Obligate multicellularity evolved at least 5 times in eukaryotes, including at the origin of Metazoa 1, 2. The molecular events leading to the acquisition of multicellularity are not understood in any lineage. Here, I present an integrated analysis into the emergence of three regulators of cytokinesis and the Metazoan kingdom. Phylogenetic analysis and structural modeling indicate that three interacting essential regulators of cytokinesis, Kif23, Cyk4, and Ect2, are highly conserved across all Metazoa. These proteins cooperate to link the plane of cell division with the position of the spindle during anaphase and subsequently nucleate the assembly of stable intercellular bridges 3-6, structures prevalent in Metazoan germlines 7. The closest relatives of Metazoa, Choanoflagellata, encode Kif23 and Ect2 orthologs. In contrast, choanoflagellate species variably encode members of paralogous sets of proteins related to Cyk4, some of which are predicted to interact with Kif23. These findings, in conjunction with prior knowledge, suggests that the evolutionary refinement of these three cytokinetic regulators was a proximal prerequisite for the evolution of defining features of Metazoa.
