Reconstitution of centrosome microtubule nucleation in Drosophila

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Reconstitution of Centrosome Microtubule Nucleation in Drosophila Michelle Moritz, Michael B. Braunfeld,* Bruce M. Alberts, t and David A. Agard* D e p a r t m e n t o f Biochemistry and Biophysics and * H H M I University o f California-San Francisco San Francisco, California 94143 t N a t i o n a l A c a d e m y o f Sciences Washington, D C 20418

I. Introduction II. Preparation of Extracts and Centrosomes from Drosophila Embryos III. In Vitro Reconstitution of Microtubule Nucleation by Salt-Inactivated Centrosomes A. Buffers, Solutions, and Special Equipment B. Inactivation of Centrosomes by Treatment with Potassium Iodide and in Vitro Complementation of Salt-Stripped Centrosomes IV. Conclusions References

I. Introduction In vitro complementation assays provide a powerful means of identifying and studying the key components of complex biological processes. Such assays involve mixing a stripped-down core element, or substrate (e.g., an organelle, a DNA, or RNA template), that is defective in the function of interest, with a cell extract or purified protein whose role in the process is in question. The end product can then be examined to determine what effect the addition of the extract or protein had on the inactive substrate. Classic examples of in vitro complementation assays include those used to study DNA replication (Barry and Alberts, 1972) and the Golgi apparatus (Fries and Rothman, 1980).

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We have successfully used the in vitro complementation assay described in this chapter to determine that the y-tubulin ring complex (y-TuRC) is required for Drosophila centrosomes to nucleate microtubules. The assay is based on earlier studies of complementation of inactivated mammalian centrosomes by Xenopus egg extract (Buendia et al., 1992; Klotz et al., 1990). The assay described here involves inactivating isolated Drosophila centrosomes with potassium iodide (KI), which leaves a salt-resistant core "scaffold," and then complementing this inactive core with an embryo extract or pure proteins (Fig. 1). Fortuitously, these experiments uncovered an unexpected requirement for an additional factor that appears to attach the y-TuRC to the centrosome (Moritz et aL, 1998). An added benefit is that the salt-stripped centrosome scaffolds are interesting in themselves: relatively few proteins remain in the structure and as yet are unknown (Fig. 2). Electron microscopy revealed that the scaffold proteins appear to be organized into ~ 10-nm filaments (data not shown). The complementation assay has thus provided a means of at least partially assembling the centrosome in vitro and of characterizing proteins involved in microtubule nucleation, as well as those to which the microtubulenucleating machinery attaches.

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Fig. 1 Complementation assay. Centrosomes isolated from Drosophila embryos are inactivated by incubating with 2 M KI. The KI-treated centrosomes are allowed to bind to a glass coverslip. The coverslip is washed and blocked with a low-salt, BSA-containing buffer and then incubated with the extract or fraction to be tested. The extract/fraction is washed away and the coverslip is incubated with rhodamine-labeled tubulin. Any resulting asters are fixed sequentially with glutaraldehyde and methanol. The asters are viewed under a fluorescence microscope. Reproduced from Moritz et aL (1998), with permission of The Rockefeller University Press.

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Fig. 2 Effects of 2 M KI treatment on centrosomes. (A) The profile of centrosomal proteins is simplitied by treatment with 2 M KI. Isolated centrosomes (~3 × 107) were mixed with an equal volume of 1X BRB80 + 4 M KI (left) or 1X BRB80 (right), incubated on ice for 10 min, and then pelleted by centrifugation at 30,000g for 1 h (16,000 rpm) in an SW41 rotor. The pellets were washed three times with 1X BRB80 and resuspended in sample buffer, boiled, and separated by SDS-PAGE on a 10% gel. The gel was silver stained. (B) Removal of CP60, CP 190, y-tubulin, and centrosomin from centrosomes by 2 M KI. Centrosomes (~5 × 106) were mixed with an equal volume of either 1X BRB80 + 4 M KI (left) or 1X BRB80 (right) and incubated on ice for 10 min. Centrosomes were pelleted by centrifugation at 30,000g for 15 min, washed with 1X BRB80, and resuspended in sample buffer for SDS-PAGE. Proteins released from centrosomes into the supernatants by the KI or buffer treatments were precipitated with 10% TCA and resuspended in sample buffer for SDS-PAGE. The presence of centrosomal proteins CP60, CP 190, y-tubulin, and centrosomin in the pellets (P) and supernatants (S) was determined by immunoblotting. (Top) CP60 was completely solubilized by KI and partly solubilized by buffer. (Middle) Most CP190 was solubilized by KI, but not by buffer. (Bottom) y-Tubulin and centrosomin were completely solubilized by KI, but not by buffer. (C) Electron micrographs of negative-stained KI- and buffer-treated centrosomes reveal that salt-inactivated centrosomes retain a core, scaffold-like structure, despite the removal of many proteins and partial extraction of the centrioles. Bar: 200 nm. Parts A and B reproduced from Moritz et al. (1998), with permission of The Rockefeller University Press.

