28 results on '"Daniela A. Sahlender"'
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2. Block Face Scanning Electron Microscopy of Fluorescently Labeled Axons Without Using Near Infra-Red Branding
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Catherine Maclachlan, Daniela A. Sahlender, Shuichi Hayashi, Zoltán Molnár, and Graham Knott
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correlative light and electron microscopy (CLEM) ,axons ,scanning electron microscopy ,neuron ultrastructure ,serial block-face electron microscopy (SBEM) ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 ,Human anatomy ,QM1-695 - Abstract
In this article, we describe the method that allows fluorescently tagged structures such as axons to be targeted for electron microscopy (EM) analysis without the need to convert their labels into electron dense stains, introduce any fiducial marks, or image large volumes at high resolution. We optimally preserve and stain the brain tissue for ultrastructural analysis and use natural landmarks, such as cell bodies and blood vessels, to locate neurites that had been imaged previously using confocal microscopy. The method relies on low and high magnification views taken with the light microscope, after fixation, to capture information of the tissue structure that can later be used to pinpoint the position of structures of interest in serial EM images. The examples shown here are td Tomato expressing cortico-thalamic axons in the posteromedial nucleus of the mouse thalamus, imaged in fixed tissue with confocal microscopy, and subsequently visualized with serial block-face EM (SBEM) and reconstructed into 3D models for analysis.
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- 2018
- Full Text
- View/download PDF
3. Correction: The Fifth Adaptor Protein Complex.
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Jennifer Hirst, Lael D. Barlow, Gabriel Casey Francisco, Daniela A. Sahlender, Matthew N. J. Seaman, Joel B. Dacks, and Margaret S. Robinson
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Biology (General) ,QH301-705.5 - Published
- 2012
- Full Text
- View/download PDF
4. Abstract CT202: Xevinapant plus avelumab in patients (pts) with advanced or metastatic non-small cell lung cancer (NSCLC): Phase 1b dose-expansion results and exploratory biomarker analyses
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Quincy Chu, Tudor Ciuleanu, Rodryg Ramlau, Daniel Renouf, Rosalyn Juergens, Ewa Kalinka, Piotr Sawrycki, Jonathan Bramson, Brad Nelson, Rafael Crabbé, Daniela A. Sahlender, Philippa Crompton, Elisabeth Rouits, Dany Spaggiari, Franck Brichory, Luke Piggott, Mike Schenker, and Glenwood Goss
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Cancer Research ,Oncology - Abstract
BACKGROUND: Combining anti-PD-1/L1 antibodies and agents that restore cancer cell susceptibility to apoptosis may enhance antitumor activity. We report results from a phase 1b dose-expansion cohort of xevinapant, a first-in-class, oral, small-molecule IAP (inhibitor of apoptosis protein) inhibitor that restores cancer cell sensitivity to apoptosis, and avelumab (anti-PD-L1) in pts with advanced NSCLC. METHODS: The recommended phase 2 dose (RP2D; 28-day cycles of xevinapant 200 mg/day [days 1-10 and 15-24] + avelumab 10 mg/kg [days 1 and 15]) was previously established during the dose-escalation part of this phase 1, open-label study. In this dose-expansion cohort, pts with advanced NSCLC who progressed on first-line (1L) platinum-based chemotherapy (CTx) or anti-PD-1/L1 ± platinum-based CTx received xevinapant (at RP2D) + avelumab for 13 cycles. The primary endpoint was objective response rate (ORR) per RECIST 1.1. Secondary endpoints included duration of response (DoR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS), safety, and pharmacokinetics (PK). RESULTS: 38 pts were treated: most had squamous cell carcinoma (45%) or adenocarcinoma (42%) of the lung, 11% had prior anti-PD-L1 therapy, 71% were men, and median age was 62 years (range, 35-75). 1 pt completed 13 cycles, and 37 permanently discontinued treatment; most common reasons were progressive disease (PD; 70%) and adverse events (AEs; 27%). ORR was 10.5% (95% CI 2.9-24.8). Best overall response (BOR): 4 pts had a confirmed partial response (PR), 19 had stable disease, and 15 had PD. Median DoR in responders was 15.9 months (95% CI 3.5-29.7); DCR was 60.5% (95% CI 43.4-76.0). Median PFS was 3.5 months (95% CI 1.9-5.1); median OS was 9.4 months (95% CI 6.7-16.2). Most pts (n=37; 97.4%) had treatment-emergent AEs (TEAEs); 21 (55.3%) had grade ≥3. Most common TEAEs were decreased appetite (n=13; 34.2%) and ALT increase (n=11; 28.9%). Nine pts died due to TEAEs; none were considered treatment-related. Xevinapant and avelumab PK were comparable to monotherapy at the same doses. In exploratory biomarker analyses, plasma IL-10 levels increased during the study treatment period. In blood, activated CD4 T cells and Tregs increased during cycle 1, and activated CD8 T cells increased during the treatment period; however, these did not correlate with antitumor activity. In tumor samples, low Ki67 expression was associated with BOR of PR (n=4), and increases in macrophages, Tregs, Th1 cells, and dendritic cells were associated with disease control. CONCLUSION: Xevinapant + avelumab had tolerable safety in pts with advanced NSCLC who progressed on 1L platinum-based CTx or anti-PD-1/L1 ± platinum-based CTx; however, the study did not meet its primary endpoint as antitumor activity was comparable to historic data for avelumab second-line monotherapy. Citation Format: Quincy Chu, Tudor Ciuleanu, Rodryg Ramlau, Daniel Renouf, Rosalyn Juergens, Ewa Kalinka, Piotr Sawrycki, Jonathan Bramson, Brad Nelson, Rafael Crabbé, Daniela A. Sahlender, Philippa Crompton, Elisabeth Rouits, Dany Spaggiari, Franck Brichory, Luke Piggott, Mike Schenker, Glenwood Goss. Xevinapant plus avelumab in patients (pts) with advanced or metastatic non-small cell lung cancer (NSCLC): Phase 1b dose-expansion results and exploratory biomarker analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT202.
