SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition

Domenico Cieri,Tullio Pozzan,Francesca Vallese,Marisa Brini,Tina Wagner,Luca Scorrano,Mattia Vicario,Marta Giacomello,Riccardo Filadi,Paola Pizzo,Tito Calì

doi:10.1038/s41418-017-0033-z

Abstract

Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8–10 nm) and wide (40–50 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells. Narrow and wide ER–mitochondria contact sites responded differently to starvation, ER stress, mitochondrial shape modifications, and changes in the levels of modulators of ER–mitochondria juxtaposition. SPLICS detected contact sites in soma and axons of D. rerio Rohon Beard (RB) sensory neurons in vivo, extending its use to analyses of organelle juxtaposition in the whole animal.

Highlights

In eukaryotic cells, organelles are often found in close proximity, leading to the generation of heterotypic membrane appositions that ensure the coordination of severalEdited by N
To generate a modular fluorescence based sensor of organelle proximity, we decided to capitalize on the ability of two nonfluorescent portions of the superfolder GFP variant [23,24,25] to restore a fully fluorescent GFP upon self-assembly
To follow short- (≈8–10 nm) and longrange (≈40–50 nm) endoplasmic reticulum (ER)–mitochondria interactions [5], two constructs that differ for the length of the spacer placed between the ER targeting sequence and the β11 fragment were created by considering the distance of 0.36 nm between two alpha-carbons in a peptide chain: a ER-Short β11 with a 29 aa spacer and a ER-Long β11 with a 146 aa spacer

Summary

Introduction

Organelles are often found in close proximity, leading to the generation of heterotypic membrane appositions that ensure the coordination of severalEdited by N. Organelles are often found in close proximity, leading to the generation of heterotypic membrane appositions that ensure the coordination of several These authors contributed : Domenico Cieri, Mattia Vicario and Marta Giacomello. A network of contact sites between membranes of different organelles guarantees their mutual communication by creating microdomains that favor different signaling and metabolic pathways [1, 2]. Due to their central role in many fundamental cell processes, the sites of apposition between mitochondria and the endoplasmic reticulum (ER), which range from 10 to 100 nm, are, so far, the best characterized [3,4,5]

Methods

Results

Conclusion