Biology subjects

Hernandez, S.

Publications and source records attributed to Hernandez, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Multiscale Cloud-based Pipeline for Neuronal Electrophysiology Analysis and Visualization

Electrophysiology offers a high-resolution method for real-time measurement of neural activity. Longitudinal recordings from high-density microelectrode arrays (HD-MEAs) can be of considerable size for local storage and of substantial complexity for extracting neural features and network dynamics. Analysis is often demanding due to the need for multiple software tools with different runtime dependencies. To address these challenges, we developed an open-source cloud-based pipeline to store, analyze, and visualize neuronal electrophysiology recordings from HD-MEAs. This pipeline is dependency agnostic by utilizing cloud storage, cloud computing resources, and an Internet of Things messaging protocol. We containerized the services and algorithms to serve as scalable and flexible building blocks within the pipeline. In this paper, we applied this pipeline on two types of cultures, cortical organoids and ex vivo brain slice recordings to show that this pipeline simplifies the data analysis process and facilitates understanding neuronal activity.

neuroscience↗

bioRxiv2024

Defense mechanisms in Lupinus luteus against Colletotrichum lupini, involving TIR-NBS-LRR protein, hypersensitive response and phenylpropanoid pathways

Lupins are key grain legumes for future crop production, providing highly sustainable protein, essential in the face of global warming, food security challenges, and the need for sustainable agriculture. Despite their potential, lupin crops are frequently devastated by Colletotrichum lupini, a member of the worlds top ten fungal pathogenic genera. In our previous study, we identified LluR1, the first C. lupini resistance gene, in a wild Lupinus luteus accession. Further research was necessary to unravel the defense mechanisms involved. Histological analysis revealed a hypersensitive response against C. lupini, while transcriptome analysis highlighted a complex network of differentially expressed genes, including TIR-NBS-LRR proteins, hypersensitive response, and phenylpropanoid pathways. SNPs were identified that distinguish the protein sequences underlying immunity. These findings, along with orthology in other lupin species, offer valuable insights for developing breeding strategies to enhance C. lupini resistance in lupins, with significant potential impacts on food, feed, and human nutrition.

plant biology↗

bioRxiv2024

Erythrocyte membrane protein 3 (EMAP3) is exposed on the surface of the Plasmodium berghei infected red blood cell

The human malaria parasite Plasmodium falciparum invades red blood cells (RBC) and exports parasite proteins to transform the host cell for its survival. These exported proteins facilitate uptake of nutrients and cytoadherence of the infected RBC (iRBC) to endothelial cells of small blood vessels, thus protecting the iRBC from splenic clearance. The parasite protein PfEMP1 and the host protein CD36 play a major role in P. falciparum iRBC cytoadherence. The murine parasite Plasmodium berghei is a widely used experimental model that combines high genetic tractability with access to in vivo studies. P. berghei iRBC also sequesters in small blood vessels, mediated by binding to CD36. However, the parasite proteins binding to CD36 are unknown and only very few parasite proteins, including EMAP1 and EMAP2, have been identified that are present at the iRBC membrane. We have identified a new protein named EMAP3 and demonstrated its export to the iRBC membrane where it interacts with EMAP1, with only EMAP3 exposed on the outer surface of the iRBC. Parasites lacking EMAP3 display no significant reduction in growth or sequestration, indicating that EMAP3 is not the major CD36-binding protein. The outer-surface location of EMAP3 offers a new scaffold for displaying P. falciparum proteins on the surface of the P. berghei iRBC, providing a platform to screen in vivo putative inhibitors of P. falciparum cytoadherence.

microbiology↗

bioRxiv2024

Mucin-induced surface dispersal of Staphylococcus aureus and Staphylococcus epidermidis via quorum sensing-dependent and independent mechanisms

