Biology subjects

Cullen, P. J.

Publications and source records attributed to Cullen, P. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Cargo levels control endosomal PI(3)P homeostasis through direct feedback between SNX17 and MTM1

The sorting of internalised cell surface proteins through the endosomal network is crucial for cellular function. Phosphatidylinositol-3-phosphate (PI(3)P) is a critical lipid identity cue for organising endosomal sorting but there is limited understanding of how PI(3)P homeostasis is regulated. Here, we show that free PI(3)P is segregated into distinct endosomal subdomains and that depletion of the disease-relevant lipid phosphatase Myotubularin1 (MTM1) leads to its accumulation and mis-segregation, impairing cargo recycling. Complementary imaging and reconstitution approaches show that MTM1 recruitment and activity are controlled by binding to the endosomal cargo adaptor SNX17. This interaction is mediated by sorting motifs in the C-Terminal tail of MTM1 and is in competition with the same sorting motifs in cargo that bind to SNX17. This competitive binding mechanistically links cargo sorting to PI(3)P depletion and because SNX17 is itself a PI(3)P effector, establishes negative feedback regulating local PI(3)P levels. Importantly, we demonstrate that this feedback couples endosomal cargo levels to PI(3)P depletion. We propose that cargo levels thereby modulate endosomal phosphoinositide identity through cargo sensitive feedback between SNX17 and MTM1.

cell biology↗

bioRxiv2026

Exploring roles for essential proteins in yeast filamentous growth identifies the WASP homolog Las17 as a regulator of the Cdc42-dependent fMAPK pathway

Cell differentiation into distinct cell types generates functional specialization in eukaryotic organisms. Many fungal species undergo filamentous growth, where cells differentiate into elongated and adhesive filaments capable of expansion and invasion into new environments. The budding yeast Saccharomyces cerevisiae also undergoes filamentous growth, and the regulatory pathways that control the response have been well characterized; however, the roles essential proteins play in this response have not been systematically explored. To address this gap in understanding, we constructed a collection of 332 temperature-sensitive (ts) alleles in 320 essential genes in a strain background that undergoes filamentous growth (called {Sigma}1278b or Sigma). The ts Sigma collection showed differences in temperature sensitivity compared to a ts laboratory strain collection, revealing unexpected phenotypic diversity in essential alleles across populations of individuals. Screening the ts Sigma collection for phenotypes related to filamentous growth uncovered new phenotypes for >35% of essential alleles. New regulators of the Mitogen-Activated Protein Kinase (MAPK) pathway that regulates filamentous growth (fMAPK) were identified, including Las17, a homolog of Wiskott-Aldrich Syndrome Protein (WASP) in humans. Las17 regulated the fMAPK pathway by promoting delivery of the sensor protein, Sho1, and Rho GTPase Cdc42 to the plasma membrane. Las17 also functioned as a hub coordinating separate parallel aspects of the filamentation response. The widespread roles for essential proteins in regulating a eukaryotic differentiation response suggest broader roles essential proteins than is currently appreciated.

genetics↗

bioRxiv2026

Global identification of neuronal and astrocytic integral membrane proteins that require Retromer for their endosomal recycling.

