AP-1 contributes to endosomal targeting of the ubiquitin ligase RNF13 via a secondary and novel non-canonical binding motif.

Cellular trafficking between organelles is typically assured by short motifs that contact carrier proteins to transport them to their destination. The ubiquitin E3 ligase RING finger protein 13 (RNF13), a regulator of proliferation, apoptosis and protein trafficking, localizes to endolysosomal compartments through the binding of a dileucine motif to clathrin adaptor protein complex AP-3. Mutations within this motif reduce the ability of RNF13 to interact with AP-3. Here, our study shows the discovery of a glutamine-based motif that resembles a tyrosine-based motif within the C-terminal region of RNF13 that binds to the clathrin adaptor protein complex AP-1, notably without a functional interaction with AP-3. Using biochemical, molecular and cellular approaches in HeLa cells, our study demonstrates that a RNF13 dileucine variant uses an AP-1-dependent pathway to be exported from the Golgi towards the endosomal compartment. Overall, this study provides mechanistic insights into the alternate route used by this variant of the dileucine sorting motif of RNF13.

Optogenetic control of kinesin-1, -2, -3 and dynein reveals their specific roles in vesicular transport.

Each cargo in a cell employs a unique set of motor proteins for its transport. To dissect the roles of each type of motor, we developed optogenetic inhibitors of endogenous kinesin-1, -2, -3 and dynein motors and examined their effect on the transport of early endosomes, late endosomes, and lysosomes. While kinesin-1, -3, and dynein transport vesicles at all stages of endocytosis, kinesin-2 primarily drives late endosomes and lysosomes. Transient optogenetic inhibition of kinesin-1 or dynein causes both early and late endosomes to move more processively by relieving competition with opposing motors. Kinesin-2 and -3 support long-range transport, and optogenetic inhibition reduces the distances that their cargoes move. These results suggest that the directionality of transport is controlled through regulating kinesin-1 and dynein activity. On vesicles transported by several kinesin and dynein motors, modulating the activity of a single type of motor on the cargo is sufficient to direct motility.

Effect of size and pH-sensitivity of liposomes on cellular uptake pathways and pharmacokinetics of encapsulated gemcitabine.

To enhance cytoplasmic delivery efficiency, pH-sensitive liposomes (PSL) have been proposed as a novel strategy. To facilitate clinical translation, this study aims to understand the impact of both size and pH-sensitivity on cellular uptake pathways, intracellular trafficking and pharmacokinetics of liposomes. The large liposomes (130-160 nm) were prepared using thin-film hydration method, while small liposomes (∼60 nm) were fabricated using microfluidics, for both PSL and non-pH-sensitive liposomes (NPSL). Cellular uptake pathways and intracellular trafficking was investigated through confocal imaging with aid of various endocytosis inhibitors. Intracellular gemcitabine delivery by various liposomal formulations was quantified using HPLC, and the cytotoxicity was assessed via cell viability assays. Pharmacokinetics of gemcitabine loaded in various liposomes was evaluated in rats following intravenous administration. Larger liposomes had a higher loading capacity for hydrophilic gemcitabine (7% vs 4%). Small PSL exhibited superior cellular uptake compared to large PSL or NPSLs. Moreover, the alkalization of endosomes significantly attenuated the cellular uptake of PSL. Large liposomes (PSL and NPSL) predominantly entered cells via clathrin-dependent pathway, whereas small liposomes partially utilized caveolae-dependent pathway. However, the long circulation of the liposomes, as measured by the encapsulated gemcitabine, was compromised by both pH-sensitivity and size reduction (9.5 h vs 5.3 h). Despite this drawback, our results indicate that small PSL holds promise as vectors for the next generation of liposomal nanomedicine, owing to their superior cytoplasmic delivery efficiency.

Large liposomes had higher loading capacity for hydrophilic gemcitabine.Reduction of liposome size enhanced drug release from pH-sensitive liposomes.The internalization efficiency of liposomes was enhanced by pH-sensitivity and size reduction.Larger liposomes (>130 nm) enter cells primarily via clathrin-dependent endocytosis, while smaller liposomes (∼60 nm) partially through caveolae-mediated pathway, regardless of the pH-sensitivity.The intracellular payload release from pH-sensitive liposomes was decreased by endosome alkalization using chloroquine.Long circulation of the encapsulated gemcitabine was compromised by the pH-sensitivity and size reduction.

A review on dendrimer-based nanoconjugates and their intracellular trafficking in cancer photodynamic therapy.

