Applying Optical Tweezers with TIRF Microscopy to Quantify Physical Interactions Between Organelles in the Plant Endomembrane System.

Plant organelles are associated with each other through tethering proteins at membrane contact sites (MCS). Methods such as total internal reflection fluorescence (TIRF) optical tweezers allow us to probe organelle interactions in live plant cells. Optical tweezers (focused infrared laser beams) can trap organelles that have a different refractive index to their surrounding medium (cytosol), whilst TIRF allows us to simultaneously image behaviors of organelles in the thin region of cortical cytoplasm. However, few MCS tethering proteins have so far been identified and tested in a quantitative manner. Automated routines (such as setting trapping laser power and controlling the stage speed and distance) mean we can quantify organelle interactions in a repeatable and reproducible manner. Here we outline a series of protocols which describe laser calibrations required to collect robust data sets, generation of fluorescent plant material (Nicotiana tabacum, tobacco), how to set up an automated organelle trapping routine, and how to quantify organelle interactions (particularly organelle interactions with the endoplasmic reticulum). TIRF-optical tweezers enable quantitative testing of putative tethering proteins to reveal their role in plant organelle associations at MCS. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Microscope system set-up and stability Basic Protocol 2: Generation of transiently expressed fluorescent tobacco tissue by Agrobacterium-mediated infiltration Basic Protocol 3: Setting up an automated organelle trapping routine Basic Protocol 4: Quantifying organelle interactions.

Arabidopsis thaliana myosin XIK is recruited to the Golgi through interaction with a MyoB receptor.

Plant cell organelles are highly mobile and their positioning play key roles in plant growth, development and responses to changing environmental conditions. Movement is acto-myosin dependent. Despite controlling the dynamics of several organelles, myosin and myosin receptors identified so far in Arabidopsis thaliana generally do not localise to the organelles whose movement they control, raising the issue of how specificity is determined. Here we show that a MyoB myosin receptor, MRF7, specifically localises to the Golgi membrane and affects its movement. Myosin XI-K was identified as a putative MRF7 interactor through mass spectrometry analysis. Co-expression of MRF7 and XI-K tail triggers the relocation of XI-K to the Golgi, linking a MyoB/myosin complex to a specific organelle in Arabidopsis. FRET-FLIM confirmed the in vivo interaction between MRF7 and XI-K tail on the Golgi and in the cytosol, suggesting that myosin/myosin receptor complexes perhaps cycle on and off organelle membranes. This work supports a traditional mechanism for organelle movement where myosins bind to receptors and adaptors on the organelle membranes, allowing them to actively move on the actin cytoskeleton, rather than passively in the recently proposed cytoplasmic streaming model.

On the move: redox-dependent protein relocation in plants.

Compartmentation of proteins and processes is a defining feature of eukaryotic cells. The growth and development of organisms is critically dependent on the accurate sorting of proteins within cells. The mechanisms by which cytosol-synthesized proteins are delivered to the membranes and membrane compartments have been extensively characterized. However, the protein complement of any given compartment is not precisely fixed and some proteins can move between compartments in response to metabolic or environmental triggers. The mechanisms and processes that mediate such relocation events are largely uncharacterized. Many proteins can in addition perform multiple functions, catalysing alternative reactions or performing structural, non-enzymatic functions. These alternative functions can be equally important functions in each cellular compartment. Such proteins are generally not dual-targeted proteins in the classic sense of having targeting sequences that direct de novo synthesized proteins to specific cellular locations. We propose that redox post-translational modifications (PTMs) can control the compartmentation of many such proteins, including antioxidant and/or redox-associated enzymes.

Stacks off tracks: a role for the golgin AtCASP in plant endoplasmic reticulum-Golgi apparatus tethering.

