Your browser doesn't support javascript.
loading
: 20 | 50 | 100
1 - 20 de 135
1.
PLoS Genet ; 20(5): e1011253, 2024 May.
Article En | MEDLINE | ID: mdl-38722918

Synaptic vesicle proteins (SVps) are transported by the motor UNC-104/KIF1A. We show that SVps travel in heterogeneous carriers in C. elegans neuronal processes, with some SVp carriers co-transporting lysosomal proteins (SV-lysosomes). LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 play a critical role in the sorting of SVps and lysosomal proteins away from each other at the SV-lysosomal intermediate trafficking compartment. Both SVp carriers lacking lysosomal proteins and SV-lysosomes are dependent on the motor UNC-104/KIF1A for their transport. In lrk-1 mutants, both SVp carriers and SV-lysosomes can travel in axons in the absence of UNC-104, suggesting that LRK-1 plays an important role to enable UNC-104 dependent transport of synaptic vesicle proteins. Additionally, LRK-1 acts upstream of the AP-3 complex and regulates its membrane localization. In the absence of the AP-3 complex, the SV-lysosomes become more dependent on the UNC-104-SYD-2/Liprin-α complex for their transport. Therefore, SYD-2 acts to link upstream trafficking events with the transport of SVps likely through its interaction with the motor UNC-104. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. SYD-2 acts in concert with AP complexes to ensure polarized trafficking & transport of SVps.


Adaptor Protein Complex 3 , Caenorhabditis elegans Proteins , Caenorhabditis elegans , Lysosomes , Nerve Tissue Proteins , Synaptic Vesicles , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Synaptic Vesicles/metabolism , Synaptic Vesicles/genetics , Adaptor Protein Complex 3/metabolism , Adaptor Protein Complex 3/genetics , Lysosomes/metabolism , Lysosomes/genetics , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Protein Transport , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Neurons/metabolism , Kinesins/metabolism , Kinesins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Axons/metabolism , Intercellular Signaling Peptides and Proteins
2.
Nat Neurosci ; 26(10): 1685-1700, 2023 10.
Article En | MEDLINE | ID: mdl-37723322

Neural systems encode information in the frequency of action potentials, which is then decoded by synaptic transmission. However, the rapid, synchronous release of neurotransmitters depletes synaptic vesicles (SVs), limiting release at high firing rates. How then do synapses convey information about frequency? Here, we show in mouse hippocampal neurons and slices that the adaptor protein AP-3 makes a subset of SVs that respond specifically to high-frequency stimulation. Neurotransmitter transporters slot onto these SVs in different proportions, contributing to the distinct properties of release observed at different excitatory synapses. Proteomics reveals that AP-3 targets the phospholipid flippase ATP8A1 to SVs; loss of ATP8A1 recapitulates the defect in SV mobilization at high frequency observed with loss of AP-3. The mechanism involves recruitment of synapsin by the cytoplasmically oriented phosphatidylserine translocated by ATP8A1. Thus, ATP8A1 enables the subset of SVs made by AP-3 to release at high frequency.


Adaptor Protein Complex 3 , Adenosine Triphosphatases , Phospholipids , Synaptic Transmission , Synaptic Vesicles , Animals , Mice , Phospholipids/metabolism , Synapses/metabolism , Synapsins/metabolism , Synaptic Vesicles/metabolism , Adaptor Protein Complex 3/metabolism , Adenosine Triphosphatases/metabolism
3.
J Cell Biol ; 221(5)2022 05 02.
Article En | MEDLINE | ID: mdl-35426896

The time course of signaling by peptide hormones, neural peptides, and other neuromodulators depends on their storage inside dense core vesicles (DCVs). Adaptor protein 3 (AP-3) assembles the membrane proteins that confer regulated release of DCVs and is thought to promote their trafficking from endosomes directly to maturing DCVs. We now find that regulated monoamine release from DCVs requires sorting nexin 5 (SNX5). Loss of SNX5 disrupts trafficking of the vesicular monoamine transporter (VMAT) to DCVs. The mechanism involves a role for SNX5 in retrograde transport of VMAT from endosomes to the TGN. However, this role for SNX5 conflicts with the proposed function of AP-3 in trafficking from endosomes directly to DCVs. We now identify a transient role for AP-3 at the TGN, where it associates with DCV cargo. Thus, retrograde transport from endosomes by SNX5 enables DCV assembly at the TGN by AP-3, resolving the apparent antagonism. A novel role for AP-3 at the TGN has implications for other organelles that also depend on this adaptor.