Nanotechnology-based cancer treatment has received considerable attention, and these treatments generally use drug-loaded nanoparticles (NPs) to target and destroy cancer cells. Nanotechnology combined with photodynamic therapy (PDT) has demonstrated positive outcomes in cancer therapy. Combining nanotechnology and PDT is effective in targeting metastatic cancer cells. Nanotechnology can also increase the effectiveness of PDT by targeting cells at a molecular level. Dendrimer-based nanoconjugates (DBNs) are highly stable and biocompatible, making them suitable for drug delivery applications. Moreover, the hyperbranched structures in DBNs have the capacity to load hydrophobic compounds, such as photosensitizers (PSs) and chemotherapy drugs, and deliver them efficiently to tumour cells. This review primarily focuses on DBNs and their potential applications in cancer treatment. We discuss the chemical design, mechanism of action, and targeting efficiency of DBNs in tumour metastasis, intracellular trafficking in cancer treatment, and DBNs' biocompatibility, biodegradability and clearance properties. Overall, this study will provide the most recent insights into the application of DBNs and PDT in cancer therapy.

DBNs' intracellular journey in cancer-PDT refines targeted therapy, boosting efficacy.DBN in PDT for tumour metastasis: targeting and drug release mechanisms.DBNs' biocompatibility, biodegradability and clearance were explored thoroughly.

Unignored intracellular journey and biomedical applications of extracellular vesicles.

The intracellular journey of extracellular vesicles (EVs) cannot be ignored in various biological pathological processes. In this review, the biogenesis, biological functions, uptake pathways, intracellular trafficking routes, and biomedical applications of EVs were highlighted. Endosomal escape is a unique mode of EVs release. When vesicles escape from endosomes, they avoid the fate of fusing with lysosomes and being degraded, thus having the opportunity to directly enter the cytoplasm or other organelles. This escape mechanism is crucial for EVs to deliver specific signals or substances. The intracellular trafficking of EVs after endosomal escape is a complex and significant biological process that involves the coordinated work of various cellular structures and molecules. Through the in-depth study of this process, the function and regulatory mechanism of EVs are fully understood, providing new dimensions for future biomedical diagnosis and treatment.

Trafficking through the blood-brain barrier is directed by core and outer surface components of layer-by-layer nanoparticles.

Drug-carrying nanoparticles are a promising strategy to deliver therapeutics into the brain, but their translation requires better characterization of interactions between nanomaterials and endothelial cells of the blood-brain barrier (BBB). Here, we use a library of 18 layer-by-layer electrostatically assembled nanoparticles (NPs) to independently assess the impact of NP core and surface materials on in vitro uptake, transport, and intracellular trafficking in brain endothelial cells. We demonstrate that NP core stiffness determines the magnitude of transport, while surface chemistry directs intracellular trafficking. Finally, we demonstrate that these factors similarly dictate in vivo BBB transport using intravital imaging through cranial windows in mice. We identify that hyaluronic acid surface chemistry increases transport across the BBB in vivo, and flow conditions are necessary to replicate this finding in vitro. Taken together, these findings highlight the importance of assay geometry, cell biology, and fluid flow in developing nanocarriers for delivery to the brain.

Making Rift Valley Fever Viral Particles Fluorescent.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that represents a significant threat to both human and veterinary public health. Since its discovery in the Great Rift Valley of Kenya in the 1930s, the virus has spread across Africa and beyond, now posing a risk of introduction into Southern Europe and Asia. Despite recent progresses, early RVFV-host cell interactions remain largely uncharacterized. In this method chapter, we delineate the procedure for labeling RVFV particles with fluorescent organic dyes. This approach makes it feasible to visualize single viral particles in both fixed and living cells and study RVFV entry into host cells. We provide additional examples with two viruses closely related to RVFV, namely, Toscana virus and Uukuniemi virus. Furthermore, we illustrate how to utilize fluorescent viral particles to examine and quantify each step of the cell entry program of RVFV, which includes state-of-the-art fluorescence-based detection techniques such as fluorescence microscopy, flow cytometry, and fluorimetry.

GGA1 interacts with the endosomal Na+/H+ exchanger NHE6 governing localization to the endosome compartment.

