cells showed at least a 25-fold increase inSAS-6 as compared with untransfected control cells (supple-mental Fig. S4A). However, �-tubulin was observed in similaramounts in the control cells and in SAS-6-GFP cells, suggestingthat elevation of SAS-6 levels does not lead to an increase inother centrosomal proteins.Immunofluorescence analysis revealed that SAS-6-GFP pre-

dominantly localized at the mitotic spindle poles or centro-somes during mitosis with little distribution in the cytoplasm(supplemental Fig. S4Bi). During interphase, most cytoplasmicSAS-6-GFP concentrated into multiple GFP foci emanatingmicrotubule asters (supplemental Fig. S4Bii), suggesting thatelevated levels of SAS-6 induce the assembly of ectopic struc-tures. Indeed, thin-section EM analysis on an interphase cellconfirmed that these structures are distinct and are absent fromuntransfected control cells (supplemental Fig. S4, C and D).Serial-sectionEManalysis (supplemental Fig. S4D) showed thatthe ectopic structures measured 200–250 nm in length andemanate microtubule asters, indicating that these structurescan function as microtubule organizing centers. Such ectopicstructures and robust microtubule asters were not detected incontrol interphase cells (n� 7). Additionally, normal centriolesin Drosophila cells at the interphase are known to lack micro-tubule-nucleating activity (30).Fractionation of SAS-6-GFP cell extract in the presence of

100 or 500 mM KCl indicated that the ectopic structures weredenser than 50 S (Fig. 4A). Although they are capable of recruit-ing �-tubulin (Fig. 4, Ai), these structures were distinct fromnormal centrosomes that are fractionated at the high density atthe end of the gradient (Fig. 4A). In the presence of 500mMKCl,�-tubulin was stripped from these structures without affectingtheir density (Fig. 4, Aiii), indicating that �-tubulin is notrequired to stabilize them.As in embryonic extracts, uponnegative stain EManalysiswe

detected tetrameric objects in the SAS-6-positive 50 S fractions(Fig. 4B). Importantly, the fractions denser than 50 S containedlarge and elongated higher order structures (Fig. 5, Ci) harbor-ing identical rings (n � 13) of �25 nm in diameter (Fig. 5, C, iiand iii). The presence of these higher order ring structures infractionated Drosophila cell extract and their absence in yeastsuggests that an unknown centriolar factor is required for theorganization of SAS-6 tetramers into stable higher order cen-tral tubule-like structures.7.4 S SAS-6 Tetramers Are Intermediates in the Disassembly

of Higher Order Structures—To evaluate directly whether ornot the 7.4 S tetrameric structures are structural intermediates

during the assembly and disassembly of higher-order struc-tures, we took a biochemical approach. We established Dro-sophila cells stably expressing SAS-6-GFP-FLAG. Cell extractswere subjected to velocity sedimentation in a linear sucrosegradient of 15–60%, allowing for isolation of low density(around 7.4 S) and high density (50 S and higher) structures, asdescribed in Fig. 4A. The low and high density pools were thenseparately subjected to affinity purification using affinity resinscoated with FLAG antibody. We then performed velocity sedi-mentation of purified SAS-6 complexes obtained fromboth lowand high density fractions (Fig. 5A). As expected, the SAS-6complex purified from the low density pool was fractionated at7.4 S, indicating that the low density SAS-6 complex is stable(Fig. 5Ai).Importantly, after velocity sedimentation the purified high

density SAS-6 complexes also fractionated at 7.4 S even thoughthey were expected to fractionate above 50 S (Fig. 5Aii). Theobservation that 7.4 S structures result when high density (50 Sand above) SAS-6 complexes are subjected to velocity sedimen-tation indicates that stable 7.4 S structures are constitutivestructural components of less stable higher order structures.We next used negative-stain EM to analyze the structures

that fractioned at 7.4 S after velocity sedimentation of affinity-purified low density complexes.We detected three populationsof objects with distinct morphology. Analysis of the objects(Fig. 5,B, i and ii) revealed the presence of tetrameric structures(Fig. 5Biii), tetrameric structures with appendages (Fig. 5Biv),and curved structures with recognizable subunits (Fig. 5Bv). SThese structures are similar to objects observed in native(Figs. 3C and 4B) and recombinant (Fig. 2, C and H) SAS-6preparations.Of significance, negative-stain EM analysis of the 7.4 S frac-

