In vitro reconstitution of the functional interplay between MCAK and EB3 at microtubule plus ends.
The kinesin-13 family member mitotic centromere-associated kinesin (MCAK) is a potent microtubule depolymerase. Paradoxically, in cells it accumulates at the growing, rather than the shortening, microtubule plus ends. This plus-end tracking behavior requires the interaction between MCAK and members of the end-binding protein (EB) family, but the effect of EBs on the microtubule-destabilizing activity of MCAK and the functional significance of MCAK accumulation at the growing microtubule tips have so far remained elusive. Here, we dissect the functional interplay between MCAK and EB3 by reconstituting EB3-dependent MCAK activity on dynamic microtubules in vitro. Whereas MCAK alone efficiently blocks microtubule assembly, the addition of EB3 restores robust microtubule growth, an effect that is not dependent on the binding of MCAK to EB3. At the same time, EB3 targets MCAK to growing microtubule ends by increasing its association rate with microtubule tips, a process that requires direct interaction between the two proteins. This EB3-dependent microtubule plus-end accumulation does not affect the velocity of microtubule growth or shortening but enhances the capacity of MCAK to induce catastrophes. The combination of MCAK and EB3 thus promotes rapid switching between microtubule growth and shortening, which can be important for remodeling of the microtubule cytoskeleton.
Montenegro Gouveia
Susana
S
Department of Cell Biology, Erasmus Medical Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
Leslie
Kris
K
Kapitein
Lukas C
LC
Buey
Rubén M
RM
Grigoriev
Ilya
I
Wagenbach
Michael
M
Smal
Ihor
I
Meijering
Erik
E
Hoogenraad
Casper C
CC
Wordeman
Linda
L
Steinmetz
Michel O
MO
Akhmanova
Anna
A
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D005456:Fluorescent Dyes / Q000378:metabolism; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D016547:Kinesins