The plant Golgi apparatus modifies and sorts incoming proteins from the endoplasmic reticulum (ER) and synthesizes cell wall matrix material. Plant cells possess numerous motile Golgi bodies, which are connected to the ER by yet to be identified tethering factors. Previous studies indicated a role for cis-Golgi plant golgins, which are long coiled-coil domain proteins anchored to Golgi membranes, in Golgi biogenesis. Here we show a tethering role for the golgin AtCASP at the ER-Golgi interface. Using live-cell imaging, Golgi body dynamics were compared in Arabidopsis thaliana leaf epidermal cells expressing fluorescently tagged AtCASP, a truncated AtCASP-ΔCC lacking the coiled-coil domains, and the Golgi marker STtmd. Golgi body speed and displacement were significantly reduced in AtCASP-ΔCC lines. Using a dual-colour optical trapping system and a TIRF-tweezer system, individual Golgi bodies were captured in planta. Golgi bodies in AtCASP-ΔCC lines were easier to trap and the ER-Golgi connection was more easily disrupted. Occasionally, the ER tubule followed a trapped Golgi body with a gap, indicating the presence of other tethering factors. Our work confirms that the intimate ER-Golgi association can be disrupted or weakened by expression of truncated AtCASP-ΔCC and suggests that this connection is most likely maintained by a golgin-mediated tethering complex.

The ureide-degrading reactions of purine ring catabolism employ three amidohydrolases and one aminohydrolase in Arabidopsis, soybean, and rice.

Several ureides are intermediates of purine base catabolism, releasing nitrogen from the purine nucleotides for reassimilation into amino acids. In some legumes like soybean (Glycine max), ureides are used for nodule-to-shoot translocation of fixed nitrogen. Four enzymes of Arabidopsis (Arabidopsis thaliana), (1) allantoinase, (2) allantoate amidohydrolase (AAH), (3) ureidoglycine aminohydrolase, and (4) ureidoglycolate amidohydrolase (UAH), catalyze the complete hydrolysis of the ureide allantoin in vitro. However, the metabolic route in vivo remains controversial. Here, in growth and metabolite analyses of Arabidopsis mutants, we demonstrate that these enzymes are required for allantoin degradation in vivo. Orthologous enzymes are present in soybean, encoded by one to four gene copies. All isoenzymes are active in vitro, while some may be inefficiently translated in vivo. Surprisingly, transcript and protein amounts are not significantly regulated by nitrogen fixation or leaf ureide content. A requirement for soybean AAH and UAH for ureide catabolism in leaves has been demonstrated by the use of virus-induced gene silencing. Functional AAH, ureidoglycine aminohydrolase, and UAH are also present in rice (Oryza sativa), and orthologous genes occur in all other plant genomes sequenced to date, indicating that the amidohydrolase route of ureide degradation is universal in plants, including mosses (e.g. Physcomitrella patens) and algae (e.g. Chlamydomomas reinhardtii).

Optical tweezers for the micromanipulation of plant cytoplasm and organelles.

Laser trapping of micron-sized particles can be achieved utilizing the radiation pressure generated by a focused infrared laser beam. Thus, it is theoretically possible to trap and manipulate organelles within the cytoplasm and remodel the architecture of the cytoplasm and membrane systems. Here we describe recent progress, using this under utilized technology, in the manipulation of cytoplasmic strands and organelles in plant cells.

Peroxisome biogenesis and positioning.

Plant peroxisomes are extremely dynamic, moving and undergoing changes of shape in response to metabolic and environmental signals. Matrix proteins are imported via one of two import pathways, depending on the targeting signal within the protein. Each pathway has a specific receptor but utilizes common membrane-bound translocation machinery. Current models invoke receptor recycling, which may involve cycles of ubiquitination. Some components of the import machinery may also play a role in proteolytic turnover of matrix proteins, prompting parallels with the endoplasmic-reticulum-associated degradation pathway. Peroxisome membrane proteins, some of which are imported post-translationally, others of which may traffic to peroxisomes via the endoplasmic reticulum, use distinct proteinaceous machinery. The isolation of mutants defective in peroxisome biogenesis has served to emphasize the important role of peroxisomes at all stages of the plant life cycle.

Motoring around the plant cell: insights from plant myosins.