tions obtained after velocity sedimentation of affinity-purifiedhigh density SAS-6 complexes also identified tetrameric objects(Fig. 5C, i and ii) and curved structures (Fig. 5Ciii), present inroughly the same ratio as before (Fig. 5D). These datastrengthen the argument that observed 7.4 S structures areindeed structural intermediates that are found after the disas-sembly of higher order structures.To check the specificity of all these 7.4 S structures, we used

silver-stain PAGE to analyze purity. The fractions used for EManalysis that sediment as low density complexes identified thepresence of SAS-6 and a few other proteins (Fig. 5E). It is pos-sible that these other proteins are SAS-6 interacting partners.Similar results were also obtained when we analyzed the frac-tions used for EM analysis that sediment as high density SAS-6complexes (Fig. 5F).7.4 S SAS-6 Tetramers Are Building Blocks of a Higher Order

Complex—Having speculated that SAS-6 tetramers are compo-nents of high density native 50 S structures, we further recipro-cally tested if the tetramers could indeed contribute to theassembly of higher order structures. To this end we mixed thepurified 7.4 S structures of recombinant SAS-6 fromE. coli (Fig.2A) and SAS-6-GFP-FLAG fromDrosophila cells (Fig. 5A) withlow density embryonic extract, and the complex was subjectedto velocity sedimentation. We found that combining SAS-6from the two different sources resulted in the formation ofcomplexes that are significantly denser than the individual



at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from








http://www.jbc.org/

components alone. Although the recombinant endogenousSAS-6 complex resulted in 11.4 S structures (Fig. 6A), the SAS-6-GFP-FLAG with embryonic extract resulted in structuresthat are fractionated around 30 S (Fig. 6B). This is more similarto the native 50 S SAS-6 core. One possible explanation is thatthe SAS-6 protein is properly folded when expressed in Dro-sophila cells plus the presence of additional factors that arerequired to enhance the structural assembly. We, however, didnot perform EM analysis of these complexes as they containedlow amounts of specific complexes in cellular extract andbecause of the instability of higher order structures for EMprocessing. Collectively, the capacity of tetramers to form highdensity complexes suggests that the SAS-6 tetramers couldserve as building blocks in the assembly of higher order struc-

tures that require endogenous factors present in embryonicextract for central tubule assembly.

DISCUSSION

Centriole biogenesis includes an early intermediateknown as the procentriole (10). The cartwheel, a conservedcentriolar scaffold, appears at the onset of procentrioleassembly (31). The absence of a cartwheel leads to the for-mation of centrioles and cilia with disrupted symmetry (11,19). To date, SAS-6 is one of the clearly conserved centrioleproteins, which is a component of the cartwheel (11, 18, 20).However, the precise function of SAS-6 in general and incartwheel formation remained unclear. The current studyprovides structural insights into the formation of a SAS-6

FIGURE 4. SAS-6 overexpression in Drosophila cells induces higher order ectopic complexes containing 25-nm ring-like substructures. A, fractionationof Drosophila cells overexpressing SAS-6-GFP at 100 mM KCl identifies a SAS-6-induced ectopic structure (marked by a rectangle) that co-fractionates with a�-tubulin peak (marked by a rectangle) (i). A measurement of the intensity of the protein signals in each fraction (graphs beneath the Western blots) showsdiscernible peaks for SAS-6 and �-tubulin. Unlike native SAS-6 structures, the SAS-6-induced ectopic structure is denser than 50 S (compare with Fig. 3B). ii, incontrol un-transfected cells the �-TuSC and �-TuRC are intact, and there was no �-tubulin enrichment in the fractions corresponding to the SAS-6-inducedectopic structure. iii, at 500 mM, SAS-6-induced structures are stable, and the fractions do not contain �-tubulin. B, negative-stain EM of the �50 S fraction(square 1 in Fig. 4Aiii) showing structures measuring �12 nm with four �6-nm stain-excluding regions (4 � 1 particles in a field of 0.45 �m, n � 5).C, negative-stain EM images of SAS-6-rich fraction (square 2 in Fig. 4Aiii) reveal large ectopic structures (13 � 1 observed in a field of 0.45 �m, n � 5) (i) thatcontain 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 toindividual subunits.



at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from


http://www.jbc.org/

structural unit with a 4-fold symmetry that could serve as abuilding block for the cartwheel central tubule.It remains unclear whether there is an upstream signal

that defines the site of procentriole assembly and triggersSAS-6 tetramers to get assembled. ZYG-1 and PLK4 kinasesare likely components of the upstream signaling cascade thatinduces centriole nucleation (21, 32). We have recentlyshown that the protein “Asterless” (Asl) is a PCM proteincritical for centriole formation and functions early in thecentriole duplication process (28). It would be interesting tofurther delineate the place of SAS-6, ZYG-1/PLK4, and Aslin the assembly of the central tubule and subsequent procen-triole formation.

