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Overview

Theo Murphy meeting organised by Professor Roy Quinlan and Professor Takekazu Kunieda.

Anhydrobiosis was first described by Van Leeuwenhoek in 1702. Both plants and animals have anhydrobiotic responses and understanding the mechanisms that support such an extreme stress response will prove invaluable for future food security and for new therapeutics to treat for example proteinopathy-based diseases. Interdisciplinary scientists at the plant-animal interface scoped the application of anhydrobiotic mechanisms in future technologies.

Organisers

Schedule


Chair

12:00-12:05
Session 1 - Animal anhydrobiosis, the proteins and their assemblies.

Abstract

Anhydrobiosis was first described by Antonie Van Leeuwenhoek in 1702 in a lecture to the Royal Society. Seeing life emerge before your very eyes from a desiccated animal or plant is one of the most incredible biological events to fascinate, bewilder and motivate scientists. The future of our planet depends on tapping into such life-preserving responses as we face man-made extremes in climate change. Whilst all life can elicit a stress response, anhydrobiosis is the ultimate. From the study of “resurrection” plants trehalose was discovered.  Both plants and animals also use proteins rich in Intrinsically Disordered Domains (IDDs) to preserve life when water is removed. Liquid-Liquid phase separation is a key to both protection strategies. Comparing the different anhydrobiotic responses of plants and animals will deliver benefits for both food security and disease prevention/treatment. There has never been a more important time to build collaborative links across disciplines and across continents to understand and exploit the biology, chemistry and physics of anhydrobiosis. 

Speakers

12:05-12:30
Unique toolbox in tardigrade anhydrobiosis

Abstract

Tardigrades are known for their anhydrobiotic ability and the resilience against various extreme stresses. The recent efforts to find the tolerance-related factors identified multiple tardigrade-unique protein families. For example, tardigrade DNA protection protein dubbed Dsup is scarcely conserved in other organisms but can protect DNA from hazardous irradiation and oxidative stress and enhances the radio resistance of human cultured cells. These findings placed tardigrade-unique proteins as important clues supporting tolerant ability of tardigrades, which are even functional in other organisms. Many of tardigrade tolerance proteins including Dsup were found to be soluble even after boiling possibly through their hydrophilic nature with resulting unstructured flexibility. Recently, the relationship between stress response and the condensation of unstructured proteins attracts attention and many tardigrade proteins have been identified through the screening of stress-dependent condensing proteins. Some of them are revealed to form cytoskeleton-like filamentous network in animal cells in response to hyperosmosis and these transitions are demonstrated to stiffen the cells and enable the cells to counteract deformation stress and to retain cell integrity during dehydration process. Stress-dependent change of the physical property of cells can be achieved through reversible protein condensation and it contributes for the resistance against stress.

Speakers

12:30-12:55
Anhydrobiotic midge and its dry-preservable cultured cell line

Abstract

Anhydrobiosis represents an extreme example of tolerance adaptation to water loss. Polypedilum vanderplanki is the unique insect known to be capable of anhydrobiosis. Trehalose, which accumulates in the larvae up to 20% of the dry body mass, is thought to replace the water in its tissues. Similarly, highly hydrophilic proteins called the late embryogenesis abundant (LEA) proteins are expressed in huge quantities and act as a protectant for biological molecules against aggregation and denaturation. However, transduction of trehalose and LEA proteins in desiccation-sensitive cells did not improve viability after long-term storage of the dried cells. These findings suggest other factors must be involved in induction of anhydrobiosis. The genome of the anhydrobiotic midge specifically contains clusters of multi-copy genes with products, which act as desiccation protectants. A cell line derived from the midge, Pv11, showing ability of complete desiccation tolerance was established. Since gene knock-down and knock-out systems for Pv11 cells have been developed, this cell line can be used as a tool to investigate the anhydrobiosis at the molecular level. Here current knowledge of molecular mechanisms underlying the anhydrobiosis in P. vanderplanki will be discussed.Anhydrobiosis represents an extreme example of tolerance adaptation to water loss. Polypedilum vanderplanki is the unique insect known to be capable of anhydrobiosis. Trehalose, which accumulates in the larvae up to 20% of the dry body mass, is thought to replace the water in its tissues. Similarly, highly hydrophilic proteins called the late embryogenesis abundant (LEA) proteins are expressed in huge quantities and act as a protectant for biological molecules against aggregation and denaturation. However, transduction of trehalose and LEA proteins in desiccation-sensitive cells did not improve viability after long-term storage of the dried cells. These findings suggest other factors must be involved in induction of anhydrobiosis. The genome of the anhydrobiotic midge specifically contains clusters of multi-copy genes with products, which act as desiccation protectants. A cell line derived from the midge, Pv11, showing ability of complete desiccation tolerance was established. Since gene knock-down and knock-out systems for Pv11 cells have been developed, this cell line can be used as a tool to investigate the anhydrobiosis at the molecular level. Here current knowledge of molecular mechanisms underlying the anhydrobiosis in P. vanderplanki will be discussed.

