Although fungi and bacteria commonly coexist within polymicrobial communities, the molecular mechanisms underlying their interactions are still not well understood. Here, we show that the fungus Candida albicans forms biofilms with the bacterium Staphylococcus aureus along a nutritional axis of mutualism and propose that “a copper economy” shapes fungal-bacterial biofilm interactions. Using in vitro biofilms formed on plastic, we found that dual species biofilms are consistently larger than single-species counterparts, indicating a cooperative interaction. Dual species proteomic analysis revealed non-reciprocal copper handling: C. albicans increased copper uptake via transporter Ctr1, while S. aureus enhanced copper export via regulator CsoR and export chaperone CopZ. Dual species biofilms exhibited specific sensitivity to both copper depletion and supplementation, with corresponding reductions in biomass. We identified fungal copper import as the crucial element in mutualistic interactions between C. albicans and staphylococcal species. Moreover, fungal hyphae served as a critical scaffold for biofilm architecture, a role that was compromised under copper-replete conditions. Notably, copper nanoparticles disrupted these dual species biofilms, highlighting a potential therapeutic avenue. Furthermore, we extend the role of C. albicans copper import to mutualistic interactions with additional bacterial species. Our findings establish copper as a central mediator of C. albicans and S. aureus cooperation and suggest that a "copper economy" underpins mutualistic interactions in biofilms.

Maintenance of mitochondrial redox homeostasis is of fundamental importance to cellular health. Mitochondria harbor a host of intrinsic antioxidant defenses, but the contribution of extrinsic, nonmitochondrial antioxidant mechanisms is less well understood. We found a direct role for peroxisomes in maintaining mitochondrial redox homeostasis through contact-mediated reactive oxygen species (ROS) transfer. We found that ACBD5 and PTPIP51 form a contact between peroxisomes and mitochondria. The percentage of these contacts increased during mitochondrial oxidative stress and helped to maintain mitochondrial health through the transfer of mitochondrial ROS to the peroxisome lumen. Our findings reveal a multiorganelle layer of mitochondrial antioxidant defense-suggesting a direct mechanism by which peroxisomes contribute to mitochondrial health-and broaden the scope of known membrane contact site functions.

Type VI Secretion Systems (T6SS) are utilised by many bacteria to deliver toxic effectors into neighbouring bacterial, fungal or host cells. Whilst many antibacterial effectors are well characterised, much less is known regarding the identity or mode-of-action of antifungal effectors. Here we combine structural modelling with proteomics and in vivo approaches, to show that the Serratia marcescens antifungal effector Tfe2 adopts a novel fold and functions as a potent inhibitor of protein translation leading to hyperactivation of the TORC1 kinase. We show that Tfe2 expression in Saccharomyces cerevisiae , or treatment with the protein translation inhibitor cycloheximide, drive identical increases in free intracellular amino acids and hyperactivation of TORC1. This, in turn, triggers the Tfe2 and cycloheximide-mediated rapid turnover of amino acid transporters through stimulating substrate-independent endocytosis. Polysome profiling, however, revealed differences in Tfe2 and cycloheximide-mediated protein translation inhibition, with Tfe2 inhibiting initiation of translation. Tfe2-mediated hyperactivation of TORC1 may also underpin adaptive responses to this effector which include significant remodelling of the lipidome and notable alterations in organelle and cell wall structures. Collectively this study has provided new insight into the mode-of-action of a structurally novel antifungal effector Tfe2. ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, https://ror.org/029chgv08, 215599/Z/19/Z

The community structure and ecological function of marine ecosystems are critically dependent on phytoplankton. However, our understanding of phytoplankton is limited due to the lack of detailed information on their morphology. To address this gap, we developed a framework that combines scanning electron microscopy (SEM) with photogrammetry to create realistic 3D (three-dimensional) models of phytoplankton. The workflow of this framework is demonstrated using two marine algal species, one dinoflagellate Prorocentrum micans and one diatom Halamphora sp. The resulting 3D models are made openly available and allow users to interact with phytoplankton and their complex structures virtually (digitally) and tangibly (3D printing). They also allow for surface area and biovolume calculations of phytoplankton, as well as the exploration of their light scattering properties, which are both important for ecosystem modeling. Additionally, by presenting these models to the public, it bridges the gap between scientific inquiry and education, promoting broader awareness on the importance of phytoplankton.

