Localization of the Kinesin-like Protein Xklp2 to Spindle Poles Requires a Leucine Zipper, a Microtubule-associated Protein, and Dynein

Abstract

Xklp2 is a plus end–directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49–59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein–dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.

Characterization of the TPX2 Domains Involved in Microtubule Nucleation and Spindle Assembly inXenopusEgg Extracts

“…Immunolocalization of the GST-Xklp2 tail on asters was performed as described previously (Wittmann et al, 1998), by using an Alexa red-coupled secondary antibody (Molecular Probes) and visualizing microtubules with Cy-5-labeled tubulin (Hyman et al, 1991). For each experiment, pictures were taken on a Leica TCS Sp2 AOBS confocal microscope at 63ϫ magnification with identical illumination settings and processed identically with Adobe Photoshop.…”

“…Spindles and asters were processed for immunofluorescence as described previously (Wittmann et al, 1998). Immunolocalization of the GST-Xklp2 tail on asters was performed as described previously (Wittmann et al, 1998), by using an Alexa red-coupled secondary antibody (Molecular Probes) and visualizing microtubules with Cy-5-labeled tubulin (Hyman et al, 1991).…”

“…CSF egg extracts were prepared according to Wittmann et al (1998). For cycled spindle assembly around sperm nuclei, rhodamine-labeled tubulin (Hyman et al, 1991) was added at 0.2 mg/ml and sperm at a concentration of ϳ500 nuclei/ l to 20 l of extract on ice.…”

“…The glutathione S-transferase (GST)-fusion protein containing the C-terminal 250 amino acids of Xklp2 tail was produced as described previously (Wittmann et al, 1998). GST-TPX (1-39) was expressed and purified as described previously (Bayliss et al, 2003).…”

Mechanisms of plant spindle formation

“…NuMA binds microtubules directly and plays a critical role in zippering microtubules into focused poles (Merdes et al 1996(Merdes et al , 2000Silk et al 2009). Similarly, TPX2 binds to microtubules and tends to concentrate at the spindle poles (Wittmann et al 1998(Wittmann et al , 2000. It has been shown that TPX2 is required for microtubule nucleation as well as spindle pole organization (Gruss et al 2002;Garrett et al 2002;Schatz et al 2003).…”