Characterizing the Dynamics of Asgard ESCRT III Homologs Expressed in Mammalian Cells
2Department of Life Science and the National Institute for Biotechnology in the Negev, Ben Gurion University, Israel
The Endosomal Sorting Complex Required for Transport (ESCRT) is an evolutionary conserved membrane-remodeling machinery, composed of five subfamilies (ESCRT- 0-III and the AAA-ATPase VPS4). Recently, ESCRTIII homologues were found in Asgard archaea, the closest archaeal relative to eukaryotes, yet the function these homologs are still unknown. As archaea are extremely challenging for laboratory work, we recently set up a heterological expression system of Asgard ESCRTs in mammalian cells. We found that archaeal ESCRTIII homologues expressed in mammalian cells interact with each other and co-localize at discrete foci located in the cytosol nucleus.
In this research, we set to characterize the dynamics of two Asgard ESCRT III homologs namely CHMP4-7 and VPS4 that results foci formation. To this end, we co-expressed fluorescently tagged versions of the archaeal homologues in mammalian cells and imaged them in a spinning disk microscope over time. We found that, VPS4 initially localizes to the cytosol while CHMP4-7 resides at the nucleus. Prior to co-localization VPS4 was found to accumulate at the rims of the nucleus. Then, VPS4 either enter to the nucleus and co-localized with CHMP4-7 in nuclear foci or continued to aggregate forming foci outside the nucleus and recruiting nuclear CHMP4-7 to these foci. Further experiments to determine the dynamics of CHMP4-7 and VPS4 in cytosolic and nuclear foci will be performed and the possibility that these foci represent a phase separation behavior of ESCRT proteins will be examined.