DrosophilaNod Protein Binds Preferentially to the Plus Ends of Microtubules and Promotes Microtubule Polymerization In Vitro

Abstract

Nod, a nonmotile kinesin-like protein, plays a critical role in segregating achiasmate chromosomes during female meiosis. In addition to localizing to oocyte chromosomes, we show that functional full-length Nod-GFP (Nod(FL)-GFP) localizes to the posterior pole of the oocyte at stages 9-10A, as does kinesin heavy chain (KHC), a plus end-directed motor. This posterior localization is abolished in grk mutants that no longer maintain the microtubule (MT) gradient in the oocyte. To test the hypothesis that Nod binds to the plus ends of MTs, we expressed and purified both full-length Nod (Nod(FL)) and a truncated form of Nod containing only the motor-like domain (Nod318) from Escherichia coli and assessed their interactions with MTs in vitro. Both Nod(FL) and Nod318 demonstrate preferential binding to the ends of the MTs, displaying a strong preference for binding to the plus ends. When Nod318-GFP:MT collision complexes were trapped by glutaraldehyde fixation, the preference for binding to plus ends versus minus ends was 17:1. Nod(FL) and Nod318 also promote MT polymerization in vitro in a time-dependent manner. The observation that Nod is preferentially localized to the plus ends of MTs and stimulates MT polymerization suggests a mechanism for its function.

The HhH(2)/NDD Domain of the Drosophila Nod Chromokinesin-like Protein Is Required for Binding to Chromosomes in the Oocyte Nucleus

“…However, as demonstrated by Cui et al (2005) and as shown in Figure 7, by stage 9 of oocyte development the cytoplasmic Nod-GFP protein accumulates at the posterior pole of the oocyte, in a fashion similar to that observed for the plusend-directed motor kinesin heavy chain (Clark et al 1997). (The significance of this localization in terms of Nod function is considered in more detail in Cui et al 2005.…”

“…Although several lines of evidence show that the motor-like domain of Nod lacks the capacity for vectorial transport (Matthies et al 2001), we have recently demonstrated that both full-length Nod protein and a construct containing only the Nod motor-like domain bind primarily to the plus ends of microtubules in vitro and stimulate polymerization of microtubules (Cui et al 2005). Thus, as noted by Cui et al (2005), one can imagine a mechanism for Nod function in which Nod binds to the plus end of microtubules (and along the length of chromosome arms).…”

“…Thus, as noted by Cui et al (2005), one can imagine a mechanism for Nod function in which Nod binds to the plus end of microtubules (and along the length of chromosome arms). Nod then ''opens'' or ''relaxes'' enough to allow the insertion of a tubulin subunit at the plus end of the microtubule, before reclamping on the new plus end of the growing fiber.…”

Microtubule polarity and axis formation in theDrosophila oocyte

“…Since Nod-␤gal accumulates at known sites of MT minus-end localization in epithelial and mitotic cell types, the localization of Nod-␤gal in the oocyte was taken as evidence of the hypothesis that minus-ends are focused primarily at the anterior cortex of stage 8 -10 oocytes (Clark et al, 1997). However, interpretation of Nod-␤gal localization is complicated by in vitro studies showing that Nod lacks directional motor activity and preferentially binds MT plus-ends, rather than minus-ends (Matthies et al, 2001;Cui et al, 2005). Furthermore, both fulllength Nod and the endogenous minus end-directed motor protein cytoplasmic Dynein accumulate at the posterior pole at stage 9, along with the plus-end directed motor protein Kinesin I (Li et al, 1994;Brendza et al, 2002;Duncan and Warrior, 2002;Januschke et al, 2002;Cui et al, 2005).…”

“…However, interpretation of Nod-␤gal localization is complicated by in vitro studies showing that Nod lacks directional motor activity and preferentially binds MT plus-ends, rather than minus-ends (Matthies et al, 2001;Cui et al, 2005). Furthermore, both fulllength Nod and the endogenous minus end-directed motor protein cytoplasmic Dynein accumulate at the posterior pole at stage 9, along with the plus-end directed motor protein Kinesin I (Li et al, 1994;Brendza et al, 2002;Duncan and Warrior, 2002;Januschke et al, 2002;Cui et al, 2005). Although the significance of this is not completely clear, it has been speculated that this reflects Kinesin-mediated recycling of Dynein (Duncan and Warrior, 2002;Januschke et al, 2002).…”