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Article ; Online: The role of the AP-1 adaptor complex in outgoing and incoming membrane traffic.

Robinson, Margaret S / Antrobus, Robin / Sanger, Anneri / Davies, Alexandra K / Gershlick, David C

2024 Volume 223, Issue 7

Abstract: 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 coexpressing tagged AP-1 and various tagged ... ...

Abstract	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 coexpressing 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 starts to sequester cargo 30 min 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 COPI's role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes.
MeSH term(s)	Humans ; Adaptor Proteins, Vesicular Transport/metabolism ; Cell Membrane/metabolism ; Endosomes/genetics ; Endosomes/metabolism ; Golgi Apparatus/genetics ; Golgi Apparatus/metabolism ; HeLa Cells ; Membrane Proteins/metabolism ; Protein Transport ; trans-Golgi Network/metabolism ; Transcription Factor AP-1/genetics ; Transcription Factor AP-1/metabolism
Chemical Substances	Adaptor Proteins, Vesicular Transport ; GGA2 protein, human ; Membrane Proteins ; Transcription Factor AP-1
Language	English
Publishing date	2024-04-05
Publishing country	United States
Document type	Journal Article
ZDB-ID	218154-x
ISSN	1540-8140 ; 0021-9525
ISSN (online)	1540-8140
ISSN	0021-9525
DOI	10.1083/jcb.202310071
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Article ; Online: Deep and fast label-free Dynamic Organellar Mapping.

Schessner, Julia P / Albrecht, Vincent / Davies, Alexandra K / Sinitcyn, Pavel / Borner, Georg H H

Nature communications

2023 Volume 14, Issue 1, Page(s) 5252

Abstract: The Dynamic Organellar Maps (DOMs) approach combines cell fractionation and shotgun-proteomics for global profiling analysis of protein subcellular localization. Here, we enhance the performance of DOMs through data-independent acquisition (DIA) mass ... ...

Abstract	The Dynamic Organellar Maps (DOMs) approach combines cell fractionation and shotgun-proteomics for global profiling analysis of protein subcellular localization. Here, we enhance the performance of DOMs through data-independent acquisition (DIA) mass spectrometry. DIA-DOMs achieve twice the depth of our previous workflow in the same mass spectrometry runtime, and substantially improve profiling precision and reproducibility. We leverage this gain to establish flexible map formats scaling from high-throughput analyses to extra-deep coverage. Furthermore, we introduce DOM-ABC, a powerful and user-friendly open-source software tool for analyzing profiling data. We apply DIA-DOMs to capture subcellular localization changes in response to starvation and disruption of lysosomal pH in HeLa cells, which identifies a subset of Golgi proteins that cycle through endosomes. An imaging time-course reveals different cycling patterns and confirms the quantitative predictive power of our translocation analysis. DIA-DOMs offer a superior workflow for label-free spatial proteomics as a systematic phenotype discovery tool.
MeSH term(s)	Humans ; HeLa Cells ; Reproducibility of Results ; Cell Fractionation ; Endosomes ; Mass Spectrometry
Language	English
Publishing date	2023-08-29
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-41000-7
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Article ; Online: Adaptor protein complexes and disease at a glance.

Sanger, Anneri / Hirst, Jennifer / Davies, Alexandra K / Robinson, Margaret S

Journal of cell science

2019 Volume 132, Issue 20

Abstract: Adaptor protein (AP) complexes are heterotetramers that select cargo for inclusion into transport vesicles. Five AP complexes (AP-1 to AP-5) have been described, each with a distinct localisation and function. Furthermore, patients with a range of ... ...

Abstract	Adaptor protein (AP) complexes are heterotetramers that select cargo for inclusion into transport vesicles. Five AP complexes (AP-1 to AP-5) have been described, each with a distinct localisation and function. Furthermore, patients with a range of disorders, particularly involving the nervous system, have now been identified with mutations in each of the AP complexes. In many cases this has been correlated with aberrantly localised membrane proteins. In this Cell Science at a Glance article and the accompanying poster, we summarize what is known about the five AP complexes and discuss how this helps to explain the clinical features of the different genetic disorders.
MeSH term(s)	Adaptor Proteins, Vesicular Transport/genetics ; Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; Genetic Diseases, Inborn/genetics ; Genetic Diseases, Inborn/metabolism ; Humans
Chemical Substances	Adaptor Proteins, Vesicular Transport
Language	English
Publishing date	2019-10-21
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2993-2
ISSN	1477-9137 ; 0021-9533
ISSN (online)	1477-9137
ISSN	0021-9533
DOI	10.1242/jcs.222992
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Article ; Online: Perforin-2 is a pore-forming effector of endocytic escape in cross-presenting dendritic cells.