Efficient transport of membrane proteins through the endosomal network is essential for brain development and function, with perturbation implicated in disease. Deficiencies in Retromer, a key regulator of endosomal transport, have been linked to aging-related neurodegenerative disorders including Alzheimers and Parkinsons disease. To better define the neuroprotective role of Retromer, we have applied cell surface restricted proteomics to identify those integral membrane proteins whose recycling to the plasma membrane is mediated by Retromer and associated cargo adaptors, sorting nexin 3 (SNX3), its paralogue sorting nexin 12 (SNX12), and sorting nexin 27 (SNX27) (data available via ProteomeXchange: PXD078277). By comparing primary rat cortical neurons and astrocytes we have identified several cargoes that require either SNX3/SNX12- or SNX27-Retromer complexes for endosomal recycling, including proteins involved in synapse organisation, synaptic signalling and Alzheimers disease pathology. We highlight that perturbed Retromer function leads to endosomal enlargement, and we establish a key role of SNX27-Retromer in modulating transport of glutamate across both neuronal and astrocytic membranes via recycling of glutamate transporters EAAT3 (SLC1A1) and EAAT1 (SLC1A3) respectively. Our study provides further mechanistic insight into the consequences of Retromer deficiency for neuronal and astrocytic function, offering new avenues of research in the treatment of neurodegenerative disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/724903v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1225eedorg.highwire.dtl.DTLVardef@1863f1dorg.highwire.dtl.DTLVardef@99c21eorg.highwire.dtl.DTLVardef@976bfd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Suppression of Retromer and the sorting nexins (SNX27, SNX3/SNX12) leads to a significant change in the surface proteome of rat cortical neurons and astrocytes. Focusing on the glutamate transporters, SLC1A1 and SLC1A3, we have validated that SNX27-Retromer is required for their trafficking, with SNX27-Retromer suppression in astrocytes leading to a loss of glutamate uptake. C_FIG

molecular biology↗

bioRxiv2026

Differential tolerance for SEA domain misfolding encodes a MAPK pathway-specific response

Signaling pathways often share components yet produce highly specialized biological responses. How signaling specificity is achieved between pathways utilizing common components is a fundamental question. In budding yeast, the same transmembrane mucin, Msb2, regulates two Mitogen-Activated Protein Kinase (MAPK) pathways controlling filamentous growth (fMAPK) and the response to osmotic stress (HOG). How this shared sensor distinguishes between stimuli and regulates different pathways is not clear. Using structure-guided analysis, we identified a conserved SEA (Sea urchin sperm protein, Enterokinase, Agrin) domain in fungal mucins and found that mutations disrupting protein folding selectively impair one pathway (fMAPK) but were tolerated by another (HOG). Mechanistically, these differences revealed distinct modes of signal transmission. The fMAPK pathway required an intact SEA domain and the cytosolic tail, consistent with a cis signaling mechanism that required structural coupling across the membrane. In contrast, the HOG pathway functioned independently of the cytosolic tail and tolerated misfolded SEA domain variants, consistent with trans signaling mediated by extracellular domains of interacting partners. The HOG pathway may detect misfolding as part of its sensing mechanism, as stressors that induce protein misfolding required Msb2 for survival. This work reveals how differential tolerance to protein deformation confers signaling specificity and identifies sensor deformation as a general feature of mechanosensory pathways that respond to environmental stress. HIGHLIGHTSO_LISignaling pathways differ in tolerance to misfolding of a sensory domain C_LIO_LIMisfolded SEA domains retain function in a stress pathway (HOG) pathway but not a cell differentiation pathway (fMAPK) O_LIMisfolded SEA domain variants showed altered protein levels, mis-localization in the secretory pathway, and turnover by ERAD C_LIO_LINon-functional variants lacked residues that stabilize the structure through intramolecular bonds C_LI C_LIO_LIDifferential tolerance for misfolding revealed distinct modes of signaling O_LITrans signaling predominated in the HOG pathway and did not require proper SEA domain folding or the mucin cytosolic tail O_LIA dominant hyperactive variant next to the SEA domain revealed basal interactions with the CR domain of tetraspanin C_LIO_LIAlphaFold modeling showed distinct interactions occur between the SEA domain and tetraspanin in the basal and activated states C_LI C_LIO_LICis signaling predominated in the fMAPK pathway O_LIRequired a properly folded SEA domain and conformational coupling to the cytosolic tail C_LIO_LIYapsin processing was required for SEA domain activation and turnover of the mucin cytosolic tail C_LI C_LI C_LIO_LIHOG pathway may sense protein misfolding as part of its activation mechanism. C_LIO_LISEA domains are conserved throughout fungal mucins and mammalian glycoprotein sensors suggesting a generalizable mechanism C_LIO_LIProtein deformation may provide information to survival pathways about environmental stress. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/723240v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1cd30f3org.highwire.dtl.DTLVardef@48c96corg.highwire.dtl.DTLVardef@9fffc2org.highwire.dtl.DTLVardef@504b1d_HPS_FORMAT_FIGEXP M_FIG C_FIG Signaling pathways often share components yet activate different effector processes through mechanisms that remain unclear. The same mucin regulates two MAPK pathways (red and green), and the discovery of a conserved SEA domain provided insights into specificity mechanisms. In the fMAPK pathway that regulates filamentous growth, the mucin works in a classical manner, where an external signal (in this case underglycosylation by glucose limitation) transduces a signal to the cytosolic domain in cis. By comparison, the HOG pathway that responds to osmotic stress displayed a remarkable tolerance for mucin and SEA domain deformation. Protein variants that caused SEA domain misfolding, mislocalization, and degradation by ERAD retained function in the HOG pathway. Truncations that removed the cytosolic tail and transmembrane anchor were also functional. These phenotypes support a trans activation mechanism with external partners that was preferential for activation of the HOG pathway. SEA domain deformation may be induced by environmental stress as a trigger for the HOG pathway. Cells may detect misfolding of protein domains to gain information about environmental stress.

