France

Changing the morphology of the plant vacuole depends on the integrity of the cytoskeleton. So far, only little is known about the nature of this relationship. The plant-specific Networked (Net) family of membrane-associated actin-binding proteins contains two members, Net4A and Net4B, which are of special interest since they localize to the tonoplast. Recently, we could show that Net4 modules the compactness of vacuoles and this in turn affects its space-filling function and eventually inhibits cell elongation and growth. Since the net4a net4b double mutant only has a mild phenotype and other NETs seems not to compensate, we expected a higher molecular complexity and searched for new interactors. Pull-down experiments followed by MS-MS identified several new interactors, potentially participating in the regulation of the vacuole-cytoskeleton interface.

SEB Annual Conference 2022

The vacuole - cytoskeleton connection: a handle to regulate growth

technical paper

Discover the art of creating extraordinary science at SEB’s Annual Conference at Le Corum Conference Center     in Montpellier, France from 5 to  8 July 2022.

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2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years - Montpellier, France

The vacuole is a hallmark of the plant cell, generating turgor pressure and thus driving growth. Seed plant embryos also contain numerous specialised Protein Storage Vacuoles (PSV) which are repositories for the nutritional content of seeds. But how do these specialised PSVs form and what mechanism drives this process? Recent work has demonstrated that PSVs are formed by the remodelling of the single embryonic vacuole into many fragmented PSVs. The main constituent of these PSV are seed storage proteins (SSP).  We have shown that SSP undergo liquid-liquid phase separation forming a droplet within the vacuole and these SSP droplets can bend the tonoplast membrane. However, can this process of SSP phase separation drive fragmentation of a single embryonic vacuole into multiple PSV? Additionally, which proteins and conditions drive phase separation of SSP in the embryonic vacuole? We have identified and are characterising protein candidates for drivers of phase separation in the embryonic vacuole and determining their role in PSV biogenesis.

Its all just a phase: Phase separation driving organelle biogenesis.

In plant cells, cargo proteins destined to the vacuoles contain sequence specific information known as vacuolar sorting determinant (VSD) that is recognized and sorted by vacuolar sorting receptors (VSRs) to the vacuoles. To understand how VSR recognizes its cargos, our group have determined the crystal structures of the PA domain of Arabidopsis VSR1 in complex with the vacuolar sorting determinants (VSDs) of a protease aleurain [1] and a 12 S globulin cruciferin (CRU) 1 [2]. Structural comparison with the apo-form of VSR1-PA suggests that cargo-binding induces conformational changes in four regions of VSR1-PA. The VSDs bind to the cargo-binding loop consisting of a conserved motif of 95RGDCYF100. The invariant Arg-95 residue was found to be essential for receptor-cargo interactions and vacuolar sorting of cargo proteins. In vitro pull-down assay showed that VSR1 interacts with the C-terminal decapeptide sequences of CRU1, CRU4 and vicilin-like seed storage protein (VL) 22, but not with those of CRU2, CRU3, VL21 and VL43. Tagging the C-terminal sequences of CRU1 and VL22 to the secretary red fluorescent proteins are sufficient in redirecting the proteins to the vacuoles in Arabidopsis protoplasts. A P453A substitution converts the C-terminal sequence of CRU2 to a C-terminal VSD that is recognized by VSR1. Structural insights on receptor-cargo obtained could be useful in targeting recombinant proteins to protein storage vacuoles in seeds.

This work was supported by grants from the Research Grants  Council  of  Hong  Kong  Special  Administrative  Region,  China (CUHK14151416, C4041-18EF, AoE/M-05/12, AoE/M403/16, and C4033-19E) and The National Natural Science Foundation of China (91854201 and31670179) and by direct grants from The Chinese University of Hong Kong.

[1] Luo F. et al. (2014) “How vacuolar sorting receptor proteins interact with their cargo proteins: crystal structures of apo and cargo-bound forms of the protease-associated domain from an Arabidopsis vacuolar sorting receptor.” Plant Cell, 26, 3693-708
[2] Tsao H.E. et al. (2022) Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins. Proc Natl Acad Sci USA, 119, e2111281119.




How cargo proteins are recognized and sorted to the vacuoles?