Importantly, in Chlamydomonas (11), Drosophila (19), andTetrahymena (20), outer centriole components do appear inthe absence of SAS-6, and in both Chlamydomonas and Dro-sophila the 9-fold symmetry is disrupted. The fact that outercentriole components, such as the microtubules, can assembleindependently of the cartwheel suggests that the primary role ofthe cartwheel is to generate symmetry. It should be noted, how-ever, that structural studies in C. elegans did not find a cart-wheel with a central tubule. Instead, a central tube measuring60 nm is connected directly to the microtubules (21). In thissystem SAS-6 physically interacts with SAS-5 (16), a proteinthat is not conserved in other organisms that possess centrioles.Both SAS-6 and SAS-5 are required for the formation of the

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 intermediatesthat 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 inAi) 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-�m 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 expressingSAS-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, shownare 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) andanti-SAS-6 (iii).



at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from


http://www.jbc.org/

central tube inC. elegans (21). It is possible that the central tubein C. elegans is a heterodimer made of both SAS-5 and SAS-6,which would account for its larger size. Future studies shouldbe able to test this hypothesis by performing in vitro experi-ments in C. elegans similar to those described here.The 7.4 S tetrameric structures formed by recombinant

SAS-6 expressed in E. coli highlight the self-oligomerizingcapacity of SAS-6 (Fig. 2C) even in a prokaryotic system thatmay not effectively facilitate the proper folding of the protein.The high density structures obtained from P. pastoris suggestthat a eukaryotic system enhances the oligomerizing propertiesof SAS-6 even in the absence of additional centriolar specificproteins. Not surprisingly, the presence of additional factors inDrosophila cells seems to further influence SAS-6 assembly, asevidenced by the successful fractionation of native 50 S SAS-6rings from Drosophila embryos. As for the stability of differentstructural intermediates of increasing complexity, the results ofaffinity purifying subunit-rich higher order structures fromDrosophila cells overexpressing SAS-6 are informative. Theenrichment of tetramers and curved structures harboring sub-unit repeats (Fig. 5C) show that the 7.4 S structures are stableintermediates that remain intact after the disassembly of higher

order structures (Fig. 5A). Impor-tantly, the ability of SAS-6 tetramersto form higher order structures invitro when combined with embry-onic extract suggest that the SAS-6tetramers serve as stable intermedi-ates in assembling higher orderstructures. This indicates that inaddition to SAS-6 tetramers, centraltubule assembly also requires otherunknown factors present in vivo.The finding that the abundance

of SAS-6 present in the cell exist astetramers (Fig. 3A and supplemen-tal Fig. S3) that tetramers re-sult from disassembly of SAS-6higher-order structures (Fig. 5A),and that tetramers assemble intohigher order structures (Fig. 6)suggest that SAS-6 tetramers andadditional factors are required toassemble into a higher order com-plex. Therefore, future biochemicaland in vitro reconstitution studiesshould be able to uncover the fac-tors that are essential for the assem-bly and stability of the centraltubule.Previously, it was proposed that

nonameric rings of SAS-6 form thecentral tubule with a one subunitthick (17). Future structural studiesare required to test this model. Ourstudies show that SAS-6 forms sta-ble tetramers, and therefore, thecentral tubule may be made of tet-

ramers. This can be tested in the future by generating muta-tions in SAS-6 that prevent the formation of tetramers and leadto defects in central tubule. These studies will require the iden-tification of amino acids that are found at the interphasebetween SAS-6 monomers.Theoretically, tetramers can be organized into the central

tubule at least in three ways. (i) A ring of tetramers where eachtetramer is placed perpendicular to the axis of the central tubuleis one possibility. In this case, the central tubule wall in thecross-section would be of two subunits thick. Precise mea-suring of the width of the central tubule wall after fine structuralstudies would test this model. (ii) A ring of tetramers that facesthe central tubule axis resulting in a one-subunit thick centraltubule wall is another possibility. This organization cannot befit into a 9-fold symmetry as circumference of 9 tetramerswould be larger than that of the central tubule; given that eachtetramer has a 12-nm in diameter, any closed disk would have aperimeter of at least 108 nm (12 nm� 9). From the experimen-tal data, we have observed that the central tubule has a perim-eter that can be estimated to be between 79nm (� � 25 nm) and94 nm (� � 30 nm) (considering the diameter of the centraltubule is 25–30 nm), which is less than theminimumperimeter

