Imaging data from the article "Deep and fast label-free Dynamic Organellar Mapping", published in Nature Communications by Schessner et al., from Fig. 6, 7 and Supp. Fig. 6c. Widefield images were captured on a Leica DMi8 inverted microscope equipped with an iTK LMT200 motorised stage, a 63x/1.47 oil objective (HC PL APO 63x/1.47 OIL) and a Leica DFC9000 GTC Camera, and controlled with LAS X (Leica Application Software X). Fig6_GLG1_TGOLN2_GALNT2_SuppFig6c_LC3B: Widefield imaging of wild-type HeLa cells cultured for 1h in either: 1) full growth medium (Control); 2) EBSS to starve the cells (Starve); 3) full growth medium plus 100 nM BafA (Control + BafA); or 4) EBSS plus 100 nM BafA (Starve + BafA). Cells were labelled with anti-GALNT2 (Alexa Fluor 488), in combination with either anti-GLG1 (Alexa Fluor 568) or anti-TGOLN2 (Alexa Fluor 680), as shown in Fig. 6, or were labelled with anti-LC3B (Alexa Fluor 488), as shown in Supp. Fig. 6c. In all images, cells were stained with DAPI to label nuclei. Fig7_GALNT2_GLG1_TM9SF2_TGOLN2_GOLIM4_SDF4: Widefield imaging of HeLa cells left untreated in full growth medium (0h) or cultured in the presence of 100 nM BafA for 0.5, 1, 2, 4, 6 or 8 hours, before fixation. Cells were labelled with anti-GALNT2 (Alexa Fluor 647) in combination with either anti-GLG1, anti-TM9SF2, anti-TGOLN2, anti-GOLIM4 or anti-SDF4 (Alexa Fluor 555). In all images, cells were stained with DAPI and phalloidin-488 to label nuclei and cytoplasm, respectively.
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.
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.
Abstract 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 Summary Pore-forming protein perforin-2 is a dedicated effector of endocytic escape specific to cross-presenting cells
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.
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 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.
Imaging data from the article "Deep and fast label-free Dynamic Organellar Mapping", published in Nature Communications by Schessner et al., from Fig. 6, 7 and Supp. Fig. 6c. Widefield images were captured on a Leica DMi8 inverted microscope equipped with an iTK LMT200 motorised stage, a 63x/1.47 oil objective (HC PL APO 63x/1.47 OIL) and a Leica DFC9000 GTC Camera, and controlled with LAS X (Leica Application Software X). Fig6_GLG1_TGOLN2_GALNT2_SuppFig6c_LC3B: Widefield imaging of wild-type HeLa cells cultured for 1h in either: 1) full growth medium (Control); 2) EBSS to starve the cells (Starve); 3) full growth medium plus 100 nM BafA (Control + BafA); or 4) EBSS plus 100 nM BafA (Starve + BafA). Cells were labelled with anti-GALNT2 (Alexa Fluor 488), in combination with either anti-GLG1 (Alexa Fluor 568) or anti-TGOLN2 (Alexa Fluor 680), as shown in Fig. 6, or were labelled with anti-LC3B (Alexa Fluor 488), as shown in Supp. Fig. 6c. In all images, cells were stained with DAPI to label nuclei. Fig7_GALNT2_GLG1_TM9SF2_TGOLN2_GOLIM4_SDF4: Widefield imaging of HeLa cells left untreated in full growth medium (0h) or cultured in the presence of 100 nM BafA for 0.5, 1, 2, 4, 6 or 8 hours, before fixation. Cells were labelled with anti-GALNT2 (Alexa Fluor 647) in combination with either anti-GLG1, anti-TM9SF2, anti-TGOLN2, anti-GOLIM4 or anti-SDF4 (Alexa Fluor 555). In all images, cells were stained with DAPI and phalloidin-488 to label nuclei and cytoplasm, respectively.
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.
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.
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