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- 2023
5. Uncoupling the functions of CALM in VAMP sorting and clathrin-coated pit formation.
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Daniela A Sahlender, Patrycja Kozik, Sharon E Miller, Andrew A Peden, and Margaret S Robinson
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Medicine ,Science - Abstract
CALM (clathrin assembly lymphoid myeloid leukemia protein) is a cargo-selective adaptor for the post-Golgi R-SNAREs VAMPs 2, 3, and 8, and it also regulates the size of clathrin-coated pits and vesicles at the plasma membrane. The present study has two objectives: to determine whether CALM can sort additional VAMPs, and to investigate whether VAMP sorting contributes to CALM-dependent vesicle size regulation. Using a flow cytometry-based endocytosis efficiency assay, we demonstrate that CALM is also able to sort VAMPs 4 and 7, even though they have sorting signals for other clathrin adaptors. CALM homologues are present in nearly every eukaryote, suggesting that the CALM family may have evolved as adaptors for retrieving all post-Golgi VAMPs from the plasma membrane. Using a knockdown/rescue system, we show that wild-type CALM restores normal VAMP sorting in CALM-depleted cells, but that two non-VAMP-binding mutants do not. However, when we assayed the effect of CALM depletion on coated pit morphology, using a fluorescence microscopy-based assay, we found that the two mutants were as effective as wild-type CALM. Thus, we can uncouple the sorting function of CALM from its structural role.
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- 2013
- Full Text
- View/download PDF
6. Ultrastructural basis of strong unitary inhibition in a binaural neuron
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Daniela A. Sahlender, Graham Knott, Clémentine Aguet, Ralf Schneggenburger, and Enida Gjoni
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0301 basic medicine ,Serial block-face scanning electron microscopy ,Proximal dendrite ,Physiology ,Chemistry ,Inhibitory postsynaptic potential ,Synapse ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,nervous system ,medicine ,Excitatory postsynaptic potential ,Soma ,Neuron ,Axon ,Neuroscience ,030217 neurology & neurosurgery - Abstract
Key points Neurons of the lateral superior olive (LSO) in the brainstem receive powerful glycinergic inhibition that originates from the contralateral ear, and that plays an important role in sound localization. We investigated the ultrastructural basis for strong inhibition of LSO neurons using serial block face scanning electron microscopy. The soma and the proximal dendrite of an LSO neuron are surrounded by a high density of inhibitory axons, whereas excitatory axons are much sparser. A given inhibitory axon establishes contacts via several large axonal thickenings, called varicosities, which typically elaborate several active zones (range 1-11). The number of active zones across inhibitory axon segments is variable. These data thus provide an ultrastructural correlate for the strong and multiquantal, but overall variable, unitary IPSC amplitude observed for inhibitory inputs to LSO neuron. Abstract Binaural neurons in the lateral superior olive (LSO) integrate sound information arriving from each ear, and powerful glycinergic inhibition of these neurons plays an important role in this process. In the present study, we investigated the ultrastructural basis for strong inhibitory inputs onto LSO neurons using serial block face scanning electron microscopy. We reconstructed axon segments that make contact with the partially reconstructed soma and proximal dendrite of a mouse LSO neuron at postnatal day 18. Using functional measurements and the Sr2+ method, we find a constant quantal size but a variable quantal content between 'weak' and 'strong' unitary IPSCs. A 3-D reconstruction of a LSO neuron and its somatic synaptic afferents reveals how a large number of inhibitory axons intermingle in a complex fashion on the soma and proximal dendrite of an LSO neuron; a smaller number of excitatory axons was also observed. A given inhibitory axon typically contacts an LSO neuron via several large varicosities (average diameter 3.7 μm), which contain several active zones (range 1-11). The number of active zones across individual axon segments was highly variable. These data suggest that the variable unitary IPSC amplitude is caused by a variable number of active zones between inhibitory axons that innervate a given LSO neuron. The results of the present study show that relatively large multi-active zone varicosities, which can be repeated many times in a given presynaptic axon, provide the ultrastructural basis for the strong multiquantal inhibition received by LSO neurons.
- Published
- 2018
7. Block face scanning electron microscopy of fluorescently labeled axons without using near infra-red branding
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Daniela A. Sahlender, Shuichi Hayashi, Zoltán Molnár, Graham Knott, and Catherine Maclachlan
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terminals ,0301 basic medicine ,Materials science ,Scanning electron microscope ,pulvinar ,Neuroscience (miscellaneous) ,axons ,in-vivo ,lateral geniculate ,lcsh:RC321-571 ,lcsh:QM1-695 ,law.invention ,neuron ultrastructure ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Optical microscope ,law ,Confocal microscopy ,medicine ,Methods ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Fixation (histology) ,serial block-face electron microscopy (SBEM) ,dynamics ,lcsh:Human anatomy ,organization ,ultrastructure ,correlative light and electron microscopy (CLEM) ,cortex ,030104 developmental biology ,medicine.anatomical_structure ,Ultrastructure ,Anatomy ,Electron microscope ,light ,Fiducial marker ,circuit ,Nucleus ,030217 neurology & neurosurgery ,scanning electron microscopy ,Biomedical engineering ,Neuroscience - Abstract
In this paper, we describe the method that allows fluorescently tagged structures such as axons to be targeted for electron microscopy analysis without the need to convert their labels into electron dense stains, introduce any fiducial marks, or image large volumes at high resolution. We optimally preserve and stain the brain tissue for ultrastructural analysis and use natural landmarks, such as cell bodies and blood vessels, to locate neurites that had been imaged previously using confocal microscopy. The method relies on low and high magnification views taken with the light microscope, after fixation, to capture information of the tissue structure that can later be used to pinpoint the position of structures of interest in serial EM images. The examples shown here are td Tomato expressing cortico-thalamic axons in the posteromedial nucleus of the mouse thalamus, imaged in fixed tissue with confocal microscopy, and subsequently visualized with serial block-face electron microscopy (SBEM) and reconstructed into 3D models for analysis.