cell biology↗

bioRxiv2025

Identification of a VPS29 isoform with restricted association to Retriever and Retromer accessory proteins through auto-inhibition.

The endosomal-lysosomal network is a hub of organelles that orchestrate the dynamic sorting of hundreds of integral membrane proteins to maintain cellular homeostasis. VPS29 is a central conductor of this network through its assembly into Retromer, Retriever and Commander endosomal sorting complexes, and its role in regulating RAB GTPase activity. Two VPS29 isoforms have been described, VPS29A and VPS29B, that differ solely in their amino-terminal sequences. Here we identify a third VPS29 isoform, which we term VPS29C, that harbours an extended amino-terminal sequence compared to VPS29A and VPS29B. Through a combination of AlphaFold predictive modelling, in vitro complex reconstitution, mass spectrometry and molecular cell biology, we find that the amino-terminal VPS29C extension constitutes an autoinhibitory sequence that limits access to a hydrophobic groove necessary for effector protein recruitment to Retromer, and association with Retriever and Commander. VPS29C is therefore unique in its ability to uncouple Retromer-dependent cargo sorting from the broader roles of VPS29A and VPS29B in regulating the endosomal-lysosomal network through accessory protein recruitment. Our identification and characterisation of VPS29C points to additional complexity in the differential subunit assembly of Retromer, an important consideration given the increasing interest in Retromer as a potential therapeutic target in neurodegenerative diseases. SIGNIFICANCE STATEMENTThe endosomal-lysosomal network is essential for normal cellular function with network defects being associated with numerous neurodegenerative diseases. Two heterotrimeric complexes, Retromer and Retriever, control transmembrane protein recycling through the network. Of these, reduced Retromer expression is observed in Alzheimers disease and Retromer mutations lead to familial Parkinsons disease. Here, we identify and characterise a new isoform of VPS29, a subunit shared between Retromer and Retriever. We reveal how this isoform, VPS29C, adopts an auto-inhibitory conformation to limit its association into Retriever and restrict the binding of VPS29C-containing Retromer to accessory proteins vital for regulating network function. By revealing added complexity in Retromer assembly and function, we provide new insight into Retromers potential as a therapeutic target in neurodegenerative diseases.

cell biology↗

bioRxiv2025

Identification of a RAB32-LRMDA-Commander membrane trafficking complex reveals the molecular mechanism of human oculocutaneous albinism type 7

