articles NATURE CELL BIOLOGY VOL 3 JULY 2001 http://cellbio.nature.com 643 Msps/XMAP215 interacts with the centrosomal protein D-TACC to regulate microtubule behaviour Michael J. Lee*, Fanni Gergely*, Kim Jeffers*, Sew Yeu Peak-Chew† and Jordan W. Raff*‡ *Department of Genetics, Wellcome/CRC Institute, Tennis Court Road, Cambridge CB2 1QR, UK †Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge, CB2 2QH, UK ‡e-mail: j.raff@welc.cam.ac.uk The XMAP215/ch-TOG/Msps family of microtubule-associated proteins (MAPs) promote microtubule growth in vitro and are concentrated at centrosomes in vivo. We show here that Msps (mini-spindles protein) interacts with the cen- trosomal protein D-TACC, and that this interaction strongly influences microtubule behaviour in Drosophila embryos. If D-TACC levels are reduced, Msps does not concentrate at the centrosomes efficiently and the centrosomal micro- tubules appear to be destabilized. If D-TACC levels are increased, both D-TACC and Msps accumulate around the centrosomes/spindle poles, and the centrosomal microtubules appear to be stabilized. We show that the interaction between D-TACC and Msps is evolutionarily conserved. We propose that D-TACC and Msps normally cooperate to stabilize centrosomal microtubules by binding to their minus ends and binding to their plus ends as they grow out from the centrosome. C entrosomes are the main microtubule-organizing centres (MTOCs) in animal cells 1 . Centrosomes have an important role in organizing many cellular processes, but surprisingly little is known about how they function at the molecular level 1–3 . In recent years, great progress has been made in understanding how centrosomes nucleate microtubules. The protein γ-tubulin is concentrated at the MTOCs, and is essential for MTOC function 4–8 . In the cell, γ-tubulin usually exists in the form of a large ring com- plex, and this complex has many of the properties expected of a microtubule-nucleating complex 9 . Ring-like structures that contain γ-tubulin have been visualized in the peri-centriolar material of centrosomes 10 , and it is now widely accepted that γ-TuRCs are directly involved in the nucleation of centrosomal microtubules 11,12 . It is clear, however, that the interaction between centrosomes and microtubules is more complex than just a simple nucleation event. In mitosis, for example, many centrosomal microtubules are released from their nucleating sites, but are then ‘captured’ by complexes of microtubule-motor proteins, such as dynein/dynactin/NuMA that function to keep these microtubules focused around the centro- somes 13–15 . Furthermore, there are several other proteins that are con- centrated at centrosomes and have been shown to interact with microtubules 16–21 . Particularly intriguing among these proteins are the XMAP215/ch-TOG family of MAPs. These proteins bind direct- ly to microtubules and seem to stabilize them by modulating their dynamics 22–25 . Surprisingly, although these proteins seem to influence mainly microtubule plus-end dynamics, they all seem to be highly concentrated at centrosomes in cells 16,24,26–29 . The function of these proteins at centrosomes, if any, is therefore unclear. We recently identified a novel Drosophila centrosomal protein, called D-TACC, that is essential for mitotic spindle function in the Drosophila embryo: when D-TACC function is perturbed by muta- tion or antibody injection, centrosomal microtubules are abnormal- ly short, and the embryos die owing to an accumulation of mitotic defects 30 . The ~200 amino acids at the carboxy terminus of D-TACC are predicted to form a coiled-coil, and this region is related to a family of mammalian proteins called the transforming acidic coiled- coil-containing (TACC) proteins. These proteins have all been implicated in cancer 31–33 , but their normal functions are unknown. We have shown that all the human TACC proteins associate with centrosomes and microtubules, at least during mitosis, indicating that the TACC domain is a conserved microtubule/centrosome- interacting domain 34 . Although D-TACC, or a glutathione S–transferase (GST)- or maltose-binding protein (MBP)-fusion protein that contains the conserved TACC domain, strongly interacts with microtubules in embryo extracts, these proteins do not strongly interact with puri- fied microtubules 30 . We suspected, therefore, that the TACC domain interacts with microtubules indirectly through another protein. We show here that D-TACC interacts with Msps, the Drosophila homologue of XMAP215, and that this interaction seems to strongly influence the stability of centrosomal micro- tubules. We show that the interaction between these families of proteins is highly conserved in evolution. Moreover, we show that the TACC domain of D-TACC can markedly stabilize microtubules in Drosophila embryos, and this process seems to require the Msps protein. We propose that D-TACC and Msps normally cooperate to regulate the stability of centrosomal microtubules. Results D-TACC interacts with Msps. We previously showed that an MBP- or GST-fusion protein that contains the conserved C-terminal TACC domain of D-TACC was concentrated at centrosomes in embryos and associated with microtubules in embryo extracts 30 . These same fusion proteins, however, did not interact significantly with purified microtubules in vitro 30 (Fig. 1a). To identify other fac- tors present in the extract that might mediate the interaction between D-TACC and microtubules, we added a purified MBP–TACC-domain fusion protein (MBP–TD, or MBP–CT in ref. 30) to embryo extracts, and then re-isolated the fusion protein on an amylose column. On Coomassie-blue-stained gels, the re-isolat- ed fusion protein co-purified with two proteins of a higher relative molecular mass of ~220,000 (M r ~220K) and ~180K (Fig. 1b). This re-purified MBP–TD fusion protein complex now strongly associ- ated with purified microtubules in spin-down experiments (Fig. 1a, lower panel). We used mass spectroscopic methods to identify the ~220K and ~180K proteins. In two separate experiments, the ~220K protein © 2001 Macmillan Magazines Ltd