Nasopharyngeal carriage of staphylococci spreads potentially pathogenic strains into (peri)oral regions and increases the chance of cross-infections. Some laboratory strains can also move rapidly on hydrated agar surfaces, but the biological relevance of these observations is not clear. Using soft-agar (0.3% w/v) plate assays, we demonstrate the rapid surface dispersal of (peri)oral isolates of Staphylococcus aureus and Staphylococcus epidermidis and closely related laboratory strains in the presence of mucin glycoproteins. Mucin-induced dispersal was a stepwise process initiated by the passive spreading of the growing colonies followed by their rapid branching (dendrites) from the colony edge. Although most spreading strains used mucin as a growth substrate, dispersal was primarily dependent on the lubricating and hydrating properties of the mucins. Using S. aureus JE2 as a genetically tractable representative, we demonstrate that mucin-induced dendritic dispersal, but not colony spreading, is facilitated by the secretion of surfactant-active Phenol-Soluble Modulins (PSMs) in a process regulated by the agr quorum sensing system. Furthermore, the dendritic dispersal of S. aureus JE2 colonies was further stimulated in the presence of surfactant-active supernatants recovered from the most robust (peri)oral spreaders of S. aureus and S. epidermidis. These findings suggest complementary roles for lubricating mucins and staphylococcal PSMs in the active dispersal of potentially pathogenic strains from perioral to respiratory mucosae, where gel-forming, hydrating mucins abound. They also highlight the impact that interspecies interactions have on the co-dispersal of S. aureus with other perioral bacteria, heightening the risk of polymicrobial infections and the severity of the clinical outcomes. ImportanceDespite lacking classical motility machinery, nasopharyngeal staphylococci spread rapidly in (peri)oral and respiratory mucosa and cause cross-infections. We describe laboratory conditions for the reproducible study of staphylococcal dispersal on mucosa-like surfaces and the identification of two dispersal stages (colony spreading and dendritic expansion) stimulated by mucin glycoproteins. The mucin type mattered, as dispersal required the surfactant activity and hydration provided by some mucin glycoproteins. While colony spreading was a passive mode of dispersal lubricated by the mucins, the more rapid and invasive form of dendritic expansion of Staphylococcus aureus and Staphylococcus epidermidis required additional lubrication by surfactant-active peptides (phenol-soluble modulins) secreted at high cell densities through quorum sensing. These results highlight a hitherto unknown role for gel-forming mucins in the dispersal of staphylococcal strains associated with cross-infections and point at perioral regions as overlooked sources of carriage and infection by staphylococci.

microbiology↗

bioRxiv2024

A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei

Many Plasmodium genes remain uncharacterised due to low genetic tractability. Previous large scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called PbHiT, which uses a single cloning step to generate targeting vectors with 100 bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide vector designs and sequences to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasites biology at scale.

molecular biology↗

bioRxiv2024

Multiple endocrine defects in adult-onset Sprouty1/2/4 triple knockout mice

Genes of the Sprouty family (Spry1-4) are feedback inhibitors of receptor tyrosine kinases, especially of Ret and the FGF receptors. As such, they play distinct and overlapping roles in embryo morphogenesis and are considered to be tumor suppressors in adult life. Genetic experiments in mice have defined in great detail the role of these genes during embryonic development, however their function in adult mice is less clearly established. Here we generate adult-onset, whole body Spry1/2/4 triple knockout mice. Tumor incidence in triple mutant mice is comparable to that of wild type littermates of up to one year of age, indicating that Sprouty loss per se is not sufficient to initiate tumorigenesis. On the other hand, triple knockout mice do not gain weight as they age, show less visceral fat, and have lower plasma glucose levels than wild type littermates, despite showing similar food intake and slightly reduced motor function. They also show alopecia, eyelid inflammation, and mild hyperthyroidism. Finally, triple knockout mice present phosphaturia and hypophosphatemia, suggesting exacerbated signaling downstream of FGF23. In conclusion, triple knockout mice develop a series of endocrine abnormalities but do not show increased tumor incidence.

physiology↗

bioRxiv2024

py_ped_sim - A flexible forward genetic simulator for complex family pedigree analysis

BackgroundLarge-scale family pedigrees are commonly used across medical, evolutionary, and forensic genetics. These pedigrees are tools for identifying genetic disorders, tracking evolutionary patterns, and establishing familial relationships via forensic genetic identification. However, there is a lack of software to accurately simulate different pedigree structures along with genomes corresponding to those individuals in a family pedigree. This limits simulation-based evaluations of methods that use pedigrees. ResultsWe have developed a python command-line-based tool called py_ped_sim that facilitates the simulation of pedigree structures and the genomes of individuals in a pedigree. py_ped_sim represents pedigrees as directed acyclic graphs, enabling conversion between standard pedigree formats and integration with the forward population genetic simulator, SLiM. Notably, py_ped_sim allows the simulation of varying numbers of offspring for a set of parents, with the capacity to shift the distribution of sibship sizes over generations. We additionally add simulations for events of misattributed paternity, which offers a way to simulate half-sibling relationships. We validated the accuracy of our software by simulating genomes onto diverse family pedigree structures, showing that the estimated kinship coefficients closely approximated expected values. Conclusionspy_ped_sim is a user-friendly and open-source solution for simulating pedigree structures and conducting pedigree genome simulations. It empowers medical, forensic, and evolutionary genetics researchers to gain deeper insights into the dynamics of genetic inheritance and relatedness within families.