Mutations in the endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome, an X-linked neurological disorder. NHE6 functions in regulation of endosome acidification and maturation in neurons. Using yeast two-hybrid screening with the NHE6 carboxyl terminus as bait, we identify Golgi-associated, gamma adaptin ear-containing, ADP-ribosylation factor (ARF) binding protein 1 (GGA1) as an interacting partner for NHE6. We corroborated the NHE6-GGA1 interaction using: coimmunoprecipitation; overexpressed constructs in mammalian cells; and coimmunoprecipitation of endogenously expressed GGA1 and NHE6 from neuroblastoma cells, as well as from the mouse brain. We demonstrate that GGA1 interacts with organellar NHEs (NHE6, NHE7, and NHE9) and that there is significantly less interaction with cell-surface localized NHEs (NHE1 and NHE5). By constructing hybrid NHE1/NHE6 exchangers, we demonstrate the cytoplasmic tail of NHE6 interacts most strongly with GGA1. We demonstrate the colocalization of NHE6 and GGA1 in cultured, primary hippocampal neurons, using super-resolution microscopy. We test the hypothesis that the interaction of NHE6 and GGA1 functions in the localization of NHE6 to the endosome compartment. Using subcellular fractionation experiments, we show that NHE6 is mislocalized in GGA1 KO cells, wherein we find less NHE6 in endosomes, but more NHE6 transport to lysosomes, and more Golgi retention of NHE6, with increased exocytosis to the surface plasma membrane. Consistent with NHE6 mislocalization, and Golgi retention, we find the intraluminal pH in Golgi to be alkalinized in GGA1-null cells. Our study demonstrates a new interaction between NHE6 and GGA1 which functions in the localization of this intracellular NHE to the endosome compartment.

The expanding clinical and genetic spectrum of DYNC1H1-related disorders.

Intracellular trafficking involves an intricate machinery of motor complexes including the dynein complex to shuttle cargo for autophagolysosomal degradation. Deficiency in dynein axonemal chains as well as cytoplasmic light and intermediate chains have been linked with ciliary dyskinesia and skeletal dysplasia. The cytoplasmic dynein 1 heavy chain protein (DYNC1H1) serves as a core complex for retrograde trafficking in neuronal axons. Dominant pathogenic variants in DYNC1H1 have been previously implicated in peripheral neuromuscular disorders (NMD) and neurodevelopmental disorders (NDD). As heavy-chain dynein is ubiquitously expressed, the apparent selectivity of heavy-chain dyneinopathy for motor neuronal phenotypes remains currently unaccounted for. Here, we aimed to evaluate the full DYNC1H1-related clinical, molecular and imaging spectrum, including multisystem features and novel phenotypes presenting throughout life. We identified 47 cases from 43 families with pathogenic heterozygous variants in DYNC1H1 (aged 0-59 years) and collected phenotypic data via a comprehensive standardized survey and clinical follow-up appointments. Most patients presented with divergent and previously unrecognized neurological and multisystem features, leading to significant delays in genetic testing and establishing the correct diagnosis. Neurological phenotypes include novel autonomic features, previously rarely described behavioral disorders, movement disorders, and periventricular lesions. Sensory neuropathy was identified in nine patients (median age of onset 10.6 years), of which five were only diagnosed after the second decade of life, and three had a progressive age-dependent sensory neuropathy. Novel multisystem features included primary immunodeficiency, bilateral sensorineural hearing loss, organ anomalies, and skeletal manifestations, resembling the phenotypic spectrum of other dyneinopathies. We also identified an age-dependent biphasic disease course with developmental regression in the first decade and, following a period of stability, neurodegenerative progression after the second decade of life. Of note, we observed several cases in whom neurodegeneration appeared to be prompted by intercurrent systemic infections with double-stranded DNA viruses (Herpesviridae) or single-stranded RNA viruses (Ross-River fever, SARS-CoV-2). Moreover, the disease course appeared to be exacerbated by viral infections regardless of age and/or severity of NDD manifestations, indicating a role of dynein in anti-viral immunity and neuronal health. In summary, our findings expand the clinical, imaging, and molecular spectrum of pathogenic DYNC1H1 variants beyond motor neuropathy disorders and suggest a life-long continuum and age-related progression due to deficient intracellular trafficking. This study will facilitate early diagnosis and improve counselling and health surveillance of affected patients.

StaufenC facilitates utilization of the ERAD pathway to transport dsRNA through the endoplasmic reticulum to the cytosol.