- Published
- 2018
8. Ultrastructural basis of strong unitary inhibition in a binaural neuron
- Author
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Enida, Gjoni, Clémentine, Aguet, Daniela A, Sahlender, Graham, Knott, and Ralf, Schneggenburger
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Mice, Inbred C57BL ,Mice ,nervous system ,Inhibitory Postsynaptic Potentials ,Presynaptic Terminals ,Animals ,Superior Olivary Complex ,Dendrites ,Neuroscience - Abstract
KEY POINTS: Neurons of the lateral superior olive (LSO) in the brainstem receive powerful glycinergic inhibition that originates from the contralateral ear, and that plays an important role in sound localization. We investigated the ultrastructural basis for strong inhibition of LSO neurons using serial block face scanning electron microscopy. The soma and the proximal dendrite of an LSO neuron are surrounded by a high density of inhibitory axons, whereas excitatory axons are much sparser. A given inhibitory axon establishes contacts via several large axonal thickenings, called varicosities, which typically elaborate several active zones (range 1–11). The number of active zones across inhibitory axon segments is variable. These data thus provide an ultrastructural correlate for the strong and multiquantal, but overall variable, unitary IPSC amplitude observed for inhibitory inputs to LSO neuron. ABSTRACT: Binaural neurons in the lateral superior olive (LSO) integrate sound information arriving from each ear, and powerful glycinergic inhibition of these neurons plays an important role in this process. In the present study, we investigated the ultrastructural basis for strong inhibitory inputs onto LSO neurons using serial block face scanning electron microscopy. We reconstructed axon segments that make contact with the partially reconstructed soma and proximal dendrite of a mouse LSO neuron at postnatal day 18. Using functional measurements and the Sr(2+) method, we find a constant quantal size but a variable quantal content between ‘weak’ and ‘strong’ unitary IPSCs. A 3‐D reconstruction of a LSO neuron and its somatic synaptic afferents reveals how a large number of inhibitory axons intermingle in a complex fashion on the soma and proximal dendrite of an LSO neuron; a smaller number of excitatory axons was also observed. A given inhibitory axon typically contacts an LSO neuron via several large varicosities (average diameter 3.7 μm), which contain several active zones (range 1–11). The number of active zones across individual axon segments was highly variable. These data suggest that the variable unitary IPSC amplitude is caused by a variable number of active zones between inhibitory axons that innervate a given LSO neuron. The results of the present study show that relatively large multi‐active zone varicosities, which can be repeated many times in a given presynaptic axon, provide the ultrastructural basis for the strong multiquantal inhibition received by LSO neurons.
- Published
- 2018
9. Computer assisted detection of axonal bouton structural plasticity in in vivo time-lapse images
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Armen Stepanyants, Graham Knott, Daniel Lebrecht, Daniela A. Sahlender, Rohan Gala, Anne Jorstad, and Anthony Holtmaat
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0301 basic medicine ,3d electron microscopy ,Mouse ,QH301-705.5 ,Science ,time-lapse imaging ,Biology ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,bouton detection ,In vivo ,Biological neural network ,Time-Lapse Imaging ,Biology (General) ,Synaptic strength ,Structural plasticity ,Laser Scanning Microscopy ,General Immunology and Microbiology ,Time-lapse imaging ,General Neuroscience ,fungi ,Bouton detection ,General Medicine ,CLEM ,Barrel cortex ,structural plasticity ,ddc:616.8 ,030104 developmental biology ,nervous system ,Biophysics ,synaptic strength ,Medicine ,bouton volume ,030217 neurology & neurosurgery ,Preclinical imaging ,Bouton volume ,Neuroscience ,Biomedical engineering - Abstract
The ability to measure minute structural changes in neural circuits is essential for long-term in vivo imaging studies. Here, we propose a methodology for detection and measurement of structural changes in axonal boutons imaged with time-lapse two-photon laser scanning microscopy (2PLSM). Correlative 2PLSM and 3D electron microscopy (EM) analysis, performed in mouse barrel cortex, showed that the proposed method has low fractions of false positive/negative bouton detections (2/0 out of 18), and that 2PLSM-based bouton weights are correlated with their volumes measured in EM (r = 0.93). Next, the method was applied to a set of axons imaged in quick succession to characterize measurement uncertainty. The results were used to construct a statistical model in which bouton addition, elimination, and size changes are described probabilistically, rather than being treated as deterministic events. Finally, we demonstrate that the model can be used to quantify significant structural changes in boutons in long-term imaging experiments.
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- 2017
10. Author response: Computer assisted detection of axonal bouton structural plasticity in in vivo time-lapse images
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Graham Knott, Anne Jorstad, Daniel Lebrecht, Daniela A. Sahlender, Rohan Gala, Armen Stepanyants, and Anthony Holtmaat
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In vivo ,Structural plasticity ,Biology ,Neuroscience - Published
- 2017
11. A human genome-wide screen for regulators of clathrin-coated vesicle formation reveals an unexpected role for the V-ATPase
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Christina Soromani, Nicola A. Hodson, Nikol Simecek, Daniela A. Sahlender, Patrycja Kozik, Jiahua Wu, Lucy M. Collinson, and Margaret S. Robinson
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Vacuolar Proton-Translocating ATPases ,Endocytic cycle ,Biology ,Endocytosis ,Clathrin ,Article ,03 medical and health sciences ,0302 clinical medicine ,RNA interference ,Gene Knockdown Techniques ,Humans ,Filipin ,030304 developmental biology ,0303 health sciences ,Gene knockdown ,Genome, Human ,Vesicle ,Clathrin-Coated Vesicles ,Coated Pits, Cell-Membrane ,Cell Biology ,Hydrogen-Ion Concentration ,Phenotype ,Cell biology ,Cholesterol ,Biochemistry ,biology.protein ,RNA Interference ,Macrolides ,030217 neurology & neurosurgery ,HeLa Cells - Abstract
Clathrin-mediated endocytosis is essential for a wide range of cellular functions. We used a multi-step siRNA-based screening strategy to identify regulators of the first step in clathrin-mediated endocytosis, formation of clathrin-coated vesicles (CCVs) at the plasma membrane. A primary genome-wide screen identified 334 hits that caused accumulation of CCV cargo on the cell surface. A secondary screen identified 92 hits that inhibited cargo uptake and/or altered the morphology of clathrin-coated structures. The hits include components of four functional complexes: coat proteins, V-ATPase subunits, spliceosome-associated proteins, and acetyltransferase subunits. Electron microscopy revealed that V-ATPase depletion caused the cell to form aberrant non-constricted clathrin-coated structures at the plasma membrane. The V-ATPase knockdown phenotype was rescued by addition of exogenous cholesterol, indicating that the knockdown blocks clathrin-mediated endocytosis by preventing cholesterol from recycling from endosomes back to the plasma membrane.
