It is known that the composition and distribution of glycolipids depend on the type and state of cells, and they are involved in various biological phenomena by control of cell function. In general, glycolipid metabolism is carried out in the Golgi apparatus, but the detailed mechanism is unknown. In the present study, I investigated the glycolipid metabolism by using live-cell imaging with BODIPY-labeled lactosylceramide (LacCer-BODIPY), which is known to undergo glycan modofications.
Previously, several reports indicated localization analysis of glycolipids using LacCer-BODIPY. However, there is no unified view regarding the transport of glycolipid probes, because the conditions vary from each experiment.Therefore, I tried to improve the condition of adding LacCer-BODIPY. As a result of various studies, I succeeded in improving the spatial temporal resolution by using a short time pulse tracking experiment. In this condition, it was demonstrated that recycling pathway between cell membrane and organella was observed (Fig. 1). Furthermore, I also confirmed this recycling process with a single cell tracking imaging system. As a result of colocalization analysis with the organella, I confirmed that LacCer-BODIPY returns to the cell membrane via early endosome and trans Golgi network.
Next, I also considered using LacCer-BODIPY as a probe to monitor membrane fluidity. In cells differentiated by nerve growth factor, I analyzed membrane fluidity in the FRAP system. Membrane fluidity increased immediately after stimulation. On the other hand, neurite outgrowth started 30 minutes after stimulation. These results suggest that increased membrane fluidity due to the stimulation initiates neurite outgrowth. Then I examined the relationship between F-actin and membrane fluidity. It was confirmed that the dynamics of F-actin was activated in response to stimulation. Therefore, It was confirmed that the change in fluidity of LacCer-BODIPY is due to the state change of actin. From the results of this study, it was shown that LacCer-BODIPY is a synthetic molecule useful for intracellular glycan conversion and membrane fluidity analysis.