bioinformatics↗

bioRxiv2024

A feedback-driven IoT microfluidic, electrophysiology, and imaging platform for brain organoid studies

The analysis of tissue cultures, particularly brain organoids, requires a sophisticated integration and coordination of multiple technologies for monitoring and measuring. We have developed an automated research platform enabling independent devices to achieve collaborative objectives for feedback-driven cell culture studies. Our approach enables continuous, communicative, non-invasive interactions within an Internet of Things (IoT) architecture among various sensing and actuation devices, achieving precisely timed control of in vitro biological experiments. The framework integrates microfluidics, electrophysiology, and imaging devices to maintain cerebral cortex organoids while measuring their neuronal activity. The organoids are cultured in custom, 3D-printed chambers affixed to commercial microelectrode arrays. Periodic feeding is achieved using programmable microfluidic pumps. We developed a computer vision fluid volume estimator used as feedback to rectify deviations in microfluidic perfusion during media feeding/aspiration cycles. We validated the system with a set of 7-day studies of mouse cerebral cortex organoids, comparing manual and automated protocols. The automated protocols were validated in maintaining robust neural activity throughout the experiment. The automated system enabled hourly electrophysiology recordings for the 7-day studies. Median neural unit firing rates increased for every sample and dynamic patterns of organoid firing rates were revealed by high-frequency recordings. Surprisingly, feeding did not affect firing rate. Furthermore, performing media exchange during a recording showed no acute effects on firing rate, enabling the use of this automated platform for reagent screening studies.

neuroscience↗

bioRxiv2023

Internet-connected cortical organoids for project-based stem cell and neuroscience education

The introduction of internet-connected technologies to the classroom has the potential to revolutionize STEM education by allowing students to perform experiments in complex models that are unattainable in traditional teaching laboratories. By connecting laboratory equipment to the cloud, we introduce students to experimentation in pluripotent stem cell-derived cortical organoids in two different settings: Using microscopy to monitor organoid growth in an introductory tissue culture course, and using high density multielectrode arrays to perform neuronal stimulation and recording in an advanced neuroscience mathematics course. We demonstrate that this approach develops interest in stem cell and neuroscience in the students of both courses. All together, we propose cloud technologies as an effective and scalable approach for complex project-based university training. HIGHLIGHTS- Development of cortical organoids as pedagogical tools for undergraduate education. - Organoids implemented in a tissue culture course through cloud-enabled microscopy. - Multielectrode arrays allow for live organoid manipulation in a mathematics course. - Students self-report increased interest in neuroscience and stem cells topics.

scientific communication and education↗

bioRxiv2023

Modulation of neuronal activity in cortical organoids with bioelectronic delivery of ions and neurotransmitters

Precise modulation of brain activity is fundamental for the proper establishment and maturation of the cerebral cortex. To this end, cortical organoids are promising tools to study circuit formation and the underpinnings of neurodevelopmental disease. However, the ability to manipulate neuronal activity with high temporal resolution in brain organoids remains limited. To overcome this challenge, we introduce a bioelectronic approach to control cortical organoid activity with the selective delivery of ions and neurotransmitters. Using this approach, we sequentially increased and decreased neuronal activity in brain organoids with the bioelectronic delivery of potassium ions (K+) and {gamma}-aminobutyric acid (GABA), respectively, while simultaneously monitoring network activity. This works highlights bioelectronic ion pumps as tools for high-resolution temporal control of brain organoid activity toward precise pharmacological studies that can improve our understanding of neuronal function.

bioengineering↗

bioRxiv2023

Open-loop lab-on-a-chip technology enables remote computer science training in Latinx life sciences students

Despite many interventions, science education remains highly inequitable throughout the world. Among all life sciences fields, Bioinformatics and Computational Biology suffer from the strongest underrepresentation of racial and gender minorities. Internet-enabled project-based learning (PBL) has the potential to reach underserved communities and increase the diversity of the scientific workforce. Here, we demonstrate the use of lab-on-a-chip (LoC) technologies to train Latinx life science undergraduate students in concepts of computer programming by taking advantage of open-loop cloud-integrated LoCs. We developed a context-aware curriculum to train students at over 8,000 km from the experimental site. We showed that this approach was sufficient to develop programming skills and increase the interest of students in continuing careers in Bioinformatics. Altogether, we conclude that LoC-based Internet-enabled PBL can become a powerful tool to train Latinx students and increase the diversity in STEM.