Adaptor Protein Complex 3 , Dense Core Vesicles , Endosomes , Sorting Nexins , Adaptor Protein Complex 3/metabolism , Biological Transport , Carrier Proteins/metabolism , Dense Core Vesicles/metabolism , Endosomes/metabolism , Neurotransmitter Agents/metabolism , Protein Transport , Sorting Nexins/metabolism
4.
Cells ; 10(11)2021 11 06.
Article En | MEDLINE | ID: mdl-34831286

Developmental and epileptic encephalopathies (DEE) are rare and serious neurological disorders characterized by severe epilepsy with refractory seizures and a significant developmental delay. Recently, DEE73 was linked to genetic alterations of the RNF13 gene, which convert positions 311 or 312 in the RNF13 protein from leucine to serine or proline, respectively (L311S and L312P). Using a fluorescence microscopy approach to investigate the molecular and cellular mechanisms affected by RNF13 protein variants, the current study shows that wild-type RNF13 localizes extensively with endosomes and lysosomes, while L311S and L312P do not extensively colocalize with the lysosomal marker Lamp1. Our results show that RNF13 L311S and L312P proteins affect the size of endosomal vesicles along with the temporal and spatial progression of fluorescently labeled epidermal growth factor, but not transferrin, in the endolysosomal system. Furthermore, GST-pulldown and co-immunoprecipitation show that RNF13 variants disrupt association with AP-3 complex. Knockdown of AP-3 complex subunit AP3D1 alters the lysosomal localization of wild-type RNF13 and similarly affects the size of endosomal vesicles. Importantly, our study provides a first step toward understanding the cellular and molecular mechanism altered by DEE73-associated genetic variations of RNF13.


Adaptor Protein Complex 3/metabolism , Endosomes/metabolism , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Amino Acid Motifs , Epidermal Growth Factor/metabolism , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , Lysosomes/metabolism , Protein Binding , Transferrin/metabolism , Ubiquitin-Protein Ligases/genetics
5.
Am J Respir Cell Mol Biol ; 65(5): 532-543, 2021 11.
Article En | MEDLINE | ID: mdl-34166600

Chronic obstructive pulmonary disease (COPD) is a common, complex disease and a major cause of morbidity and mortality. Although multiple genetic determinants of COPD have been implicated by genome-wide association studies (GWASs), the pathophysiological significance of these associations remains largely unknown. From a COPD protein-protein interaction network module, we selected a network path between two COPD GWAS genes for validation studies: FAM13A (family with sequence similarity 13 member A)-AP3D1-CTGF- TGFß2. We find that TGFß2, FAM13A, and AP3D1 (but not CTGF) form a cellular protein complex. Functional characterization suggests that this complex mediates the secretion of TGFß2 through an AP-3 (adaptor protein 3)-dependent pathway, with FAM13A acting as a negative regulator by targeting a late stage of this transport that involves the dissociation of coat-cargo interaction. Moreover, we find that TGFß2 is a transmembrane protein that engages the AP-3 complex for delivery to the late endosomal compartments for subsequent secretion through exosomes. These results identify a pathophysiological context that unifies the biological network role of two COPD GWAS proteins and reveal novel mechanisms of cargo transport through an intracellular pathway.