Rodríguez-Silvestre, Pablo / Laub, Marco / Krawczyk, Patrycja A / Davies, Alexandra K / Schessner, Julia P / Parveen, Reejuana / Tuck, Benjamin J / McEwan, William A / Borner, Georg H H / Kozik, Patrycja

Science (New York, N.Y.)

2023 Volume 380, Issue 6651, Page(s) 1258–1265

Abstract: During initiation of antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Cross-presentation relies on the unusual "leakiness" of ... ...

Abstract	During initiation of antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Cross-presentation relies on the unusual "leakiness" of endocytic compartments in DCs, whereby internalized 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 assay suitable for genetic screening and identified a pore-forming protein, perforin-2 (
MeSH term(s)	Animals ; Mice ; Antigen Presentation ; Antigens/immunology ; CD8-Positive T-Lymphocytes/immunology ; Cross-Priming/genetics ; Cross-Priming/immunology ; Dendritic Cells/immunology ; Endocytosis/genetics ; Endocytosis/immunology ; Genetic Testing ; Histocompatibility Antigens Class I ; Pore Forming Cytotoxic Proteins/genetics ; Pore Forming Cytotoxic Proteins/metabolism ; Proteolysis
Chemical Substances	Antigens ; Histocompatibility Antigens Class I ; Mpeg1 protein, mouse ; Pore Forming Cytotoxic Proteins
Language	English
Publishing date	2023-06-22
Publishing country	United States
Document type	Journal Article
ZDB-ID	128410-1
ISSN	1095-9203 ; 0036-8075
ISSN (online)	1095-9203
ISSN	0036-8075
DOI	10.1126/science.adg8802
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Article ; Online: High-content screening identifies a small molecule that restores AP-4-dependent protein trafficking in neuronal models of AP-4-associated hereditary spastic paraplegia.

Saffari, Afshin / Brechmann, Barbara / Böger, Cedric / Saber, Wardiya Afshar / Jumo, Hellen / Whye, Dosh / Wood, Delaney / Wahlster, Lara / Alecu, Julian E / Ziegler, Marvin / Scheffold, Marlene / Winden, Kellen / Hubbs, Jed / Buttermore, Elizabeth D / Barrett, Lee / Borner, Georg H H / Davies, Alexandra K / Ebrahimi-Fakhari, Darius / Sahin, Mustafa

Nature communications

2024 Volume 15, Issue 1, Page(s) 584

Abstract: Unbiased phenotypic screens in patient-relevant disease models offer the potential to detect therapeutic targets for rare diseases. In this study, we developed a high-throughput screening assay to identify molecules that correct aberrant protein ... ...

Abstract	Unbiased phenotypic screens in patient-relevant disease models offer the potential to detect therapeutic targets for rare diseases. In this study, we developed a high-throughput screening assay to identify molecules that correct aberrant protein trafficking in adapter protein complex 4 (AP-4) deficiency, a rare but prototypical form of childhood-onset hereditary spastic paraplegia characterized by mislocalization of the autophagy protein ATG9A. Using high-content microscopy and an automated image analysis pipeline, we screened a diversity library of 28,864 small molecules and identified a lead compound, BCH-HSP-C01, that restored ATG9A pathology in multiple disease models, including patient-derived fibroblasts and induced pluripotent stem cell-derived neurons. We used multiparametric orthogonal strategies and integrated transcriptomic and proteomic approaches to delineate potential mechanisms of action of BCH-HSP-C01. Our results define molecular regulators of intracellular ATG9A trafficking and characterize a lead compound for the treatment of AP-4 deficiency, providing important proof-of-concept data for future studies.
MeSH term(s)	Humans ; Spastic Paraplegia, Hereditary/drug therapy ; Spastic Paraplegia, Hereditary/genetics ; Spastic Paraplegia, Hereditary/metabolism ; Proteomics ; Neurons/metabolism ; Protein Transport ; Proteins/metabolism ; Mutation
Chemical Substances	Proteins
Language	English
Publishing date	2024-01-17
Publishing country	England
Document type	Journal Article
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-44264-1
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Article ; Online: From the Cover: Does the Assessment of Nondisjunction Provide a More Sensitive Assay for the Detection of Aneugens?

Elloway, Joanne M / Davies, Alexandra K / Hayes, Julie E / Doherty, Ann T

Toxicological sciences : an official journal of the Society of Toxicology

2017 Volume 157, Issue 1, Page(s) 20–29

Abstract: The detection of aneugenic chemicals is important due to the implications of aneuploidy for human health. Aneuploidy can result from chromosome loss or nondisjunction due to chromosome mis-segregation at anaphase. Frequently, aneugens are detected using ... ...

