Aebi, and T.E. CLIP-170 is closely linked to tubulin polymerization. oocytes, increase in the polymerization rate is restricted specifically to microtubule plus ends (Gard and Kirschner, 1987; Vasquez et al., 1994), the main assembly site in vivo. Such an interaction would also promote association of the MBP preferentially at the elongating end. Localization of an MBP to the plus ends of microtubules by either of these mechanisms could allow both regulation of assembly of microtubules and promotion of interaction of microtubule ends with cytoplasmic organelles. In this study, we have examined the association of CLIP-170 with microtubules, to characterize the basis for its in vivo localization. We have found that the NH2-terminal microtubule-binding domain of CLIP-170 alone can localize to microtubule plus ends in cells, indicating that interactions of the tail domain with other structures are not essential for its correct targeting. We show that the association of CLIP-170 with the plus ends of microtubules is coordinated with polymer assembly, since the protein localizes preferentially to growing microtubules, both in vivo and in vitro. Finally, we provide evidence suggesting that of the two possible mechanisms for the plus end targeting of the protein that we considered, the more likely is copolymerization of CLIP-170 with tubulin oligomers, rather than specific recognition of a transient conformational cap at the ends of microtubules. The specific localization of CLIP-170 with the dynamic plus ends of microtubules raises the possibility that it may assist microtubules to explore the cellular space, helping them to find and capture organelles for subsequent transport. Materials and Methods Materials Paclitaxel was a gift of the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD). Nocodazole (and were stored as stock solutions in water at ?20C. MES was purchased from for 15 min at 30C, disassembling the polymers in buffer at 4C and clarifying this solution Mouse monoclonal to MPS1 by ultracentrifugation. The final concentrations of tubulin and GDP were 10 and 50 M, respectively. Purification and characterization of H1 and H2 recombinant proteins will be reported elsewhere (Scheel, J., P. Pierre, J.E. Rickard, G.S. Diamantopoulos, C. Valetti, F.G. van der Goot, M. H?ner, U. Aebi, and T.E. Kreis, manuscript in preparation). In brief, the cDNAs encoding H1 and the first 481 amino acids of CLIP-170 (H2) were cloned in the pET19 vector (Novagen) for expression in bacteria with an NH2-terminal histidine tag. The histidine-tagged fusion proteins were purified from bacterial lysates using nickel chelate chromatography according to the Novagen protocol. The cDNA encoding MAP2C cloned in the pET3d vector (Novagen) was kindly provided by A. Matus (Friedrich Miescher Institute, Basel, Switzerland). The protein expressed in bacteria was purified using its property of heat stability (Takeuchi et al., 1992). In brief, the bacterial lysates were boiled for 5 min and then left on ice for another 10 min. Heat-stable MAP2C remained in the supernatant after centrifugation of the boiled sample at 90,000 for Norisoboldine 30 min and was then concentrated, followed by dialysis. Porcine brain MAP2 was prepared according to Drubin and Kirschner (1986) with a slight modification at the gel filtration step, which was performed on Sepharose CL-4B using an XK16 (16-mm diam, 1-m length) column (inverted fluorescence microscope (model Axiovert TV 135). Images were recorded with a cooled charge-coupled device camera (model CH250, 1,317 1,035 pixels; Photometrics), controlled by a Power Macintosh 8100/100 (Apple). Images were recorded with the software package IPLab spectrum V2.3 (Signal Analytics) and Norisoboldine processed using Adobe Photoshop 3.0 (Adobe Systems). Cross-linking Experiments Reactions containing proteins (2.5 M) as indicated in Fig. ?Fig.77 were mixed on ice in BRB80 buffer containing 1 mM GTP before addition of the cross-linker 1-ethyl-3-(3-[dimethyl-amino]propyl) carbodiimide as previously reported (Song and Mandelkow, 1993). Tubulin was cycled immediately before use in this assay, and both cycled tubulin and other proteins were centrifuged at 150,000 for 20 min to remove aggregates before cross-linking. After Norisoboldine 2 h on ice, reactions were quenched by the addition of 10 mM hydroxylamine followed by dilution in standard SDS sample buffer containing DTT, and separated by SDS-PAGE using the Laemmli buffer system (Laemmli, 1970) except that the separating gel buffer was at pH 9.5 to maximize separation of the tubulin subunits (Melki et al., 1991). Cross-linked products were detected with specific antibodies against H1 (anti-KRKV and anti-GSIK; Pierre et al., 1992), -tubulin (1A2), or -tubulin (JDR.3B8; buffer were centrifuged either at 25,000 or 60,000 rpm.