During the reductive evolution of obligate intracellular parasites called microsporidia, a tiny remnant mitochondrion (mitosome) lost its typical cristae, organellar genome, and most canonical functions. Here, we combine electron tomography, stereology, immunofluorescence microscopy, and bioinformatics to characterise mechanisms of growth, division, and inheritance of this minimal mitochondrion in two microsporidia species (grown within a mammalian RK13 culture-cell host). Mitosomes of Encephalitozoon cuniculi (2-12/cell) and Trachipleistophora hominis (14-18/nucleus) displayed incremental/non-phasic growth and division and were closely associated with an organelle identified as equivalent to the fungal microtubule-organising centre (microsporidian spindle pole body; mSPB). The mitosome-mSPB association was resistant to treatment with microtubule-depolymerising drugs nocodazole and albendazole. Dynamin inhibitors (dynasore and Mdivi-1) arrested mitosome division but not growth, whereas bioinformatics revealed putative dynamins Drp-1 and Vps-1, of which, Vps-1 rescued mitochondrial constriction in dynamin-deficient yeast (Schizosaccharomyces pombe). Thus, microsporidian mitosomes undergo incremental growth and dynamin-mediated division and are maintained through ordered inheritance, likely mediated via binding to the microsporidian centrosome (mSPB).

Transmission electron microscopy (TEM) has long been a vital technology to visualize the interaction of cellular compartments at the highest possible resolution. While this paved the way to describing organelles within the cellular context in detail, TEM has long been underused to generate quantitative data, analyzing those interactions as well as underlying mechanisms leading to their formation and modification. Here we describe a simple stereological method to unbiasedly assess the extent of organelle-organelle membrane contact sites, able to efficiently generate accurate and reproducible quantitative data from cultured mammalian cells prepared for TEM.

Peroxisomes and the endoplasmic reticulum (ER) are intimately linked subcellular organelles, physically connected at membrane contact sites. While collaborating in lipid metabolism, for example, of very long-chain fatty acids (VLCFAs) and plasmalogens, the ER also plays a role in peroxisome biogenesis. Recent work identified tethering complexes on the ER and peroxisome membranes that connect the organelles. These include membrane contacts formed via interactions between the ER protein VAPB (vesicle-associated membrane protein-associated protein B) and the peroxisomal proteins ACBD4 and ACBD5 (acyl-coenzyme A-binding domain protein). Loss of ACBD5 has been shown to cause a significant reduction in peroxisome-ER contacts and accumulation of VLCFAs. However, the role of ACBD4 and the relative contribution these two proteins make to contact site formation and recruitment of VLCFAs to peroxisomes remain unclear. Here, we address these questions using a combination of molecular cell biology, biochemical, and lipidomics analyses following loss of ACBD4 or ACBD5 in HEK293 cells. We show that the tethering function of ACBD5 is not absolutely required for efficient peroxisomal β-oxidation of VLCFAs. We demonstrate that loss of ACBD4 does not reduce peroxisome-ER connections or result in the accumulation of VLCFAs. Instead, the loss of ACBD4 resulted in an increase in the rate of β-oxidation of VLCFAs. Finally, we observe an interaction between ACBD5 and ACBD4, independent of VAPB binding. Overall, our findings suggest that ACBD5 may act as a primary tether and VLCFA recruitment factor, whereas ACBD4 may have regulatory functions in peroxisomal lipid metabolism at the peroxisome-ER interface.

While bacteriophages (“phages”) are often cited as some of the key drivers of microbial diversity, evidence for this is greatly mixed. We demonstrate, in silico and experimentally, that the impact of phages, an example of a mobile genetic element (MGE), on community structure can diminish with increasing MGE diversity.

Zymoseptoria tritici is an economically damaging fungal pathogen of wheat which is able to survive for long periods on the leaf surface. In this environment, the fungus is exposed to many stresses, including fluctuations in temperature, UV radiation, drying, and foliar fungicide applications. We observed biofilm-like cell aggregations on the surface of wheat leaves infected with Z. tritici in both compatible and non-compatible interactions. A literature search revealed few examples of plant pathogenic fungi forming biofilms, but demonstrated that such biofilms have a number of key characteristics, which are shared with other biofilmforming microbes. These include adhesion of cells to the substrate, production of extracellular matrix, altered gene expression and increased tolerance to various stresses. We used a combination of microscopy, qRT-PCR and stress tolerance assays to determine whether putative Z. tritici biofilms possessed these diagnostic characteristics. We show that Z. tritici biofilms resemble in form and function those formed by other filamentous and dimorphic fungi, producing extra-cellular matrix in which cells become embedded, creating layers of hyphal bundles and blastospores. These biofilms show increased tolerance to drying and high temperature. There is also isolate-dependent resistance to reactive oxygen stress and the fungicide carboxin. Taken together, these findings demonstrate that Z. tritici is indeed able to produce genuine biofilms.