Abstract	The detection of aneugenic chemicals is important due to the implications of aneuploidy for human health. Aneuploidy can result from chromosome loss or nondisjunction due to chromosome mis-segregation at anaphase. Frequently, aneugens are detected using the in vitro micronucleus assay (IVM), with either centromere or kinetochore labeling. However, this method does not consider nondisjunction, the suggested predominant mechanism of spindle poison induced aneugenicity in primary human lymphocytes. Therefore, the IVM may be relatively insensitive in detecting aneuploidy. To investigate whether chromosome distribution analysis, specifically of nondisjunction, using chromosome-specific centromeric probes provides a more sensitive assay for aneugen detection, six reference aneugens with differing modes of action were tested on human lymphoblastoid TK6 cells. The results show that chromosome loss is a substantial part of the process leading to aneuploidy in TK6 cells. This differs from previous studies on human lymphocytes where nondisjunction has been described as the major mechanism of aneugenicity. However, in the current study more cells and types of aneugenic damage were analyzed. Although compound specific effects on nondisjunction were identified, chromosome distribution analysis did not provide increased sensitivity for the detection of aneugens: For the six reference aneugens examined, chromosome loss was shown at the same concentrations or lower than nondisjunction, even when nondisjunction levels were comparatively high. Therefore, in TK6 cells methods that detect chromosome loss, eg, the IVM, provide a more sensitive technique for the detection of aneugens than the measurement of nondisjunction.
Language	English
Publishing date	2017--01
Publishing country	United States
Document type	Journal Article
ZDB-ID	1420885-4
ISSN	1096-0929 ; 1096-6080
ISSN (online)	1096-0929
ISSN	1096-6080
DOI	10.1093/toxsci/kfx014
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Article ; Online: AP-4-mediated axonal transport controls endocannabinoid production in neurons.

Davies, Alexandra K / Alecu, Julian E / Ziegler, Marvin / Vasilopoulou, Catherine G / Merciai, Fabrizio / Jumo, Hellen / Afshar-Saber, Wardiya / Sahin, Mustafa / Ebrahimi-Fakhari, Darius / Borner, Georg H H

Nature communications

2022 Volume 13, Issue 1, Page(s) 1058

Abstract: The adaptor protein complex AP-4 mediates anterograde axonal transport and is essential for axon health. AP-4-deficient patients suffer from a severe neurodevelopmental and neurodegenerative disorder. Here we identify DAGLB (diacylglycerol lipase-beta), ... ...

Abstract	The adaptor protein complex AP-4 mediates anterograde axonal transport and is essential for axon health. AP-4-deficient patients suffer from a severe neurodevelopmental and neurodegenerative disorder. Here we identify DAGLB (diacylglycerol lipase-beta), a key enzyme for generation of the endocannabinoid 2-AG (2-arachidonoylglycerol), as a cargo of AP-4 vesicles. During normal development, DAGLB is targeted to the axon, where 2-AG signalling drives axonal growth. We show that DAGLB accumulates at the trans-Golgi network of AP-4-deficient cells, that axonal DAGLB levels are reduced in neurons from a patient with AP-4 deficiency, and that 2-AG levels are reduced in the brains of AP-4 knockout mice. Importantly, we demonstrate that neurite growth defects of AP-4-deficient neurons are rescued by inhibition of MGLL (monoacylglycerol lipase), the enzyme responsible for 2-AG hydrolysis. Our study supports a new model for AP-4 deficiency syndrome in which axon growth defects arise through spatial dysregulation of endocannabinoid signalling.
MeSH term(s)	Adaptor Protein Complex 4/metabolism ; Animals ; Axonal Transport ; Axons/metabolism ; Endocannabinoids/metabolism ; Humans ; Mice ; Monoacylglycerol Lipases/genetics ; Monoacylglycerol Lipases/metabolism ; Neurons/metabolism
Chemical Substances	Adaptor Protein Complex 4 ; Endocannabinoids ; Monoacylglycerol Lipases (EC 3.1.1.23)
Language	English
Publishing date	2022-02-25
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-022-28609-w
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Article ; Online: AP-4 vesicles contribute to spatial control of autophagy via RUSC-dependent peripheral delivery of ATG9A.

Davies, Alexandra K / Itzhak, Daniel N / Edgar, James R / Archuleta, Tara L / Hirst, Jennifer / Jackson, Lauren P / Robinson, Margaret S / Borner, Georg H H

Nature communications

2018 Volume 9, Issue 1, Page(s) 3958

Abstract: 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 ' ... ...