Cells typically remain relatively small, because if cells grow beyond a critical limit, the surface of the plasma membrane eventually ceases to accommodate intracellular needs. Compared to animals, plant cells can dramatically increase their size without the apparent need for surface furcation. The vacuole is the biggest plant organelle and has a space-filling function, allowing to occupy up to 90% of the cell volume (Löfke et al., 2015; Scheuring et al., 2016; Dünser et al., 2019). The dynamic regulation of vacuolar size allows plant cells to expand with little increase of the cytosol (Dünser et al., 2019; Dünser et al., 2022), maintaining a favorable cell surface to cytosol ratio during growth. Here we discuss subcellular mechanisms that ensure the coordination of cellular and vacuolar size expansions.

Coordination of growth in plants

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.

The centriole is an evolutionary conserved organelle that coordinates fundamental biological processes including cell division, cellular signalling and cell motility. This organelle, of 500 nm long and 250 nm in diameter is composed of about 100 different proteins, some of which have been associated with human diseases. How these proteins are organized at the level of the centriole architecture and how associated mutations could be involved in pathologies is still poorly understood. I will present the latest work from my laboratory, which tackle these fundamental structural cell biology questions using cryo-tomography, cell biology and our newly developed ultrastructure expansion microscopy (U-ExM) method.

Expansion microscopy for Structural cell biology: from centriole architecture to human diseases

Determining how plasticity of developmental traits respond to environmental conditions is a challenge that must combine evolutionary sciences, ecology and developmental biology. Developmental plasticity is defined as the ability of an organism to adjust its development depending on environmental signals, thus producing alternative phenotypes precisely adjusted to the environment. Yet the mechanisms underlying developmental plasticity are not fully understood yet. Determining how plastic developmental changes that occur in response to environmental conditions are coordinated at the physiological, cellular, and molecular levels is a challenge that must combine ecology with developmental biology. The mechanisms that underlie the development of alternative phenotypes are still unclear for many systems and is one major goal of ecological developmental biology or Eco-Evo-Devo. We observed that clownfish (Amphiprion percula) young juveniles have a different rate of white bar formation depending on the sea anemone species, their obligate symbiotic partner, in which they are recruited: white bars develop more rapidly when fish are recruited in Stichodactyla gigantea than in Heteractis magnifica. Because the sister species A. ocellaris acquire their adult color pattern during metamorphosis under thyroid hormone (TH) control, we asked whether developmental plasticity in bar formation was associated with alteration in TH status as these are the main hormones triggering metamorphosis in vertebrates. We found that thyroid hormones regulate white bar formation and that a shift in hormone levels, associated with ecological differences, results in divergent color patterns in different sea anemone species in which the young fish is recruited. Taken together our results suggest that TH control the timing of adult color pattern formation and that shifts in gene expression and TH levels are associated with ecological differences resulting in divergent ontogenetic trajectories in color pattern development. We recently observed that the phenotypic change observed not only concerns pigmentation but also energy metabolism. The evolutionary implications of these observations will be discussed

Phenotypic plasticity in anemonefishes: An Eco/Evo/Devo analysis

As poikilotherms, fish exposed to environmental extremes temperatures experience a stressful metabolic challenge, which elicits physiological responses required to maintain cellular homeostasis. These responses include modified glucose or lipid metabolism, altered gene expression and alternative splicing, and endocrine and immune system activity. Additionally, heat seeking may not resolve the homeostatic needs of tropical poikilotherms under unpredictable extreme cold events, which occur frequently due to global climate changes. In spite of the physiological responses that occur in poikilothermic vertebrates, the prevailing notion is that their reduction in metabolic rate is passive. Here, we explored molecular hypothalamic and physiological responses to cold stress in the tropical cichlid, Nile tilapia (Oreochromis niloticus). When exposed to low temperatures, tilapia exhibited complex homeostatic responses, including increased plasma glucose and cortisol concomitant with reduced plasma lactate and metabolic rate. Hypothalamic transcriptome analysis revealed increased oxytocin expression. Blockage of oxytocin signaling using zebrafish CRISPR-knockout lines for oxytocin or its receptors, and pharmacological antagonist in tilapia, further affected temperature-dependent metabolic rate in two cold-exposed tropical fish species. This indicates that oxytocin, a known thermoregulator in homeotherms, actively regulates temperature-related homeostasis in fish. Overall, our findings show that the fish brain actively responds to cold temperature by regulating metabolic physiology. Moreover, we identify oxytocin signaling as an adaptive and evolutionarily conserved metabolic regulator of temperature-related homeostasis. 

