Structural And Mechanistic Insights Into Cytokinesis Related Proteins Mid1 And Centralspindlin

Mitosis is an important process conserved from yeast to humans.The final step in the cell cycle is the formation of two genetically identical daughter cells by cytokinesis.A sophisticated signaling network is involved in regulation of the anchorage of contractile ring,expecially,the Centralspindlin-Rho GTPase signaling pathway,so that each daughter cell inherits an intact set of genetic materials.In order to understand how the contractile ring is anchored at the cleavage furrow through Mid1,and how the central spindle is organized by Centralspindlin,we determined crystal structure of Mid1 and Centralspindlin.Mid1 are scaffold proteins that play key roles in anchorage of the contractile ring at the cell equator during cytokinesis in fungi.Here,we report crystal structure and functional analysis of S.Pombe Mid1.The combined data show Mid1 binds to the membrane through a cryptic C2 domain.N-terminal domain of Mid1 directly binds to multiple components of the contractile machinery,dimerization of Mid1 C-terminal domain leads to high affinity and preference for PI(4,5)P2,which stably anchors Mid1 at the division plane,bypassing the requirement for Rho GTPase.These findings uncover the regulatory logic for the anchorage of the contractile ring through the Mid1 from yeast.Centralspindlin complex is comprised of two proteins,a kinesin-like protein,Zen4,and a Rho GTPase activating protein,Cyk4.Mitotic kinesin Zen4 from C.elegans is kinesin-6 family proteins.Kinesins hydrolyze ATP to transport intracellular cargoes along microtubules.Kinesin neck linker(NL)functions as the central mechano-chemical coupling element by changing its conformation through the ATPase cycle.Here,we report the crystal structure of kinesin-6 Zen4 and enlightened analyses on kinesin-1.The structure shows Zen4 is in a nucleotide-free,apo state,with the NL initial segment(NIS)adopting a backward-docked conformation and the preceding ?6 helix partially melted.Single molecule fluorescence resonance energy transfer(smFRET)analyses indicate the NIS of kinesin-1 undergoes similar conformational changes under tension in the two-head bound(2HB)state,whereas it is largely disordered without tension.The backward-docked structure of NIS is essential for the motility of the motor.Our findings reveal a key missing conformation of kinesins,which provides the structural basis of the stable 2HB state and offers a tension-based rationale for the optimal NL length to ensure processivity of the motor.We also determined crystal structure of Centralspindlin.By the weak interaction between N-terminal coil-coiled domain of Cyk4,the Centralspindlin forms an octamer in the crystal structure.In order to explore the bio-function of the octamer protein,we reconstitute the central spindle in vitro.As the same with our hypothesis,the N-terminal coiled-coil domain of Cyk4 regulates the assemblement of the central spindle.Besides,we also find that the clustering domain of Zen4 can bind to the MT.The hypothetical model provides a new insight into the mechanism of Centralspindlin facilitating the formation of the central spindle.