Adaptor Protein Complex 3/metabolism , Adaptor Protein Complex delta Subunits/metabolism , GTPase-Activating Proteins/metabolism , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Disease, Chronic Obstructive/metabolism , Transforming Growth Factor beta2/metabolism , Adaptor Protein Complex 3/genetics , Adaptor Protein Complex delta Subunits/genetics , Cell Line , Exosomes/metabolism , GTPase-Activating Proteins/genetics , Genome-Wide Association Study , HEK293 Cells , Humans , Protein Interaction Maps/genetics , Protein Transport , Reproducibility of Results , Transforming Growth Factor beta2/genetics
6.
Proc Natl Acad Sci U S A ; 118(20)2021 05 18.
Article En | MEDLINE | ID: mdl-33990468

Lamellar bodies (LBs) are lysosome-related organelles (LROs) of surfactant-producing alveolar type 2 (AT2) cells of the distal lung epithelium. Trafficking pathways to LBs have been understudied but are likely critical to AT2 cell homeostasis given associations between genetic defects of endosome to LRO trafficking and pulmonary fibrosis in Hermansky Pudlak syndrome (HPS). Our prior studies uncovered a role for AP-3, defective in HPS type 2, in trafficking Peroxiredoxin-6 to LBs. We now show that the P4-type ATPase ATP8A1 is sorted by AP-3 from early endosomes to LBs through recognition of a C-terminal dileucine-based signal. Disruption of the AP-3/ATP8A1 interaction causes ATP8A1 accumulation in early sorting and/or recycling endosomes, enhancing phosphatidylserine exposure on the cytosolic leaflet. This in turn promotes activation of Yes-activating protein, a transcriptional coactivator, augmenting cell migration and AT2 cell numbers. Together, these studies illuminate a mechanism whereby loss of AP-3-mediated trafficking contributes to a toxic gain-of-function that results in enhanced and sustained activation of a repair pathway associated with pulmonary fibrosis.


Adaptor Protein Complex 3/genetics , Adaptor Proteins, Signal Transducing/genetics , Adenosine Triphosphatases/genetics , Alveolar Epithelial Cells/metabolism , Hermanski-Pudlak Syndrome/genetics , Phospholipid Transfer Proteins/genetics , Pulmonary Fibrosis/genetics , Transcription Factors/genetics , Adaptor Protein Complex 3/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adenosine Triphosphatases/metabolism , Alveolar Epithelial Cells/cytology , Animals , Biological Transport , Cell Line , Cell Movement , Disease Models, Animal , Endosomes/metabolism , Female , Gene Expression Regulation , Hermanski-Pudlak Syndrome/metabolism , Hermanski-Pudlak Syndrome/pathology , Humans , Lung/metabolism , Lung/pathology , Lysosomes/metabolism , Male , Mice , Mice, Inbred C57BL , Peroxiredoxin VI/genetics , Peroxiredoxin VI/metabolism , Phosphatidylserines/metabolism , Phospholipid Transfer Proteins/metabolism , Primary Cell Culture , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/pathology , Signal Transduction , Transcription Factors/metabolism , YAP-Signaling Proteins , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism
7.
Eur J Hum Genet ; 29(8): 1312-1315, 2021 08.
Article En | MEDLINE | ID: mdl-33867526

Critically ill coronavirus disease 2019 (COVID-19) is characterized by severe cytokine storms, a hyperinflammatory condition intimately related to the development of fatal outcomes. Why some individuals seem particularly vulnerable to severe cytokine storms is still unknown. Primary immunodeficiency (PID)-related genes are inherited factors that dysregulate host inflammatory responses to infection, especially hemophagocytic lymphohistiocytosis (HLH)-related genes, established as contributors to the development of excessive cytokine storms. We analyzed the association between PID gene variants with severe cytokine storms in COVID-19. We conducted whole-exome sequencing in 233 hospitalized COVID-19 patients and identified four PID gene (UNC13D, AP3B1, RNF168, DHX58) variants were significantly enriched in COVID-19 patients experiencing severe cytokine storms. The total percentage of COVID-19 patients with variants in UNC13D or AP3B1, two typical HLH genes, was dramatically higher in high-level cytokine group than in low-level group (33.3 vs. 5.7%, P < 0.001). Germline variants in UNC13D and AP3B1 were associated with the development of severe cytokine storms, fatal outcomes in COVID-19. These findings advance the understanding of individual susceptibility to severe cytokine storms and help optimize the current management of COVID-19.