II. Preparation o f Extracts and C e n t r o s o m e s f r o m Drosophila E m b r y o s R e c o n s t i t u t i o n o f m i c r o t u b u l e n u c l e a t i o n b y s a l t - s t r i p p e d c e n t r o s o m e s b e g i n s w i t h the p r e p a r a t i o n o f active c e n t r o s o m e s a n d c o n c e n t r a t e d e x t r a c t f r o m 0- to 3-h Drosophila e m b r y o s . D e t a i l e d m e t h o d s for the i s o l a t i o n o f Drosophila c e n t r o s o m e s ( M o r i t z a n d A l b e r t s , 1999) a n d the p r e p a r a t i o n o f e m b r y o n i c extract ( M o r i t z , 2 0 0 0 ) h a v e b e e n

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published and will not be repeated here, except to note that large quantities (--~1-5 × 107) of centrosomes can be isolated, flash frozen in liquid nitrogen, and stored as small aliquots for many months at -80°C. The frozen centrosomes can be thawed, treated with salt (see Section HI,B), and used in the complementation assay. Similarly, the complementing extract can be prepared in advance, and a low-speed (1500g) postnuclear supernatant can be stored at -80°C for months. This semicrude extract is simply thawed and centrifuged (e.g., at 100,000g) a second time to generate a high-speed supernatant that can be used in the complementation assay. Alternatively, fractions of the extract or purified proteins may be tested for their ability to restore microtubule-nucleating activity.

III. In Vitro Reconstitution o f Microtubule Nucleation by Salt-Inactivated Centrosomes In this procedure, isolated centrosomes are incubated with 2 M KI, which removes most proteins from the structure, including y-tubulin, centrosomin, CP60, and CP190 (Figs. 1 and 2) (Kellogg and Alberts, 1992; Kellogg et al., 1989; Li and Kaufman, 1996; Moritz et al., 1998; Oakley and Oakley, 1989; Whitfield et al., 1988). The remaining salt-resistant core scaffold is inactive for microtubule nucleation, but this ability can be restored by incubation with the high-speed supernatant of an embryo extract, or fractions thereof [Figs. 1 and 3 (Moritz et al., 1998)]. Other studies have shown that centrosomes can also be inactivated by treatment with KC1, NaC1, urea, or proteases (Buendia et al., 1992; Klotz et al., 1990; Kuriyama, 1984; Mitchison and Kirschner, 1984; Ohta et al., 1993).

A. Buffers, Solutions, and Special Equipment 1. Buffers and Solutions Isolated Drosophila centrosomes: freshly prepared or from a frozen stock (see Section II) 5X BRB80:400 mM K-PIPES, pH 6.8, 5 mM MgC12, 5 rnM Na2EGTA 4 M KI in 1X BRB80; prepare fresh from solid KI just before use GTP stock: 0.5 M GTP (Sigma Chemical Co., St. Louis, MO) in 1X BRB80; store at -20°C in small aliquots. HEPES block: 50 mM K-HEPES, pH 7.6, 100 mM KC1, 1 mM MgClz, 1 mM Na3EGTA, 10 mg/ml bovine serum albumin (BSA; fraction V, Sigma Chemical Co.), 1 mM/%mercaptoethanol Tubulin dilution buffer (TDB): 1X BRB80, 10% glycerol, 1 mM GTP (add GTP from 0.5 M GTP stock just before use) TDB wash: TDB + 10 mg/ml BSA (fraction V, Sigma Chemical Co.) + 1 mM GTP (add GTP from 0.5 M GTP stock just before use)

9. Reconstitution of Centrosome Microtubule Nucleation

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Tubulin: Purified from bovine brain (Mitchison and Kirschner, 1984) Fluorescenfly labeled tubulin prepared as described previously (Hyman et al., 1991) or antibodies against ot-tubulin (DM1A: monoclonal anti-u-tubulin, T-9026, Sigma Immuno Chemicals, St. Louis, MO) and fluorescently labeled antimouse secondary antibodies 1% glutaraldehyde in 1X BRB80, made from a 50% stock (Electron Microscopy Grade, Ted Pella, Redding, CA); prepare fresh just before use Methanol at - 2 0 ° C Mounting medium: 80% glycerol in PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 136 mM NaC1, 2.6 mM KC1, pH 7.2) + 1 mg/ml p-phenylenediamine; store in the dark at - 2 0 ° C or - 8 0 ° C 2. Special Equipment Acid-washed, poly-L-lysine-coated, 12-mm round glass coverslips: Prepare large batches of these by incubating the coverslips in a large glass beaker with 1 N HC1 at 65°C for 4 h to overnight with occasional swirling. Rinse the coverslips extensively in dH20 until the pH is neutral (check rinse water with pH paper) and then incubate in 0.1% (w/v) poly-L-lysine for 20 min. Rinse the coverslips in dH20 again. Dry the coverslip's in a drying oven or by laying them out on a large piece of filter paper. Be careful not to let lint or dust collect on them. After drying, store the coverslips in a large, covered petri dish. Water bath at 30°C with a support to hold a Petri dish so that its bottom is immersed in the water. Humid petri dish to hold coverslips during the assay: Cover the bottom of the dish with a piece of Parafilm "M" (American National Can, Chicago, IL). Twist a Kimwipe and use it to line the edge of the bottom of the dish. Wet the Kimwipe with water. Immerse the bottom of the dish in the 30°C water bath.

