Mutations in the gene for β-catenin cause liver cancer cells to release fewer exosomes, which reduces the number of immune cells infiltrating the tumor.
Intercellular distribution of nutrients and coordination of responses to internal and external cues via endogenous signaling molecules are hallmarks of multicellular organisms. Vegetative mycelia of ...multicellular fungi are syncytial networks of interconnected hyphae resulting from hyphal tip growth, branching, and fusion. Such mycelia can reach considerable dimensions and, thus, different parts can be exposed to quite different environmental conditions. Our knowledge about the mechanisms by which fungal mycelia can adjust nutrient gradients or coordinate their defense response to fungivores is scarce, in part due to limitations in technologies currently available for examining different parts of a mycelium over longer time periods at the microscopic level. Here, we combined a tailor-made microfluidic platform with time-lapse fluorescence microscopy to visualize the dynamic response of the vegetative mycelium of a basidiomycete to two different stimuli. The microfluidic platform allows simultaneous monitoring at both the colony and single-hypha level. We followed the dynamics of the distribution of a locally administered nutrient analog and the defense response to spatially confined predation by a fungivorous nematode. Although both responses of the mycelium were constrained locally, we observed long-distance propagation for both the nutrient analog and defense response in a subset of hyphae. This propagation along hyphae occurred in both acropetal and basipetal directions and, intriguingly, the direction was found to alternate every 3 hr in an individual hypha. These results suggest that multicellular fungi have, as of yet, undescribed mechanisms to coordinate the distribution of nutrients and their behavioral response upon attack by fungivores.
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•Transport of signals and nutrients in fungal mycelia occurs via specialized hyphae•Transport in these hyphae is bidirectional•Direction of transport in these hyphae oscillates between acropetal and basipetal
Schmieder, Stanley et al. provide experimental evidence for the bidirectional propagation of signals and nutrients along specialized hyphae. These results, collected on a microfluidic chip, suggest that multicellular fungi use undescribed mechanisms to coordinate the distribution of nutrients and their behavioral response upon attack by fungivores.
Resistance of fungi to predation is thought to be mediated by toxic metabolites and proteins. Many of these fungal defense effectors are highly abundant in the fruiting body and not produced in the ...vegetative mycelium. The defense function of fruiting body-specific proteins, however, including cytoplasmically localized lectins and antinutritional proteins such as biotin-binding proteins, is mainly based on toxicity assays using bacteria as a heterologous expression system, with bacterivorous/omnivorous model organisms as predators. Here, we present an ecologically more relevant experimental setup to assess the toxicity of potential fungal defense proteins towards the fungivorous, stylet-feeding nematodes
and
As a heterologous expression host, we exploited the filamentous fungus
Using this new system, we assessed the toxicity of six previously characterized, cytoplasmically localized, potential defense proteins from fruiting bodies of different fungal phyla against the two fungivorous nematodes. We found that all of the tested proteins were toxic against both nematodes, albeit to various degrees. The toxicity of these proteins against both fungivorous and bacterivorous nematodes suggests that their targets have been conserved between the different feeding groups of nematodes and that bacterivorous nematodes are valid model organisms to assess the nematotoxicity of potential fungal defense proteins.
Our results support the hypothesis that cytoplasmic proteins abundant in fungal fruiting bodies are involved in fungal resistance against predation. The toxicity of these proteins toward stylet-feeding nematodes, which are also capable of feeding on plants, and the abundance of these proteins in edible mushrooms, may open possible avenues for biological crop protection against parasitic nematodes, e.g., by expression of these proteins in crops.
The complex sphingolipids exhibit a diversity of ceramide acyl chain structures that influence their trafficking and intracellular distributions, but it remains unclear how the cell discerns among ...the different ceramides to affect such sorting. To address the mechanism, we synthesize a library of GM1 glycosphingolipids with naturally varied acyl chains and quantitatively assess their sorting among different endocytic pathways. We find that a stretch of at least 14 saturated carbons extending from C1 at the water-bilayer interface dictate lysosomal sorting by exclusion from endosome sorting tubules. Sorting to the lysosome by the C14∗ motif is cholesterol dependent. Perturbations of the C14∗ motif by unsaturation enable GM1 entry into endosomal sorting tubules of the recycling and retrograde pathways independent of cholesterol. Unsaturation occurring beyond the C14∗ motif in very long acyl chains rescues lysosomal sorting. These results define a structural motif underlying the membrane organization of sphingolipids and implicate cholesterol-sphingolipid nanodomain formation in sorting mechanisms.
