陰道滴蟲ARF蛋白調控PIP2訊息之功能探討

Abstract

高等真核細胞ADP ribosylation factor (ARF) 結合三磷酸鳥苷 (Guanosine triphosphate) 成為活化態，是真核細胞中細胞膜運輸與細胞骨架之關鍵調節因子，其功能之一可活化磷脂醯肌醇-4-磷酸5-激酶 (phosphatidylinositol 4-phosphate 5-kinase, PI4P5K) 並與PI4P5K形成複合體共同移動至細胞膜以產生Phosphatidylinositol 4,5-bisphosphate (PIP2)。在陰道滴蟲細胞內，前人發現鐵離子刺激後能活化ARF6同源蛋白(TvARF220)，與TvPI4P5K形成複合體促進PIP2 產生，並調控下游鈣離子訊號。本研究目標在探討鐵如何在陰道滴蟲透過活化TvARF220來調控PIP2訊息傳導及其下游細胞功能。利用免疫螢光分析發現，TvPI4P5K與PIP2在陰道滴蟲細胞中共同定位在細胞膜上，並與TvARF220細胞內分布成正相關。同時，鐵刺激會增加TvPI4P5K與TvARF220共同轉移到細胞膜上，趨勢與鐵刺激後細胞膜上PIP2含量增加一致。相反地，當利用布雷非德菌素A (brefeldin A, BFA) 抑制陰道滴蟲TvARF220活化，TvARF220與TvPI4P5K共同轉移至細胞膜周邊的趨勢則下降，同時造成細胞膜上PIP2訊號降低。這些發現表明TvARF220功能可促進TvPI4P5K轉移到細胞膜上並增加PIP2產生。值得注意的是，鈣離子螢光指示劑顯示BFA處理造成陰道滴蟲內鈣訊號量降低的同時，也減緩肌動蛋白細胞骨架重組幅度，並造成細胞骨架主導之阿米巴型態遷移能力下降，從而確認TvARF220可作為PIP2與鈣離子等訊號的上游因子，最終調控肌動蛋白相關作用。本研究確認TvARF220作為引導特定蛋白細胞膜定位的關鍵功能，為陰道滴蟲寄生與宿主交互作用時的分子機制提供新見解。

Small GTPases of the ADP-ribosylation factor (ARF) family are key regulators of membrane trafficking and cytoskeletal dynamics in eukaryotic cells. In the protozoan parasite Trichomonas vaginalis, iron was previously found to activate an ARF6 homolog (TvARF220) and trigger its complex formation with TvPI4P5K, regulating downstream calcium signaling. This study aims to investigate the mechanism of how iron induces PIP2 signaling and its downstream cellular functions through TvARF220 activation in this parasite. Immunofluorescence analysis revealed that the plasma membrane colocalization of TvPI4P5K and its lipid product PIP2 strongly correlated with TvARF220 cellular distribution. Iron challenge enhanced TvPIP4P5K and TvARF220 enrichment at the plasma membrane, coinciding with increased PIP2 levels. In contrast, ARF inhibitor brefeldin A (BFA) diminished the plasma membrane localization of TvPI4P5K and TvARF220, resulting in reduced PIP2 production. These findings suggest that TvARF220 facilitates TvPI4P5K trafficking to the plasma membrane, thereby promoting localized PIP2 synthesis. Notably, BFA treatment also led to decreased intracellular calcium levels, impaired actin dynamics, and slowed down actin cytoskeleton-dependent amoeboid motility, supporting that TvARF220 acts upstream of a PIP2-calcium axis critical for actin-dependent processes. Together, this study highlights TvARF220 as a key regulator of membrane-localization signaling, offering new insights into the molecular basis of parasite colonization and host interaction.