None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. ...Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous β₂-adrenoreceptor (β₂-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated β₂-AR (Nb80) is highly immobile and organized in nanoclusters. The Gαs−GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated β₂-AR (Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.
Dynamin-like proteins (DLPs) mediate various membrane fusion and fission processes within the cell, which often require the polymerization of DLPs. An IFN-inducible family of DLPs, the ...guanylate-binding proteins (GBPs), is involved in antimicrobial and antiviral responses within the cell. Human guanylate-binding protein 1 (hGBP1), the founding member of GBPs, is also engaged in the regulation of cell adhesion and migration. Here, we show how the GTPase cycle of farnesylated hGBP1 (hGBP1F) regulates its self-assembly and membrane interaction. Using vesicles of various sizes as a lipid bilayer model, we show GTP-dependent membrane binding of hGBP1F. In addition, we demonstrate nucleotide-dependent tethering ability of hGBP1F. Furthermore, we report nucleotide-dependent polymerization of hGBP1F, which competes with membrane binding of the protein. Our results show that hGBP1F acts as a nucleotide-controlled molecular switch by modulating the accessibility of its farnesyl moiety, which does not require any supportive proteins.
Dicotyledonous plants growing under limited iron availability initiate a response resulting in the solubilization, reduction, and uptake of soil iron. The protein factors responsible for these steps ...are transmembrane proteins, suggesting that the intracellular trafficking machinery may be involved in iron acquisition. In search for components involved in the regulation of Arabidopsis thaliana iron deficiency responses, we identified the members of the SORTING NEXIN (SNX) protein family. SNX loss-of-function plants display enhanced susceptibility to iron deficiency in comparison to the wild type. The absence of SNX led to reduced iron import efficiency into the root. SNX1 showed partial colocalization with the principal root iron importer IRON-REGULATED TRANSPORTER1 (IRT1). In SNX loss-of-function plants, IRT1 protein levels were decreased compared with the wild type due to enhanced IRT1 degradation. This resulted in diminished amounts of the IRT1 protein at the plasma membrane, snx mutants exhibited enhanced iron deficiency responses compared with the wild type, presumably due to the lower iron uptake through IRT1. Our results reveal a role of SNX1 for the correct trafficking of IRT1 and, thus, for modulating the activity of the iron uptake machinery.
The IRON-REGULATED TRANSPORTER1 (IRT1) is the principal importer of soil iron in Arabidopsis thaliana. It has a complex intracellular trafficking behavior, including continuous cycling between plasma ...membrane and endosomes. SORTING NEXIN1 is required for the recycling of endosome-localized IRT1. In its absence, IRT1 is mistargeted for degradation, resulting in reduced plant iron-uptake efficiency. Consequently, IRT1 promoter activity gets limited to a specific portion of the root. We tested the influence of two hormones known to positively affect iron uptake on IRT1 spatial regulation. We found that ethylene treatment in wild-type background mimics the effects of the SNX-loss-of-function situation. Conversely, auxin splits the IRT1 expression zone and forces it toward the two extremities of the root. This shows that IRT1 expression along the root is modulated by ethylene-auxin interplay.
Abstract
Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the ...dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function.
The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic ...vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live‐cell super‐resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor‐binding‐deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG‐synaptotagmin‐1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1‐SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A‐ and BoNT/E‐induced neurointoxication as quantified by SNAP‐25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.
Synopsis
Synaptic targeting of botulinum neurotoxins is mediated by interaction with toxin‐type‐specific receptors and complex gangliosides, such as polysialoganglioside (PSG). This study shows that botulinum neurotoxin type A (BoNT/A) uptake and sorting into synaptic vesicles requires a tripartite nanocluster on the neuronal plasma membrane.
Live‐cell super‐resolution and electron microscopy in primary hippocampal neurons shows BoNT/A synaptic vesicle targeting depends on a tripartite PSG‐synaptotagmin 1 (Syt1)‐synaptic vesicle protein 2 (SV2) nanocluster on the plasma membrane.
