Abstract
Many organelles from the secretory pathway fuse to the plasma membrane, to exocytose different cargoes. Their proteins are then retrieved from the plasma membrane by endocytosis, and the organelles are re-formed. It is generally unclear whether the organelle proteins colocalize when they are on the plasma membrane, or whether they disperse. To address this, we generated here a new approach, which we tested on synaptic vesicles, organelles that are known to exo- and endocytose frequently. We tagged the synaptotagmin molecules of newly exocytosed vesicles using clusters of primary and secondary antibodies targeted against the luminal domains of these molecules. The antibody clusters are too large for endocytosis, and thus sequestered the synaptotagmin molecules on the plasma membrane. Immunostainings for other synaptic molecules then revealed whether they colocalized with the sequestered synaptotagmin molecules. We suggest that such assays may be in the future extended to other cell types and other organelles.