Developmental assembly of contractile actomyosin networks in the C. elegans spermatheca requires anillin and successful cytokinesis

Proper contractility of the Caenorhabditis elegans spermatheca relies on the coordinated developmental assembly of actomyosin bundles, yet the mechanisms that organize these structures remain poorly understood. From an RNAi screen targeting 117 F-actin regulators, we uncovered an essential role for ANI-1/anillin in spermatheca morphogenesis. Live imaging revealed that ANI-1 depletion can prevent organ contractility due to disrupted F-actin organization, impaired Rho activation, and absent calcium signaling, leading to ovulation failure. We show that a primary defect of ANI-1 depletion is furrow regression during spermatheca cell cytokinesis, which leads to multinucleation during early spermatheca development. Following failed cytokinesis, F-actin bundle formation is severely impaired, preventing robust F-actin network assembly especially in the spermatheca distal neck. Depletion of the cytokinetic regulators AIR-2/Aurora B and the centralspindlin component ZEN-4 produced identical F-actin defects in the mature spermatheca. We conclude that successful cell division is a prerequisite for subsequent F-actin network assembly that supports organ contractility, and identify ANI-1 as a key regulator of spermatheca function.