B. Inactivation o f C e n t r o s o m e s by T r e a t m e n t w i t h P o t a s s i u m I o d i d e and in Vitro C o m p l e m e n t a t i o n o f Salt-Stripped C e n t r o s o m e s

1. Set one acid-washed, poly-L-lysine-coated coverslip on Parafilm in a humidified, 30°C petri dish (see Section III,A,2) for each complementation assay or control to be performed. 2. Place HEPES block and TDB wash in the 30°C water bath to warm. 3. To destroy the microtubule-nucleating activity of centrosomes, mix equal volumes 4 M KI in 1X BRB80 and centrosomes and incubate on ice for 10 rain. The volume needed depends on the number of assays to be performed: each assay requires 20 Id of inactivated centrosomes. 4. Apply 20/zl of salt-inactivated centrosomes to each coverslip. Allow the centrosomes to bind to the coverslip for 5 min.

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Fig. 3 Examples of complementation of KI-treated centrosomes. The F-tubulin ring complex (F-TuRC) is necessary but not sufficient for the complementation of salt-stripped centrosomes. The complementation assay was carried out as outlined in Fig. 1 and Section III. (A) Microtubule asters regrew on buffer-treated centrosomes that were incubated with rhodamine-labeled tubulin. (B) Microtubules, but no asters, formed when a 228,000g supernatant from a 0- to 2-h embryo extract was incubated with rhodamine-tubulin in the absence of centrosomes. (C) When KI-treated centrosomes were incubated with buffer instead of extract, few microtubules and no asters formed. (D) KI-treated centrosomes were complemented by extract and formed asters in the presence of rhodamine-tubnlin. (E) KI-treated centrosomes were not complemented by F-tubulin-depleted extract. (F) KI-treated centrosomes were not complemented by immunoaffinity-purified F-TuRC, although microtubules could form. (G) Asters formed after incubation of KI-treated centrosomes with a 1:1 mixture of immunoaffinity-purified F-TuRC and F-tubulin-depleted extract. (H) Microtubules but no asters formed when a 1:1 mixture of immunoaffinity-purified F-TuRC and F-tubulin-depleted extract were incubated in the absence of centrosomes. Bar: 10/zm. Reproduced from Moritz et al. (1998), with permission of The Rockefeller University Press.

9. Reconstitution of Centrosome Microtubule Nucleation

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5. Wash briefly by pipetting on and aspirating off 3 x 60/zl 30°C HEPES block. For controls in which the centrosomes are omitted, wash the coverslips in the same way. Incubate the coverslip in the final wash 5 min. Note: If proteins to be tested for complementing ability are in a buffer other than HEPES, the HEPES buffer in the HEPES block should be replaced with this buffer. 6. Apply 10-60/~1 of the complementing extract, control buffer, or other sample (e.g,, purified protein) to be tested. Incubate for 10 min and then wash briefly with 3 × 60/xl 30°C TDB wash. 7. Apply a 25/xl a 1:7 ratio, but this lin) diluted to 2-2.5 bate with unlabeled (see step 8).

mixture of fluorescently labeled and unlabeled tubulin (usually in must be determined empirically for each batch of labeled tubumg/ml in TDB. Incubate for 10 min at 30°C. Alternatively, incutubulin and stain with antibodies against a-tubulin after fixation

