Biology subjects

Davies, A. K.

Publications and source records attributed to Davies, A. K..

6 recordsLinked to original sources

bioRxiv2025

Arrayed CRISPR/Cas9 Loss-Of-Function Screen in a Neuronal Model of Adaptor Protein Complex 4 Deficiency Identifies Modulators of ATG9A Trafficking

Biallelic loss-of-function variants in the adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including the autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the druggable genome, in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans-Golgi network, suggesting they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.

cell biology↗

bioRxiv2023

The role of the AP-1 adaptor complex in outgoing and incoming membrane traffic

The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we generated stably transduced HeLa cells co-expressing tagged AP-1 and various tagged membrane proteins. Live cell imaging showed that AP-1 is recruited onto tubular carriers trafficking from the Golgi apparatus to the plasma membrane, as well as onto transferrin-containing early/recycling endosomes. Analysis of single AP-1 vesicles showed that they are a heterogeneous population, which start to sequester cargo by 30 minutes after exit from the ER. Vesicle capture showed that AP-1 vesicles contain transmembrane proteins found at the TGN and early/recycling endosomes, as well as lysosomal hydrolases, but very little of the anterograde adaptor GGA2. Together, our results support a model in which AP-1 retrieves proteins from post-Golgi compartments back to the TGN, analogous to COPIs role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes. SummaryRobinson et al. investigate AP-1 function using complementary approaches on cells co-expressing tagged AP-1 and cargo proteins. Their results support a model in which AP-1 is recruited onto post-Golgi carriers and early/recycling endosomes, where it retrieves selected proteins back to the TGN.

cell biology↗

bioRxiv2023

Perforin-2 is a pore-forming effector of endocytic escape in cross- presenting dendritic cells

During initiation of antiviral and antitumour T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on MHC class I. Cross-presentation relies on the unique leakiness of endocytic compartments in DCs, whereby internalised proteins escape into the cytosol for proteasome-mediated generation of MHC I-binding peptides. Given that type 1 conventional DCs excel at cross-presentation, we searched for cell-type specific effectors of endocytic escape. We devised an escape assay suitable for genetic screening and identified a pore-forming protein, perforin-2, as a dedicated effector exclusive to cross-presenting cells. Perforin-2 is recruited to antigen-containing compartments, where it undergoes maturation, releasing its pore-forming domain. Mpeg1-/- mice fail to efficiently prime CD8+ T cells to cell-associated antigens, revealing an important role of perforin-2 in cytosolic entry of antigens during cross-presentation. One-Sentence SummaryPore-forming protein perforin-2 is a dedicated effector of endocytic escape specific to cross-presenting cells

cell biology↗

bioRxiv2021

Data independent acquisition enables deep and fast label-free dynamic organellar mapping

The Dynamic Organellar Maps (DOMs) approach combines cell fractionation and shotgun-proteomics for global profiling analysis of protein subcellular localization. Here, we have drastically enhanced the performance of DOMs through data-independent acquisition (DIA) mass spectrometry (MS). DIA-DOMs achieve twice the depth of our previous workflow in the same MS runtime, and substantially improve profiling precision and reproducibility. We leveraged this gain to establish flexible map formats scaling from rapid analyses to ultra-deep coverage. Our fastest format takes only [~]2.5h/map and enables high-throughput experimental designs. Furthermore, we introduce DOM-QC, an open-source software tool for in-depth standardized analysis of DOMs and other profiling data. We then applied DIA-DOMs to capture subcellular localization changes in response to starvation and disruption of lysosomal pH in HeLa cells, which revealed a subset of Golgi proteins that cycle through endosomes. DIA-DOMs offer a superior workflow for label-free spatial proteomics as a systematic phenotype discovery tool.

biochemistry↗

bioRxiv2020

AP-4 mediates vesicular transport of the 2-AG endocannabinoid producing enzyme DAGLB

The adaptor protein complex AP-4 mediates anterograde axonal transport and is essential for axon health. AP-4-deficient patients suffer from a severe neurological disorder, which encompasses neurodevelopmental and neurodegenerative features. While impaired autophagy has been suggested to account for axon degeneration in AP-4 deficiency, axon growth defects occur through an unknown mechanism. Here we use orthogonal proteomic and imaging methods to identify DAGLB (diacylglycerol lipase-beta) as a cargo of AP-4 vesicles. DAGLB is a key enzyme for the generation of 2-AG (2-arachidonoylglycerol), the most abundant endocannabinoid in brain. During normal development, DAGLB is targeted to the axon, where 2-AG signalling drives axonal growth. We show that DAGLB accumulates at the TGN of AP-4-deficient cells, including in iPSC-derived neurons from a patient with AP-4 deficiency syndrome. Our data thus support that AP-4 mediates axonal targeting of DAGLB, and we propose that axon growth defects in AP-4 deficiency may arise through spatial dysregulation of endocannabinoid signalling.

cell biology↗

bioRxiv2017

AP-4 vesicles unmasked by organellar proteomics to reveal their cargo and machinery

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including Dynamic Organellar Maps, to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the TGN to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the \"ATG9A reservoir\" required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

cell biology↗