- Published
- 2012
12. Distinct and Overlapping Roles for AP-1 and GGAs Revealed by the 'Knocksideways' System
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Daniela A. Sahlender, Nicola A. Hodson, Georg H. H. Borner, Robin Antrobus, Andrew A. Peden, Jennifer Hirst, and Margaret S. Robinson
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Adaptor Protein Complex 1 ,Vesicular Transport Proteins ,Plasma protein binding ,Biology ,Clathrin ,General Biochemistry, Genetics and Molecular Biology ,R-SNARE Proteins ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Report ,Humans ,RNA, Small Interfering ,030304 developmental biology ,0303 health sciences ,Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,Peripheral membrane protein ,Signal transducing adaptor protein ,Clathrin-Coated Vesicles ,Transmembrane protein ,Cell biology ,Adaptor Proteins, Vesicular Transport ,biology.protein ,RNA Interference ,Clathrin adaptor proteins ,SNARE Proteins ,General Agricultural and Biological Sciences ,030217 neurology & neurosurgery ,HeLa Cells ,Protein Binding - Abstract
Summary Although adaptor protein complex 1 (AP-1) and Golgi-localized, γ ear-containing, ADP-ribosylation factor-binding proteins (GGAs) are both adaptors for clathrin-mediated intracellular trafficking, the pathways they mediate and their relationship to each other remain open questions [1]. To tease apart the functions of AP-1 and GGAs, we rapidly inactivated each adaptor using the “knocksideways” system [2] and then compared the protein composition of clathrin-coated vesicle (CCV) fractions from control and knocksideways cells. The AP-1 knocksideways resulted in a dramatic and unexpected loss of GGA2 from CCVs. Over 30 other peripheral membrane proteins and over 30 transmembrane proteins were also depleted, including several mutated in genetic disorders, indicating that AP-1 acts as a linchpin for intracellular CCV formation. In contrast, the GGA2 knocksideways affected only lysosomal hydrolases and their receptors. We propose that there are at least two populations of intracellular CCVs: one containing both GGAs and AP-1 for anterograde trafficking and another containing AP-1 for retrograde trafficking. Our study shows that knocksideways and proteomics are a powerful combination for investigating protein function, which can potentially be used on many different types of proteins., Graphical Abstract Highlights ► AP-1 knocksideways depletes ∼100 proteins from clathrin-coated vesicles (CCVs) ► GGA2 knocksideways mainly depletes hydrolases and their receptors ► GGA2 depends on AP-1 for incorporation into CCVs ► AP-1 acts as a linchpin for intracellular CCV formation and is bidirectional
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- 2012
- Full Text
- View/download PDF
13. Spatial and Functional Relationship of GGAs and AP-1 inDrosophilaand HeLa Cells
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Rita Sinka, Daniela A. Sahlender, Michael D.J. Parkinson, Maja K. Choma, Jennifer Hirst, Margaret S. Robinson, and Michael E. Harbour
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ADP ribosylation factor ,Immunoelectron microscopy ,Green Fluorescent Proteins ,Coated vesicle ,Transfection ,Major histocompatibility complex ,Biochemistry ,Downregulation and upregulation ,Structural Biology ,RNA interference ,Genetics ,Animals ,Humans ,RNA, Small Interfering ,Molecular Biology ,Gene knockdown ,biology ,Clathrin-Coated Vesicles ,Cell Biology ,Cell biology ,Transcription Factor AP-1 ,Adaptor Proteins, Vesicular Transport ,Protein Transport ,biology.protein ,Drosophila ,RNA Interference ,Intracellular ,HeLa Cells - Abstract
The GGAs [Golgi-localised, gamma-ear containing, ARF (ADP ribosylation factor)-binding proteins] and the AP-1 (adaptor protein-1) complex are both adaptors for clathrin-mediated intracellular trafficking, but their relationship to each other is unclear. We have used two complementary systems, HeLa cells and Drosophila Dmel2 cells, to investigate GGA and AP-1 function. Immunoelectron microscopy of endogenous AP-1 and GGA in Dmel2 cells shows that they are predominantly associated with distinct clathrin-coated structures. Depletion of either GGA or AP-1 by RNAi does not affect the incorporation of the other adaptor into clathrin-coated vesicles (CCVs), and the cargo protein GFP-LERP (green fluorescent protein-lysosomal enzyme receptor protein) is lost from CCVs only when both adaptors are depleted. Similar results were obtained using HeLa cells treated with siRNA to deplete all three GGAs simultaneously. AP-1 was still incorporated into CCVs after GGA depletion and vice versa, and both needed to be depleted for a robust inhibition of receptor-mediated sorting of lysosomal hydrolases. In contrast, downregulation of major histocompatibility complex (MHC) class I by HIV-1 Nef, which requires AP-1, was not affected by a triple GGA knockdown. Thus, our results indicate that the two adaptors can function independently of each other.
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- 2009
14. Auxilin Depletion Causes Self-Assembly of Clathrin into Membraneless CagesIn Vivo
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Daniela A. Sahlender, Nienke B. Lubben, Sam Li, Jennifer Hirst, Margaret S. Robinson, and Georg H. H. Borner
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Auxilins ,Endocytic cycle ,Coated vesicle ,Auxilin ,Biology ,plasma membrane ,Endocytosis ,Models, Biological ,Biochemistry ,Clathrin ,Cell membrane ,03 medical and health sciences ,Cytosol ,0302 clinical medicine ,Structural Biology ,Genetics ,medicine ,Humans ,RNA, Small Interfering ,GGA ,Molecular Biology ,030304 developmental biology ,Neurons ,0303 health sciences ,auxilin ,Vesicle ,Cell Membrane ,HSC70 Heat-Shock Proteins ,Fluorescence recovery after photobleaching ,intracellular membranes ,Clathrin-Coated Vesicles ,Original Articles ,Cell Biology ,AP-1 ,AP-2 ,coated vesicle ,Cell biology ,Protein Transport ,medicine.anatomical_structure ,TGN ,biology.protein ,RNA Interference ,030217 neurology & neurosurgery ,Fluorescence Recovery After Photobleaching ,HeLa Cells - Abstract
Auxilin is a cofactor for Hsc70-mediated uncoating of clathrin-coated vesicles (CCVs). However, small interfering RNA (siRNA) knockdown of the ubiquitous auxilin 2 in HeLa cells only moderately impairs clathrin-dependent trafficking. In this study, we show that HeLa cells also express auxilin 1, previously thought to be neuron specific, and that both auxilins need to be depleted for inhibition of clathrin-mediated endocytosis and intracellular sorting. Depleting both auxilins cause an approximately 50% reduction in the number of clathrin-coated pits at the plasma membrane but enhances the association of clathrin and adaptors with intracellular membranes. CCV fractions isolated from auxilin-depleted cells have an approximately 1.5-fold increase in clathrin content and more than fivefold increase in the amount of AP-2 adaptor complex and other endocytic machinery, with no concomitant increase in cargo. In addition, the structures isolated from auxilin-depleted cells are on average smaller than CCVs from control cells and are largely devoid of membrane, indicating that they are not CCVs but membraneless clathrin cages. Similar structures are observed by electron microscopy in intact auxilin-depleted HeLa cells. Together, these findings indicate that the two auxilins have overlapping functions and that they not only facilitate the uncoating of CCVs but also prevent the formation of nonproductive clathrin cages in the cytosol.
