In Vivo Quantification of Peroxisome Tethering to Chloroplasts in Tobacco Epidermal Cells Using Optical Tweezers

Peroxisomes are highly motile organelles that display a range of motions within a short time frame. In static snapshots, they can be juxtaposed to chloroplasts, which has led to the hypothesis that they are physically interacting. Here, using optical tweezers, we tested the dynamic physical interaction in vivo. Using near-infrared optical tweezers combined with TIRF microscopy, we were able to trap peroxisomes and approximate the forces involved in chloroplast association in vivo in tobacco (Nicotiana tabacum) and observed weaker tethering to additional unknown structures within the cell. We show that chloroplasts and peroxisomes are physically tethered through peroxules, a poorly described structure in plant cells. We suggest that peroxules have a novel role in maintaining peroxisome-organelle interactions in the dynamic environment. This could be important for fatty acid mobilization and photorespiration through the interaction with oil bodies and chloroplasts, highlighting a fundamentally important role for organelle interactions for essential biochemistry and physiological processes.A combination of genetically encoded fluorescent probes, advances in light microscopy, and interdisciplinary approaches has revolutionized our understanding of organelle transport. Organelle movement in highly vacuolated leaf epidermal cells appears erratic, with individual organelles undergoing a range of movements within a relatively short time frame: they stop-go, change direction (trajectory), and move at varying speeds. The use of pharmacological inhibitors indicated a role for actin, and therefore myosins, in this process; however, myosin-organelle specificity is poorly characterized (Madison and Nebenführ, 2013; Tamura et al., 2013; Buchnik et al., 2015). Therefore, we are still at a relatively rudimentary stage in the understanding of the molecular and physical control, and interaction, of organelles in plant cells compared with that known in other model systems (Hammer and Sellers, 2012; Prinz, 2014). However, it is clear that organelle movement plays important roles in physological processes in plants; reduced movement effects growth and development, and movement is correlated with responses to extracellular stresses such as pathogens and heavy metals (for refs., see Sparkes, 2011; Madison and Nebenführ, 2013; Buchnik et al., 2015). Organelle interactions in other systems have important roles in calcium and lipid exchange, setting a precedent for physiologically important roles in plants (Prinz, 2014). However, characterization of the molecular factors required to physically tether organelles, as opposed to those that function in the exchange of molecules at the interaction site, is challenging. Monitoring organelle interactions in highly vacuolated plant epidermal cells is further complicated by the constraints imposed by the large central vacuole. Static snapshots provided through electron microscopy of highly vacuolated cells, where the vacuole can effectively push organelles together, giving the impression of direct interaction between organelles, is not a suitable method to determine dynamic interactions. Other techniques, such as the laser-induced shockwave by explosion method used by Oikawa et al. (2015), works globally without directly manipulating the individual organelle. Here, using optical tweezers with submicron precision, we provide a means to assess and quantify the dynamic interaction between peroxisomes and chloroplasts in vivo in leaf epidermal cells.Peroxisomes are responsible for several biochemical reactions, including the glyoxylate cycle and β-oxidation, which provides an energy source for germination in oilseeds. They also produce and scavenge free radicals, synthesize jasmonic acid and indole-3-acetic acid, and are required for photorespiration (for refs., see Hu et al., 2012). The photorespiratory pathway spans peroxisomes, chloroplasts, and mitochondria, where phosphoglycolate produced in the chloroplast is converted back to 3-phosphoglycerate. It has been suggested that functional connectivity between these organelles accounts for the close association observed in ultrastructural micrographs (Frederick and Newcomb, 1969). Several Arabidopsis (Arabidopsis thaliana) pex10 (peroxisomal membrane protein) mutants show altered chloroplast-peroxisome juxtaposition with a defect in photorespiration, while others do not (Schumann et al., 2007; Prestele et al., 2010). Both CLUMPED CHLOROPLASTS1 (CLMP1) and CHLOROPLAST UNUSUAL POSITIONING1 (CHUP1) encode for proteins that localize to the chloroplast, with CHUP1 playing a role in chloroplast-actin formation (Oikawa et al., 2003, 2008; Schmidt von Braun and Schleiff, 2008; Yang et al., 2011). While CHUP1 and CLMP1 affect chloroplast positioning, they have differential effects on peroxisome and mitochondrial location; clmp1 causes chloroplast clustering without affecting mitochondria or peroxisome location (Yang et al., 2011), whereas chup1 was reported to affect peroxisome location (Oikawa et al., 