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•Glycosphingolipids are sorted by a C14∗ motif in fatty acid of the ceramide•C14∗ motif consists of 14 plus 1 saturated hydrocarbons extending from the amide bond•Glycosphingolipids with C14∗ motif are exclusively sorted to lysosome•Sorting by C14∗ motif is cholesterol dependent
Lipids display structural diversity in their acyl chains. To elucidate the effect this diversity has on lipid trafficking, Schmieder et al. synthesize a glycosphingolipid (GM1) library with varied fatty acids. They find that GM1 is endosomally sorted by a C14∗ motif within the fatty acid of the ceramide moiety.
Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 ...glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1.
Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how ...they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.
Effector proteins of innate immune systems recognize specific non-self epitopes. Tectonins are a family of β-propeller lectins conserved from bacteria to mammals that have been shown to bind ...bacterial lipopolysaccharide (LPS). We present experimental evidence that two Tectonins of fungal and animal origin have a specificity for O-methylated glycans. We show that Tectonin 2 of the mushroom Laccaria bicolor (Lb-Tec2) agglutinates Gram-negative bacteria and exerts toxicity toward the model nematode Caenorhabditis elegans , suggesting a role in fungal defense against bacteria and nematodes. Biochemical and genetic analysis of these interactions revealed that both bacterial agglutination and nematotoxicity of Lb-Tec2 depend on the recognition of methylated glycans, namely O-methylated mannose and fucose residues, as part of bacterial LPS and nematode cell-surface glycans. In addition, a C. elegans gene, termed samt-1 , coding for a candidate membrane transport protein for the presumptive donor substrate of glycan methylation, S-adenosyl-methionine, from the cytoplasm to the Golgi was identified. Intriguingly, limulus lectin L6, a structurally related antibacterial protein of the Japanese horseshoe crab Tachypleus tridentatus , showed properties identical to the mushroom lectin. These results suggest that O-methylated glycans constitute a conserved target of the fungal and animal innate immune system. The broad phylogenetic distribution of O-methylated glycans increases the spectrum of potential antagonists recognized by Tectonins, rendering this conserved protein family a universal defense armor.
IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates ...IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge β-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.
Polarized epithelial cells form an essential barrier against infection at mucosal surfaces. Many pathogens breach this barrier to cause disease, often by co-opting cellular endocytosis mechanisms to ...enter the cell through the lumenal (apical) cell surface. We recently discovered that the loss of the cell polarity gene PARD6B selectively diminishes apical endosome function. Here, we find that in response to the entry of certain viruses and bacterial toxins into the epithelial cells via the apical membrane, PARD6B and aPKC, two components of the PARD6B-aPKC-Cdc42 apical polarity complex, undergo rapid proteasome-dependent degradation. The perturbation of apical membrane glycosphingolipids by toxin- or virus-binding initiates degradation of PARD6B. The loss of PARD6B causes the depletion of apical endosome function and renders the cell resistant to further infection from the lumenal cell surface, thus enabling a form of cell-autonomous host defense.
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•Apical membranes of mucosal epithelial cells sense virus and enterotoxin binding•Virus and toxin sensing causes rapid degradation of the polarity complex components•Loss of function in the polarity complex inhibits apical endosome function•Depletion of apical endosome blocks further virus and toxin entry
Maeda et al. discover that intestinal epithelial cells sense the apical membrane binding of cholera toxin and rotavirus to induce proteasome-dependent degradation of the PARD6B/aPKC complex. This leads to the downregulation of the apical endosome, which blocks further infection, representing a form of cell-autonomous host defense.
We introduce a new family of fungal protease inhibitors with β-trefoil fold from the mushroom
, named cocaprins, which inhibit both cysteine and aspartic proteases. Two cocaprin-encoding genes are ...differentially expressed in fungal tissues. One is highly transcribed in vegetative mycelium and the other in the stipes of mature fruiting bodies. Cocaprins are small proteins (15 kDa) with acidic isoelectric points that form dimers. The three-dimensional structure of cocaprin 1 showed similarity to fungal β-trefoil lectins. Cocaprins inhibit plant C1 family cysteine proteases with
in the micromolar range, but do not inhibit the C13 family protease legumain, which distinguishes them from mycocypins. Cocaprins also inhibit the aspartic protease pepsin with
in the low micromolar range. Mutagenesis revealed that the β2-β3 loop is involved in the inhibition of cysteine proteases and that the inhibitory reactive sites for aspartic and cysteine proteases are located at different positions on the protein. Their biological function is thought to be the regulation of endogenous proteolytic activities or in defense against fungal antagonists. Cocaprins are the first characterized aspartic protease inhibitors with β-trefoil fold from fungi, and demonstrate the incredible plasticity of loop functionalization in fungal proteins with β-trefoil fold.
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