Commander is an endosome associated sixteen protein assembly that associates with the sorting nexin 17 (SNX17) cargo adaptor to regulate cell surface recycling of internalised integral membrane proteins including integrins and lipoprotein receptors. Mutations in Commander are causative for Ritscher-Schinzel syndrome (RSS), a multiorgan developmental disease associated with a core triad of cerebellar-cardiac-craniofacial malformation. Here, using unbiased proteomics and computational modelling, we identify leucine rich melanocyte differentiation associated (LRMDA) as a novel Commander binding protein. Using recombinant protein reconstitution, we show that LRMDA simultaneously associates with Commander and active RAB32, and, by revealing that LRMDA and SNX17 share a common mechanism of Commander association, establish the mutually exclusive nature of RAB32-LRMDA-Commander and SNX17-Commander assemblies. From functional analysis in human melanocytes, we establish an essential role for RAB32-LRMDA-Commander in melanosome biogenesis and pigmentation and reveal a distinct functional role for SNX17-Commander in this organelle biogenesis pathway. We reveal how LRMDA mutations, causative for oculocutaneous albinism type 7 (OCA7), a hypopigmentation disorder accompanied by poor visual acuity, uncouple RAB32 and Commander binding thereby establishing the mechanistic basis of this disease. Our discovery and characterisation of this alternative Commander assembly establishes an unrecognised plasticity of Commander function within a highly complex organelle biogenesis pathway. This extends Commander function beyond the confines of SNX17-mediated cell surface recycling into RAB32-family mediated biogenesis of lysosome-related organelles and, potentially, other RAB32 regulated pathways including host-pathogen defence mechanisms. Our work also extends the breath of Commander pathway dysfunction for human disease.

cell biology↗

bioRxiv2024

Mapping the endosomal proximity proteome reveals Retromer as a hub for RAB GTPase regulation.

Endosomal retrieval and recycling of integral cargo proteins is essential for cell, tissue and organism-level development and homeostasis and is orchestrated through a specialised retrieval sub-domain on the endosomal vacuole. However, although sub-domain dysfunction is associated with human disease our appreciation of the molecular details and functional components of the retrieval sub-domain(s) remains poorly described. Here, using comparative proximity proteomics of critical retrieval sub-domain components Retromer and Retriever, their cargo adaptors, and a component of the opposing ESCRT-degradative sub-domain, we provide a data-rich resource that identifies new molecular details of retrieval sub-domain composition and organization, including an unrecognised complexity in the interface of Retromer with RAB GTPases. Combining X-ray crystallography and in silico predictions with extensive biochemical and cellular analysis, we dissect the direct association of Retromer with RAB10 regulators DENND4A, DENND4C, TBC1D1, and TBC1D4, and the RAB35 regulator TBC1D13. Overall, we conclude that the Retromer retrieval sub-domain constitutes a major hub for the regulated switching of selected RAB GTPases and propose that this constitutes a major component of the role of Retromer in neuroprotection.

cell biology↗

bioRxiv2024

Antimicrobial mechanism of in-situ plasma activated water treatment of pathogenic Escherichia coli and Staphylococcus aureus biofilms

AimsThis study investigated the efficacy and mechanisms of inactivation of against Escherichia coli UTI89 and Staphylococcus aureus NCTC8325 through an in-situ plasma-activated water (PAW) treatment. Methods and ResultsPAW was prepared by discharging atmospheric pressure cold plasma beneath the surface of sterile distilled water. The study investigated the inactivation of biofilm cells and biofilm matrix. A complete killing of biofilm cells was achieved on both of E. coli (6.76 {+/-} 0.01 log CFU/mL) and S. aureus (6.82 {+/-} 0.02 log CFU/mL). This process happened earlier in S. aureus. Simultaneously, PAW treatment disrupted the biofilm structure, inducing a significant reduction in general biofilm biomass and extracellular polymer substances (EPS) matrix. With the disruption of EPS, PAW was enabled to further interact with the bacterial membrane, causing a significant increase in membrane permeability and disrupted membrane structure. Finally, PAW treatment led to a significant accumulation of intracellular reactive oxygen and nitrogen species within the biofilm cells. ConclusionsCollectively, these findings indicate that PAW effectively inactivates biofilms by mechanically targeting the biofilm EPS matrix and biofilm cells in both gram-negative and gram-positive bacteria. Impact statementThis study contributes novel insights into plasma-activated waters mechanisms of action, particularly its impact on the biofilm extracellular polymeric substances matrix (exopolysaccharides, extracellular DNA, and protein), cell membrane permeability, depolarization, and intracellular ROS and RNS accumulation in both of Gram-positive and Gram-negative species. These findings highlight PAW-based treatments against biofilm-related challenges in antimicrobial development and water system decontamination.