2003). In vitro analysis through density centrifugation highlighted chloroplast sedimentation with peroxisomes under certain conditions (Schnarrenberger and Burkhard, 1977), although this does not necessarily reflect the organelle interaction in live cells. Peroxisome proteomics studies have been hampered by difficulties in isolating pure peroxisomal fractions (Bussell et al., 2013). This could be indicative of interaction, where associated membranes are isolated together, or sticky nonspecific contaminating chloroplast membranes. The work by Oikawa et al. (2015) provides insight into the physiological processes controlling peroxisome-chloroplast interaction (photosynthesis dependent), but they did not determine the effective baseline force required to move peroxisomes that were not next to chloroplasts under control or altered environmental conditions. Comparisons between the relative forces required to move peroxisomes next to chloroplasts versus those that are not next to chloroplasts are critical in understanding and probing the physical interaction between the two organelles, the hypothesis being that tethering would increase the force required to move peroxisomes compared with organelles that are not tethered. Since peroxisomes have diverse biochemical roles that affect a wide range of physiological processes throughout the plant life cycle (Hu et al., 2012), an understanding of if and how peroxisomes may interact with other subcellular structures is likely to be an important consideration for efficient peroxisome function.Peroxisomes are highly pleomorphic, dynamic organelles bounded by a single membrane (Hu et al., 2012), whose movement is driven by acto-myosin-dependent processes (Jedd and Chua, 2002; Mano et al., 2002; Mathur et al., 2002; Avisar et al., 2008; Sparkes et al., 2008). Tubular emanations termed peroxules (Scott et al., 2007) can extend from the main peroxisome body, yet it is unclear what function they may play. Formation is quite frequent in hypocotyl cells (Cutler et al., 2000; Mano et al., 2002; Sinclair et al., 2009), can occur around chloroplasts in cotyledonary leaf pavement cells (Sinclair et al., 2009), and is not always from the trailing edge of the peroxisome (Sinclair et al., 2009). Exogenous addition of hydroxyl reactive oxygen species (ROS), or exposure to UV light, induces peroxule formation (Sinclair et al., 2009). It has been suggested that they represent an increased surface area for increased biochemical function or might represent a morphological precursor for peroxisome division (Jedd and Chua, 2002). Based on subcellular coalignment, a retro-flow model for the potential exchange of luminal content between the endoplasmic reticulum (ER) and peroxisome through the peroxule has been suggested (Sinclair et al., 2009; Barton et al., 2013). However, these studies, as with many others, interpret the close association between organelles to indicate physical connectivity between organelles, whereas, in fact, in highly vacuolated leaf epidermal cells, organelles can be closely packed within the cytoplasm due to mere spatial constrictions generated through the large central vacuole. This is further complicated by the highly motile, and seemingly stochastic, nature of acto-myosin-driven organelle movement, resulting in frequent apparent organelle collisions that may not reflect a functional requirement for organelle interaction.Optical trapping provides a highly specific and sensitive means to measure physical connectivity between organelles. By focusing an infrared beam, it allows the user to trap objects that have a significantly different refractive index from the surrounding medium. Upon trapping, the user can then move the trapped object relative to its original position to gain an understanding of whether the movement affects the position and motion of other structures (such as other organelles) that may be physically attached to the trapped organelle. For example, unlike the ER, Golgi bodies are amenable to trapping. By trapping and micromanipulating (i.e. precisely moving) the Golgi, a physical association between the ER and the Golgi was determined in a qualitative manner (Sparkes et al., 2009b). Here, we have developed a system to generate quantitative measures for organelle interaction by standardizing and automating how far we move the trapped organelle (which we call the translation step) at a defined speed and assessing how trapping efficiency alters in response to the power of the laser trap itself. By using these parameters, we can then model the forces imparted on the organelle, providing further insight into the tethering processes.Our results indicate that peroxisomes are amenable to being trapped, that they physically interact with chloroplasts in leaf epidermal cells, and, surprisingly, that peroxisomes are also tethered to other unknown structures within the cell. This approach highlights that organelle interactions within plant cells are not random but regulated through tethering. In addition, we provide a novel role for peroxules and a simple biophysical model to describe peroxisome motion during the trapping process.