- Published
- 2008
15. Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking
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Daniela A. Sahlender, Georg H. H. Borner, Robin Antrobus, Jennifer Hirst, Sam Li, James R. Edgar, Margaret S. Robinson, Hirst, Jennifer [0000-0001-9063-8494], Edgar, James [0000-0001-7903-8199], Robinson, Margaret [0000-0003-0631-0053], and Apollo - University of Cambridge Repository
- Subjects
Endosome ,Coated vesicle ,Endosomes ,Endocytosis ,Clathrin ,Humans ,Molecular Biology ,N-Ethylmaleimide-Sensitive Proteins ,biology ,Membrane Proteins ,Clathrin-Coated Vesicles ,Articles ,Cell Biology ,Transport protein ,Cell biology ,Adaptor Proteins, Vesicular Transport ,Protein Transport ,Membrane protein ,Membrane Trafficking ,biology.protein ,Intracellular ,HeLa Cells ,Protein Binding ,Subcellular Fractions - Abstract
EpsinR and gadkin are two components of intracellular clathrin-coated vesicles whose precise functions are unclear. Rapid depletion of each protein from the available pool using the knocksideways method strongly inhibited the production of intracellular clathrin-coated vesicles, providing new insights into the functions of both proteins., The precise functions of most of the proteins that participate in clathrin-mediated intracellular trafficking are unknown. We investigated two such proteins, epsinR and gadkin, using the knocksideways method, which rapidly depletes proteins from the available pool by trapping them onto mitochondria. Although epsinR is known to be an N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)-specific adaptor, the epsinR knocksideways blocked the production of the entire population of intracellular clathrin-coated vesicles (CCVs), suggesting a more global function. Using the epsinR knocksideways data, we were able to estimate the copy number of all major intracellular CCV proteins. Both sides of the vesicle are densely covered, indicating that CCVs sort their cargo by molecular crowding. Trapping of gadkin onto mitochondria also blocked the production of intracellular CCVs but by a different mechanism: vesicles became cross-linked to mitochondria and pulled out toward the cell periphery. Both phenotypes provide new insights into the regulation of intracellular CCV formation, which could not have been found using more conventional approaches.
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- 2015
16. Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles
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Georg H. H. Borner, Gary S. Bhumbra, Robin Antrobus, Patrycja Kozik, Daniela A. Sahlender, Jennifer Hirst, Lauren P. Jackson, Margaret S. Robinson, Hirst, Jennifer [0000-0001-9063-8494], Robinson, Margaret [0000-0003-0631-0053], and Apollo - University of Cambridge Repository
- Subjects
Proteomics ,Coated vesicle ,Tandem mass spectrometry ,Clathrin ,Mass Spectrometry ,Tools ,03 medical and health sciences ,0302 clinical medicine ,Tandem Mass Spectrometry ,Stable isotope labeling by amino acids in cell culture ,Humans ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,biology ,Proteomic Profiling ,Vesicle ,Signal transducing adaptor protein ,Computational Biology ,Clathrin-Coated Vesicles ,Cell Biology ,Cell biology ,Adaptor Proteins, Vesicular Transport ,biology.protein ,030217 neurology & neurosurgery ,Cell Division ,Chromatography, Liquid ,HeLa Cells - Abstract
A multivariate proteomics approach identified numerous new clathrin-coated vesicle proteins as well as the first AP-4 accessory protein, and also revealed how auxilin depletion causes mitotic arrest through sequestration of spindle proteins in clathrin cages., Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a “profiling” cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions.
- Published
- 2012
17. Correction: The Fifth Adaptor Protein Complex
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Joel B. Dacks, Lael D. Barlow, Daniela A. Sahlender, Jennifer Hirst, Matthew N.J. Seaman, Gabriel Casey Francisco, and Margaret S. Robinson
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General Immunology and Microbiology ,QH301-705.5 ,Section (archaeology) ,General Neuroscience ,Library science ,Signal transducing adaptor protein ,Correction ,Biology (General) ,Biology ,General Agricultural and Biological Sciences ,Futures contract ,General Biochemistry, Genetics and Molecular Biology - Abstract
The following information was missing from the Funding section: This study was supported by a grant from Alberta Innovates Technology Futures (AITF).
- Published
- 2012
18. A targeted siRNA screen to identify SNAREs required for constitutive secretion in mammalian cells
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Daniela A. Sahlender, Lisa M. Bond, David E. Gordon, and Andrew A. Peden
- Subjects
Biology ,Syntaxin 17 ,Biochemistry ,symbols.namesake ,Structural Biology ,Genetics ,Syntaxin ,Animals ,Humans ,RNA, Small Interfering ,Molecular Biology ,Qa-SNARE Proteins ,Reverse Transcriptase Polymerase Chain Reaction ,Vesicle ,Endoplasmic reticulum ,Cell Biology ,Golgi apparatus ,Membrane transport ,Flow Cytometry ,Syntaxin 3 ,Cell biology ,Transport protein ,Protein Transport ,nervous system ,symbols ,biological phenomena, cell phenomena, and immunity ,SNARE Proteins ,Signal Transduction - Abstract
The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry-based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein-specific siRNA screen (38 SNAREs, 4 SNARE-like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post-Golgi SNAREs SNAP-29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP-29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP-29-depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19-interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP-23, SNAP-25, SNAP-29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post-Golgi-specific R-SNARE in this process.