RNA interference (RNAi) is more efficient in coleopteran insects than other insects. StaufenC (StauC), a coleopteran-specific double-stranded RNA (dsRNA)-binding protein, is required for efficient RNAi in coleopterans. We investigated the function of StauC in the intracellular transport of dsRNA into the cytosol, where dsRNA is digested by Dicer enzymes and recruited by Argonauts to RNA-induced silencing complexes. Confocal microscopy and cellular organelle fractionation studies have shown that dsRNA is trafficked through the endoplasmic reticulum (ER) in coleopteran Colorado potato beetle (CPB) cells. StauC is localized to the ER in CPB cells, and StauC-knockdown caused the accumulation of dsRNA in the ER and a decrease in the cytosol, suggesting that StauC plays a key role in the intracellular transport of dsRNA through the ER. Using immunoprecipitation, we showed that StauC is required for dsRNA interaction with ER proteins in the ER-associated protein degradation (ERAD) pathway, and these interactions are required for RNAi in CPB cells. These results suggest that StauC works with the ERAD pathway to transport dsRNA through the ER to the cytosol. This information could be used to develop dsRNA delivery methods aimed at improving RNAi.

Pyrogallol intermediates elicit beta-amyloid secretion via radical formation and alterations in intracellular trafficking, distinct from pyrogallol-generated superoxide.

This study unveils a novel role of pyrogallol (PG), a recognized superoxide generator, in inducing beta-amyloid (Aß) secretion in an Alzheimer's disease (AD) cellular model. Contrary to expectations, the analysis of dihydroethidium fluorescence and UV-VIS spectrum scanning reveals that Aß secretion arises from PG reaction intermediates rather than superoxide or other by-products. Investigation into Aß secretion mechanisms identifies dynasore-dependent endocytosis and BFA-dependent exocytosis as independent pathways, regulated by tiron, tempol, and superoxide dismutase. Cell-type specificity is observed, with 293sw cells showing both pathways, while H4sw cells and primary astrocytes from an AD animal model exclusively exhibit the Aß exocytosis pathway. This exploration contributes to understanding PG's chemical reactions and provides insights into the interplay between environmental factors, free radicals, and AD, linking occupational PG exposure to AD risk as reported in the literature.

Modulating Endoplasmic Reticulum Chaperones and Mutant Protein Degradation in GABRG2(Q390X) Associated with Genetic Epilepsy with Febrile Seizures Plus and Dravet Syndrome.

A significant number of patients with genetic epilepsy do not obtain seizure freedom, despite developments in new antiseizure drugs, suggesting a need for novel therapeutic approaches. Many genetic epilepsies are associated with misfolded mutant proteins, including GABRG2(Q390X)-associated Dravet syndrome, which we have previously shown to result in intracellular accumulation of mutant GABAA receptor γ2(Q390X) subunit protein. Thus, a potentially promising therapeutic approach is modulation of proteostasis, such as increasing endoplasmic reticulum (ER)-associated degradation (ERAD). To that end, we have here identified an ERAD-associated E3 ubiquitin ligase, HRD1, among other ubiquitin ligases, as a strong modulator of wildtype and mutant γ2 subunit expression. Overexpressing HRD1 or knockdown of HRD1 dose-dependently reduced the γ2(Q390X) subunit. Additionally, we show that zonisamide (ZNS)-an antiseizure drug reported to upregulate HRD1-reduces seizures in the Gabrg2+/Q390X mouse. We propose that a possible mechanism for this effect is a partial rescue of surface trafficking of GABAA receptors, which are otherwise sequestered in the ER due to the dominant-negative effect of the γ2(Q390X) subunit. Furthermore, this partial rescue was not due to changes in ER chaperones BiP and calnexin, as total expression of these chaperones was unchanged in γ2(Q390X) models. Our results here suggest that leveraging the endogenous ERAD pathway may present a potential method to degrade neurotoxic mutant proteins like the γ2(Q390X) subunit. We also demonstrate a pharmacological means of regulating proteostasis, as ZNS alters protein trafficking, providing further support for the use of proteostasis regulators for the treatment of genetic epilepsies.

Functional Divergence of the Paralog Salmonella Effector Proteins SopD and SopD2 and Their Contributions to Infection.