8. Fix any resulting microtubules or asters by a 3-min incubation with 60 /zl 1% glutaraldehyde in 1X BRB80, move the petri dish to the bench top (room temperature), and then perform a 3-min incubation in 60 # 1 - 2 0 ° C methanol. If unlabeled tubulin was used, stain the asters as follows: a. Rehydrate the structures by washing the coverslips with 3 x 60/zl PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 136 mM NaCI, 2.6 mM KC1, pH 7.2). b. Inactivate residual glutaraldehyde by incubating in 60 #10.1% sodium borohydride for 7 min. c. Wash briefly with 3 x 60/zl PBS + 3% BSA. Incubate in final wash 5 min. d. Incubate for 1 h in anti-a-tubulin diluted 1:1000 in PBS + 3% BSA and wash 3 × 60 #1 for 5 min each in PBS + 0.1% Tween 20. e. Incubate for 1 h in fluorescently labeled secondary antibodies diluted in PBS + 0.1% Tween 20. f. Wash 3 x 60 #1 for 5 min each in PBS + 0.1% Tween 20. Wash briefly in 2 × 60/zl PBS. 9. Invert the coverslips onto small drops (~2 mm in diameter) of mounting medium on slides for viewing in the fluorescence microscope (view at 100X). Examples of assay results are shown in Fig. 3. The assay can also be quantitative (data not shown, see Moritz et al., 1998).

IV. Conclusions The complementation assay described here can be used to study proteins involved in microtubule nucleation as well as centrosome composition and assembly in a very powerful way. Several studies have shown that the components of this system are functionally highly conserved; components from a variety of organisms can be mixed, resulting in the restoration of microtubule-nucleating activity (Buendia et al., 1992; Klotz et al., 1990;

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Schnackenberg et al., 2000). Thus, the assay should be useful for studying centrosomes from a variety of organisms. Acknowledgments We thank Hector Aldaz for helpful comments on the manuscript. This work was supported by the Howard Hughes Medical Institute and NIH Grants GM31627 to D.A.A. and GM23928 to B.M.A.

References Barry, J., and Alberts, B. (1972). In vitro complementation as an assay for new proteins required for bacteriophage T4 DNA replication: Purification of the complex specified by T4 genes 44 and 62. Proc. Natl. Acad. Sci. USA 69, 2717-2721. Buendia, B., Draetta, G., and Karsenti, E. (1992). Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: Role of cyclin A-associated protein kinase. J. Cell Biol. 116, 1431-1442. Fries, E., and Rothman, J. E. (1980). Transport of vesicular stomatitis virus glycoprotein in a cell-free extract. Proc. Natl. Acad. Sci. USA 77, 3870-3874. Hyman, A., Drechsel, D., Kellogg, D., Salser, S., Sawin, K., et al. (1991). Preparation of modified tubulins. Methods Enzymol. 196, 478-487. Kellogg, D. R., and Alberts, B. M. (1992). Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies. Mol. Biol. Cell 3, 1-11. Kellogg, D. R., Field, C. M., and Alberts, B. M. (1989). Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo. J. Cell BioL 109, 2977-91. Klotz, C., Dabauvalle, M.-C., Paintrand, M., Weber, T., Bornens, M., and Karsenti, E. (1990). Parthenogenesis in Xenopus eggs requires centrosomal integrity. J. Cell Biol. 110, 405--415. Kuriyama, R. (1984). Activity and stability of centrosomes in Chinese hamster ovary cells in nucleation of microtubules in vitro. J. Cell Sci. 66, 277-295. Li, K., and Kaufman, T. C. (1996). The homeotic target gene centrosomin encodes an essential centrosomal component. Cell 85, 585-596. Mitchison, T. J., and Kirschner, M. W. (1984). Microtubule assembly nucleated by isolated centrosomes. Nature 312, 232-237. Moritz, M. (2000). Preparing cytoplasmic extracts from Drosophila embryos. In "Drosophila Protocols" (W. Sullivan, M. Ashburner, and R. S. Hawley, eds.), pp. 571-575. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Moritz, M., and Alberts, B .M. (1999). Isolation of centrosomes from Drosophila embryos. In "Methods in Cell Biology" (C. L. Rieder, ed.), Vol. 61. pp. 1-12. Academic Press, San Diego. Moritz, M., Zheng, Y., Alberts, B. M., and Oegema, K. (1998). Recruitment of the y-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. J. Cell Biol. 142, 775-786. Oakley, C. E., and Oakley, B. R. (1989). Identification of gamma-tubulin, a new member of the tubulin superfamily encoded by mipA gene ofAspergillus nidulans. Nature 338, 662-664. Ohta, K., Shiina, N., Okumura, E., Hisanaga, S.-I., Kishimoto, T., et al. (1993). Microtubule nucleating activity of centrosomes in cell-free extracts from Xenopus eggs: Involvement of phosphorylation and accumulation of pericentriolar material. J. Cell Sci. 104, 125-137. Schnackenberg, B. J., Balczon, R. D., Hull, D. R., and Palazzo, R. E. (2000). Reconstitution of microtubule nucleation potential in centrosomes isolated from Spisula solidissima oocytes. J. Cell Sci. 113, 943-953. Whitfield, W. G., Millar, S. E., Saumweber, H., Frasch, M., and Glover, D. M. (1988). Cloning of a gene encoding an antigen associated with the centrosome in Drosophila. J. Cell Sci, 89, 467-480.