Antarctic krill (Euphausia superba) plays a central role in the Antarctic marine food web and biogeochemical cycles and has been identified as a species that is potentially vulnerable to plastic pollution. While plastic pollution has been acknowledged as a potential threat to Southern Ocean marine ecosystems, the effect of nanoplastics (<1000 nm) is poorly understood. Deleterious impacts of nanoplastic are predicted to be higher than that of larger plastics, due to their small size which enables their permeation of cell membranes and potentially provokes toxicity. Here, we investigated the intergenerational impact of exposing Antarctic krill to nanoplastics. We focused on whether embryonic energy resources were affected when gravid female krill were exposed to nanoplastic by determining lipid and fatty acid compositions of embryos produced in incubation. Embryos were collected from females who had spawned under three different exposure treatments (control, nanoplastic, nanoplastic + algae). Embryos collected from each maternal treatment were incubated for a further 6 days under three nanoplastic exposure treatments (control, low concentration nanoplastic, and high concentration nanoplastic). Nanoplastic additions to seawater did not impact lipid metabolism (total lipid or fatty acid composition) across the maternal or direct embryo treatments, and no interactive effects were observed. The provision of a food source during maternal exposure to nanoplastic had a positive effect on key fatty acids identified as important during embryogenesis, including higher total polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) when compared to the control and nanoplastic treatments. Whilst the short exposure time was ample for lipids from maternally digested algae to be incorporated into embryos, we discuss why the nanoplastic-fatty acid relationship may be more complex. Our study is the first to scope intergeneration effects of nanoplastic on Antarctic krill lipid and fatty acid reserves. From this, we suggest directions for future research including long term exposures, multi-stressor scenarios and exploring other critical energy reserves such as proteins.

Peroxisome membrane dynamics and division are essential to adapt the peroxisomal compartment to cellular needs. The peroxisomal membrane protein PEX11β (also known as PEX11B) and the tail-anchored adaptor proteins FIS1 (mitochondrial fission protein 1) and MFF (mitochondrial fission factor), which recruit the fission GTPase DRP1 (dynamin-related protein 1, also known as DNML1) to both peroxisomes and mitochondria, are key factors of peroxisomal division. The current model suggests that MFF is essential for peroxisome division, whereas the role of FIS1 is unclear. Here, we reveal that PEX11β can promote peroxisome division in the absence of MFF in a DRP1- and FIS1-dependent manner. We also demonstrate that MFF permits peroxisome division independently of PEX11β and restores peroxisome morphology in PEX11β-deficient patient cells. Moreover, targeting of PEX11β to mitochondria induces mitochondrial division, indicating the potential for PEX11β to modulate mitochondrial dynamics. Our findings suggest the existence of an alternative, MFF-independent pathway in peroxisome division and report a function for FIS1 in the division of peroxisomes. This article has an associated First Person interview with the first authors of the paper.

