Microsphere super-resolution microscopy has recently received a lot of attention due to its simplicity and its capability to resolve features beyond the diffraction limit for living systems, in real time, without being invasive. Current systems which are able to image small biological structures are destructive, invasive (they use fluorescent dyes) or they provide reconstructed images (i.e. not a real time imaging of living organisms). The authors designed and built a bio-microscope tailored around the SMAL® (Super-Resolution Microsphere Amplifying Lens) objective lens which overcomes these drawbacks. The novelty of the setup is the ability to study cell’s structures and viruses which previously were not possible to be imaged using conventional white light microscopy. In this work, along various plant cells, label-free tobacco mosaic virus was employed for imaging in this work due to its stability, reliability, and its dimensions beyond the diffraction limit. The results were compared with images obtained using an X100/1.40 NA oil immersion objective lens fitted onto a white light microscope, in the same location. Microsphere assisted microscopy is a novel type of optical microscopy with great potential for high resolution imaging of in vivo biological structures and pathogens.

Optical super-resolution imaging of tissues and tobacco mosaic virus (TMV) using microsphere technology

Polyploids are of common occurrence among the plants. Several studies have been conducted to understand the impact of polyploidy on the evolution of plant genome. Natural polyploids do occur in the wild, but it is difficult to decipher which changes are due to polyploidy as the plants accumulate variation over the history of their existence. It is important to study the impact of polyploidy away from natural variation. Therefore, we generated synthetic neoautopolyploids in two species of Vigna L. using colchicine-based cotton swab method. The polyploids were screened via flowcytometry and confirmed polyploids were taken to the field to study biological parameters. The two species were found to be behaving differently based on various parameters like photosynthetic efficiency, cell density, stomatal count, cell size and nucleus size. We will be studying inheritance pattern of these traits in subsequent generations. Our study provides a proof that polyploidy does not always have a positive scaling effect on plants at various biological parameters and the changes are species specific. We will be studying genomic changes in future and will correlate them with morphological changes observed.  

Keywords: Polyploids, WGD, Autopolyploid, Flowcytometry, morphometery

Whole Genome Duplication Show Species-Specific Morphological Changes In Neoautopolyploids

Programmed cell death (PCD) is a genetically managed biological process that results in the targeted sacrifice of unwanted or diseased cells. Plants use PCD for various purposes such as development, defence against pathogens, response to abiotic stress caused by UV radiation or heat shock. Several proteinases are involved in the PCD process; among them, cathepsin B (CathB) which plays an important role in pathogen defence, plant development, germination, senescence, microspore embryogenesis and abiotic stress response. A greater understanding of the molecular mechanisms underlying the processes downstream of CathB is the next important step for new discoveries in cell biology and applications in biotechnology. To achieve this goal, it is important to discover and test substrates cleaved by plant CathB. Up to 13 substrates for CathB were identified in animal cells, and we found orthologous genes in Arabidopsis thaliana for eight of them. Among these, we are testing some candidate substrates for CathB. To experimentally validate the cleavage site, we used leaf agro-infiltration to co-expressed substrate GFP-fusion and CathB in tobacco cells and carried out cleavage assays. in vivo and in vitro results will be presented. 
















Characterising the proteolytic network of plant cathepsin B

Mammalian cell culture is a fundamental tool used to study living cells. Presently, the standard protocol for performing cell culture involves the use of commercial media that contain an excess of nutrients to prevent depletion during experiments. While this reduces the likelihood of cell starvation, it creates non-physiological culture conditions that have been shown to ‘re-wire’ cellular metabolism and broadly influence cell physiology. Recently, researchers have developed physiological media like Plasmax, formulated to approximate the nutrient composition of human blood plasma. Although this represents an improvement in cell culture practice, such physiologic media may be vulnerable to nutrient depletion over the course of an experiment  extending over several days. In this study we directly addressed this concern by measuring the rates of glucose and amino acid depletion from Plasmax by several cancer cell lines (PC3, LNCaP, MCF-7 and SH-SY5Y) over 48 hours. In all cell lines, depletion of glucose from Plasmax was rapid such that, by 48h, cells were hypoglycemic (less than 2mM glucose). Most amino acids were similarly rapidly depleted to sub-physiological levels by 48h. In contrast, glucose and most amino acids remained within the physiological range at 24h. Using RNA sequencing, we show that this nutrient depletion is associated with enrichment of cellular starvation responses, amino acid biosynthesis, apoptotic signalling, and endoplasmic reticulum stress response. Alterations in mitochondrial respiration using Seahorse extracellular flux analysis were also observed. Taken together, these results exemplify the metabolic considerations for Plasmax use, highlighting that cell culture in Plasmax requires daily media exchange.