microbiology↗

bioRxiv2024

Transcriptional Signatures Associated with the Survival of Escherichia coli Biofilm During Treatment with Plasma-Activated Water

Biofilm formation on surfaces, tools and equipment can damage their quality and lead to high repair or replacement costs. Plasma-activated water (PAW), a new technology, has shown promise in killing biofilm and non-biofilm bacteria due to its mix of reactive oxygen and nitrogen species (RONS), and in particular superoxide. However, the specific genetic mechanisms behind PAWs effectiveness, especially against biofilms, are not yet fully understood. Here, we examined the stress responses of Escherichia coli biofilms when exposed to sub-lethal PAW treatment with and without superoxide (by adding the scavenger Tiron to remove it). A 40% variation in gene expression was observed for PAW treated biofilms when compared to PAW-Tiron and controls. Specifically, PAW treatment resulted in 478 upregulated genes (> 1.5 log2FC) and 186 downregulated genes (< -1.5 log2FC) compared to the control. Pathway enrichment and biological process enrichment analysis revealed significant upregulation of sulfur metabolism, ATP-binding cassette transporter genes, amino acid metabolic/biosynthesis pathways, hypochlorite response systems and oxidative phosphorylation for biofilms treated with PAW compared to control. Knockout mutants of significantly upregulated genes associated with these pathways trxC (4.23-fold), cysP (1.58-fold) and nuoM (1.74-fold) were compared to the wild-type (WT) for their biofilm viability and intracellular RONS accumulation. Relative to PAW-treated WT,{Delta} trxC and{Delta} nuoM knockout mutants displayed significantly reduced biofilm viability (P [≤] 0.05) confirming their role in PAW-mediated response. Interestingly,{Delta} trxC biofilms had the highest intracellular ROS accumulation, as revealed by DCFDA staining after PAW treatment. This study gives a detailed insight into how E. coli biofilms respond to oxidative stress induced by PAW. It highlights the significance of superoxide in PAWs bactericidal effects. Overall, our findings shed light on the specific genes and pathways that help E. coli biofilms survive and respond to PAW treatment, offering a new understanding of plasma technology and its anti-biofilm mechanisms.

microbiology↗

bioRxiv2024

Mechanism and regulation of cargo entry into the Commander recycling pathway

Commander is a multiprotein complex that orchestrates endosomal recycling of diverse integral cargo proteins and in humans is required for normal skeletal, brain, kidney, and cardiovascular development. While the structure of this complex has recently been described, the central question of how cargo proteins are selected for entry into the Commander recycling pathway remains unclear. Here using recombinant protein reconstitution and in silico predictions we identify the evolutionary conserved mechanism through which the unstructured carboxy-terminal tail of the integral protein adaptor sorting nexin-17 (SNX17) directly binds to the Retriever sub-complex of Commander. SNX17 adopts an autoinhibited conformation where its carboxy-terminal tail occupies the cargo binding groove. Competitive cargo binding overcomes this autoinhibition, promoting SNX17 endosomal residency and the release of the carboxy tail for Retriever association. Using molecular cell biology and high-resolution microscopy, we establish the central importance of SNX17-Retriever association in the handover of integrin and lipoprotein receptor cargoes into pre-existing endosomal retrieval sub-domains for entry into the recycling pathway. In describing the principal mechanism of cargo entry into the Commander recycling pathway we provide key insight into the function and regulation of this evolutionary conserved sorting complex.

cell biology↗

bioRxiv2023

Modulators of MAPK pathway activity during filamentous growth in Saccharomyces cerevisiae

