超音波對於Septin-2磷酸化與其絲狀結構解離之影響

Abstract

Septins在許多文獻中被歸類為第四種細胞骨架，有別於微管、微絲、與中間絲。雖然研究表明Septins常被發現存在於細胞中感應力敏感的區域，如細胞膜、初級纖毛、和應力纖維，且與許多感應力敏感的行為相關，但Septins對超音波這樣的機械力的反應尚未被證明。本研究開發一個偵測Septin-2在S218位點的單磷酸化的兔多克隆抗體，經測試後將其應用於西方墨點法和免疫螢光染色。利用這個抗體，我們觀察到當輸入電壓超過一定閾值時，超音波刺激引起的Septin-2磷酸化會呈現時間依賴性和劑量依賴性。此外，當Septin-2纖維被光漂白後，超音波加速其螢光恢復的速度，顯示其對Septin-2動態行為的影響。此外，Septin-2與肌動蛋白具有高度的共定位，並在穩定肌動蛋白方面發揮作用，然而超音波刺激則降低了磷酸化的Septin-2與肌動蛋白的共定位。從磷酸化現象、運動學、和亞細胞定位等方面的變化，我們證明了Septin-2對超音波的敏感性，更驗證了S218磷酸化Septin-2抗體作為用於監測超音波誘導的細胞變化的標記物的潛力。

Septins are newly categorized as the fourth component of the cytoskeleton, which conventionally comprises microtubules, microfilaments, and intermediate filaments. While studies have suggested that septins are often found close to mechanosensitive compartments, like cell membrane, primary cilia, and stress fiber, and are correlated with mechanosensitive behavior, septins are ultrasound-responsive has not been demonstrated. In this study, we developed a rabbit polyclonal antibody specific to the mono-phosphorylation at S218 of Septin-2, and characterized it for Western blot and Immunofluorescence purposes. Using this antibody, we observed that ultrasound exposure leads to time-dependent and dose-dependent phosphorylation of Septin-2, particularly when the input voltage exceeds a certain threshold. Additionally, ultrasound accelerates the recovery time of Septin-2 filaments after photobleaching. Furthermore, ultrasound stimulation reduces the colocalization between phosphorylated Septin-2 and actin. These observations demonstrate the responsiveness of Septin-2 to ultrasound, as evidenced by alterations in its phosphorylation level, kinematics, and subcellular localization. Moreover, the findings offer evidence that supports the S218 phospho-Septin-2 antibody as a reliable marker for monitoring ultrasound-induced cellular changes.