UNRAVELING THE ULTRASTRUCTURE OF THE FUSOGENIC SYNAPSE WITH CLEM

The term Synapse, originally coined by Charles Sherrington in 1897 to describe the junctions between neurons, has since expanded to describe a range of cell-to-cell interfaces, mediating complex interactions. From the neuronal synapses, (500-1000 nm) which mediate neuron-neuron or neuron-muscle communication, to immunological synapses (>1 micron) which mediate interactions between immune cells and antigen presenting cells during the immune response. More recently, fusogenic synapses, which mediate fusion between muscle cells have been described in Drosophila melanogaster. However, little is known about the fusogenic synapse and it is unclear whether it is conserved in vertebrates muscle. Moreover, it is poorly understood how the fusogenic synapse forms and how information is exchanged across it. In order to gain a molecular level understanding of the fusogenic synapse high-resolution information at different stages of its development is needed. Obtaining this information is challenging because the fusogenic synapse is at the interface between two cells, so large fields of view need to be captured. Serial block face scanning electron microscopy after focused ion beam milling (FIB-SEM) is an ideal choice for this task. This instrument enables the 3-dimentional visualization of biological specimens with unprecedented detail and with up to 5 nm resolution along all three axes. However, in order to capture cells in the middle of a transient event, such as cell-to-cell fusion, we must have molecular or morphological determinants as reporters for the dynamic state of the event. Alternatively, we need to capture cells in act of fusion by live cell imaging and image the same cells by FIB-SEM. Therefore, we have developed a correlative light and electron microscopy workflow for targeting cells by fluorescence microscopy for FIB-SEM imaging. Our workflow is compatible with ambient- and cryo- conditions, thus providing information from both resin-embedded as well as vitrified cells. Moreover, we show that it can be used to target fluorescence signals in large organisms such as C. elegans and D. melanogaster.