Fine-scale foraging ecology and habitat use of sympatric green and hawksbill turtles in the Western Indian ocean.

Using stable isotope analysis of carbon and nitrogen of turtle tissues and putative prey items, we investigated the diet of immature green turtles and hawksbill turtles foraging in the lagoon of Aldabra Atoll, a relatively undisturbed atoll in the southern Seychelles. Aldabra offers a unique environment for understanding sea turtle ecology. Green turtles mostly consumed seagrass and brown algae while hawksbill turtles mainly consumed mangroves and invertebrates. Green turtles showed a dietary shift with size (a proxy for age). There was minimal niche overlap between species and evidence of small-scale foraging site fidelity with turtle tissue reflecting site-specific prey. This highlights the ecological importance of seagrass and mangrove habitats and suggests that turtles play a role in controlling algal biomass at Aldabra. This study is the first to closely examine the foraging ecology of these sympatric turtle species in the Western Indian Ocean, a globally important region for both species.

Trait-based approaches reveal that deep reef ecosystems in the Western Indian Ocean are functionally distinct.

Tropical deep reefs (>30 m) are biologically and ecologically unique ecosystems with a higher geographic reach to shallow (<30 m) reefs. Yet they are poorly understood and rarely considered in conservation practices. Here, we characterise benthic and fish communities across a depth gradient (10-350 m) in remote coral atolls in Seychelles, Western Indian Ocean. Using taxonomic and trait-based approaches we present the taxonomic and functional composition of shallow and deep reef communities, with distinct communities and traits dominating different depths. Depth-related changes in community metrics (taxa richness, abundance and biomass) and functional diversity metrics (richness, dispersion, and evenness) indicate complex relationships across different biological components (fish, benthos) that differ between shallow and deep reefs. These in turn translate into different patterns of reef resilience against disturbance or species invasions with depth. Notably, deep reefs host on average fewer and less abundant taxa but with higher functional contribution and originality scores, some of which are of conservation concern. Overall, the results highlight the unique nature of deep reefs that requires their explicit consideration in conservation and management activities.

Fast and Multiplexed Super Resolution Imaging of Fixed and Immunostained Cells with DNA-PAINT-ERS.

Recent advances in super resolution microscopy have enabled imaging at the 10-20 nm scale on a light microscope, providing unprecedented details of native biological structures and processes in intact and hydrated samples. Of the existing strategies, DNA points accumulation in imaging nanoscale topography (DNA-PAINT) affords convenient multiplexing, an important feature in interrogating complex biological systems. A practical limitation of DNA-PAINT, however, has been the slow imaging speed. In its original form, DNA-PAINT imaging of each target takes tens of minutes to hours to complete. To address this challenge, several improved implementations have been introduced. These include DNA-PAINT-ERS (where E = ethylene carbonate; R = repeat sequence; S = spacer), a set of strategies that leads to both accelerated DNA-PAINT imaging speed and improved image quality. With DNA-PAINT-ERS, imaging of typical cellular targets such as microtubules takes only 5-10 min. Importantly, DNA-PAINT-ERS also facilitates multiplexing and can be easily integrated into current workflows for fluorescence staining of biological samples. Here, we provide a detailed, step-by-step guide for fast and multiplexed DNA-PAINT-ERS imaging of fixed and immunostained cells grown on glass substrates as adherent monolayers. The protocol should be readily extended to biological samples of a different format (for example tissue sections) or staining mechanisms (for example using nanobodies). © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of probes for DNA-PAINT-ERS Basic Protocol 2: Sample preparation for imaging membrane targets with DNA-PAINT-ERS in fixed cells Alternate Protocol: Immunostaining of extracted U2OS cells Basic Protocol 3: Super resolution image acquisition and analysis.

GLUT4 translocation and dispersal operate in multiple cell types and are negatively correlated with cell size in adipocytes.