Organelle movement in plants cells is extremely dynamic. Movement is driven by the acto-myosin system. Higher plant myosins fall into two classes: classes XI and VIII. Localization studies have highlighted that myosins are present throughout the cytosol, label motile puncta and decorate the nuclear envelope and plasma membrane. Functional studies through expression of dominant-negative myosin variants, RNAi (RNA interference) and T-DNA insertional analysis have shown that class XI myosins are required for organelle movement. Intriguingly, organelle movement is also linked to Arabidopsis growth and development. The present review tackles current findings relating to plant organelle movement and the role of myosins.

Salt stress causes peroxisome proliferation, but inducing peroxisome proliferation does not improve NaCl tolerance in Arabidopsis thaliana.

The PEX11 family of peroxisome membrane proteins have been shown to be involved in regulation of peroxisome size and number in plant, animals, and yeast cells. We and others have previously suggested that peroxisome proliferation as a result of abiotic stress may be important in plant stress responses, and recently it was reported that several rice PEX11 genes were up regulated in response to abiotic stress. We sought to test the hypothesis that promoting peroxisome proliferation in Arabidopsis thaliana by over expression of one PEX11 family member, PEX11e, would give increased resistance to salt stress. We could demonstrate up regulation of PEX11e by salt stress and increased peroxisome number by both PEX11e over expression and salt stress, however our experiments failed to find a correlation between PEX11e over expression and increased peroxisome metabolic activity or resistance to salt stress. This suggests that although peroxisome proliferation may be a consequence of salt stress, it does not affect the ability of Arabidopsis plants to tolerate saline conditions.

The plant endoplasmic reticulum: a cell-wide web.

The ER (endoplasmic reticulum) in higher plants forms a pleomorphic web of membrane tubules and small cisternae that pervade the cytoplasm, but in particular form a polygonal network at the cortex of the cell which may be anchored to the plasma membrane. The network is associated with the actin cytoskeleton and demonstrates extensive mobility, which is most likely to be dependent on myosin motors. The ER is characterized by a number of domains which may be associated with specific functions such as protein storage, or with direct interaction with other organelles such as the Golgi apparatus, peroxisomes and plastids. In the present review we discuss the nature of the network, the role of shape-forming molecules such as the recently described reticulon family of proteins and the function of some of the major domains within the ER network.

A comparative study of the involvement of 17 Arabidopsis myosin family members on the motility of Golgi and other organelles.

Gene families with multiple members are predicted to have individuals with overlapping functions. We examined all of the Arabidopsis (Arabidopsis thaliana) myosin family members for their involvement in Golgi and other organelle motility. Truncated fragments of all 17 annotated Arabidopsis myosins containing either the IQ tail or tail domains only were fused to fluorescent markers and coexpressed with a Golgi marker in two different plants. We tracked and calculated Golgi body displacement rate in the presence of all myosin truncations and found that tail fragments of myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K were the best inhibitors of Golgi body movement in the two plants. Tail fragments of myosins XI-B, XI-F, XI-H, and ATM1 had an inhibitory effect on Golgi bodies only in Nicotiana tabacum, while tail fragments of myosins XI-G and ATM2 had a slight effect on Golgi body motility only in Nicotiana benthamiana. The best myosin inhibitors of Golgi body motility were able to arrest mitochondrial movement too. No exclusive colocalization was found between these myosins and Golgi bodies in our system, although the excess of cytosolic signal observed could mask myosin molecules bound to the surface of the organelle. From the preserved actin filaments found in the presence of enhanced green fluorescent protein fusions of truncated myosins and the motility of myosin punctae, we conclude that global arrest of actomyosin-derived cytoplasmic streaming had not occurred. Taken together, our data suggest that the above myosins are involved, directly or indirectly, in the movement of Golgi and mitochondria in plant cells.

Grab a Golgi: laser trapping of Golgi bodies reveals in vivo interactions with the endoplasmic reticulum.