Mitogen-activated protein kinase (MAPK) pathways control the response to intrinsic and extrinsic stimuli. In the budding yeast Saccharomyces cerevisiae, cells undergo filamentous growth, which is regulated by the fMAPK pathway. To better understand the regulation of the fMAPK pathway, a genetic screen was performed to identify spontaneous mutants with elevated activity of an fMAPK-pathway dependent growth reporter (ste4 FUS1-HIS3). In total, 159 mutants were isolated and analyzed by secondary screens for invasive growth by the plate-washing assay, and filament formation by microscopy. Thirty-two mutants were selected for whole-genome sequencing, which identified new alleles in genes encoding known regulators of the fMAPK pathway. These included gain-of-function alleles in STE11, which encodes the MAPKKK, as well as loss-of-function alleles in KSS1, which encodes the MAP kinase, and RGA1, which encodes a GTPase activating protein (GAP) for CDC42. New alleles in previously identified pathway modulators were also uncovered in ALY1, AIM44, RCK2, IRA2, REG1 and in genes that regulate protein folding (KAR2), glycosylation (MNN4), and turnover (BLM10). C-terminal truncations in the transcription factor Ste12p were also uncovered that resulted in elevated reporter activity, presumably identifying an inhibitory domain in the C-terminus of the protein. We also show that a wide variety of filamentous growth phenotypes result from mutations in different regulators of the response. The alleles identified here expand the connections surrounding MAPK pathway regulation and reveal new features of proteins that function in the signaling cascade. ARTICLE SUMMARYSignaling pathways control the response to stimuli. In yeast, a signaling (MAPK) pathway controls a fungal behavioral response called filamentous growth. A genetic screen was performed to identify spontaneous mutants that show hyperactivity of a MAPK pathway-dependent reporter. Select mutants were analyzed by whole-genome sequencing. New alleles in known regulatory proteins were identified. A potential inhibitory domain in the C-terminus of the transcription factor Ste12p was also uncovered. Our results indicate that filamentous growth is determined by the combinatorial effects of multiple positive and negative regulatory inputs.

genetics↗

bioRxiv2023

Structural basis for coupling of the WASH subunit FAM21 with the endosomal SNX27-Retromer complex

Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the WASH complex which nucleates actin filaments establishing the endosomal reycling domain. Crystal structures, modelling, biochemical and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling, however it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains. Significance StatementCell surface transmembrane proteins are regulated by a constant cycle of internalization and recycling from intracellular endosomal compartments. The Retromer protein complex and the sorting nexin adaptor protein play a critical role in the retrieval of hundreds of proteins responsible for ion transport, glucose metabolism, neurotransmission, and other cell functions. We have defined the mechanism by which both Retromer and SNX27 engage the actin-nucleating complex called WASH through multiple repeated sequences in the subunit FAM21. Dysfunction in WASH, Retromer and SNX27 are implicated in various disorders including Parkinsons disease, Alzheimers disease, hereditary spastic paraplegia, and this work provides important insights into the assembly of these essential endosomal sorting machineries.

cell biology↗

bioRxiv2023

Plasma Activated Water as a Pre-Treatment Strategy in the Context of Biofilm-Infected Chronic Wounds

Healing and treatment of chronic wounds are often complicated due to biofilm formation by pathogens. Here, the efficacy of Plasma Activated Water (PAW) as a pre-treatment strategy has been investigated prior to the application of topical antiseptics polyhexamethylene biguanide, povidone iodine, and MediHoney, which are routinely used to treat chronic wounds. The efficacy of this treatment strategy was determined against biofilms of Escherichia coli formed on a plastic substratum and on a human keratinocyte monolayer substratum used as an in vitro biofilm-skin epithelial cell model. PAW pre-treatment greatly increased the killing efficacy of all the three antiseptics to eradicate the E. coli biofilms formed on the plastic and keratinocyte substrates. However, the efficacy of the combined PAW-antiseptic treatment and single treatments using PAW or antiseptic alone was lower for biofilms formed in the in vitro biofilm-skin epithelial cell model compared to the plastic substratum. Scavenging assays demonstrated that reactive species present within the PAW were largely responsible for its anti-biofilm activity. PAW treatment resulted in significant intracellular RONS accumulation within the E. coli biofilms, while also rapidly acting on the microbial membrane leading to outer membrane permeabilisation and depolarisation. Together, these factors contribute to significant cell death, potentiating the antibacterial effect of the assessed antiseptics.