- Published
- 2010
19. Coiled-coil interactions are required for post-Golgi R-SNARE trafficking
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David E. Gordon, Daniela A. Sahlender, Andrew A. Peden, Myriam Mirza, and Jovana Jakovleska
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Coiled coil ,VAMP3 ,Chemistry ,media_common.quotation_subject ,Q-SNARE Proteins ,Scientific Report ,Golgi Apparatus ,Golgi apparatus ,Biochemistry ,Models, Biological ,Cell biology ,R-SNARE Proteins ,symbols.namesake ,Protein Transport ,Membrane protein ,Genetics ,symbols ,Animals ,Humans ,Internalization ,Molecular Biology ,media_common ,Protein Binding ,trans-Golgi Network - Abstract
The sorting of post-Golgi R-SNAREs (vesicle-associated membrane protein (VAMP)1, 2, 3, 4, 7 and 8) is still poorly understood. To address this, we developed a system to investigate their localization, trafficking and cell-surface levels. Here, we show that the distribution and internalization of VAMPs 3 and 8 are determined solely through a new conserved mechanism that uses coiled-coil interactions, and that VAMP4 does not require these interactions for its trafficking. We propose that VAMPs 3 and 8 are trafficked while in a complex with Q-SNAREs. We also show that the dileucine motif of VAMP4 is required for both its internalization and retrieval to the trans-Golgi network. However, when the dileucine motif is mutated, the construct can still be internalized potentially through coiled-coil interactions with Q-SNAREs.
- Published
- 2009
20. Sdmg1 is a conserved transmembrane protein associated with germ cell sex determination and germline-soma interactions in mice
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Daniela A. Sahlender, Andrew A. Peden, Ian R. Adams, Diana Best, and Norbert Walther
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Homeobox protein NANOG ,Male ,Cell type ,endocrine system ,Sex Differentiation ,Disorders of Sex Development ,Vesicular Transport Proteins ,Biology ,Germline ,Article ,Cell Line ,Mice ,Pregnancy ,Testis ,medicine ,Animals ,Cloning, Molecular ,Molecular Biology ,DNA Primers ,Granulosa Cells ,Sertoli Cells ,Base Sequence ,urogenital system ,Gene Expression Profiling ,Ovary ,Gene Expression Regulation, Developmental ,Membrane Proteins ,Sex Determination Processes ,Sertoli cell ,Embryonic stem cell ,Molecular biology ,medicine.anatomical_structure ,Germ Cells ,Cell culture ,Female ,RNA Interference ,Germ line development ,Germ cell ,Developmental Biology - Abstract
In mammals, the supporting cell lineage in an embryonic gonad communicates the sex-determining decision to various sexually dimorphic cell types in the developing embryo, including the germ cells. However, the molecular nature of the sex-determining signals that pass from the supporting cells to the germ cells is not well understood. We have identified a conserved transmembrane protein, Sdmg1, owing to its male-specific expression in mouse embryonic gonads. Sdmg1 is expressed in the Sertoli cells of embryonic testes from 12.5 dpc, and in granulosa cells of growing follicles in adult ovaries. In Sertoli cells, Sdmg1 is localised to endosomes, and knock-down of Sdmg1 in Sertoli cell lines causes mis-localisation of the secretory SNARE Stx2 and defects in membrane trafficking. Upregulation of Sdmg1appears to be part of a larger programme of changes to membrane trafficking pathways in embryonic Sertoli cells, and perturbing secretion in male embryonic gonads in organ culture causes male-to-female germ cell sex reversal. These data suggest that changes that occur in the cell biology of embryonic Sertoli cells may facilitate the communication of male sex-determining decisions to the germ cells during embryonic development.
- Published
- 2008
21. HIV-1 Nef-induced Down-Regulation of MHC Class I Requires AP-1 and Clathrin but Not PACS-1 and Is Impeded by AP-2
- Author
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Philippe Benaroch, Margaret S. Robinson, Nienke B. Lubben, Daniela A. Sahlender, Alison M. Motley, and Paul J. Lehner
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Adaptor Protein Complex 1 ,Blotting, Western ,Adaptor Protein Complex 2 ,Vesicular Transport Proteins ,Down-Regulation ,Major histocompatibility complex ,Clathrin ,Gene Products, nef ,Downregulation and upregulation ,MHC class I ,HLA-A2 Antigen ,Humans ,nef Gene Products, Human Immunodeficiency Virus ,RNA, Small Interfering ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,biology ,RNA ,Signal transducing adaptor protein ,Cell Biology ,Articles ,Virology ,Transport protein ,Protein Transport ,Vacuoles ,biology.protein ,HIV-1 ,HeLa Cells - Abstract
Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells. However, knocking down PACS-1 has no effect, not only on Nef-induced down-regulation of HLA-A2 but also on the localization of other proteins containing acidic cluster motifs. Surprisingly, knocking down AP-2 actually enhances Nef activity. Immuno-electron microscopy labeling of Nef-expressing cells indicates that HLA-A2 is rerouted not to the TGN, but to endosomes. In AP-2–depleted cells, more of the HLA-A2 localizes to the inner vesicles of multivesicular bodies. We propose that depleting AP-2 potentiates Nef activity by altering the membrane composition and dynamics of endosomes and causing increased delivery of HLA-A2 to a prelysosomal compartment.
- Published
- 2007
22. Functional Analysis of AP-2 α and μ2 Subunits
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Jennifer Hirst, David J. Owen, Daniela A. Sahlender, Margaret S. Robinson, Nicola Berg, Marcus J. Taylor, and Alison M. Motley
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Recombinant Fusion Proteins ,Protein subunit ,Green Fluorescent Proteins ,Biology ,Transfection ,Endocytosis ,Protein Structure, Secondary ,Adaptor Protein Complex alpha Subunits ,Mice ,Animals ,Humans ,RNA, Small Interfering ,Binding site ,Molecular Biology ,chemistry.chemical_classification ,Functional analysis ,Vesicle ,Transferrin ,Articles ,Cell Biology ,Adaptor Protein Complex mu Subunits ,Cell biology ,Protein Subunits ,chemistry ,HeLa Cells - Abstract
The AP-2 adaptor complex plays a key role in cargo recognition and clathrin-coated vesicle formation at the plasma membrane. To investigate the functions of individual binding sites and domains of the AP-2 complex in vivo, we have stably transfected HeLa cells with wild-type and mutant small interfering RNA-resistant alpha and mu2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of alpha, the phosphorylation site of mu2, or the YXXPhi binding site of mu2 impairs AP-2 function, as assayed by transferrin uptake. In contrast, removing the C-terminal appendage domain of alpha, or mutating the PtdIns(4,5)P2 binding site of mu2, has no apparent effect. However, adding a C-terminal GFP tag to alpha renders it completely nonfunctional. These findings demonstrate that there is some functional redundancy in the binding sites of the various AP-2 subunits, because no single mutation totally abolishes function. They also help to explain why GFP-tagged AP-2 never appears to leave the plasma membrane in some live cell imaging studies. Finally, they establish a new model system that can be used both for additional structure-function analyses, and as a way of testing tagged constructs for function in vivo.