Rapid nutrient depletion by cancer cells cultured in the physiologic medium Plasmax  

Mitochondria are most known for their role in energy production, but they are also implicated in many other cellular functions (apoptosis, ROS production, cell proliferation, aging). In humans, the mitochondrial DNA was believed to encode only 37 genes involved in energy production and mitochondrial translation. However, recent studies have shown that within these genes could hide several small alternative genes (genes with translation initiation or termination sequences distinct from the reference gene sequences in which they are found) coding for micropeptides with a broad spectrum of functions that can be newly added to the large list of functions in which mitochondria are involved.

Following the discovery of the mitochondrial alternative gene mtaltnd4, we try to elucidate the functions of the gene and its peptide (MTALTND4) by studying: (1) the expression pattern of the peptide in different human tissues and blood plasma, (2) the involvement of the gene in the major mitochondrial functions by exposing cells to various treatments, (3) the interaction partners of MTALTND4 by mass spectrometry and (4) the impact of the peptide on cell transcriptome. 

We have found that mtaltnd4 may potentially produce a signaling protein in response to stress that would regulate certain cell metabolic processes and there is surely more to discover.











The alternative gene mtaltnd4: uncovering the mitochondria’s hidden functions

Sessile benthic species are submitted to fluctuating oxygen and temperature conditions that can negatively affect their energy and redox balance. Single effects of temperature and oxygen fluctuations have been extensively studied, but their combined effects are not yet well understood. We studied the bioenergetics and oxidative stress markers in the Pacific oyster Crassostrea gigas exposed for 10 days to daily immersion-emersion cycles under the control (15°C) (H), elevated (30°C) (HW), and fluctuating (15°C in water and 30°C in air) (HF) temperature. Oysters maintained under normoxia and 15°C were used as controls. Mortality increased in the oysters exposed to hypoxia and elevated temperature indicating acute thermal stress. Oysters co-exposed to fluctuating oxygen and temperature showed a decrease in the mitochondrial aerobic capacity but no onset of anaerobiosis shown by the lack of succinate accumulation. Glycogen content decreased in the digestive gland, whereas protein content increased in the gill of the HF group indicating higher protein deposition combined with higher energy demand during reoxygenation. In contrast, the HW group showed decreased protein levels reflecting heat-induced suppression of protein synthesis. Lipid peroxidation levels were elevated in the gills of the oysters from HW (but not H or HF group) indicating that antioxidant systems of oysters can cope with hypoxia-induced oxidative stress under the normal or fluctuating temperature but not during constant warming. Thus, long-term warming combined with intermittent hypoxia can lead to breakdown of physiological functions and higher summer mortality like already observed in the German Wadden Sea.

Effects of fluctuating oxygen and temperature regime on bioenergetics and oxidative stress of the pacific oyster Crassostrea gigas

So called ‘drop-in’ Biofuels produced from plants are becoming increasingly important for the aviation and shipping industries, which urgently need to move away from fossil fuels in order to limit their impact on the climate. Algae farms provide a number of benefits over traditional biofuel crops, such as sugar cane, by alleviating competition for arable land. When exposed to high light, the green alga Chlamydomonas reinhardtii produces triacylglycerols (TAGs) which can be converted to drop-in biodiesel. Excess light results in the over-reduction of the photosynthetic electron transport chain, and it has been suggested that the production of TAGs is in response to this redox imbalance. A deeper understanding, therefore, of how Chlamydomonas cells address redox imbalance will help to inform future work aiming to maximise TAG production in these conditions. Our work has highlighted a potential role for the phenomenon known as light enhanced respiration (LER) in responding to redox stress induced by high light. We show that presence of acetate in the growth media may allow cells to better acclimate to high light by increasing the capacity of the cells for LER. Autotrophic cells (cells grown without acetate) appear to be more susceptible to photodamage than mixotrophic (grown with acetate) when grown in high light. Our results display the complexity of the high light response and provide preliminary evidence for the potential of the mitochondria as a tool for addressing redox imbalance. 