The regulated translocation of the glucose transporter, GLUT4, to the surface of adipocytes and muscle is a key action of insulin. This is underpinned by the delivery and fusion of GLUT4-containing vesicles with the plasma membrane. Recent studies have revealed that a further action of insulin is to mediate the dispersal of GLUT4 molecules away from the site of GLUT4 vesicle fusion with the plasma membrane. Although shown in adipocytes, whether insulin-stimulated dispersal occurs in other cells and/or is exhibited by other proteins remains a matter of debate. Here we show that insulin stimulates GLUT4 dispersal in the plasma membrane of adipocytes, induced pluripotent stem cell-derived cardiomyocytes and HeLa cells, suggesting that this phenomenon is specific to GLUT4 expressed in all cell types. By contrast, insulin-stimulated dispersal of TfR was not observed in HeLa cells, suggesting that the mechanism may be unique to GLUT4. Consistent with dispersal being an important physiological mechanism, we observed that insulin-stimulated GLUT4 dispersal is reduced under conditions of insulin resistance. Adipocytes of different sizes have been shown to exhibit distinct metabolic properties: larger adipocytes exhibit reduced insulin-stimulated glucose transport compared to smaller cells. Here we show that both GLUT4 delivery to the plasma membrane and GLUT4 dispersal are reduced in larger adipocytes, supporting the hypothesis that larger adipocytes are refractory to insulin challenge compared to their smaller counterparts, even within a supposedly homogeneous population of cells.

Nanoscopic Spatial Association between Ras and Phosphatidylserine on the Cell Membrane Studied with Multicolor Super Resolution Microscopy.

Recent work suggests that Ras small GTPases interact with the anionic lipid phosphatidylserine (PS) in an isoform-specific manner, with direct implications for their biological functions. Studies on PS-Ras associations in cells, however, have relied on immuno-EM imaging of membrane sheets. To study their spatial relationships in intact cells, we have combined the use of Lact-C2-GFP, a biosensor for PS, with multicolor super resolution imaging based on DNA-PAINT. At ~20 nm spatial resolution, the resulting super resolution images clearly show the nonuniform molecular distribution of PS on the cell membrane and its co-enrichment with caveolae, as well as with unidentified membrane structures. Two-color imaging followed by spatial analysis shows that KRas-G12D and HRas-G12V both co-enrich with PS in model U2OS cells, confirming previous observations, yet exhibit clear differences in their association patterns. Whereas HRas-G12V is almost always co-enriched with PS, KRas-G12D is strongly co-enriched with PS in about half of the cells, with the other half exhibiting a more moderate association. In addition, perturbations to the actin cytoskeleton differentially impact PS association with the two Ras isoforms. These results suggest that PS-Ras association is context-dependent and demonstrate the utility of multiplexed super resolution imaging in defining the complex interplay between Ras and the membrane.

Fleshy red algae mats act as temporary reservoirs for sessile invertebrate biodiversity.

Many coastal ecosystems, such as coral reefs and seagrass meadows, currently experience overgrowth by fleshy algae due to the interplay of local and global stressors. This is usually accompanied by strong decreases in habitat complexity and biodiversity. Recently, persistent, mat-forming fleshy red algae, previously described for the Black Sea and several Atlantic locations, have also been observed in the Mediterranean. These several centimetre high mats may displace seagrass meadows and invertebrate communities, potentially causing a substantial loss of associated biodiversity. We show that the sessile invertebrate biodiversity in these red algae mats is high and exceeds that of neighbouring seagrass meadows. Comparative biodiversity indices were similar to or higher than those recently described for calcifying green algae habitats and biodiversity hotspots like coral reefs or mangrove forests. Our findings suggest that fleshy red algae mats can act as alternative habitats and temporary sessile invertebrate biodiversity reservoirs in times of environmental change.

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

Ras Multimers on the Membrane: Many Ways for a Heart-to-Heart Conversation.

Formation of Ras multimers, including dimers and nanoclusters, has emerged as an exciting, new front of research in the 'old' field of Ras biomedicine. With significant advances made in the past few years, we are beginning to understand the structure of Ras multimers and, albeit preliminary, mechanisms that regulate their formation in vitro and in cells. Here we aim to synthesize the knowledge accrued thus far on Ras multimers, particularly the presence of multiple globular (G-) domain interfaces, and discuss how membrane nanodomain composition and structure would influence Ras multimer formation. We end with some general thoughts on the potential implications of Ras multimers in basic and translational biology.

First insights into coral recruit and juvenile abundances at remote Aldabra Atoll, Seychelles.