- Published
- 2006
23. Uncoupling the Functions of CALM in VAMP Sorting and Clathrin-Coated Pit Formation
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Margaret S. Robinson, Sharon E. Miller, Andrew A. Peden, Patrycja Kozik, and Daniela A. Sahlender
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Gene Expression ,Golgi Apparatus ,lcsh:Medicine ,Coated Pit ,Biochemistry ,R-SNARE Proteins ,0302 clinical medicine ,Molecular Cell Biology ,RNA, Small Interfering ,lcsh:Science ,Extracellular Matrix Proteins ,0303 health sciences ,Multidisciplinary ,Vesicle ,Coated Pits, Cell-Membrane ,Flow Cytometry ,Endocytosis ,Cell biology ,Transport protein ,Protein Transport ,Monomeric Clathrin Assembly Proteins ,Cytochemistry ,Membranes and Sorting ,Signal Transduction ,Research Article ,Protein Sorting Signals ,Biology ,Clathrin ,03 medical and health sciences ,Humans ,Amino Acid Sequence ,Transport Vesicles ,030304 developmental biology ,Organelles ,Cell Membrane ,lcsh:R ,Membrane Proteins ,Proteins ,Regulatory Proteins ,Microscopy, Fluorescence ,Mutation ,biology.protein ,lcsh:Q ,Sequence Alignment ,Cytometry ,030217 neurology & neurosurgery ,HeLa Cells - Abstract
CALM (clathrin assembly lymphoid myeloid leukemia protein) is a cargo-selective adaptor for the post-Golgi R-SNAREs VAMPs 2, 3, and 8, and it also regulates the size of clathrin-coated pits and vesicles at the plasma membrane. The present study has two objectives: to determine whether CALM can sort additional VAMPs, and to investigate whether VAMP sorting contributes to CALM-dependent vesicle size regulation. Using a flow cytometry-based endocytosis efficiency assay, we demonstrate that CALM is also able to sort VAMPs 4 and 7, even though they have sorting signals for other clathrin adaptors. CALM homologues are present in nearly every eukaryote, suggesting that the CALM family may have evolved as adaptors for retrieving all post-Golgi VAMPs from the plasma membrane. Using a knockdown/rescue system, we show that wild-type CALM restores normal VAMP sorting in CALM-depleted cells, but that two non-VAMP-binding mutants do not. However, when we assayed the effect of CALM depletion on coated pit morphology, using a fluorescence microscopy-based assay, we found that the two mutants were as effective as wild-type CALM. Thus, we can uncouple the sorting function of CALM from its structural role.
- Published
- 2013
24. The fifth adaptor protein complex
- Author
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Jennifer Hirst, Margaret S. Robinson, Gabriel Casey Francisco, Daniela A. Sahlender, Matthew N.J. Seaman, Joel B. Dacks, Lael D. Barlow, Hirst, Jennifer [0000-0001-9063-8494], Seaman, Matthew [0000-0001-9916-3245], Robinson, Margaret [0000-0003-0631-0053], and Apollo - University of Cambridge Repository
- Subjects
QH301-705.5 ,Endosome ,Protein subunit ,Sequence Homology ,Endosomes ,Biology ,Clathrin ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,Humans ,Biology (General) ,Protein Structure, Quaternary ,Phylogeny ,030304 developmental biology ,0303 health sciences ,General Immunology and Microbiology ,Spastic Paraplegia, Hereditary ,General Neuroscience ,Adaptor Protein Complex Subunits ,Signal transducing adaptor protein ,16. Peace & justice ,Molecular biology ,Endocytosis ,Cell biology ,Transport protein ,Adaptor Proteins, Vesicular Transport ,Protein Transport ,Synopsis ,biology.protein ,Clathrin adaptor proteins ,Apoptosis Regulatory Proteins ,General Agricultural and Biological Sciences ,030217 neurology & neurosurgery ,HeLa Cells - Abstract
Adaptor protein (AP) complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolutionarily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.
- Published
- 2011
25. The Molecular Basis for the Endocytosis of Small R-SNAREs by the Clathrin Adaptor CALM
- Author
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Andrew A. Peden, Stephen C. Graham, Stefan Höning, Margaret S. Robinson, David J. Owen, Sharon E. Miller, and Daniela A. Sahlender
- Subjects
Models, Molecular ,Endosome ,Endocytic cycle ,Endocytosis ,Crystallography, X-Ray ,Clathrin ,General Biochemistry, Genetics and Molecular Biology ,Exocytosis ,Article ,R-SNARE Proteins ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Animals ,Humans ,Transport Vesicles ,030304 developmental biology ,0303 health sciences ,biology ,Biochemistry, Genetics and Molecular Biology(all) ,Cell Membrane ,3. Good health ,Cell biology ,Rats ,Endocytic vesicle ,Monomeric Clathrin Assembly Proteins ,biology.protein ,Clathrin adaptor proteins ,biological phenomena, cell phenomena, and immunity ,SNARE complex ,SNARE Proteins ,030217 neurology & neurosurgery ,HeLa Cells - Abstract
Summary SNAREs provide a large part of the specificity and energy needed for membrane fusion and, to do so, must be localized to their correct membranes. Here, we show that the R-SNAREs VAMP8, VAMP3, and VAMP2, which cycle between the plasma membrane and endosomes, bind directly to the ubiquitously expressed, PtdIns4,5P2-binding, endocytic clathrin adaptor CALM/PICALM. X-ray crystallography shows that the N-terminal halves of their SNARE motifs bind the CALMANTH domain as helices in a manner that mimics SNARE complex formation. Mutation of residues in the CALM:SNARE interface inhibits binding in vitro and prevents R-SNARE endocytosis in vivo. Thus, CALM:R-SNARE interactions ensure that R-SNAREs, required for the fusion of endocytic clathrin-coated vesicles with endosomes and also for subsequent postendosomal trafficking, are sorted into endocytic vesicles. CALM's role in directing the endocytosis of small R-SNAREs may provide insight into the association of CALM/PICALM mutations with growth retardation, cognitive defects, and Alzheimer's disease., Graphical Abstract Highlights ► Binding to CALM selects VAMPs 8, 3, and 2 for incorporation into endocytic CCVs ► The CALM ANTH domain binds VAMPs and PtdIns4,5P2 simultaneously ► Helical N-terminal halves of VAMP SNARE motifs displace the CALM ANTH final helix ► VAMP endocytosis is blocked by mutation of residues in the CALM:SNARE interface, CALM recognizes the SNARE motif of small R-SNARE proteins as a sorting signal to direct R-SNARE endocytosis and trafficking to the appropriate intracellular compartment while simultaneously shielding the SNARE motif from inappropriate interactions. This unique role for CALM, distinct from other clathrin adaptors, may explain the genetic association of the CALM/PICALM gene with neurological disorders.
