Drosophila Nod protein binds preferentially to the plus ends of microtubules and promotes microtubule polymerization in vitro.
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.
Cui
Wei
W
Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
Sproul
Lisa R
LR
Gustafson
Susan M
SM
Matthies
Heinrich J G
HJ
Gilbert
Susan P
SP
Hawley
R S
RS
D000818:Animals; D002875:Chromosomes / Q000648:ultrastructure; D029721:Drosophila Proteins / Q000378:metabolism* / Q000502:physiology*; D066298:In Vitro Techniques; D016547:Kinesins; D008868:Microtubule Proteins / Q000378:metabolism* / Q000502:physiology*; D008870:Microtubules / Q000378:metabolism*; D009865:Oocytes / Q000648:ultrastructure; D011108:Polymers / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050257:Tubulin Modulators / Q000378:metabolism
D029721:Drosophila Proteins; D008868:Microtubule Proteins; D011108:Polymers; D050257:Tubulin Modulators; C080298:Nod protein, Drosophila; D016547:Kinesins