Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Cláudia Pereira; Rita M. Reis; José B. Gama; Dhanya K. Cheerambathur; Ana X. Carvalho; Reto Gassmann

The RZZ complex is well-established as an outer kinetochore component required for the recruitment of Mad1-Mad2 and dynein [11, [33] [34] [35] [36] [37] . Here, we present evidence that RZZ additionally functions as a structural precursor for the assembly of the dynamic outermost kinetochore domain, which expands in early prometaphase to accelerate spindle assembly [22] . Recent work has revealed that the RZZ subunit Rod is evolutionarily related to coat proteins that form higher order assemblies on membranes [16, 29] . We demonstrate in C. elegans that Rod proteins are also capable of selfassembly. Although additional components may be required in vivo, we show in vitro that a complex consisting of ROD-1 without its b-propeller and CZW-1 Zw10 is sufficient to form µm-scale filaments, which have an invariant diameter of ~50 nm. Based on the human RZZ structure [16] , the precursor of these filaments likely corresponds to a ROD-1(D1-372)-CZW-1 Zw10 dimer. Similarly, the precursor of the GFP::RZZ filaments we observe in vivo is likely a dimer of a trimer, containing two copies each of ROD-1, CZW-1 Zw10 , and ZWL-1 Zwilch . Deletion of the b-propeller greatly favors oligomerization of ROD-1 in vitro and in vivo. The ROD-1 b-propeller forms a tight complex with ZWL-1 Zwilch [15, 32] , raising the possibility that the presence of ZWL-1 Zwilch in RZZ has an inhibitory effect on self-assembly.