Salmonella enterica is a leading cause of bacterial food-borne illness in humans and is responsible for millions of cases annually. A critical strategy for the survival of this pathogen is the translocation of bacterial virulence factors termed effectors into host cells, which primarily function via protein-protein interactions with host proteins. The Salmonella genome encodes several paralogous effectors believed to have arisen from duplication events throughout the course of evolution. These paralogs can share structural similarities and enzymatic activities but have also demonstrated divergence in host cell targets or interaction partners and contributions to the intracellular lifecycle of Salmonella. The paralog effectors SopD and SopD2 share 63% amino acid sequence similarity and extensive structural homology yet have demonstrated divergence in secretion kinetics, intracellular localization, host targets, and roles in infection. SopD and SopD2 target host Rab GTPases, which represent critical regulators of intracellular trafficking that mediate diverse cellular functions. While SopD and SopD2 both manipulate Rab function, these paralogs display differences in Rab specificity, and the effectors have also evolved multiple mechanisms of action for GTPase manipulation. Here, we highlight this intriguing pair of paralog effectors in the context of host-pathogen interactions and discuss how this research has presented valuable insights into effector evolution.

Pathogenic and Apathogenic Strains of Lymphocytic Choriomeningitis Virus Have Distinct Entry and Innate Immune Activation Pathways.

Lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV) share many genetic and biological features including subtle differences between pathogenic and apathogenic strains. Despite remarkable genetic similarity, the viscerotropic WE strain of LCMV causes a fatal LASV fever-like hepatitis in non-human primates (NHPs) while the mouse-adapted Armstrong (ARM) strain of LCMV is deeply attenuated in NHPs and can vaccinate against LCMV-WE challenge. Here, we demonstrate that internalization of WE is more sensitive to the depletion of membrane cholesterol than ARM infection while ARM infection is more reliant on endosomal acidification. LCMV-ARM induces robust NF-κB and interferon response factor (IRF) activation while LCMV-WE seems to avoid early innate sensing and failed to induce strong NF-κB and IRF responses in dual-reporter monocyte and epithelial cells. Toll-like receptor 2 (TLR-2) signaling appears to play a critical role in NF-κB activation and the silencing of TLR-2 shuts down IL-6 production in ARM but not in WE-infected cells. Pathogenic LCMV-WE infection is poorly recognized in early endosomes and failed to induce TLR-2/Mal-dependent pro-inflammatory cytokines. Following infection, Interleukin-1 receptor-associated kinase 1 (IRAK-1) expression is diminished in LCMV-ARM- but not LCMV-WE-infected cells, which indicates it is likely involved in the LCMV-ARM NF-κB activation. By confocal microscopy, ARM and WE strains have similar intracellular trafficking although LCMV-ARM infection appears to coincide with greater co-localization of early endosome marker EEA1 with TLR-2. Both strains co-localize with Rab-7, a late endosome marker, but the interaction with LCMV-WE seems to be more prolonged. These findings suggest that LCMV-ARM's intracellular trafficking pathway may facilitate interaction with innate immune sensors, which promotes the induction of effective innate and adaptive immune responses.

Bacterial Pathogenesis: Assessment of Intracellular Positioning of Pathogen-Containing Vacuoles During Infection.

Intracellular bacterial pathogens implement a diverse array of strategies to target host cells and establish infection. For vacuolar pathogens, the process of pathogen-containing vacuole movement within host cells, termed intracellular trafficking, is central to both pathogen survival and infection progression. Typically a process mediated by secreted virulence factors that manipulate the host cytoskeletal machinery, internalized pathogen-containing vacuoles traffic to the site of replication to establish a unique replicative niche, and if applicable, traffic back toward the host cell periphery for cell-to-cell spread. As such, the intracellular positioning of pathogen-containing vacuoles represents a fundamental measure of infection progression. Here, we describe a fluorescence microscopy-based method to quantitatively assess bacterial intracellular positioning, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This experimental approach can be modified to study infection in diverse host cell types, and with a broad array of pathogens. The system can also be adapted to examine the kinetics of infection, identify secreted virulence factors that mediate host trafficking, investigate host factors that are targeted by the pathogen for trafficking, and assess functional domains within a virulence factor responsible for mediating the phenotype. Collectively, these tools can provide fundamental insight into the pathogenesis of a diverse array of intracellular bacterial pathogens, and new host factors that are hijacked to mediate infection. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture and preparation of host cells Alternate Protocol: Culture and preparation of host cells to assess host factor contribution to bacterial positioning Basic Protocol 2: Infection of epithelial cells with S. Typhimurium Basic Protocol 3: Fluorescence staining for analysis of bacterial positioning Basic Protocol 4: Fluorescence microscopy analysis of bacterial positioning.

Bacterial Pathogen-Mediated Suppression of Host Trafficking to Lysosomes: Fluorescence Microscopy-Based DQ-Red BSA Analysis.