scientific communication and education↗

bioRxiv2023

Whole genome sequencing of Borrelia burgdorferi isolates reveals linked clusters of plasmid-borne accessory genome elements associated with virulence.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 patient-derived B. burgdorferi sensu stricto (Bbss) isolates from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bbss isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bbss isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of [~]800 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, are associated with increased rates of dissemination. OspC type A strains possess a unique constellation of strongly linked genetic changes including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. The patterns of OspC type A strains typify a broader paradigm across Bbss isolates, in which genetic structure is defined by correlated groups of strain-variable genes located predominantly on plasmids, particularly for expression of surface-exposed lipoproteins. These clusters of genes are inherited in blocks through strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

microbiology↗

bioRxiv2023

Chemo-sEVs release in cisplatin-resistance ovarian cancer cells are regulated by the lysosomal function

Ovarian cancer (OvCa) is an aggressive disease usually treated with cisplatin (CDDP)-based therapy. However, among the different types of cancers treated with CDDP, OvCa commonly develops chemoresistance to this treatment. The small extracellular vesicles (sEVs) play a central role in chemoresistance. In response to chemotherapy, resistant cells secrete sEVs named chemo-sEVs characterized by specific cargo landscape content involved in the transfer of chemoresistance to recipient cells. sEVs encompass a variety of vesicle types, including exosomes, and are formed as intraluminal vesicles (ILVs) within multivesicular endosomes (MVEs). MVEs follow at least two trafficking pathways regulated by RAB GTPase family members; 1) a secretory pathway where MVEs fuse with the plasma membrane (PM) for sEVs secretion, where RAB27A is the most studied; 2) a degradative pathway where MVEs fuse with lysosomes, an event controlled by RAB7. There is growing evidence suggesting that a loss of lysosomal function can increase sEVs secretion; however, whether sEVs secretion and the transfer of CDDP chemoresistance in OvCa is the result of a fine regulation between these two MVEs trafficking pathways is unknown. In this work, we study the status of these two pathways, between CDDP-sensitive (A2780) and CDDP-resistant (A2780cis) OvCa cells. We found A2780cis cells have an increased number of MVEs and ILVs structures, together with higher levels of ESCRTs machinery components and RAB27A, compared to A2780 cells. Moreover, CDDP promotes the secretion of chemo-sEVs in A2780cis cells. Interestingly, chemo-sEVs contain a high number of proteins related to DNA damage response. In addition, we determine A2780cis cells have a poor lysosomal function with reduced levels of RAB7. Surprisingly, silencing of RAB27A in A2780cis cells was found to be sufficient to restore lysosomal function and levels of RAB7 in A2780cis cells, switching into an A2780-like cellular phenotype. Next, we found rapamycin, a potent enhancer of lysosomal function, reduced the secretion of chemo-sEVs. Taken together, these results indicate that the secretion of chemo-sEVs in OvCa cells is determined by the balance between secretory MVEs and MVEs that are destined for lysosomal degradation. Thus, our results suggest that adjusting this balance between these two MVEs trafficking pathways could be a promising strategy for overcoming CDDP chemoresistance in OvCa. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/526974v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@167227corg.highwire.dtl.DTLVardef@91c6forg.highwire.dtl.DTLVardef@29e997org.highwire.dtl.DTLVardef@1a6bebe_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

FH variant pathogenicity promotes purine salvage pathway dependence in kidney cancer

The tricarboxylic citric acid cycle enzyme fumarate hydratase (FH) is a tumor suppressor. When lost in cells, its substrate fumarate accumulates to mM levels and drives oncogenic signaling and transformation. Germline alterations lead to an autosomal dominant condition known as hereditary leiomyomatosis and renal cell cancer (HLRCC) where patients are predisposed to various benign tumors and an aggressive form of kidney cancer. FH alterations of unclear significance are frequently observed with germline testing; thus, there is an unmet need to classify FH variants by their cancer-associated risk, allowing for screening, early diagnosis and treatment. Here we quantify catalytic efficiency of 74 FH variants of uncertain significance. Over half were enzymatically inactive which is strong evidence of pathogenicity. We generated a panel of HLRCC cell lines expressing FH variants with a range of catalytic activities, then correlated fumarate levels with metabolic features. We found that fumarate accumulation blocks purine biosynthesis, rendering FH-deficient cells reliant on purine salvage to maintain purine nucleotide pools. Genetic or pharmacologic inhibition of the purine salvage pathway reduced HLRCC tumor growth in vivo. Together, these findings suggest pathogenicity of many patient-associated FH variants and reveal purine salvage as a targetable vulnerability in FH-deficient tumors. Statement of SignificanceThis study functionally characterizes patient-associated FH variants with unknown significance for pathogenicity. This study also reveals nucleotide salvage pathways as a targetable feature of FH-deficient cancers, which are shown to be sensitive to the purine salvage pathway inhibitor 6-mercaptopurine. This presents a new rapidly translatable treatment strategy for FH-deficient cancers.