Coincidental binding of BoNT/A to a preassembled PSG‐Syt1 complex and SV2 facilitates Syt1‐SV2 nanoclustering on the neuronal plasma membrane and endocytic sorting of the toxin into synaptic vesicles.
Syt1 is required for the BoNT/A and BoNT/E neurointoxication, suggesting a general mechanism of entry for clostridial botulinum neurotoxins.
Live‐cell super‐resolution imaging and electron microscopy of botulinum toxin type A in hippocampal neurons reveal the mechanism for its synaptic vesicle hijacking and neurointoxication.
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•3D SIM revealed differences in localization patterns of fungal cytosolic enzymes.•The cytosolic trichodiene synthase Tri5 was enriched at the endoplasmic reticulum.•Conversely, Hms1 ...was not significantly enriched at the endoplasmic reticulum.•Localized accumulation of Tri5 may allow for interaction with ER proteins.
Trichothecene mycotoxin synthesis in the phytopathogen Fusarium graminearum involves primarily endoplasmic reticulum (ER)-localized enzymes of the mevalonate- and trichothecene biosynthetic pathways. Two exceptions are 3-hydroxy-3-methylglutaryl CoA synthase (Hms1) and trichodiene synthase (Tri5), which are known cytosolic enzymes. Using 3D structured illumination microscopy (3D SIM), GFP-tagged Tri5 and Hms1 were tested for preferential localization in the cytosol proximal to the ER. Tri5 protein was significantly enriched in cytosolic regions within 500 nm of the ER, but Hms1 was not. Spatial organization of enzymes in the cytosol has potential relevance for pathway efficiency and metabolic engineering in fungi and other organisms.
•Novel high-throughput mutant screen for Fusarium spores was established using FACS.•Confirmation through the identification and characterisation of the g8 mutant.•The g8 mutant over-produces DON ...under DON production repressive conditions.•A mutation in the FAC1 adenylyl cyclase causes the DON over-production.•The mutant FAC1 is a gain-of-function allele resulting in increased cAMP production.
Fusarium head blight and crown rot, caused by the fungal plant pathogen Fusarium graminearum, impose a major threat to global wheat production. During the infection, plants are contaminated with mycotoxins such as deoxynivalenol (DON), which can be toxic for humans and animals. In addition, DON is a major virulence factor during wheat infection. However, it is not fully understood how DON production is regulated in F. graminearum. In order to identify regulators of DON production, a high-throughput mutant screen using Fluorescence Activated Cell Sorting (FACS) of a mutagenised TRI5-GFP reporter strain was established and a mutant over-producing DON under repressive conditions identified. A gain-of-function mutation in the F. graminearum adenylyl cyclase (FAC1), which is a known positive regulator of DON production, was identified as the cause of this phenotype through genome sequencing and segregation analysis. Our results show that the high-throughput mutant screening procedure developed here can be applied for identification of fungal proteins involved in diverse processes.
Fusarium pseudograminearum is an agronomically important fungus, which infects many crop plants, including wheat, where it causes Fusarium crown rot. Like many other fungi, the Fusarium genus ...produces a wide range of secondary metabolites of which only few have been characterized. Recently a novel gene cluster was discovered in F. pseudograminearum, which encodes production of cytokinin-like metabolites collectively named Fusarium cytokinins. They are structurally similar to plant cytokinins and can activate cytokinin signalling in vitro and in planta. Here, the regulation of Fusarium cytokinin production was analysed in vitro. This revealed that, similar to deoxynivalenol (DON) production in Fusariumgraminearum, cytokinin production can be induced in vitro by specific nitrogen sources in a pH-dependent manner. DON production was also induced in both F. graminearum and F. pseudograminearum in cytokinin-inducing conditions. In addition, microscopic analyses of wheat seedlings infected with a F. pseudograminearum cytokinin reporter strain showed that the fungus specifically induces its cytokinin production in hyphae, which are in close association with the plant, suggestive of a function of Fusarium cytokinins during infection.
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