Current and Emerging Approaches for Studying Inter-Organelle Membrane Contact Sites

Abstract

Inter-organelle membrane contact sites (MCSs) are classically defined as areas of close proximity between heterologous membranes and established by specific proteins (termed tethers). The interest on MCSs has rapidly increased in the last years, since MCSs play a crucial role in the transfer of cellular components between different organelles and have been involved in important cellular functions such as apoptosis, organelle division and biogenesis, and cell growth. Recently, an unprecedented depth and breadth in insights into the details of MCSs have been uncovered. On one hand, extensive MCSs (organelles interactome) are revealed by comprehensive analysis of organelle network with high temporal-spatial resolution at the system level. On the other hand, more and more tethers involving in MCSs are identified and further works are focusing on addressing the role of these tethers in regulating the function of MCSs at the molecular level. These enormous progresses largely depend on the powerful approaches, including several different types of microscopies and various biochemical techniques. These approaches have greatly accelerated recent advances in MCSs at the system and molecular level. In this review, we summarize the current and emerging approaches for studying MCSs, such as various microscopies, proximity-driven fluorescent signal generation and proximity-dependent biotinylation. In addition, we highlight the advantages and disadvantages of the techniques to provide a general guidance for the study of MCSs.

Flavonoids: A complementary approach to conventional therapy of COVID-19?

“…In FRET-based assays, the proteolytic activity is detected through cleavage of a fluorogenic peptide and measuring the increase in fluorescence intensity by continuously monitoring the reaction (Park et al 2017 ). FRET is a non-destructive method, widely exploited to study protein interactions (Margineanu et al 2016 ), and is also applied to detect signals in living cells (Huang et al 2020 ). While the cell-free FRET assays can provide easy and fast access to proteins including some difficult targets (Sierecki et al 2013 ), the cell-based FRET methods enable in situ analysis of a variety of biological targets and protein–protein interactions in a more natural environment (Silverman et al 1998 ).…”

Micro‐ and Nano‐Devices for Studying Subcellular Biology

“…In addition to enabling insight into the biophysical and chemical details of individual organelles, future work using similar micro‐ and nano‐traps could potentially be used to study interorganelle interactions through visualization of supramolecular structures tethered to or associated with the trapped organelle. [ 54 ]…”

“…Such organelle interactions have proven to be important in mitochondrial fusion and fission, ER remodeling, and other subcellular processes. [ 41,54,56 ]…”

“…For the interested reader, there are several excellent reviews and resources on super resolution microscopy, fluorescence techniques, optogenetics, atomic force microscopy, and acoustic manipulations. [53][54][55][56][57][58] Though not the focus here, the interested reader is also directed to recent reviews of technology for processing exosomes and extracellular vesicles. [59,60]…”

Maintaining social contacts: The physiological relevance of organelle interactions

“…between internal organelle membranes), and considerable variation in their composition, size, distance between organelles and stability have been described [ 3 ]. Guidelines have been delineated to define MCSs and their tethers [ 8 , 9 ], and new approaches are being developed to study and quantify MCSs, and to distinguish them from stochastic interactions [ [10] , [11] , [12] ]. Consequently, new contact sites are being discovered and the number of tethers and proteins associated with MCS is constantly expanding [ 9 ].…”