microbiology↗

bioRxiv2023

Five inhibitory receptors display distinct vesicular distributions in T cells

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3 and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and a biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.

immunology↗

bioRxiv2023

Ecological Inducers of the Yeast Filamentous Growth Pathway Reveal Environment-Dependent Roles for Pathway Components

Signaling modules, such as MAPK pathways, are evolutionarily conserved drivers of cell differentiation and stress responses. In many fungal species including pathogens, MAPK pathways control filamentous growth, where cells differentiate into an elongated cell type. The convenient model budding yeast Saccharomyces cerevisiae undergoes filamentous growth by the filamentous growth (fMAPK) pathway; however, the inducers of the pathway remain unclear, perhaps because pathway activity has been mainly studied in laboratory conditions. To address this knowledge gap, an ecological framework was employed, which uncovered new fMAPK pathway inducers, including pectin, a material found in plants, and the metabolic byproduct ethanol. We also show that induction by a known inducer of the pathway, the non-preferred carbon source galactose, required galactose metabolism and induced the pathway differently than glucose limitation or other non-preferred carbon sources. By exploring fMAPK pathway function in fruit, we found induction of the pathway led to visible digestion of fruit rind through a known target, PGU1, which encodes a pectolytic enzyme. Different stimuli revealed different modes of pathway signaling. For example, combinations of inducers (galactose and ethanol) stimulated the pathway to near maximal levels, which showed dispensability of several fMAPK pathway components (e.g. mucin sensor, PAK), but not others (e.g. adaptor, MAPKKK) and required the Ras2-PKA pathway. This included a difference between the transcription factor binding partners for the pathway, as Tec1p, but not Ste12p, was partly dispensable for fMAPK pathway activity. Thus, by exploring ecologically-relevant stimuli, new modes of MAPK pathway signaling were uncovered, perhaps revealing how a pathway can respond differently to specific environments. Data Availability StatementAll data are in the manuscript and/or supporting information files.

microbiology↗

bioRxiv2023

New Features Surrounding the Cdc42-Ste20 Module that Regulates MAP Kinase Signaling in Yeast

Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular responses, including the response to stress and cell differentiation, and are highly conserved across eukaryotes from yeast to humans. In yeast, the canonical activation of several MAPK pathways includes the interaction of the small GTPase Cdc42p with the p21-activated kinase (PAK) Ste20p. We recently found that the active conformation of Cdc42p is regulated by turnover, which impacts the activity of the pathway that regulates filamentous growth (fMAPK). Here, we show that Ste20p is turned over by the 26S proteasome. Ste20p was stabilized when bound to Cdc42p, presumably to sustain MAPK pathway signaling. Ste20p is a major conduit by which signals flow through the fMAPK pathway; however, by genetic approaches we also identified a Ste20p-independent branch of the fMAPK pathway. Ste20p-dependent signaling required the 14-3-3 proteins, Bmh1p and Bmh2p, while Ste20p-independent signaling required the fMAPK pathway adaptor and Cdc42p-interacting protein, Bem4p. Ste20p-independent signaling was inhibited by one of the GTPase-activating proteins for Cdc42p in the fMAPK pathway, Rga1p, which also dampened basal but not active fMAPK pathway activity. Finally, the polarity adaptor and Cdc42p-interacting protein, Bem1p, which also regulates the fMAPK pathway, interacts with the tetra-span protein Sho1p, connecting a sensor at the plasma membrane to a protein that regulates the GTPase module. Collectively, these data reveal new regulatory features surrounding a Rho-PAK module that may extend to other pathways that control cell differentiation.