Speakers

12:55-13:20
Poster session
13:20-13:25
Break
13:25-13:50
A novel tertiary structure model for ab-initio interpretation of BioSaxs data

Abstract

Small angle x-ray scattering is one of the most flexible and readily available experimental methods for obtaining information on the structure of proteins in solution. In the advent of powerful predictive methods such as the alphaFold and rossettaFold algorithms, this information has become increasingly in demand, owing to the need to characterise the more flexible and varying components of proteins which resist characterisation by these and more standard experimental techniques. To deal with structures about little of which is known a parsimonious method of representing the tertiary fold of a protein backbone as a discrete curve has been developed. It represents the fundamental Ramachandran constraints through a pair of parameters and is able to generate millions of potentially realistic portent geometries from only a sequence in a short space of time. The method for using this model to interpret BioSaxs data will be introduced. A particular focus will be given to how the model can additionally be linked to established protein modelling techniques and additional data sources of ab-intio protein structure, in order to boost the methods predictive efficacy.

Speakers

13:50-14:15
Biophysical principles of molecular chaperone self- and co-assembly

Abstract

We have been developing and applying quantitive mass-measurement approaches to interrogate directly the structure and dynamics of proteins. Here I will focus on the insights this has enabled in studying the evolution of specificity in assembly of molecular chaperone proteins, and the implications this has with respect to their key role in protecting the cell under times of acute and chronic stress. 

Speakers

14:15-14:40
Phase separation as a stress survival strategy

Abstract

Biomolecular condensates formed by phase separation are membraneless compartments in the cytoplasm and nucleoplasm of cells, which have major roles in cellular organization and physiology. RNP granules are a specific type of condensate that assemble from RNA-binding proteins and RNA. In this talk, I will discuss how the concept of biomolecular condensates has expanded our view of RNP granules and their link to the cellular stress response. I will introduce in vitro reconstitution systems based on the concept of phase separation that now allow us to reconstruct RNP granules in the test tube. Using these reconstitution systems as well as cell biological and genetic approaches, we have gained important insights into the molecular rules of RNP granule assembly, such as the driving forces and amino acids that govern condensation, the conformational changes underlying assembly and molecular mechanisms of condensate regulation and control. I will further discuss how the concept of phase separation has allowed us to dissect the functions of RNP granules, and I will demonstrate how condensate formation can be used by cells to sense and respond to changes in the environment and regulate fundamental cellular processes such as protein synthesis.

Speakers

14:40-15:05
Liquid-Liquid Phase Separation of Anhydrobiosis-Related Proteins from Artemia franciscana

Abstract

The brine shrimp Artemia franciscana expresses Late Embryogenesis Abundant proteins from three groups in their anhydrobiotic life-history stage and is the only animal known to express LEA proteins belonging to groups 1 (LEA_5) and 6 (SMP) in addition to group 3 (LEA_4) proteins. The reason for the more extensive LEA repertoire in brine shrimp compared to other anhydrobiotic members in this kingdom of life is unknown. The proteins AfrLEA6 and AfLEA1 are mostly intrinsically disordered in the hydrated state, and both proteins form biomolecular condensates driven by the consensus sequence motifs attributed to the LEA_5 and SMP families. Interestingly, RNA promotes LLPS only for AfLEA1 while AfrLEA6 droplets incorporate target proteins based on their surface charge. The role of proteinaceous LLPS in cellular stress responses has been firmly established in several models and includes heat shock and osmotic stress, but its role in animal anhydrobiosis is less well defined. Based on these findings, it is still premature to conclude that most proteins from the LEA_5 and SMP families will form biomolecular condensates during desiccation stress. Nevertheless, forming target-specific protective compartments via LLPS constitutes an additional feature in the growing protection repertoire of this exciting family of proteins.