A role for mitochondria during high light stress in Chlamydomonas reinhardtii

Membrane trafficking and organelle biogenesis play important roles in plant growth and development, as well as responses to external signals. The plant endomembrane system contains several functionally distinct membrane-enclosed organelles, including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN) or early endosomes (EE), prevacuolar compartment (PVC) or multivesicular body (MVB) and vacuole. One of our major research programs has been focused on illustrating the underlying mechanisms of vacuolar trafficking and vacuole biogenesis and function in plant cells. One major approach has been the whole-cell electron tomography (ET) analysis with nanometer resolution of vacuole formation and distribution in different cell types of developmental stages, including root cells, stomatal lineage cells and developing pollens in Arabidopsis. Here I will present our recent work and models of vacuole biogenesis and function in plants from the ET analysis. Supported by grants from the Research Grants Council of Hong Kong and CUHK. 

Whole-cell Electron Tomography Analysis of Vacuoles in Plant Cells

Reactive oxygen species (ROS) are by-products of oxidative metabolism, and while low levels contribute to cell signalling, high levels can be toxic. In mammals, ischaemia-reperfusion leads to production of toxic levels of ROS, which cause oxidative tissue damage. In contrast, anoxia-tolerant species, such as the freshwater turtle, do not sustain oxidative tissue damage after anoxia and reoxygenation. How anoxia-tolerant animals are able to prevent ROS dysregulation and oxidative damage with anoxia is unclear. We investigated ROS production and antioxidant capacity in the turtle heart upon anoxia-reoxygenation. Using the in vivo mass-spectrometric H2O2-probe MitoB, we found that there is no excessive production of ROS in the turtle heart after whole animal anoxia-reoxygenation. We hypothesized that this was due to the fact that turtles avoid large accumulation of succinate in the heart and preserve energy levels. This would prevent reverse electron transfer and extensive ROS production from complex I after anoxia. However, addition of exogenous succinate and manipulation of membrane potential with oligomycin did not induce excessive ROS production in turtle heart strips in vitro. This suggests that other factors, such as S-nitrosation of complex I, supercomplex stability or high antioxidant capacity may also be involved in preventing ROS after anoxia in the turtle heart. Together, this study shows that turtles are able to avoid toxic levels of ROS in the heart and oxidative tissue damage after anoxia and reoxygenation. 

Low in vivo production of reactive oxygen species after anoxia and reoxygenation prevents oxidative damage in the turtle heart

Surviving in low oxygen environments requires complex and integrated physiological responses, many of which center on economizing energy budgets. At the cellular level, significant reductions in protein synthesis and even translational arrest (in anoxia) can save as much as 90% of routine ATP demand. This suggests strategic investment in the protein complement of essential tissues, like the heart, that must maintain some level of function for the animal to survive through and recover from a hypoxic bout. The use of quantitative proteomics in animal models of hypoxia tolerance has unveiled new ideas about how the cardiac proteome is influenced by hypoxia exposure, as well as the ontogenic constraints on this aspect of cardiac plasticity. For example, developmental hypoxia exposure of American alligators (Alligator mississippiensis) induces persistent shifts in the cardiac proteome that, paradoxically, reflect an increased capacity for translation, proteolysis, and metabolic capacity. Concomitant with these changes was an equitable down-regulation of seemingly random cardiac proteins, which may offset the cellular energy budget. In Western painted turtles (Chrysemys picta bellii), the cold acclimation required to prepare tissues for chronic anoxia during winter dormancy induced general down-regulation of electron transport system proteins in adult but not hatchling turtles. Interestingly, both studies revealed 
‘age’ as a key explanatory variable in the cardiac proteome. This suggests a robust developmental program in the ectotherm heart that is resilient even to potent environmental stressors like oxygen and temperature, and warrants further investigations into the developmental thresholds of hypoxia tolerance.