cancer biology↗

bioRxiv2022

An atlas of epithelial cell states and plasticity in lung adenocarcinoma

Understanding cellular processes underlying early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8+ alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8+ cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation.

cancer biology↗

bioRxiv2022

Population structure and genetic connectivity of the scalloped hammerhead shark (Sphyrna lewini) across nursery grounds from the Eastern Tropical Pacific: implications for management and conservation

Defining demographically independent units and understanding gene flow between them is essential for managing and conserving exploited populations. The scalloped hammerhead shark, Sphyrna lewini, is a coastal semi-oceanic species found worldwide in tropical and subtropical waters. Pregnant females give birth in shallow coastal estuarine habitats that serve as nursery grounds for neonates and small juveniles, and adults move offshore and become highly migratory. We evaluated the population structure and connectivity of S. lewini in coastal areas across the Eastern Tropical Pacific (ETP) using both sequences of the mitochondrial DNA control region (mtCR) and nuclear-encoded microsatellite loci. The mtCR defined two genetically discrete geographic groups: the Mexican Pacific and the central-southern Eastern Tropical Pacific (Guatemala, Costa Rica, Panama, and Colombia). Overall, the mtCR data showed low levels of haplotype diversity ranged from 0.000 to 0.608, while nucleotide diversity ranged from 0.000 to 0.0015. A more fine-grade population structure analysis was detected using microsatellite loci where Guatemala, Costa Rica, and Panama differed significantly. Genetic diversity analysis with nuclear markers revealed an observed heterozygosity ranging from 0.68 to 0.71 and an allelic richness from 5.89 to 7.00. Relatedness analysis revealed that individuals within nursery areas were more closely related than expected by chance, suggesting that S. lewini may exhibit reproductive philopatric behaviour within the ETP. Findings of at least two different management units, and evidence of philopatric behaviour call for intensive conservation actions for this critically endangered species in the ETP.

genetics↗

bioRxiv2021

Improving Lab Culture Through Self-Assessment: A Case Study

PurposeMotivated by perceived dissatisfaction within our labs changed working environment brought about by the COVID-19 pandemic, we performed a self-assessment of our lab culture through anonymous surveys and live sessions. MethodsIn Survey 1, we asked each lab member to identify and rank up to 10 values that are important for a healthy lab environment. They were then asked to rate how well the lab embodied those values at two time points: before the COVID-19 pandemic while working onsite, and at the time of the survey while working remotely (10 months into the pandemic). In a series of live group sessions, we reviewed relevant literature and the survey results to finalize ten themes. We then reflected on each theme and proposed action items to address any deficiencies. Finally, we conducted Survey 2 after the self-assessment to judge the groups finalized themes, implemented changes, and overall satisfaction with the assessment process. ResultsThemes identified were attitude, accountability, teamwork/collaboration, communication, diversity/inclusion, emotional intelligence, integrity, training, well-being, and adaptability in crisis-management. All lab members liked the self-assessment process and felt their voices were heard. On average, there was a 12% increase in satisfaction across all themes from the start to end of the lab assessment. ConclusionWe successfully assessed the culture of our lab and subsequently improved lab member satisfaction. The success of this team project suggests that other scientific labs could benefit from similar interactive self-assessments.

scientific communication and education↗

bioRxiv2020

The proteasomal deubiquitinating enzyme PSMD14 regulates macroautophagy by controlling Golgi-to-ER retrograde transport

Ubiquitination regulates several biological processes. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1,187 genes of the human "ubiquitinome" using Amyloid Precursor Protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel key regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 caused a robust and rapid inhibition of Golgi-to-ER retrograde transport which leads to a potent blockage of macroautophagy by a mechanism associated with the retention of Atg9A and Rab1A at the Golgi apparatus. Because pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and their K-63-Ub chains, act as a crucial regulator factor for macroautophagy by controlling Golgi-to-ER retrograde transport.

cell biology↗