In many vacuolate plant cells, individual Golgi bodies appear to be attached to tubules of the pleiomorphic cortical endoplasmic reticulum (ER) network. Such observations culminated in the controversial mobile secretory unit hypothesis to explain transport of cargo from the ER to Golgi via Golgi attached export sites. This proposes that individual Golgi bodies and an attached-ER exit machinery move over or with the surface of the ER whilst collecting cargo for secretion. By the application of infrared laser optical traps to individual Golgi bodies within living leaf cells, we show that individual Golgi bodies can be micromanipulated to reveal their association with the ER. Golgi bodies are physically attached to ER tubules and lateral displacement of individual Golgi bodies results in the rapid growth of the attached ER tubule. Remarkably, the ER network can be remodelled in living cells simply by movement of laser trapped Golgi dragging new ER tubules through the cytoplasm and new ER anchor sites can be established. Finally, we show that trapped Golgi ripped off the ER are 'sticky' and can be docked on to and attached to ER tubules, which will again show rapid growth whilst pulled by moving Golgi.

Truncated myosin XI tail fusions inhibit peroxisome, Golgi, and mitochondrial movement in tobacco leaf epidermal cells: a genetic tool for the next generation.

Although organelle movement in higher plants is predominantly actin-based, potential roles for the 17 predicted Arabidopsis myosins in motility are only just emerging. It is shown here that two Arabidopsis myosins from class XI, XIE, and XIK, are involved in Golgi, peroxisome, and mitochondrial movement. Expression of dominant negative forms of the myosin lacking the actin binding domain at the amino terminus perturb organelle motility, but do not completely inhibit movement. Latrunculin B, an actin destabilizing drug, inhibits organelle movement to a greater extent compared to the effects of AtXIE-T/XIK-T expression. Amino terminal YFP fusions to XIE-T and XIK-T are dispersed throughout the cytosol and do not completely decorate the organelles whose motility they affect. XIE-T and XIK-T do not affect the global actin architecture, but their movement and location is actin-dependent. The potential role of these truncated myosins as genetically encoded inhibitors of organelle movement is discussed.

Identification, biochemical characterization, and subcellular localization of allantoate amidohydrolases from Arabidopsis and soybean.

Allantoate amidohydrolases (AAHs) hydrolize the ureide allantoate to ureidoglycolate, CO(2), and two molecules of ammonium. Allantoate degradation is required to recycle purine-ring nitrogen in all plants. Tropical legumes additionally transport fixed nitrogen via allantoin and allantoate into the shoot, where it serves as a general nitrogen source. AAHs from Arabidopsis (Arabidopsis thaliana; AtAAH) and from soybean (Glycine max; GmAAH) were cloned, expressed in planta as StrepII-tagged variants, and highly purified from leaf extracts. Both proteins form homodimers and release 2 mol ammonium/mol allantoate. Therefore, they can truly be classified as AAHs. The kinetic constants determined and the half-maximal activation by 2 to 3 microm manganese are consistent with allantoate being the in vivo substrate of manganese-loaded AAHs. The enzymes were strongly inhibited by micromolar concentrations of fluoride as well as by borate, and by millimolar concentrations of L-asparagine and L-aspartate but not D-asparagine. L-Asparagine likely functions as competitive inhibitor. An Ataah T-DNA mutant, unable to grow on allantoin as sole nitrogen source, is rescued by the expression of StrepII-tagged variants of AtAAH and GmAAH, demonstrating that both proteins are functional in vivo. Similarly, an allantoinase (aln) mutant is rescued by a tagged AtAln variant. Fluorescent fusion proteins of allantoinase and both AAHs localize to the endoplasmic reticulum after transient expression and in transgenic plants. These findings demonstrate that after the generation of allantoin in the peroxisome, plant purine degradation continues in the endoplasmic reticulum.

Overexpression of a plant reticulon remodels the lumen of the cortical endoplasmic reticulum but does not perturb protein transport.