biochemistry↗

bioRxiv2023

An effective sanitizer for fresh produce production: In situ plasma activated water treatment inactivates pathogenic bacteria and maintains the quality of cucurbit fruit

The effect of plasma activated water (PAW) generated with a dielectric barrier discharge diffusor (DBDD) system on microbial load and organoleptic quality of cucamelons was investigated and compared to the established sanitizer, sodium hypochlorite (NaOCl). Pathogenic serotypes of Escherichia coli, Salmonella enterica, and Listeria monocytogenes were inoculated onto the surface of cucamelons (6.5 log CFU g-1) and into the wash water (6 log CFU mL-1). PAW treatment involved 2 minutes in situ with water activated at 1500 Hz and 120 V, and air as the feed gas; NaOCl treatment was a wash with 100 ppm total chlorine; and the control treatment was a wash with tap water. PAW treatment produced a 3 log CFU g-1 reduction of pathogens on the cucamelon surface without negatively impacting quality or shelf life. NaOCl treatment reduced the pathogenic bacteria on the cucamelon surface by 3-4 log CFU g-1, however, this treatment also reduced fruit shelf life and quality. Both systems reduced 6 log CFU ml-1 pathogens in the wash water to below detectable limits. The critical role of superoxide anion radical ({middle dot}O2-) in the antimicrobial power of DBDD-PAW was demonstrated through a scavenger assay, and chemistry modelling confirmed that {middle dot}O2- generation readily occurs in DBDD-PAW generated with the employed settings. Modelling of the physical forces produced during plasma treatment showed that bacteria likely experience strong local electric fields and polarization. We hypothesize that these physical effects synergise with reactive chemical species to produce the acute antimicrobial activity seen with the in situ PAW system. ImportancePlasma activated water (PAW) is an emerging sanitizer in the fresh food industry, where food safety must be achieved without a thermal kill step. Here we demonstrate PAW generated in situ to be a competitive sanitizer technology, providing a significant reduction of pathogenic and spoilage micro-organisms while maintaining the quality and shelf life of the produce item. Our experimental results are supported by modelling of the plasma chemistry and applied physical forces, which show that the system can generate highly reactive superoxide radicals and strong electric fields that combine to produce potent antimicrobial power. In situ PAW has promise in industrial applications as it only requires low power (12 W), tap water and air. Moreover, it does not produce toxic by-products or hazardous effluent waste, making it a sustainable solution for fresh food safety.

microbiology↗

bioRxiv2022

CHC22 clathrin membrane recruitment uses SNX5 in bipartite interaction with secretory tether p115

The two clathrin isoforms, CHC17 and CHC22, mediate separate intracellular transport routes. CHC17 performs endocytosis and housekeeping membrane traffic in all cells. CHC22, expressed most highly in skeletal muscle, transports the glucose transporter GLUT4 from the endoplasmic-reticulum-to-Golgi intermediate compartment (ERGIC) directly to an intracellular GLUT4 storage compartment (GSC) from where GLUT4 can be mobilized to the plasma membrane by insulin. Here, the molecular determinants distinguishing CHC22 from CHC17 trafficking are defined. The C-terminal trimerization domain of CHC22 binds SNX5, which also binds the ERGIC tether p115. SNX5, and the functionally redundant SNX6, are required for CHC22 localization independently of their participation in the endosomal ESCPE-1 complex. In tandem, an isoform-specific patch in the CHC22 N-terminal domain separately mediates binding to p115. This dual mode of clathrin recruitment, involving interactions at both heavy chain termini, is required for CHC22 targeting to ERGIC membranes to mediate the Golgi bypass route for GLUT4 traffic. Interference with either interaction inhibits GLUT4 targeting to the GSC, defining a bipartite mechanism regulating a key pathway in human glucose metabolism.

cell biology↗