FIP5 phosphorylation during mitosis regulates apical trafficking and lumenogenesis

Apical lumen formation is a critical process in epithelial development. This process requires precise coordination of membrane dynamics and cytoskeletal changes. Rab11/FIP5 endosomes play a central role in delivering components to the apical membrane initiation site. However, the mechanisms controlling their targeting remain unclear. Prior research has shown that endocytic transport is essential for polarized cell organization. The role of phosphorylation in regulating these events has not been fully explored. This gap motivated the current investigation into how FIP5 activity is controlled. Understanding these regulatory steps could clarify how epithelial polarity is established.

This study aimed to identify how FIP5 activity is regulated during apical lumen formation. The researchers focused on the role of phosphorylation in controlling FIP5 function. They examined whether FIP5 phosphorylation affects its interaction with SNX18. The study also investigated the timing of this modification during the cell cycle. The goal was to determine how phosphorylation influences endosome targeting to the AMIS. The researchers hypothesized that phosphorylation at a specific site could control FIP5 activity. They sought to link this modification to the fidelity of lumen formation. This work aimed to clarify the spatiotemporal regulation of apical trafficking.

The researchers used cell culture models to study epithelial morphogenesis. They performed live-cell imaging to track endosome movement during mitosis. Phosphorylation status of FIP5 was analyzed using mass spectrometry. The role of GSK-3 was tested using pharmacological inhibitors. They also used CRISPR to generate FIP5 mutants for functional studies. The interaction between FIP5 and SNX18 was assessed through co-immunoprecipitation. Time-lapse imaging captured the dynamics of endosome targeting to the AMIS. The study combined biochemical and imaging approaches to test their hypotheses.

FIP5 phosphorylation at T276 was detected during metaphase and anaphase. This modification was mediated by GSK-3 activity. Phosphorylation reduced the interaction between FIP5 and SNX18. This disruption affected the formation of apical endocytic carriers. Endosome targeting to the AMIS was delayed in phosphorylated FIP5 mutants. The midbody formed during cytokinesis marked the AMIS location. Phosphorylation ensured precise timing of endosome delivery to the AMIS. These findings suggest a regulatory mechanism for apical trafficking during mitosis.

The study shows that FIP5 phosphorylation at T276 is a mitotic event. This modification inhibits the FIP5-SNX18 interaction during cell division. The researchers propose that this inhibition controls endosome trafficking to the AMIS. The timing of phosphorylation aligns with the cell cycle to ensure lumen formation. The midbody serves as a spatial cue for AMIS localization. These findings suggest a link between mitotic events and apical polarity. The study highlights the role of phosphorylation in regulating endocytic transport. The authors suggest that this mechanism contributes to epithelial morphogenesis.