Generation and characterization of genetically encoded fluorescent probes to visualize mitochondria-endoplasmic reticulum contact sites.

openEndoplasmic reticulum (ER) physically contacts mitochondria via its specialized subdomain called mitochondria-associated membranes (MAMs) at sites of mitochondria-ER contacts (MERCs). Interaction between these organelles at the MERCs plays essential roles in lipid and calcium transfer and ultimately in the homeostasis of the two individual organelles. Since the width of the MERCs could be as narrow as 10 nm, which is below the diffraction limit, a handful of probes have been developed to evaluate MERCs formation and dynamics. However, the current probes are dim, or artificially induce tethering, or require complicated imaging procedures that are not compatible with the common imaging setups available to most of the labs. To circumvent these problems, we are developing “STACCATO”, a new generation of probes to visualize MERCs that are based on split Fluorescence-Activating and absorption-Shifting Tag (FAST). STACCATO capitalizes on the reversible nature of split FAST complementation so the probe itself does not work as an artificial tether. In this Thesis we report the generation of a STACCATO probe for MERCs and its initial characterization and we show that STACCATO can report areas of proximity between mitochondria and the endoplasmic reticulum.Endoplasmic reticulum (ER) physically contacts mitochondria via its specialized subdomain called mitochondria-associated membranes (MAMs) at sites of mitochondria-ER contacts (MERCs). Interaction between these organelles at the MERCs plays essential roles in lipid and calcium transfer and ultimately in the homeostasis of the two individual organelles. Since the width of the MERCs could be as narrow as 10 nm, which is below the diffraction limit, a handful of probes have been developed to evaluate MERCs formation and dynamics. However, the current probes are dim, or artificially induce tethering, or require complicated imaging procedures that are not compatible with the common imaging setups available to most of the labs. To circumvent these problems, we are developing “STACCATO”, a new generation of probes to visualize MERCs that are based on split Fluorescence-Activating and absorption-Shifting Tag (FAST). STACCATO capitalizes on the reversible nature of split FAST complementation so the probe itself does not work as an artificial tether. In this Thesis we report the generation of a STACCATO probe for MERCs and its initial characterization and we show that STACCATO can report areas of proximity between mitochondria and the endoplasmic reticulum