Using Optical Tweezers to Characterize Physical Tethers at Membrane Contact Sites: Grab It, Pull It, Set It Free?

Compartmentalisation is a defining feature of eukaryotic life. Effective communication between organelles is essential for cell maintenance, growth and response to external stimuli. Static snapshots provided through ultrastructural studies of preserved tissue highlight that certain organelles are in intimate contact at membrane contact sites (MCSs), also referred to as inter-organellar tethering sites. However, live cell imaging indicates that these interactions are not necessarily stable with organelles frequently " colliding, " moving in unison and then separating. This dramatic intracellular " waltz " between organelles with ever changing partners (organelles) indicates that the molecular factors controlling MCSs are highly regulated. Key questions therefore relate to defining which organelles physically interact, deciphering the molecular components that control MCS formation, and ultimately deciphering the specific functional role that the interaction provides to the cell (Figure 1). provide an overview of the techniques used to interrogate MCSs and how optical tweezers could provide a future platform for characterizing the biophysical nature of MCSs. Tethers have been isolated using multiple techniques, however a difficulty has been being able to discriminate between a role in physical tethering versus a role in transferring components at the MCS itself. For example, tethering sites are required for signaling, trafficking and biogenesis. Proteins located at MCSs could provide physical stability and MCSs formation, whereas others could collocate to and function in the actual transfer of molecules such as lipids and calcium. These generic roles are not necessarily mutually exclusive as evidenced by studies with OSBP (Mesmin et al., 2013). One way to discriminate between these two generic roles is if disruption of the potential tether affects the subsequent physical association between organelles. A clear example of this is observed during division of budding yeast where tethering can play a role in organelle inheritance into the bud cell. Dependent on the organism and cell type, organelles can be highly motile during interphase. Here, organelles are not clearly partitioned in a similar manner during cell division, and so seemingly random motion could result in organelles occupying similar physical regions without it being as a result of direct physical interaction per se. Organelle movement in higher plants is even further complicated by fast cytoplasmic streaming events. Attempts to isolate and probe the nature of physical tethers include biochemical fractionation, genetic screens and microscopy. Applications of these techniques to investigate MCSs are covered in more depth in the review by Helle et al. (2013). …