US scientists have performed an advanced study of structures inside live cells using total internal reflection fluorescence (TIRF) microscopy. The experiment, which was carried out by two postgraduates from Yale University, captured the first time-lapse pictures of the APPL1 endosome, a type of vesicle that carries signals inside a cell from its surface. By tracking the vesicle using time-lapse imaging, they found APPL1 helps cells communicate with each other. Previously, scientists had believed most intercellular communication occurred at a cell’s surface. The research shows that signaling between cells is much more complex than originally thought and it provides insight into diseases associated with APPL1, such as Dent disease, a rare kidney disease, and Lowe syndrome, a rare eye, kidney and brain condition. The APPL1 vesicle also could have a role in cancer and metabolic diseases such as diabetes. “TIRF is designed to image things close to the surface of the cell with unmatched definition and signal-to-noise,” said Roberto Zoncu, one of the postgraduates who performed the experiment. “But it wasn’t thought to be ideal for endosomes.” The researchers predicted that some vesicles would park close to the cell surface after they entered the cell, meaning they could be observed using TIRF. They captured time-lapse images of the vesicles, which were labeled with fluorescing particles, as they moved through the cell using an Andor iXon 897 electron-multiplying CCD camera. The fluorescent dots indicating each vesicle were tracked from image to image using Andor’s iQ image-analysis software. “We have proved we can image things moving from the surface of the cell inside using TIRF,” Zoncu said. “And that’s an advantage because it’s the most sensitive technique of all.” The time-lapse results suggest APPL1 transforms into an early endosome – a compartment that acts like a sorting office, dispatching signals to different parts of the cell. The team also discovered that a phospholipid (fat) molecule called phosphatidylinositol-3-phosphate is the molecular switch that turns the APPL1 vesicle into an endosome. Zoncu is optimistic for future research on APPL1. “Thanks to this technology, we can focus further studies on diseases where this endosome is likely to be involved.” The research was published in the journal Cell last year. For more information, visit: www.andor.com