Abstract	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 trans-Golgi network 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.
MeSH term(s)	Adaptor Protein Complex 4/metabolism ; Adaptor Proteins, Signal Transducing/metabolism ; Autophagy ; Autophagy-Related Proteins/metabolism ; Carrier Proteins/metabolism ; HeLa Cells ; Humans ; Membrane Proteins/metabolism ; Microtubules/metabolism ; Microtubules/ultrastructure ; Models, Biological ; Phagosomes/metabolism ; Phagosomes/ultrastructure ; Phenotype ; Protein Binding ; Proteomics ; Transport Vesicles/metabolism ; Transport Vesicles/ultrastructure ; Vesicular Transport Proteins/metabolism ; trans-Golgi Network/metabolism ; trans-Golgi Network/ultrastructure
Chemical Substances	Adaptor Protein Complex 4 ; Adaptor Proteins, Signal Transducing ; ATG9A protein, human ; Autophagy-Related Proteins ; Carrier Proteins ; Membrane Proteins ; RUSC1 protein, human ; RUSC2 protein, human ; SERINC1 protein, human ; Vesicular Transport Proteins
Language	English
Publishing date	2018-09-27
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-018-06172-7
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Article ; Online: Clathrin heavy chain 22 contributes to the control of neuropeptide degradation and secretion during neuronal development.

Nahorski, Michael S / Borner, Georg H H / Shaikh, Samiha S / Davies, Alexandra K / Al-Gazali, Lihadh / Antrobus, Robin / Woods, C Geoffrey

Scientific reports

2018 Volume 8, Issue 1, Page(s) 2340

Abstract: The repertoire of cell types in the human nervous system arises through a highly orchestrated process, the complexity of which is still being discovered. Here, we present evidence that CHC22 has a non-redundant role in an early stage of neural precursor ... ...

Abstract	The repertoire of cell types in the human nervous system arises through a highly orchestrated process, the complexity of which is still being discovered. Here, we present evidence that CHC22 has a non-redundant role in an early stage of neural precursor differentiation, providing a potential explanation of why CHC22 deficient patients are unable to feel touch or pain. We show the CHC22 effect on neural differentiation is independent of the more common clathrin heavy chain CHC17, and that CHC22-dependent differentiation is mediated through an autocrine/paracrine mechanism. Using quantitative proteomics, we define the composition of clathrin-coated vesicles in SH-SY5Y cells, and determine proteome changes induced by CHC22 depletion. In the absence of CHC22 a subset of dense core granule (DCG) neuropeptides accumulated, were processed into biologically active 'mature' forms, and secreted in sufficient quantity to trigger neural differentiation. When CHC22 is present, however, these DCG neuropeptides are directed to the lysosome and degraded, thus preventing differentiation. This suggests that the brief reduction seen in CHC22 expression in sensory neural precursors may license a step in neuron precursor neurodevelopment; and that this step is mediated through control of a novel neuropeptide processing pathway.
MeSH term(s)	Autocrine Communication ; Cell Differentiation ; Cell Line, Tumor ; Clathrin Heavy Chains/genetics ; Clathrin Heavy Chains/metabolism ; Gene Knockdown Techniques ; Humans ; Lysosomes ; Neurons ; Neuropeptides/metabolism ; Paracrine Communication ; Protein Transport ; Proteolysis
Chemical Substances	CLTCL1 protein, human ; Neuropeptides ; Clathrin Heavy Chains (114899-12-6)
Language	English
Publishing date	2018-02-05
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-018-19980-0
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Article ; Online: Molecular Basis for the Interaction Between AP4 β4 and its Accessory Protein, Tepsin.

Frazier, Meredith N / Davies, Alexandra K / Voehler, Markus / Kendall, Amy K / Borner, Georg H H / Chazin, Walter J / Robinson, Margaret S / Jackson, Lauren P

Traffic (Copenhagen, Denmark)

2016 Volume 17, Issue 4, Page(s) 400–415

Abstract: The adaptor protein 4 (AP4) complex (ϵ/β4/μ4/σ4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin ...

Abstract	The adaptor protein 4 (AP4) complex (ϵ/β4/μ4/σ4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para- or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 β4 C-terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C-terminus, which binds directly and specifically to the C-terminal β4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the β4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on β4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4-coated vesicles in cell biology, and especially in neuronal function.
MeSH term(s)	Adaptor Protein Complex 4/chemistry ; Adaptor Protein Complex 4/genetics ; Adaptor Protein Complex 4/metabolism ; Binding Sites ; HEK293 Cells ; HeLa Cells ; Humans ; Point Mutation ; Protein Binding
Chemical Substances	Adaptor Protein Complex 4
Language	English
Publishing date	2016-03-04
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1483852-7
ISSN	1600-0854 ; 1398-9219
ISSN (online)	1600-0854
ISSN	1398-9219
DOI	10.1111/tra.12375
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