FIGURE 6. In vitro assembly of higher order structures from 7.4 S SAS-6 structures. A, the 7.4 S recombinantSAS-6 from E. coli (Fig. 2A) and SAS-6-GFP from low density embryonic extract (fractions around 7.4 S from Fig.3B) form an 11.3 S complex. B, in the presence of embryonic extract, purified 7.4 S SAS-6-GFP-FLAG fromDrosophila cells (A) assembled into higher order structures that are fractionated at around 30 S. Such a shift indensity is not observed for Misato or when the SAS-6 from E. coli or Drosophila cells was incubated with theembryonic extract buffer. An equivalent amount of purified SAS-6 from E. coli and Drosophila cells (6 �g/ml)was mixed with (3 mg/ml) of low density embryonic extract. The mixture was incubated for 2 h at 4 °C beforebeing subjected to velocity sedimentation in a 15– 60% gradient.



at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from




http://www.jbc.org/

required for a stack of disks (108 nm). (iii) A helice of tetramersis a third possibility. If a 9-fold symmetrical central tubule onesubunit thick is assumed, then the central tubulewould be orga-nized into 4.5 tetramers per turn so that one turn correspondsto a nine-subunit repeat. Future detailed structural studies arerequired to elucidate the internal organization of SAS-6 in thecentral tubule.It was also proposed that SAS-6 organizes the procentriole by

orienting and holding a preassembled structure that corre-sponds to 1/9 of the centriole, which are named as enatosomes(19, 22). It is possible that SAS-6 tetramers and their assemblyto higher order structures are the basic structural units medi-ating the organization of enatosomes.It has been decades since EM was first used to reveal the

centriole structure, andmultiple RNAi and genetic studies haveindicated a role for SAS-6 early in centriole formation (11,15–17, 19, 21), but technical challenges have prevented analysisof the central tubule and its internal organization. The resultsfrom the current study suggest that SAS-6 self-assembles intotetramers that serve as building blocks during assembly of acartwheel central tubule.

Acknowledgments—We thank Dr. Janet Iwasa for scientific illustra-tions, Prof. F. Mckeon forMyc antibody, and Dr. M. Bettencourt-Diasfor technical discussions and the generous gift of chicken SAS-6 anti-body and a SAS-6 bacterial plasmid.We thank Prof.Moazed from theDepartment of Cell Biology for reading and commenting on themanuscript. We are grateful to members of the Department of CellBiology Drs. Karim Mekhail, Aaron Johnson, Daniel Nedelcu, EricaGerace, Yougen Zhan, Hari, Saravanan, and collegues Stephanie Bla-chon, Kela Roberts, and Marcus Basiri for technical help and discus-sions. We also thank Dr. Dirk Linke, MPI-Tubingen, Germany, fortechnical suggestions regarding protein purification and Prof. Sus-umu Ito and the EM facility at the Harvard Medical School for gen-erous help with EM analysis.

REFERENCES1. Azimzadeh, J., and Bornens, M. (2004) in Centrosomes in Development

and Disease (Nigg, E. A., ed.) pp 93–116, Wiley-VCH, Weinheim,Germany

2. Dippell, R. V. (1968) Proc. Natl. Acad. Sci. U.S.A. 61, 461–4683. Vorobjev, I. A., and Chentsov, Yu. S. (1982) J. Cell Biol. 93, 938–9494. Rosenbaum, J. (2002) Curr. Biol. 12, R1255. Scholey, J. M., and Anderson, K. V. (2006) Cell 125, 439–4426. Badano, J. L., Teslovich, T. M., and Katsanis, N. (2005)Nat. Rev. Genet. 6,

194–2057. Gonzalez, C., Tavosanis, G., and Mollinari, C. (1998) J. Cell Sci. 111,

2697–27068. Phillips, D. M. (1967) J. Cell Biol. 33, 73–92

9. Rodrigues-Martins, A., Riparbelli, M., Callaini, G., Glover, D. M., andBettencourt-Dias, M. (2007) Science 316, 1046–1050

