Proposed mechanism for KDM3A in ciliary regulation. (A) Collapsed time frames (3 h) of live RPE1^WT and KDM3A^null cultures transiently transfected with ARL13B-mKate2 and IFT81-mGFP showing consistently (arrows) increased accumulations and/recruitment of IFT81-EGFP at the mutant basal body. (B) Enrichment of Halo-KDM3A in centrosomes in ∼2% of transfected cells. (C) Centrosomal localization of KDM3A confirmed by construct containing its zinc-finger and nuclear receptor–binding domains fused to GFP, colocalizing with IFT81. See also Video 5. These findings support KDM3A regulating both global and local actin dynamics at specific subcellular compartments with a direct impact on ciliogenesis. (D) Summary of KDM3A mutant cellular phenotype: In the absence of KDM3A, the actin cytoskeleton contains reduced abundance of actin filaments, wide lamellipodia at all cellular edges and reduced migration. Without an actin gate, ciliogenesis is facilitated, and these cilia are long but unstable. (E) Summary of serum (+FCS or low serum [LS]) and pharmacogenetic modulation of actin at the pericentriolar periciliary compartment (PPC) illustrates that promoting actin polymerization either phenocopies ciliary growth in wild-type cells or rescues the abnormal resorption of KDM3A mutant cilia. CK-869 treatment of cells pinpoints the actin nucleation activity of ARP2/3 to be necessary to maintain normal IFT within cilia. IFT81 accumulations suggest unbalanced anterograde-retrograde transport (represented by different arrow size) when actin is perturbed. Together, these results indicate that the ciliary traits of KDM3A mutants stem from perturbations of actin dynamics, which upset IFT.