Oxytocin centrally regulates temperature-dependent metabolism in fish

Animals respond to environmental challenges with a suite of physiological responses – the stress response. As high temperatures become more frequent and intense globally, determining whether and how individuals vary in their stress response to heat is urgent. While there is an emerging interest in understanding avian stress response to heat, very often, only glucocorticoids are quantified. Here, we tested whether early-life conditions affect multiple traits of nestling stress physiology in response to heat. In the arid-adapted zebra finch, parents emit “heat-calls” when incubating under high temperatures, which adaptively alter offspring growth. We exposed nestlings to prenatal heat-calls or control-calls, and manipulated postnatal nest temperatures. At 13-day old, we collected blood samples in two experiments: under undisturbed “in-nest” conditions, or following a 2.5-hour heat-challenge. As biomarkers of acute, cellular and chronic stress, we measured, respectively, levels of corticosterone (CORT), constitutive heat-shock cognate 70 (HSC70) and stress-inducible heat-shock protein 90 (HSP90), and, the heterophil to lymphocyte (H/L) ratio. We found that none of the stress biomarkers co-varied in either experiment, likely due to distinct time-courses. After the heat-challenge, only the H/L ratio was higher than in the in-nest experiment. Furthermore, in the in-nest experiment, control-call nestlings had a marginally higher H/L ratio than heat-calls nestlings, and both HSPs increased with nest temperatures, in control-call individuals only. These results suggest a higher sensitivity of control-calls nestlings to summer nest temperatures. Overall, our study shows for the first time that a prenatal acoustic signal for heat affects physiological stress responses to postnatal thermal conditions. 

Prenatal acoustic signals and postnatal heat shape nestling stress physiology

Many pesticides are endocrine-active, even if the intended mechanism of action is not specified to directly interfere with outcomes such as development, reproduction, or sex determination. Pyrethroid pesticides, which are commonly used in both North America and the European Union, are based on toxins naturally produced by the chrysanthemum plant, but are chemically enhanced to increase persistence and toxicity to target organisms. These modifications make pyrethroids more likely to interact with endocrine targets such as the nuclear and membrane estrogen receptors, to modify downstream responses like the activity of steroidogenic enzymes, and to ultimately induce organismal responses indicative of endocrine disruption in fish (e.g. changes in gonad size, sex ratio). Previous work from our group confirmed these interactions for specific pyrethroid types, demonstrating that early life exposure can result in impacts that span multiple generations, and that increased temperature can exacerbate these effects. Recent findings indicate that other abiotic factors, such as salinity, can influence the direction and degree of endocrine-related responses, likely due to a combination of osmoregulatory modulation and differential chemical behavior that is dependent upon aqueous salinity. These observations are highly relevant in terms of predicted changes in temperature and salinity regimes over the coming decades, and our findings underscore the importance of understanding responses to endocrine-active compounds in the context of altered environmental conditions.

Endocrine disruption in the context of climate change: understanding responses in fish

On the upper surface of its leafs salvinia molesta has pillar shaped structures which are hydrophilic at the tip and hydrophobic at the shaft. When submerged under water, air is retained between the surface structure. Beside the storage of oxygen this feature enables a variety of further applications. The air layer could serve as barrier to prevent the settlement of biological organisms (antifouling) and reduce wall shear stress if water flows along the surface. These phenomena are from higher interest in case of ships or liquid transport in pipes. If an air retaining structure could be realised on such surfaces, a reduction of frictional drag, fuel consumption and CO2 production could follow. A requirement for this is the understanding of the physics and the influence of fundamental parameters such as structure geometry, size, contact angle of the structure material, pressure, or air saturation of the liquid. In this work the effect of different parameters is presented and discussed using numerical simulation, analytical approaches and experiments.

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