Proteomes & Juveniles: A synthesis of ideas on the ontogeny of hypoxia tolerance

Stomata are anatomical structures at the surface of leaves controlling gas exchanges between the plant and the atmosphere. The stomatal pore aperture is regulated by two specialized cells, the guard cells, having the capacity to reversibly change their volume and shape. The changes of the guard cells’ volume are under tight control of signaling cascades regulating the ionic transport processes across the plasma and the vacuolar membranes. In guard cells the vacuole can reversibly change its volume during the opening and closure of stomata. We have used a 3D reconstruction approach to visualize the dynamics changes subcellular compartments volumes in living guard cells. The method we developed allowed us to simultaneously quantify the volumetric modifications of the cytosol and the vacuole. Notably, we could follow modification of the subcellular organization in parallel with the ion transport dynamics at the plasma and vacuolar membranes. Our data show that the vacuole accounts for the whole guard cell volume changes. This suggests that the turgor changes of the guard cells are driven by the vacuole during stomata aperture. 

Subcellular volume changes in stomata opening: vacuolar vs cytosolic volume changes.

Birds metabolically remodel their flight muscle prior to migration to meet the high energetic demands of migratory flight. The degree of plasticity mediated by changes in mitochondrial function is poorly understood but could be explained by two non-mutually exclusive hypotheses: variation in mitochondrial quantity or individual mitochondrial function. For each hypothesis, we predicted higher mitochondrial abundance and predict higher substrate oxidative capacity and reduced reactive oxygen species formation during migration, respectively. We evaluated both hypotheses using yellow-rumped warblers (Setophaga coronata) captured during their autumn migration at Long Point, Ontario, Canada. Half of the birds were sampled in the migratory phenotype and the remaining birds were transferred to a short day photoperiod (9L:15D) and sampled in a non-migratory phenotype. We measured mitochondrial abundance via citrate synthase maximal activity in the pectoralis major flight muscle. We investigated mitochondrial function via high-resolution fluororespirometry on isolated pectoralis mitochondria. We measured O2 consumption and H2O2 formation rates from oxidation of lipid and carbohydrate substrates. We also measured oxygen consumption specific to electron transport system protein complexes using complex-specific substrates and inhibitors. Citrate synthase activity and mitochondrial respiration for each substrate and complexes II, III and IV was higher in the migratory phenotype, yet H2O2 formation rates were similar between phenotypes. These data suggest that pectoralis mitochondrial abundance and oxidative capacity are elevated during migration without increasing reactive oxygen species formation. Our findings support both hypotheses and indicate that mitochondrial function mediates seasonal metabolic plasticity in the pectoralis flight muscle of migratory songbirds.

Underline digital video library

Optical super-resolution imaging of tissues and tobacco mosaic virus (TMV) using microsphere technology

Whole Genome Duplication Show Species-Specific Morphological Changes In Neoautopolyploids

Characterising the proteolytic network of plant cathepsin B

Rapid nutrient depletion by cancer cells cultured in the physiologic medium Plasmax

The alternative gene mtaltnd4: uncovering the mitochondria’s hidden functions

Effects of fluctuating oxygen and temperature regime on bioenergetics and oxidative stress of the pacific oyster Crassostrea gigas

A role for mitochondria during high light stress in Chlamydomonas reinhardtii

Whole-cell Electron Tomography Analysis of Vacuoles in Plant Cells

Low in vivo production of reactive oxygen species after anoxia and reoxygenation prevents oxidative damage in the turtle heart

Proteomes & Juveniles: A synthesis of ideas on the ontogeny of hypoxia tolerance

Subcellular volume changes in stomata opening: vacuolar vs cytosolic volume changes.

Flight muscle mitochondria are seasonally plastic in a migratory songbird

Stay up to date with the latest Underline news!

PRESENTATIONS

CONFERENCES

COMPANY

RESOURCES