IDR-driven LLPS of GAS2L3 scaffolds CHMP4B condensates to accelerate cytokinesis in hepatocellular carcinoma cells.

INTRODUCTION

Dysregulated cell cycle progression is a hallmark of cancer, promoting uncontrolled cell proliferation. Growth arrest-specific 2 like 3 (GAS2L3), which is highly expressed in hepatocellular carcinoma (HCC) cells, correlates with poor clinical outcomes. However, the role of GAS2L3 in HCC pathogenesis remains unclear.

OBJECTIVES

This study aimed to investigate the molecular mechanisms by which GAS2L3 contributes to cytokinesis and hyperproliferation of HCC cells.

METHODS

Loss-of-function assays of GAS2L3 were performed to assess midbody assembly, a process essential for cytokinesis. By analyzing protein structure, we predicted liquid-liquid phase separation (LLPS) potential of GAS2L3, which was validated via fluorescence recovery after photobleaching (FRAP), time-lapse imaging, and in vitro droplet formation assays. Additionally, we generated an intrinsically disordered region (IDR)-deficient model to investigate the role of IDR in LLPS and its effects on cytokinesis, cell cycle progression, and HCC tumorigenicity.

RESULTS

Highly expressed GAS2L3 localizes to the midbody and accelerates the cytokinesis of HCC cells by facilitating the recruitment of the charged multivesicular body protein 4B (CHMP4B). Notably, EGFP-GAS2L3 aggregates in the midbody exhibit dynamic behavior. Structural analysis and experimental evidence confirm GAS2L3 undergoes phase separation via its IDR. GAS2L3 scaffolds the CHMP4B condensate formation at midbody through phase separation, thereby accelerating cytokinesis in HCC cells. Moreover, GAS2L3 knockdown or IDR deletion in HCC cells leads to defective cytokinesis, G1 arrest, and significant tumorigenicity inhibition.

CONCLUSION

GAS2L3 critically regulates cytokinesis in HCC through LLPS-mediated remodeling of midbody abscission microenvironment. These findings reveal novel insights into HCC hyperproliferation and suggest GAS2L3 as a potential therapeutic target.