10. Azimzadeh, J., and Bornens, M. (2007) J. Cell Sci. 120, 2139–214211. Nakazawa, Y.,Hiraki,M., Kamiya, R., andHirono,M. (2007)Curr. Biol.17,

2169–217412. Strnad, P., and Gonczy, P. (2008) Trends Cell Biol. 18, 389–39613. Cavalier-Smith, T. (1974) J. Cell Sci. 16, 529–55614. Satir, P., and Satir, B. (1964) J. Theor. Biol. 7, 123–12815. Dammermann, A., Muller-Reichert, T., Pelletier, L., Habermann, B., De-

sai, A., and Oegema, K. (2004) Dev. Cell 7, 815–82916. Leidel, S., Delattre, M., Cerutti, L., Baumer, K., and Gonczy, P. (2005)Nat.

Cell Biol. 7, 115–12517. Strnad, P., Leidel, S., Vinogradova, T., Euteneuer, U., Khodjakov, A., and

Gonczy, P. (2007) Dev. Cell 13, 203–21318. Kilburn, C. L., Pearson, C. G., Romijn, E. P., Meehl, J. B., Giddings, T. H.,

Jr., Culver, B. P., Yates, J. R., 3rd, and Winey, M. (2007) J. Cell Biol. 178,905–912

19. Rodrigues-Martins, A., Bettencourt-Dias, M., Riparbelli, M., Ferreira, C.,Ferreira, I., Callaini, G., and Glover, D. M. (2007) Curr. Biol. 17,1465–1472

20. Culver, B. P., Meehl, J. B., Giddings, T. H., Jr., and Winey, M. (2009)Mol.Biol. Cell 20, 1865–1877

21. Pelletier, L.,O’Toole, E., Schwager, A.,Hyman,A.A., andMuller-Reichert,T. (2006) Nature 444, 619–623

22. Rodrigues-Martins, A., Riparbelli, M., Callaini, G., Glover, D. M., andBettencourt-Dias, M. (2008) Cell Cycle 7, 11–16

23. Alvey, P. L. (1986) Cell Biol. Int. Rep. 10, 589–59824. Hiraki,M.,Nakazawa, Y., Kamiya, R., andHirono,M. (2007)Curr. Biol.17,

1778–178325. Callaini, G., Whitfield, W. G., and Riparbelli, M. G. (1997) Exp. Cell Res.

234, 183–19026. Moritz, M., Braunfeld, M. B., Fung, J. C., Sedat, J. W., Alberts, B. M., and

Agard, D. A. (1995) J. Cell Biol. 130, 1149–115927. Riparbelli, M. G., Dallai, R., Mercati, D., Bu, Y., and Callaini, G. (2009)Cell

Motil. Cytoskeleton 66, 1100–110528. Blachon, S., Gopalakrishnan, J., Omori, Y., Polyanovsky, A., Church, A.,

Nicastro, D., Malicki, J., and Avidor-Reiss, T. (2008) Genetics 180,2081–2094

29. Moritz, M., Zheng, Y., Alberts, B. M., and Oegema, K. (1998) J. Cell Biol.142, 775–786

30. Rogers, G. C., Rusan, N. M., Peifer, M., and Rogers, S. L. (2008)Mol. Biol.Cell 19, 3163–3178

31. Anderson, R. G., and Brenner, R. M. (1971) J. Cell Biol. 50, 10–3432. Kleylein-Sohn, J., Westendorf, J., Le Clech, M., Habedanck, R., Stierhof,

Y. D., and Nigg, E. A. (2007) Dev. Cell 13, 190–20233. Abramoff, M., Magelhaes, P., and Ram, S. (2004) Biophoton Int 11, 36–4234. Sorzano, C. O., Marabini, R., Velazquez-Muriel, J., Bilbao-Castro, J. R.,

Scheres, S. H., Carazo, J. M., and Pascual-Montano, A. (2004) J. Struct.Biol. 148, 194–204

35. Fernandez, J. J., and Li, S. (2003) J. Struct. Biol. 144, 152–16136. Crowther, R. A., and Amos, L. A. (1971) J. Mol. Biol. 60, 123–13037. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt,

D. M., Meng, E. C., and Ferrin, T. E. (2004) J. Comput. Chem. 25,1605–1612



at UC

SF

Library & C

KM

, on March 12, 2010

ww

w.jbc.org

Dow

nloaded from


http://www.jbc.org/

self-assemblingsas-6multimerisacorecentriole … · self-assemblingsas-6multimerisacorecentriole...

Documents