Adaptor Protein Complex 3/genetics , Adaptor Protein Complex beta Subunits/genetics , COVID-19/genetics , COVID-19/pathology , Membrane Proteins/genetics , Adaptor Protein Complex 3/metabolism , Adaptor Protein Complex beta Subunits/metabolism , Aged , COVID-19/immunology , COVID-19/metabolism , Cytokine Release Syndrome/genetics , Humans , Lymphohistiocytosis, Hemophagocytic/genetics , Membrane Proteins/metabolism , Middle Aged
8.
Autophagy ; 17(4): 837-839, 2021 04.
Article En | MEDLINE | ID: mdl-32543335

The common view of art and science as polar opposites along the educational spectrum can sometimes mask the degree to which they inform one another. In fact, art can also serve as a way to foster interest in querying the natural world, ultimately allowing us to recruit highly creative individuals to join the scientific community. We have experienced firsthand how cellular processes, such as autophagy, which are not usually highlighted or described in detail in foundational cell biology textbooks, have served as an on-ramp for artists at the undergraduate and high school levels in the context of scientific research and science outreach, respectively. We discuss our experiences in this article and highlight the ways in which art's many dimensions are well-suited, not only for forging connections between scientists and their communities but also for encouraging creativity in the way scientists engage with visually and conceptually complex phenomena, such as autophagy.Abbreviations: AP-3: adaptor protein complex 3; Atg27: autophagy related protein 27; STEAM: science, technology, engineering, arts, and mathematics; STEM: science, technology, engineering and math.


Autophagy , Science , Adaptor Protein Complex 3/metabolism , Autophagosomes/metabolism , Creativity , Humans , Lipids/biosynthesis
9.
Proc Natl Acad Sci U S A ; 116(36): 18132-18141, 2019 09 03.
Article En | MEDLINE | ID: mdl-31431522

Stomatal movement is essential for plant growth. This process is precisely regulated by various cellular activities in guard cells. F-actin dynamics and vacuole morphology are both involved in stomatal movement. The sorting of cargoes by clathrin adaptor protein (AP) complexes from the Golgi to the vacuole is critical for establishing a normal vacuole morphology. In this study, we demonstrate that the medium subunit of the AP3 complex (AP3M) binds to and severs actin filaments in vitro and that it participates in the sorting of cargoes (such as the sucrose exporter SUC4) to the tonoplast, and thereby regulates stomatal closure in Arabidopsis thaliana Defects in AP3 or SUC4 led to more rapid water loss and delayed stomatal closure, as well as hypersensitivity to drought stress. In ap3m mutants, the F-actin status was altered compared to the wild type, and the sorted cargoes failed to localize to the tonoplast. AP3M contains a previously unidentified F-actin binding domain that is conserved in AP3M homologs in both plants and animals. Mutations in the F-actin binding domain of AP3M abolished its F-actin binding activity in vitro, leading to an aberrant vacuole morphology and reduced levels of SUC4 on the tonoplast in guard cells. Our findings indicate that the F-actin binding activity of AP3M is required for the precise localization of AP3-dependent cargoes to the tonoplast and for the regulation of vacuole morphology in guard cells during stomatal closure.


Actin Cytoskeleton/metabolism , Adaptor Protein Complex 3/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Plant Stomata/metabolism , Vacuoles/metabolism , Actin Cytoskeleton/genetics , Adaptor Protein Complex 3/genetics , Arabidopsis/cytology , Arabidopsis Proteins/genetics , Plant Stomata/cytology , Plant Stomata/genetics , Vacuoles/genetics
10.
Neurology ; 93(10): e954-e963, 2019 09 03.
Article En | MEDLINE | ID: mdl-31371564

