title: Evidence for reciliation of RPE1 cells in late G1 phase, and ciliary localisation of cyclin B1.
abstract: The primary cilium, an organelle that transduces extracellular signals important for development and tissue homeostasis, is typically assembled upon cell cycle exit and disassembled upon cell cycle re-entry. Cilium assembly is thought to be suppressed in cycling cells, however the extent of suppression is not clear. For example, primary cilia are present in certain proliferating cells during development, and a period of reciliation has been reported to occur in late G1 in murine 3T3 cells released from serum starvation-induced quiescence. Human retinal pigmented epithelial (hTERT-RPE1; herein, RPE1) cells are commonly used to investigate pathways regulating cilium disassembly, however the ciliary disassembly profile of these cells remains uncertain. A period of reciliation has not been observed. Here, we analyse the ciliary disassembly profile of RPE1 cells by immunofluorescence microscopy. The results suggest a profile similar to 3T3 cells, including a period of reciliation in late G1 and a second wave of deciliation in S phase. We present evidence that arresting cells in early S phase with hydroxyurea or excess thymidine prevents the second wave of deciliation, and that deciliation is initiated shortly after release from a thymidine block, consistent with coupling to DNA replication. These findings support the often overlooked notion that cilium formation can occur in late G1, and suggest that RPE1 cells could serve as a model system for studying the molecular pathways that direct this process, in addition to those that stimulate cilium disassembly. We also present immunofluorescence data indicating that cyclin B1 localises to primary cilia.
authors.affiliation: Human Development and Health, University of Southampton, UK.
authors.forename: Cosma
authors.initials: C
authors.lastname: Spalluto
authors.forename: David I
authors.initials: DI
authors.lastname: Wilson
authors.forename: Tom
authors.initials: T
authors.lastname: Hearn
keywords: AurA, Aurora kinase A; Aurora A; BrdU, bromodeoxyuridine; CDK, cyclin-dependent kinase; CDK1; Cilium disassembly; Cyclin B1; DAPI, 4’,6-diamidino-2-phenylindole; DNA replication; FBS, fetal bovine serum; HU, hydroxyurea; Mim, mimosine; Primary cilia; SS, serum-starved; Thy, thymidine; siRNA, short interfering RNA