The development of a method for the visualisation of intra-Golgi vesicle trafficking

The Golgi apparatus relies on the retrograde movement of intra-Golgi vesicles to maintain the cisternal locations of resident glycosylation enzymes. Previous studies have ascertained the structures and functions of tethering factors within the Golgi which mediate and control the movement of retrograde intra-Golgi vesicles containing the glycosylation enzymes. However, the specific interactions between the vesicles and target membranes, as well as the vesicle tethering and fusion processes, are still largely unknown. Here I present a method for the identification of the number of fluorescent molecules present in isolated intra-Golgi vesicles with TIRFM, as well as the generation of an intensity/distance from slide graph to quantify a relationship between the recorded fluorescence of a fluorescently tagged glycosylation enzyme with TIRFM and its distance from the slide surface. This data will be useful in future work to examine the transport, tethering and binding of intra-Golgi vesicles in a cell free assay. Furthermore, I attempted to develop a stable cell expressing tagged B4GALT1 and MGAT1 glycosylation enzymes for use in future cell-free experiments, however confocal microscopy revealed that HaloTag-B4GALT1 does not localise to the Golgi apparatus. From the methods described in this project, a cell line successfully expressing tagged glycosylation enzymes could be developed in the future.