We have cloned a member of the reticulon (RTN) family of Arabidopsis thaliana (RTNLB13). When fused to yellow fluorescent protein (YFP) and expressed in tobacco leaf epidermal cells, RTNLB13 is localized in the endoplasmic reticulum (ER). Coexpression of a soluble ER luminal marker reveals that YFP-tagged, myc-tagged or untagged RTNLB13 induces severe morphological changes to the lumen of the ER. We show, using fluorescence recovery after photobleaching (FRAP) analysis, that RTNLB13 overexpression greatly reduces diffusion of soluble proteins within the ER lumen, possibly by introducing constrictions into the membrane. In spite of this severe phenotype, Golgi shape, number and dynamics appear unperturbed and secretion of a reporter protein remains unaffected.

Arabidopsis PEX19 is a dimeric protein that binds the peroxin PEX10.

Peroxisomes are organelles found in all eukaryotic cells. Peroxisomes import integral membrane proteins post-translationally, and PEX19 is a predominantly cytosolic, farnesylated protein of mammalian and yeast cells that binds multiple peroxisome membrane proteins and is required for their correct targeting/insertion to the peroxisome membrane. We report the characterisation of the Arabidopsisthaliana homologue of PEX19 which is a predominantly cytosolic protein. AtPEX19 is encoded by two genes (designated AtPEX19-1 and AtPEX19-2) that are expressed in all tissues and at all developmental stages of the plant. Quantitative real time PCR shows that AtPEX19-1 and AtPEX19-2 have distinct expression profiles. Using in vitro translation and co-immunoprecipitation AtPEX19-1 was shown to bind to the Arabidopsis peroxisomal membrane protein PEX10. Additionally, bacterially expressed recombinant AtPEX19-1 was able to bind a fusion protein consisting of the C-terminus of PEX10 and glutathione S-transferase in pull-down assays, thereby demonstrating that non-farnesylated AtPEX19 can interact with the C-terminus of AtPEX10. Purified recombinant AtPEX19-1 was analysed by gel filtration chromatography and was found to have a molecular weight consistent with it forming a dimer and a dimer was detected in Arabidopsis cell extracts that was slightly destabilised in the presence of DTT. Moreover, cross-linking studies of native AtPEX19 suggest that in vivo it is the dimeric species of the protein that preferentially forms complexes with other proteins.

Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.

Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.

An Arabidopsis pex10 null mutant is embryo lethal, implicating peroxisomes in an essential role during plant embryogenesis.

Peroxisomes participate in many important functions in plants, including seed reserve mobilization, photorespiration, defense against oxidative stress, and auxin and jasmonate signaling. In mammals, defects in peroxisome biogenesis result in multiple system abnormalities, severe developmental delay, and death, whereas in unicellular yeasts, peroxisomes are dispensable unless required for growth of specific substrates. PEX10 encodes an integral membrane protein required for peroxisome biogenesis in mammals and yeast. To investigate the importance of PEX10 in plants, we characterized a Ds insertion mutant in the PEX10 gene of Arabidopsis (AtPEX10). Heterozygous AtPEX10::dissociation element mutants show normal vegetative phenotypes under optimal growth conditions, but produce about 20% abnormal seeds. The embryos in the abnormal seeds are predominantly homozygous for the disruption allele. They show retarded development and some morphological abnormalities. No viable homozygous mutant plants were obtained. AtPEX10 fused to yellow fluorescent protein colocalized with green fluorescent protein-serine-lysine-leucine, a well-documented peroxisomal marker, suggesting that AtPEX10 encodes a peroxisomal protein that is essential for normal embryo development and viability.

Peroxisome biogenesis and protein import in plants, animals and yeasts: enigma and variations? (Review).

Peroxisomes are found in virtually all eukaryotic cells, where they play varied but essential metabolic roles, exemplified by the catastrophic effects of mutations that compromise peroxisome biogenesis and function. This review will aim to provide an accessible introduction to peroxisome biogenesis and protein import for the non-specialist, and draws together recent advances in peroxisome protein targeting and import in plants, animals and yeasts, seeking to define common themes and highlight variations. Despite much progress, many aspects of peroxisome biology remain an enigma, and current questions and controversies in the field are highlighted and discussed.