Septin and actin contributions to endothelial cell-cell junctions and monolayer integrity

Endothelial cell monolayers form barriers regulating tissue homeostasis. These barriers rely on dynamic junctions between cells. Prior research has shown that junctional stability involves cadherins and actin networks. However, the role of septins in junctional mechanics remains unclear. Septins are cytoskeletal proteins known to associate with membranes in other cell types. No prior work had resolved how septins interact with actin at endothelial junctions. This gap motivated investigations into septin localization and function. Understanding septin-actin interactions could clarify how junctions remain stable amid membrane fluctuations. The absence of clear evidence for septin roles in endothelial junctions highlights the need for targeted studies. This uncertainty drove the current analysis of septin contributions to junctional integrity.

This study aimed to determine how septins influence endothelial cell junctions and monolayer stability. The specific problem addressed is the lack of understanding about septin localization and function in endothelial junctions. Researchers sought to clarify whether septins contribute to junctional mechanics. They focused on septin-membrane interactions and their relationship with actin structures. The motivation stems from prior findings on septin roles in other cell types. This work seeks to establish whether similar mechanisms apply in endothelial cells. The study also aimed to identify how septin depletion affects junctional dynamics. By examining septin-actin interplay, the researchers hoped to reveal new insights into endothelial barrier function.

The study used endothelial cell cultures and genetic manipulation to deplete septins. Fluorescence microscopy tracked septin localization at cell junctions. Researchers analyzed membrane curvature and septin accumulation patterns. Biochemical assays tested septin-lipid interactions using site-specific mutations. Actin distribution was assessed using F-actin staining techniques. Transendothelial electric resistance (TEER) measured monolayer integrity. Cell migration assays evaluated transendothelial migration (TEM) of immune and cancer cells. The experimental approach combined imaging, biochemical assays, and functional measurements to assess septin roles.

Septin depletion disrupted endothelial cell-cell junctions, as shown by VE-cadherin mislocalization. Septins localized at membrane regions with positive curvature, adjacent to actin-rich protrusions. Loss of septins reduced the number and breadth of cell-cell contacts. Septin depletion also decreased membrane retractions and protrusions at cell edges. F-actin levels dropped at the cortical membrane but increased in cytoplasmic stress fibers. TEER values declined, indicating compromised monolayer integrity. TEM increased due to gaps between cells after septin depletion. Septin filaments appear to provide a mechanical base for actin-based protrusive forces.

The authors propose that septins contribute to junctional stability by interacting with membrane curvature and actin structures. Septin depletion leads to junctional gaps and reduced TEER, as observed in the study. These findings suggest septins help maintain endothelial barrier function. The results support a model where septins provide a mechanical footing for actin protrusions. Septin-actin interactions may involve molecular feedback loops, as suggested by the data. The study highlights the importance of septin-lipid interactions in junctional dynamics. Septin depletion disrupts both junctional and actin cytoskeletal organization. The findings imply that septins are part of a regulatory network maintaining endothelial integrity.