Coral recruitment and successive growth are essential for post-disturbance reef recovery. As coral recruit and juvenile abundances vary across locations and under different environmental regimes, their assessment at remote, undisturbed reefs improves our understanding of early life stage dynamics of corals. Here, we first explored changes in coral juvenile abundance across three locations (lagoon, seaward west and east) at remote Aldabra Atoll (Seychelles) between 2015 and 2019, which spanned the 2015/16 global coral bleaching event. Secondly, we measured variation in coral recruit abundance on settlement tiles from two sites (lagoon, seaward reef) during August 2018-August 2019. Juvenile abundance decreased from 14.1 ± 1.2 to 7.4 ± 0.5 colonies m-2 (mean ± SE) during 2015-2016 and increased to 22.4 ± 1.2 colonies m-2 during 2016-2019. Whilst juvenile abundance increased two- to three-fold at the lagoonal and seaward western sites during 2016-2018 (from 7.7-8.3 to 17.3-24.7 colonies m-2), increases at the seaward eastern sites occurred later (2018-2019; from 5.8-6.9 to 16.6-24.1 colonies m-2). The composition of coral recruits on settlement tiles was dominated by Pocilloporidae (64-92% of all recruits), and recruit abundance was 7- to 47-fold higher inside than outside the lagoon. Recruit abundance was highest in October-December 2018 (2164 ± 453 recruits m-2) and lowest in June-August 2019 (240 ± 98 recruits m-2). As Acroporid recruit abundance corresponded to this trend, the results suggest that broadcast spawning occurred during October-December, when water temperature increased from 26 to 29°C. This study provides the first published record on coral recruit abundance in the Seychelles Outer Islands, indicates a rapid (2-3 years) increase of juvenile corals following a bleaching event, and provides crucial baseline data for future research on reef resilience and connectivity within the region.

Early trajectories of benthic coral reef communities following the 2015/16 coral bleaching event at remote Aldabra Atoll, Seychelles.

Documenting post-bleaching trajectories of coral reef communities is crucial to understand their resilience to climate change. We investigated reef community changes following the 2015/16 bleaching event at Aldabra Atoll, where direct human impact is minimal. We combined benthic data collected pre- (2014) and post-bleaching (2016-2019) at 12 sites across three locations (lagoon, 2 m depth; seaward west and east, 5 and 15 m depth) with water temperature measurements. While seaward reefs experienced relative hard coral reductions of 51-62%, lagoonal coral loss was lower (- 34%), probably due to three-fold higher daily water temperature variability there. Between 2016 and 2019, hard coral cover did not change on deep reefs which remained dominated by turf algae and Halimeda, but absolute cover on shallow reefs increased annually by 1.3% (east), 2.3% (west) and 3.0% (lagoon), reaching, respectively, 54%, 68% and 93% of the pre-bleaching cover in 2019. Full recovery at the shallow seaward locations may take at least five more years, but remains uncertain for the deeper reefs. The expected increase in frequency and severity of coral bleaching events is likely to make even rapid recovery as observed in Aldabra's lagoon too slow to prevent long-term reef degradation, even at remote sites.

Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes.

Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)-containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA-GLUT4-GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA-GLUT4-GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells.

Boosting recovery rather than buffering reactivity: Higher stress-induced oxytocin secretion is associated with increased cortisol reactivity and faster vagal recovery after acute psychosocial stress.

Animal models and human studies using paradigms designed to stimulate endogenous oxytocin release suggest a stress-buffering role of oxytocin. We here examined the involvement of stress-induced peripheral oxytocin secretion in reactivity and recovery phases of the human psychosocial stress response. Healthy male and female participants (N=114) were subjected to a standardized laboratory stressor, the Trier Social Stress Test. In addition to plasma oxytocin, cortisol was assessed as a marker of hypothalamic-pituitary-adrenal (HPA-) axis activity, alpha-amylase and heart rate as markers of sympathetic activity, high frequency heart rate variability as a marker of vagal tone and self-rated anxiety as an indicator of subjective stress experience. On average, oxytocin levels increased by 51% following psychosocial stress. The stress-induced oxytocin secretion, however, did not reduce stress reactivity. To the contrary, higher oxytocin secretion was associated with greater cortisol reactivity and peak cortisol levels in both sexes. In the second phase of the stress response the opposite pattern was observed, with higher oxytocin secretion associated with faster vagal recovery. We suggest that after an early stage of oxytocin and HPA-axis co-activation, the stress-reducing action of oxytocin unfolds. Due to the time lag it manifests as a recovery-boosting rather than a reactivity-buffering effect. By reinforcing parasympathetic autonomic activity, specifically during stress recovery, oxytocin may provide an important protective function against the health-compromising effects of sustained stress.