Intracellular bacterial pathogens have evolved to be adept at manipulating host cellular function for the benefit of the pathogen, often by means of secreted virulence factors that target host pathways for modulation. The lysosomal pathway is an essential cellular response pathway to intracellular pathogens and, as such, represents a common target for bacterial-mediated evasion. Here, we describe a method to quantitatively assess bacterial pathogen-mediated suppression of host cell trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This live-cell imaging assay involves the use of a BODIPY TR-X conjugate of BSA (DQ-Red BSA) that traffics to and fluoresces in functional lysosomes. This method can be adapted to study infection with a broad array of pathogens in diverse host cell types. It is capable of being applied to identify secreted virulence factors responsible for a phenotype of interest as well as domains within the bacterial protein that are important for mediating the phenotype. Collectively, these tools can provide invaluable insight into the mechanisms of pathogenesis of a diverse array of pathogenic bacteria, with the potential to uncover virulence factors that may be suitable targets for therapeutic intervention. Key features • Infection-based analysis of bacterial-mediated suppression of host trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of human epithelial cells as a model. • Live microscopy-based analysis allows for the visualization of individually infected host cells and is amenable to phenotype quantification. • Assay can be adapted to a broad array of pathogens and diverse host cell types. • Assay can identify virulence factors mediating a phenotype and protein domains that mediate a phenotype.

Two roads diverge for cellulose synthase complex trafficking.

Cellulose, an abundant and essential component of plant cell walls, is made by cellulose synthase complexes at the plasma membrane (PM). Recently, Liu et al. uncovered molecular mechanisms that suggest the existence of two distinct pathways for cellulose synthase trafficking from the Golgi apparatus to the PM.

Integrated transcriptomics uncovers an enhanced association between the prion protein gene expression and vesicle dynamics signatures in glioblastomas.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor that exhibits resistance to current treatment, making the identification of novel therapeutic targets essential. In this context, cellular prion protein (PrPC) stands out as a potential candidate for new therapies. Encoded by the PRNP gene, PrPC can present increased expression levels in GBM, impacting cell proliferation, growth, migration, invasion and stemness. Nevertheless, the exact molecular mechanisms through which PRNP/PrPC modulates key aspects of GBM biology remain elusive. METHODS: To elucidate the implications of PRNP/PrPC in the biology of this cancer, we analyzed publicly available RNA sequencing (RNA-seq) data of patient-derived GBMs from four independent studies. First, we ranked samples profiled by bulk RNA-seq as PRNPhigh and PRNPlow and compared their transcriptomic landscape. Then, we analyzed PRNP+ and PRNP- GBM cells profiled by single-cell RNA-seq to further understand the molecular context within which PRNP/PrPC might function in this tumor. We explored an additional proteomics dataset, applying similar comparative approaches, to corroborate our findings. RESULTS: Functional profiling revealed that vesicular dynamics signatures are strongly correlated with PRNP/PrPC levels in GBM. We found a panel of 73 genes, enriched in vesicle-related pathways, whose expression levels are increased in PRNPhigh/PRNP+ cells across all RNA-seq datasets. Vesicle-associated genes, ANXA1, RAB31, DSTN and SYPL1, were found to be upregulated in vitro in an in-house collection of patient-derived GBM. Moreover, proteome analysis of patient-derived samples reinforces the findings of enhanced vesicle biogenesis, processing and trafficking in PRNPhigh/PRNP+ GBM cells. CONCLUSIONS: Together, our findings shed light on a novel role for PrPC as a potential modulator of vesicle biology in GBM, which is pivotal for intercellular communication and cancer maintenance. We also introduce GBMdiscovery, a novel user-friendly tool that allows the investigation of specific genes in GBM biology.

Matrix metalloproteinases at a glance.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that belong to the group of endopeptidases or matrixins. They are able to cleave a plethora of substrates, including components of the extracellular matrix and cell-surface-associated proteins, as well as intracellular targets. Accordingly, MMPs play key roles in a variety of physiological and pathological processes, such as tissue homeostasis and cancer cell invasion. MMP activity is exquisitely regulated at several levels, including pro-domain removal, association with inhibitors, intracellular trafficking and transport via extracellular vesicles. Moreover, the regulation of MMP activity is currently being rediscovered for the development of respective therapies for the treatment of cancer, as well as infectious, inflammatory and neurological diseases. In this Cell Science at a Glance article and the accompanying poster, we present an overview of the current knowledge regarding the regulation of MMP activity, the intra- and extra-cellular trafficking pathways of these enzymes and their diverse groups of target proteins, as well as their impact on health and disease.