Speakers

15:05-15:10
Break
15:10-15:25
Discussion

Speakers


Chair

12:00-12:25
Calcium signalling in response to stress in plants

Abstract

Abiotic and biotic environmental stimuli are sensed and transduced by signalling networks in plants leading to an appropriate pattern of protective gene expression.  My lab is interested in how calcium, involved in response to so many different primary signals, can encode specific information to elicit the correct downstream responses.  The calcium signature hypothesis states that different external stimuli elicit unique spatiotemporal patterns of elevations in cellular calcium concentration and thus encode stimulus-specific information that is “read” by plant cells. Through a combination of experimental and mathematical approaches, we have determined how calcium signatures are “decoded” by specific transcription factors to lead to appropriate specific gene expression responses. Our most recent work has found that unique calcium signatures occur when different stresses are applied simultaneously or sequentially, or when a single stress is applied under different environmental conditions. These signatures represent integrated information obtained from different environmental cues together, and are decoded to produce unique gene expression “decisions”.

Speakers

12:25-12:50
How do angiosperm plants survive desiccation of vegetative tissues using Craterostigma plantagineum as a model plant?

Abstract

The objective is to understand how angiosperm plants can survive long periods without water. Resurrection plants are a small group of angiosperm plants which  tolerate severe water loss, and adjust their water content with the relative humidity in the environment. The plants can remain in the desiccated state for several months. When rainfall occurs, the plants recover and assume full physiological activity. The studies are focused on the desiccation tolerant resurrection plants Craterostigma plantagineum and Lindernia brevidens. Genome and transcriptome sequences are available and are compared with the sequences from the desiccation sensitive close relative Lindernia subrasemosa. This comparative approach demonstrates that expression levels of genes encoding protective molecules including proteins and carbohydrate metabolites are differentially regulated in desiccation tolerant and non-tolerant plants. These genes are excellent candidates to identify essential molecular components of desiccation tolerance in angiosperm plants. It is suggested that desiccation tolerance has evolved through expressing components of seed desiccation pathways in vegetative tissues.The objective is to understand how angiosperm plants can survive long periods without water. Resurrection plants are a small group of angiosperm plants which  tolerate severe water loss, and adjust their water content with the relative humidity in the environment. The plants can remain in the desiccated state for several months. When rainfall occurs, the plants recover and assume full physiological activity. The studies are focused on the desiccation tolerant resurrection plants Craterostigma plantagineum and Lindernia brevidens. Genome and transcriptome sequences are available and are compared with the sequences from the desiccation sensitive close relative Lindernia subrasemosa. This comparative approach demonstrates that expression levels of genes encoding protective molecules including proteins and carbohydrate metabolites are differentially regulated in desiccation tolerant and non-tolerant plants. These genes are excellent candidates to identify essential molecular components of desiccation tolerance in angiosperm plants. It is suggested that desiccation tolerance has evolved through expressing components of seed desiccation pathways in vegetative tissues.

Speakers

12:50-13:20
Identification of genes important for drought tolerance in non-model species

Abstract

Exploitation of comparative genomics in an evolutionary framework has allowed us to understand the modes of genome and gene family evolution to identify novel stress response genes across the plant kingdom. We have shown that the evolutionary history of plants was shaped by two bursts of unprecedented genomic novelty linked to multicellularity and terrestrialisation, and that gene expansion and co-option are the more common mechanisms of biological innovation in the evolution of features enabling water uptake and transport. Specifically, by incorporating trait evolution (drought adaptation) into a comparative genomics framework, we identified different patterns of gene family evolution in species with opposing stress phenotypes. For example, the C3 desert species, Rhazya stricta (R. stricta) maintains high photosynthetic rates despite extreme environmental conditions. Gene families specific to R. stricta, such as photosynthesis and respiration associated genes responded to diurnal temperature changes and water limitation. By combining transcriptomics and evolutionary genomics, we showed that specific genes have diverged from their homologs in other species.  Furthermore, we quantified gene loss in drought sensitive compared to closely related drought tolerant species, and together these analyses may provide useful targets for improving tolerance to extreme environments.

