利用單分子追蹤揭示FlhA與FliF的構型動態調控大腸桿菌基底周圍的聚集

Abstract

大腸桿菌的鞭毛雖為全表面型分布，長期以來被認為是隨機定位，但觀察到鞭毛蛋白FlhA與FliF在細胞膜上會形成穩定聚集，顯示其分布可能受控於尚未明確的機制。本研究利用螢光蛋白與單分子追蹤技術，將PAmCherry與mTurquoise2結合至FlhA與FliF蛋白，觀察其在膜上的動態行為，透過統計分析及模型擬合，解析其運動狀態與分布變化。結果顯示，FlhA與FliF在膜上皆顯示雙態擴散運動，且擴散係數一致，然而，由於兩者單體結構的跨膜螺旋數量不同，推測兩個蛋白以不同的構型存在，特別的是，FlhA似乎以半嵌入的狀態存在。提高誘導濃度後，發現兩者的快速擴散群的比例顯著下降，且快速擴散群皆變得更快，我們推測FliF在高濃度條件下可能通過自由能驅動的重分配機制，促使更多分子形成慢擴散群並整合至基底結構，FlhA則可能成為自己和FliF的催化劑。FliF在增加自身表達時，FliF參與聚集的比例不變，但額外的快速群減少，表示FliF更傾向進入聚集。儘管對於基底結構生成的調控機制仍存在未解問題，例如FlhA或FliF表達量交互影響對方運動狀態的機制，或者兩者在聚集內外擴散時的構型差異等，但無論如何，我們的研究更進一步揭示了大腸桿菌形成鞭毛基底結構的動態調控機制，為相關機制的理解提供了新線索。Although Escherichia coli flagella are distributed across the entire cell surface, they have long been considered to be randomly positioned. However, the observation that the flagellar proteins FlhA and FliF form stable clusters on the cell membrane suggests that their distribution may be regulated by an as-yet-uncharacterized mechanism. In this study, we employed fluorescent proteins and single-molecule tracking techniques by fusing PAmCherry and mTurquoise2 to FlhA and FliF, respectively, to investigate their dynamic behavior on the membrane. Through statistical analysis and model fitting, we characterized their motion states and spatial distribution.Our results show that both FlhA and FliF exhibit biphasic diffusion on the membrane with similar diffusion coefficients. However, due to differences in the number of transmembrane helix in their monomeric structures, we hypothesize that the two proteins exist in different conformations. When induction levels were increased, we observed a marked reduction in the proportion of the fast diffusing FliF population, whereas FlhA’s fast diffusing proportion remained stable. Concurrently, both proteins exhibited higher diffusion coefficients in their fast diffusing states. We propose that under high expression conditions, FliF undergoes a free energy–driven redistribution, driving more molecules into the slow diffusing population and promoting their incorporation into the basal body structure, FlhA could potentially serve as a catalyst for both itself and FliF. Importantly, although the overall clustering proportion of FliF remained unchanged, the unclustered fast diffusing subpopulation decreased, indicating that newly synthesized FliF preferentially integrates into existing clusters.Although key questions remain—such as how the expression levels of FlhA and FliF may cross-regulate each other’s diffusion dynamics, or how their conformations differ between clustered and non-clustered states—our study provides new insights into the dynamic regulatory mechanisms underlying the formation of the flagellar basal body in E. coli, and offers valuable clues for further understanding of the process.