Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity.
To uncover their contrasting mechanisms, antimitotic drugs that inhibit Eg5 (kinesin-5) were analyzed in mixed-motor gliding assays of kinesin-1 and Eg5 motors in which Eg5 "braking" dominates motility. Loop-5 inhibitors (monastrol, STLC, ispinesib, and filanesib) increased gliding speeds, consistent with inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, consistent with locking Eg5 in a rigor state. Biochemical and single-molecule assays demonstrated that BRD9876 acts as an ATP- and ADP-competitive inhibitor with 4 nM KI. Consistent with its microtubule polymerase activity, Eg5 was shown to stabilize microtubules against depolymerization. This stabilization activity was eliminated in monastrol but was enhanced by BRD9876. Finally, in metaphase-arrested RPE-1 cells, STLC promoted spindle collapse, whereas BRD9876 did not. Thus, different Eg5 inhibitors impact spindle assembly and architecture through contrasting mechanisms, and rigor inhibitors may paradoxically have the capacity to stabilize microtubule arrays in cells.
Department of Biomedical Engineering, Pennsylvania State University , University Park, Pennsylvania 16802, United States.
Geng-Yuan
GY
Chen
Department of Biomedical Engineering, Pennsylvania State University , University Park, Pennsylvania 16802, United States.
You Jung
YJ
Kang
Department of Cell and Developmental Biology, Vanderbilt University , Nashville, Tennessee 37203, United States.
A Sophia
AS
Gayek
Department of Physics, Worcester Polytechnic Institute , Worcester, Massachusetts 01609, United States.
Wiphu
W
Youyen
Department of Physics, Worcester Polytechnic Institute , Worcester, Massachusetts 01609, United States.
Erkan
E
Tüzel
Department of Cell and Developmental Biology, Vanderbilt University , Nashville, Tennessee 37203, United States.
Ryoma
R
Ohi
Department of Biomedical Engineering, Pennsylvania State University , University Park, Pennsylvania 16802, United States.
William O
0000-0001-5547-8755
WO
Hancock
D000818:Animals; D002460:Cell Line; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors*; D008677:Metaphase / Q000187:drug effects*; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D008941:Spindle Apparatus / Q000187:drug effects* / Q000378:metabolism
C470243:KIF11 protein, human; D016547:Kinesins