Speakers

13:20-13:45
Poster session
13:45-13:50
Break
13:50-14:15
Phase behaviours of unfolded proteins reveal novel twists to the unfolded protein response

Abstract

Protein homeostasis involves regulation of the concentrations of unfolded states of globular proteins. Dysregulation can cause phase separation leading to protein-rich deposits. Mutations within a model protein are used in cellular studies to understand the connection between protein stability and phase separation. Protein unfolding is necessary but insufficient to drive phase separation. Instead, only those unfolded states that possess a requisite sequence grammar can drive both protein destabilization, to increase the concentration of unfolded states and enable cohesive interactions among unfolded proteins. The formation of unfolded protein deposits (UPODs) allows for specificity in targeting by molecular chaperones such as Hsp40 and Hsp70. These chaperones destabilize UPODs by binding preferentially to and processing unfolded proteins in the dilute phase. Proteomic analysis of UPODs reveal surprises that can be explained based on physico-chemical principles but are confounding from a biological perspective. 

Speakers

14:15-14:40
Ionic strength influences interactions between intrinsically disordered proteins and curved membrane surfaces

Abstract

Intrinsically disordered proteins (IDPs) are a class of proteins that lack substantial regions of tertiary structure and have dynamic behaviour that can be well characterized by polymer theory. In addition, IDPs often contain high degrees of net charge, giving rise to polyampholytic properties as well as electrostatic interactions with anionic membranes. Zeno et al. uses quantitative fluorescence approaches to quantify intramolecular and intermolecular interactions between IDPs and charged membrane surfaces. By modulating the ionic strength of the solution and to control the level of electrostatic screening, they discover that IDPs are potent sensors of membrane curvature. These findings are applicable to a wide range of proteins, especially those involved in membrane trafficking and remodelling. 

Speakers

14:40-15:05
Phase Transitions and Biomedical Applications

Abstract

Proteostasis imbalances lead to cell dysfunction and disease. The main source of misfolded proteins in cells are defective ribosomal products (DRiPs), resulting from premature translation termination, damaged mRNAs, DNA mutations. DRiPs are recognized by protein quality control (PQC) machineries, including the chaperones HSP70s, small HSPs and VCP. These chaperones bind to DRiPs and promote their proteasomal degradation. 

DRiPs can accumulate inside biomolecular condensates called stress granules (SGs) and promote their conversion from a liquid-like state into a solid-like aggregated state. DRiPs also rapidly accumulate in nucleoli and PML bodies, which are biomolecular condensates similar to SGs. By targeting misfolded proteins and DRiPs for degradation, the PQC prevents the conversion of SGs, PML and nucleoli into an irreversibly aggregated state. Thus, targeting aggregation-prone proteins to condensates upon stress is emerging as a general mechanism to prevent irreversible protein aggregation. 

Our data have important pathological implications, since alteration of SG dynamics and nucleolar stress are implicated in the pathogenesis of Amyotrophic Lateral Sclerosis. Moreover, mutations of chaperones that alter their ability to undergo dynamic phase transitions and chaperone activity are linked to neuromuscular diseases. We propose that boosting chaperone targeting to condensates may maintain phase transition dynamics, offering new therapeutic opportunities.

Speakers

15:05-15:10
Break
15:10-15:25
Discussion

Speakers


Chair

12:00-12:25
Evolution of stress resistance in metazoans

Abstract

Water is essential for life, and a significant reduction of body water usually leads to physiological malfunctioning and eventually death. Nonetheless, some organisms have evolved strategies to survive at least a limited lack of water, and a few taxa have evolved the ability to survive complete dehydration. In particular, microscopic invertebrates like tardigrades, rotifers and nematodes seem to have discovered and adopted a range of strategies to survive stressful conditions which would be lethal for other animals.

Despite the importance of understanding the basic principles of how these organisms can cope with stress associated with lack of water and their potential biotechnological applications, the exact mechanisms (behavioural, physiological, cellular and molecular) are still poorly understood, but recent advances in genomics are allowing comparative analyses of both common and unique mechanisms which different taxa employ to survive challenging conditions. This comparative survey of how taxonomically different organisms cope with lack of water highlights similarities and differences and can help informing further studies to understand principles and help developing practical applications.

