Quantitatively Assessing Co-Localization of Golgi Proteins by Distance Analysis Using the DiAna Software.

This method determines if two fluorescently labeled proteins are in close proximity to each other in situ. It is an alternative to commonly used co-localization assays and is based on measuring distances between pairs of objects representative of the two proteins. It makes use of a relatively recently developed ImageJ plugin called DiAna, which employs semi-automated object recognition and subsequent distance analysis of the recognized objects. The advantages of this method are that it is largely independent of the actual pixel intensity values, quite robust against background noise, and not reliant on arbitrarily set intensity threshold values. We present here a use case for the DiAna plugin in the context of plant cells with fluorescently labeled subcellular structures, such as proteins associated with the plant Golgi apparatus.

EPSIN1 and MTV1 define functionally overlapping but molecularly distinct trans-Golgi network subdomains in Arabidopsis.

The plant trans-Golgi network (TGN) is a central trafficking hub where secretory, vacuolar, recycling, and endocytic pathways merge. Among currently known molecular players involved in TGN transport, three different adaptor protein (AP) complexes promote vesicle generation at the TGN with different cargo specificity and destination. Yet, it remains unresolved how sorting into diverging vesicular routes is spatially organized. Here, we study the family of Arabidopsis thaliana Epsin-like proteins, which are accessory proteins to APs facilitating vesicle biogenesis. By comprehensive molecular, cellular, and genetic analysis of the EPSIN gene family, we identify EPSIN1 and MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1) as its only TGN-associated members. Despite their large phylogenetic distance, they perform overlapping functions in vacuolar and secretory transport. By probing their relationship with AP complexes, we find that they define two molecularly independent pathways: While EPSIN1 associates with AP-1, MTV1 interacts with AP-4, whose function is required for MTV1 recruitment. Although both EPSIN1/AP-1 and MTV1/AP-4 pairs reside at the TGN, high-resolution microscopy reveals them as spatially separate entities. Our results strongly support the hypothesis of molecularly, functionally, and spatially distinct subdomains of the plant TGN and suggest that functional redundancy can be achieved through parallelization of molecularly distinct but functionally overlapping pathways.