OBJECTIVE: To describe phenotypes, treatment response, and outcomes of autoimmunity targeting a synaptic vesicle coat protein, the neuronal (B2) form of adaptor protein-3 (AP3). METHODS: Archived serum and CSF specimens (from 616,025 screened) harboring unclassified synaptic antibodies mimicking amphiphysin-immunoglobulin G (IgG) on tissue-based indirect immunofluorescence assay (IFA) were re-evaluated for novel IgG staining patterns. Autoantigens were identified by western blot and mass spectrometry. Recombinant western blot and cell-binding assay (CBA) were used to confirm antigen specificity. Clinical data were obtained retrospectively. RESULTS: Serum (10) and CSF (6) specimens of 10 patients produced identical IFA staining patterns throughout mouse nervous system tissues, most prominently in cerebellum (Purkinje neuronal perikarya, granular layer synapses, and dentate regions), spinal cord gray matter, dorsal root ganglia, and sympathetic ganglia. The antigen revealed by mass spectrometry analysis and confirmed by recombinant assays (western blot and CBA) was AP3B2 in all. Of 10 seropositive patients, 6 were women; median symptom onset age was 42 years (range 24-58). Clinical information was available for 9 patients, all with subacute onset and rapidly progressive gait ataxia. Neurologic manifestations were myeloneuropathy (3), peripheral sensory neuropathy (2), cerebellar ataxia (2), and spinocerebellar ataxia (2). Five patients received immunotherapy; none improved, but they did not worsen over the follow-up period (median 36 months; range 3-94). Two patients (both with cancer) died. One of 50 control sera was positive by western blot only (but not by IFA or CBA). CONCLUSION: AP3B2 (previously named ß-neuronal adaptin-like protein) autoimmunity appears rare, is accompanied by ataxia (sensory or cerebellar), and is potentially treatable.


Adaptor Protein Complex 3/metabolism , Adaptor Protein Complex beta Subunits/metabolism , Autoimmunity/physiology , Gait Disorders, Neurologic/diagnostic imaging , Gait Disorders, Neurologic/metabolism , Immunoglobulin G/metabolism , Adult , Animals , Autoantibodies/blood , Autoantibodies/cerebrospinal fluid , Biomarkers/blood , Biomarkers/cerebrospinal fluid , Cells, Cultured , Cerebellar Ataxia/diagnostic imaging , Cerebellar Ataxia/metabolism , Female , Humans , Male , Mice , Middle Aged , Rats
11.
Front Immunol ; 10: 1449, 2019.
Article En | MEDLINE | ID: mdl-31293596

Adaptor proteins contribute to the selection, differentiation and activation of natural killer T (NKT) cells, an innate(-like) lymphocyte population endowed with powerful immunomodulatory properties. Distinct from conventional T lymphocytes NKT cells preferentially home to the liver, undergo a thymic maturation and differentiation process and recognize glycolipid antigens presented by the MHC class I-like molecule CD1d on antigen presenting cells. NKT cells express a semi-invariant T cell receptor (TCR), which combines the Vα14-Jα18 chain with a Vß2, Vß7, or Vß8 chain in mice and the Vα24 chain with the Vß11 chain in humans. The avidity of interactions between their TCR, the presented glycolipid antigen and CD1d govern the selection and differentiation of NKT cells. Compared to TCR ligation on conventional T cells engagement of the NKT cell TCR delivers substantially stronger signals, which trigger the unique NKT cell developmental program. Furthermore, NKT cells express a panoply of primarily inhibitory NK cell receptors (NKRs) that control their self-reactivity and avoid autoimmune activation. Adaptor proteins influence NKT cell biology through the integration of TCR, NKR and/or SLAM (signaling lymphocyte-activation molecule) receptor signals or the variation of CD1d-restricted antigen presentation. TCR and NKR ligation engage the SH2 domain-containing leukocyte protein of 76kDa slp-76 whereas the SLAM associated protein SAP serves as adaptor for the SLAM receptor family. Indeed, the selection and differentiation of NKT cells selectively requires co-stimulation via SLAM receptors. Furthermore, SAP deficiency causes X-linked lymphoproliferative disease with multiple immune defects including a lack of circulating NKT cells. While a deletion of slp-76 leads to a complete loss of all peripheral T cell populations, mutations in the SH2 domain of slp-76 selectively affect NKT cell biology. Furthermore, adaptor proteins influence the expression and trafficking of CD1d in antigen presenting cells and subsequently selection and activation of NKT cells. Adaptor protein complex 3 (AP-3), for example, is required for the efficient presentation of glycolipid antigens which require internalization and processing. Thus, our review will focus on the complex contribution of adaptor proteins to the delivery of TCR, NKR and SLAM receptor signals in the unique biology of NKT cells and CD1d-restricted antigen presentation.