Speakers

12:25-12:50
Resurrection plants as models for climate smart agriculture

Abstract

Drought is the greatest threat to world agriculture and due to global warming, increased aridification is predicted in most current food producing areas. This is particularly significant for Africa, where 95% of agriculture is rain fed.  Current crops are intolerant of even moderate water loss and while improved resistance to water loss has been achieved, such mechanisms fail under severe and prolonged drought. There are some 240 Angiosperm species (resurrection plants) that display vegetative desiccation tolerance; a phenomenon which if fully understood, could ultimately be used in the production of crops with increased tolerance to water deficit stress. My group has systematically, using a multidisciplinary approach, investigated mechanisms whereby several different resurrection plants, each as a model for a crop to be transformed,  tolerate these extreme conditions. Recent studies include investigations of resurrection plant associated root microbiomes and their potential roles in plant drought tolerance.  In this presentation an overview of  molecular physiological processes associated with DT in a range of resurrection plants will be given and current and future potential applied outputs discussed. 

Speakers

12:50-13:20
Poster session
13:20-13:25
Break
13:25-13:50
Sorghum’s Solutions to Drought

Abstract

Sorghum is a crop adapted for growth in arid climates characterised by water scarcity and extreme heat. Its productivity remains undiminished under harsh drought conditions where other cereals fail. A recent discovery is that the superior stress tolerance of sorghum also exists at the single cell level in primary stem cells prior to differentiation and tissue formation. This provides a useful experimental system for identifying protein/gene networks underpinning sorghum drought stress tolerance. A remarkable finding is that signals secreted by sorghum cells into the extracellular matrix can be used to stimulate Arabidopsis cells to behave as if they were sorghum cells – they become extremely osmotic stress-tolerant to similar levels displayed by sorghum cells. This aligns with the global hypothesis in the Chivasa lab, that extracellular matrix signals are key drivers of collective “decision-making” to synchronise adaptive responses and optimise energy distribution between stress metabolism and productive growth. How nature has solved the drought problem in sorghum is being investigated using this hypothesis and the latest results revealing some of the key players will be presented.

Speakers

13:50-14:15
Leveraging evolutionary innovations to improve plant resilience

Abstract

Drought is the most pervasive issue we face in agriculture today and there is considerable interest to produce new crop varieties that can thrive on marginal soils and use less water. Work in the VanBuren Lab focuses on understanding natural adaptations to overcome extreme drought such as the desiccation tolerance found in resurrection plants. To answer fundamental and applied research questions, the lab employs systems-level approaches across diverse model and crop species. Much of this work focuses on wild grasses and orphan cereals in the Chloridoideae subfamily of C4 grasses, which have superior heat, drought, and salinity tolerance compared to other grasses. Comparative genomics, systems biology, physiology, and modelling-based approaches are leveraged to understand the genetic basis of stress tolerance in these unique grasses. The long-term goal of this work is to use natural adaptations to engineer improved stress tolerance and resilience into crop plants.

Speakers

14:15-14:40
Desiccation as an alternative to cold-storage of biologic pharmaceuticals

Abstract

The past two decades have seen an explosion in the development and use of ‘biologics,’ drugs derived from or containing components of living organisms. This includes protein and nucleic acid-based pharmaceuticals, anti-venoms, allergens, blood, blood components, and cells. While biologics have proven both effective and efficient, they have one major drawback, many are inherently unstable. Currently, the most widespread method of biologic stabilization is cold storage. Cold storage can be effective, but in remote or developing parts of the world, under austere and crisis conditions, or during prolonged space missions, the cold storage represents an enormous economic and logistic burden. To unlock the full potential of biologics, economic and effective means of stabilization must be developed for these life-saving therapeutics. Building on the past contributions of researchers in the desiccation tolerance field, and our own fundamental research on anhydrobiosis, we are developing and testing innovations for storing diverse biologics in a dry, unrefrigerated state. We seek to understand the detrimental effects desiccation imposes on diverse biomolecules, what types of natural protectants are most efficient at preventing these effects, and how we can use genetic, protein-, and chemical-engineering to tune protection for specific biologic targets.

Speakers

14:40-14:45
Break
14:45-15:45
Closing Discussion

Speakers