Candida albicans exists as a commensal of mucosal surfaces and the gastrointestinal tract without causing pathology. However, this fungus is also a common cause of mucosal and systemic infections when antifungal immune defenses become compromised. The activation of antifungal host defenses depends on the recognition of fungal pathogen-associated molecular patterns (PAMPs), such as β-1,3-glucan. In C. albicans, most β-1,3-glucan is present in the inner cell wall, concealed by the outer mannan layer, but some β-1,3-glucan becomes exposed at the cell surface. In response to host signals, such as lactate, C. albicans induces the Xog1 exoglucanase, which shaves exposed β-1,3-glucan from the cell surface, thereby reducing phagocytic recognition. We show here that β-1,3-glucan is exposed at bud scars and punctate foci on the lateral wall of yeast cells, that this exposed β-1,3-glucan is targeted during phagocytic attack, and that lactate-induced masking reduces β-1,3-glucan exposure at bud scars and at punctate foci. β-1,3-Glucan masking depends upon protein kinase A (PKA) signaling. We reveal that inactivating PKA, or its conserved downstream effectors, Sin3 and Mig1/Mig2, affects the amounts of the Xog1 and Eng1 glucanases in the C. albicans secretome and modulates β-1,3-glucan exposure. Furthermore, perturbing PKA, Sin3, or Mig1/Mig2 attenuates the virulence of lactate-exposed C. albicans cells in Galleria. Taken together, the data are consistent with the idea that β-1,3-glucan masking contributes to Candida pathogenicity. IMPORTANCE Microbes that coexist with humans have evolved ways of avoiding or evading our immunological defenses. These include the masking by these microbes of their "pathogen-associated molecular patterns" (PAMPs), which are recognized as "foreign" and used to activate protective immunity. The commensal fungus Candida albicans masks the proinflammatory PAMP β-1,3-glucan, which is an essential component of its cell wall. Most of this β-1,3-glucan is hidden beneath an outer layer of the cell wall on these microbes, but some can become exposed at the fungal cell surface. Using high-resolution confocal microscopy, we examine the nature of the exposed β-1,3-glucan at C. albicans bud scars and at punctate foci on the lateral cell wall, and we show that these features are targeted by innate immune cells. We also reveal that downstream effectors of protein kinase A (Mig1/Mig2, Sin3) regulate the secretion of major glucanases, modulate the levels of β-1,3-glucan exposure, and influence the virulence of C. albicans in an invertebrate model of systemic infection. Our data support the view that β-1,3-glucan masking contributes to immune evasion and the virulence of a major fungal pathogen of humans.

Nanoplastics (NPs; <1 μm) have greater availability to marine organisms than microplastics (1-5000 μm). Understanding NP uptake and depuration in marine organisms intended for human consumption is imperative for food safety, but until now it has been limited due to analytical constraints. Oysters (Crassostrea gigas) were exposed to polystyrene NPs doped with palladium (Pd), allowing the measurements of their uptake into tissues by inductively coupled plasma mass spectrometry (ICP-MS) combined with electron microscopy. Oysters were exposed for 6 days (d) to "Smooth" or "Raspberry" NPs, followed by 30 d of depuration with the aim of assessing the NP concentration in C. gigas following exposure, inferring the accumulation and elimination rates, and understanding the clearance of Pd NPs during the depuration period. After 6 d, the most significant accumulation was found in the digestive gland (106.6 and 135.3 μg g-1 dw, for Smooth and Raspberry NPs, respectively) and showed the most evident depuration (elimination rate constant KSmooth = 2 d-1 and KRaspberry = 0.2 d-1). Almost complete depuration of the Raspberry NPs occurred after 30 d. While a post-harvesting depuration period of 24-48 h for oysters could potentially reduce the NP content by 75%, more research to validate these findings, including depuration studies of oysters from the field, is required to inform practices to reduce human exposure through consumption.

Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 [acyl-coenzyme A-binding domain protein 5] and the ER-resident protein VAPB [vesicle-associated membrane protein-associated protein B]. ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like [two phenylalanines (FF) in an acidic tract] motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB and thus, peroxisome-ER contact sites differently. Moreover, we demonstrate that GSK3β [glycogen synthase kinase-3 beta] regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome-ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction. SUMMARY Kors et al. reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation and GSK3β in mammalian cells. Phosphorylation sites in the FFAT-like motif of ACBD5 affect the binding to VAPB and thus, peroxisome-ER contact sites, differently.

