Cali 2015 Abstract MiPschool London 2015
|Complementable green fluorescent protein based sensors of organelle proximity.|
Event: MiPschool London 2015
Due to the dynamic nature of organelle contact sites, it is difficult to monitor their behavior in living cells in a simple, one-step manner. Organelle proximity represents an important mechanism employed by the cells to ensure the tight coordination of several cellular activities. Indeed, a network of contact sites between the membranes of different organelles guarantee their mutual communication by creating microdomains that favor both signaling and the passage of lipids and ions. Because of their central role in the most essential cellular activities, the contact sites between mitochondria and the endoplasmic reticulum (ER) are, so far, the best characterized. However, the lack of a general and reliable method to properly monitor their proximity often prevented the possibility to draw clear conclusions. Here we describe a split-GFP based method for quick and reliable monitoring of organelle proximity adapted for ER-mitochondria contact sites evaluation. It is based on the ability of two non-fluorescent portions (the GFP 1-10 moiety and the GFP β-strand 11) of the extremely well folded superfolder GFP variant to restore a fully fluorescent GFP upon self-assembly.
To this aim we targeted the 16-amino-acid GFP β-strand 11 spaced by a 13 amino-acid (Sac Short β-11) and a 130 amino-acid long (Sac Long β-11) linker, to the cytosolic face of the ER and the GFP 1-10 detector fragment the outer mitochondrial membrane (OMM) in order to follow the appearance of tight (<15 nm) as well as loose contacts (<100 nm). We reasoned that upon co-expression in mammalian cells this one-step imaging technique would result in fluorescence emission specifically in the sites of apposition of the two organelles lying in a range between 15 and 100 nm.
Labels: MiParea: Instruments;methods
1-Dept Biol, Univ Padova, Italy. - [email protected] 2-Dept Biomed Sc, Univ Padova