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26. Rapid Inactivation of Proteins by Rapamycin-Induced Rerouting to Mitochondria
- Author
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Daniela A. Sahlender, Margaret S. Robinson, and Samuel D. Foster
- Subjects
Resource ,PROTEINS ,Protein subunit ,Biological Transport, Active ,Mitochondrion ,Endocytosis ,Transfection ,Clathrin ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Receptor, IGF Type 2 ,Cell Line ,Tacrolimus Binding Proteins ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Animals ,Humans ,RNA, Small Interfering ,Receptor ,Molecular Biology ,030304 developmental biology ,Sirolimus ,0303 health sciences ,biology ,Base Sequence ,Transferrin ,Signal transducing adaptor protein ,Clathrin-Coated Vesicles ,Cell Biology ,Recombinant Proteins ,Cell biology ,Mitochondria ,Transcription Factor AP-1 ,Kinetics ,FKBP ,Transcription Factor AP-2 ,Multiprotein Complexes ,biology.protein ,CELLBIO ,030217 neurology & neurosurgery ,Developmental Biology ,HeLa Cells - Abstract
Summary We have developed a method for rapidly inactivating proteins with rapamycin-induced heterodimerization. Cells were stably transfected with siRNA-resistant, FKBP-tagged subunits of the adaptor protein (AP) complexes of clathrin-coated vesicles (CCVs), together with an FKBP and rapamycin-binding domain-containing construct with a mitochondrial targeting signal. Knocking down the endogenous subunit with siRNA, and then adding rapamycin, caused the APs to be rerouted to mitochondria within seconds. Rerouting AP-2 to mitochondria effectively abolished clathrin-mediated endocytosis of transferrin. In cells with rerouted AP-1, endocytosed cation-independent mannose 6-phosphate receptor (CIMPR) accumulated in a peripheral compartment, and isolated CCVs had reduced levels of CIMPR, but normal levels of the lysosomal hydrolase DNase II. Both observations support a role for AP-1 in retrograde trafficking. This type of approach, which we call a “knocksideways,” should be widely applicable as a means of inactivating proteins with a time scale of seconds or minutes rather than days., Graphical Abstract Highlights ► We have developed a way of getting rid of proteins quickly (i.e., minutes not days) ► This approach provides new insights into the function of the coat protein AP-1
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27. Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport
- Author
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Els Pardon, Daniela A. Sahlender, Laura Kunz, Yvonne Neldner, Saša Štefanić, Iaroslav Savtchouk, Toshiharu Suzuki, Stephan Schenck, Eric R. Geertsma, Andrea Volterra, Jan Steyaert, Raimund Dutzler, Department of Bio-engineering Sciences, Structural Biology Brussels, University of Zurich, and Schenck, Stephan
- Subjects
Models, Molecular ,10078 Institute of Parasitology ,0301 basic medicine ,1303 Biochemistry ,Phage display ,Recombinant Fusion Proteins ,Green Fluorescent Proteins ,Glutamic Acid ,Nerve Tissue Proteins ,610 Medicine & health ,Biology ,Neurotransmission ,Synaptic Transmission ,Biochemistry ,Synaptic vesicle ,Mice ,03 medical and health sciences ,Peptide Library ,Membrane Transport Modulators ,10019 Department of Biochemistry ,Animals ,Humans ,Cells, Cultured ,Cerebral Cortex ,Neurons ,HEK 293 cells ,Glutamate receptor ,Central Nervous System Depressants ,food and beverages ,Biological Transport ,Single-Domain Antibodies ,Embryo, Mammalian ,Fusion protein ,Rats ,Cell biology ,Cytosol ,HEK293 Cells ,030104 developmental biology ,Vesicular Glutamate Transport Protein 1 ,570 Life sciences ,biology ,Synaptic Vesicles ,Camelids, New World ,Intracellular - Abstract
The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.
28. Computer assisted detection of axonal bouton structural plasticity in in vivo time-lapse images
- Author
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Rohan Gala, Daniel Lebrecht, Daniela A Sahlender, Anne Jorstad, Graham Knott, Anthony Holtmaat, and Armen Stepanyants
- Subjects
bouton detection ,bouton volume ,structural plasticity ,time-lapse imaging ,synaptic strength ,CLEM ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The ability to measure minute structural changes in neural circuits is essential for long-term in vivo imaging studies. Here, we propose a methodology for detection and measurement of structural changes in axonal boutons imaged with time-lapse two-photon laser scanning microscopy (2PLSM). Correlative 2PLSM and 3D electron microscopy (EM) analysis, performed in mouse barrel cortex, showed that the proposed method has low fractions of false positive/negative bouton detections (2/0 out of 18), and that 2PLSM-based bouton weights are correlated with their volumes measured in EM (r = 0.93). Next, the method was applied to a set of axons imaged in quick succession to characterize measurement uncertainty. The results were used to construct a statistical model in which bouton addition, elimination, and size changes are described probabilistically, rather than being treated as deterministic events. Finally, we demonstrate that the model can be used to quantify significant structural changes in boutons in long-term imaging experiments.
- Published
- 2017
- Full Text
- View/download PDF
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