Abstract Background Rodent and human β-cells are differentially susceptible to the “lipotoxic” effects of long-chain saturated fatty acids (LC-SFA) but the factors accounting for this are unclear. Here, we have studied the intracellular disposition of the LC-SFA palmitate in human vs rodent β–cells and present data that reveal new insights into the factors regulating β-cell lipotoxicity. Methods The subcellular distribution of the LC-SFA palmitate was studied in rodent (INS-1E and INS-1 823/13 cells) and human (EndoC-βH1) β-cells using confocal fluorescence and electron microscopy (EM). Protein expression was assessed by Western blotting and cell viability, by vital dye staining. Results Exposure of INS-1 cells to palmitate for 24 h led to loss of viability, whereas EndoC-βH1 cells remained viable even after 72 h of treatment with a high concentration (1 mM) of palmitate. Use of the fluorescent palmitate analogue BODIPY FL C16 revealed an early localisation of the LC-SFA to the Golgi apparatus in INS-1 cells and this correlated with distention of intracellular membranes, visualised under the EM. Despite this, the PERK-dependent ER stress pathway was not activated under these conditions. By contrast, BODIPY FL C16 did not accumulate in the Golgi apparatus in EndoC-βH1 cells but, rather, co-localised with the lipid droplet-associated protein, PLIN2, suggesting preferential routing into lipid droplets. When INS-1 cells were treated with a combination of palmitate plus oleate, the toxic effects of palmitate were attenuated and BODIPY FL C16 localised primarily with PLIN2 but not with a Golgi marker. Conclusion In rodent β-cells, palmitate accumulates in the Golgi apparatus at early time points whereas, in EndoC- βH1 cells, it is routed preferentially into lipid droplets. This may account for the differential sensitivity of rodent vs human β-cells to “lipotoxicity” since manoeuvres leading to the incorporation of palmitate into lipid droplets is associated with the maintenance of cell viability in both cell types.

Peroxisomes are highly dynamic subcellular compartments with important functions in lipid and ROS metabolism. Impaired peroxisomal function can lead to severe metabolic disorders with developmental defects and neurological abnormalities. Recently, a new group of disorders has been identified, characterised by defects in the membrane dynamics and division of peroxisomes rather than by loss of metabolic functions. However, the contribution of impaired peroxisome plasticity to the pathophysiology of those disorders is not well understood. Mitochondrial fission factor (MFF) is a key component of both the peroxisomal and mitochondrial division machinery. Patients with MFF deficiency present with developmental and neurological abnormalities. Peroxisomes (and mitochondria) in patient fibroblasts are highly elongated as a result of impaired organelle division. The majority of studies into MFF-deficiency have focused on mitochondrial dysfunction, but the contribution of peroxisomal alterations to the pathophysiology is largely unknown. Here, we show that MFF deficiency does not cause alterations to overall peroxisomal biochemical function. However, loss of MFF results in reduced import-competency of the peroxisomal compartment and leads to the accumulation of pre-peroxisomal membrane structures. We show that peroxisomes in MFF-deficient cells display alterations in peroxisomal redox state and intra-peroxisomal pH. Removal of elongated peroxisomes through induction of autophagic processes is not impaired. A mathematical model describing key processes involved in peroxisome dynamics sheds further light into the physical processes disturbed in MFF-deficient cells. The consequences of our findings for the pathophysiology of MFF-deficiency and related disorders with impaired peroxisome plasticity are discussed.

We describe a rule-based voice dialog system for in-vehicle infotainment. The system is specified by a declarative model comprising the dialog definition, dialog management logic, and domain knowledge, which are backed by an RDFS ontology. The approach simplifies coping with dialog phenomena such as anaphora or implicit confirmation, that can be addressed without writing code. We introduce the concept of a virtual knowledge base to accommodate the diversity of dialog-relevant knowledge sources in a car. It unifies the knowledge access for the dialog logic, no matter whether an internal knowledge base or an external source like a web service is being used. The virtual KB thus supports the model’s portability and re-usability. Dialog models can be exported and deployed to on-market voice assistants like Amazon Alexa and Google Assistant, which essentially means to extend their built-in dialog management capabilities. The dialog management run-time system is deployed on a separate cloud server and acts as a back-end for the respective voice assistant.

The Arabian killifish, Aphanius dispar, is a small tropical teleost fish living in wide range of habitats in sea water and freshwater in theMiddle East. Here, we report extraordinary fluorescent pigment cells in the Arabian killifish embryo. These cells appear brown in transmitted light, yellowish white in reflected light, and as strong fluorescence in GFP and RFP filters. TEM and confocal microscopy analyses show the fluorescence emanates from leucosome-like pigment organelles. The cells express the gene encoding GTP cyclohydrolase (gch), a marker for leucophores and xanthophore. Gene knockdown and knockout of gch usingmorpholino or CRISPR-Cas9 induced loss of fluorescence in these embryos, indicating a crucial role of the enzyme and the associated pterine biosynthesis pathway in the generation of the fluorescence. We concluded that these cells are a highly fluorescent subtype of leucophores and have named them as fluoroleucophores.