Adaptor Proteins, Signal Transducing/immunology , Antigens, CD1d/immunology , Natural Killer T-Cells/immunology , Phosphoproteins/immunology , Receptors, Antigen, T-Cell/immunology , Receptors, Natural Killer Cell/immunology , Signaling Lymphocytic Activation Molecule Family/immunology , Adaptor Protein Complex 3/immunology , Adaptor Protein Complex 3/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antigen Presentation/immunology , Antigens, CD1d/metabolism , Humans , Lymphocyte Activation/immunology , Mice , Natural Killer T-Cells/metabolism , Phosphoproteins/metabolism , Receptors, Antigen, T-Cell/metabolism , Receptors, Natural Killer Cell/metabolism , Signaling Lymphocytic Activation Molecule Family/metabolism
12.
Mol Biol Cell ; 30(18): 2422-2434, 2019 08 15.
Article En | MEDLINE | ID: mdl-31268833

Planar cell polarity (PCP) regulates coordinated cellular polarity among neighboring cells to establish a polarity axis parallel to the plane of the tissue. Disruption in PCP results in a range of developmental anomalies and diseases. A key feature of PCP is the polarized and asymmetric localization of several membrane PCP proteins, which is essential to establish the polarity axis to orient cells coordinately. However, the machinery that regulates the asymmetric partition of PCP proteins remains largely unknown. In the present study, we show Van gogh-like 2 (Vangl2) in early and recycling endosomes as made evident by colocalization with diverse endosomal Rab proteins. Vangl2 biochemically interacts with adaptor protein-3 complex (AP-3). Using short hairpin RNA knockdown, we found that Vangl2 subcellular localization was modified in AP-3-depleted cells. Moreover, Vangl2 membrane localization within the cochlea is greatly reduced in AP-3-deficient mocha mice, which exhibit profound hearing loss. In inner ears from AP-3-deficient mocha mice, we observed PCP-dependent phenotypes, such as misorientation and deformation of hair cell stereociliary bundles and disorganization of hair cells characteristic of defects in convergent extension that is driven by PCP. These findings demonstrate a novel role of AP-3-mediated sorting mechanisms in regulating PCP proteins.


Nerve Tissue Proteins/metabolism , Adaptor Protein Complex 3/metabolism , Animals , Cell Movement/physiology , Cell Polarity/physiology , Ear, Inner/cytology , Ear, Inner/metabolism , Endosomes/genetics , Endosomes/metabolism , Hair Cells, Auditory/metabolism , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Plakins/metabolism , Protein Transport , Transcription Factors/metabolism
13.
Neuron ; 102(4): 786-800.e5, 2019 05 22.
Article En | MEDLINE | ID: mdl-31003725

In contrast to temporal coding by synaptically acting neurotransmitters such as glutamate, neuromodulators such as monoamines signal changes in firing rate. The two modes of signaling have been thought to reflect differences in release by different cells. We now find that midbrain dopamine neurons release glutamate and dopamine with different properties that reflect storage in different synaptic vesicles. The vesicles differ in release probability, coupling to presynaptic Ca2+ channels and frequency dependence. Although previous work has attributed variation in these properties to differences in location or cytoskeletal association of synaptic vesicles, the release of different transmitters shows that intrinsic differences in vesicle identity drive different modes of release. Indeed, dopamine but not glutamate vesicles depend on the adaptor protein AP-3, revealing an unrecognized linkage between the pathway of synaptic vesicle recycling and the properties of exocytosis. Storage of the two transmitters in different vesicles enables the transmission of distinct signals.


Adaptor Protein Complex 3/metabolism , Calcium Channels/metabolism , Dopamine/metabolism , Dopaminergic Neurons/metabolism , Exocytosis , Glutamic Acid/metabolism , Synaptic Vesicles/metabolism , Animals , Mesencephalon/cytology , Mice , Neurons/metabolism , Neurotransmitter Agents/metabolism
14.
Reprod Fertil Dev ; 31(5): 972-982, 2019 Apr.
Article En | MEDLINE | ID: mdl-30786955

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder in humans and mice. Pale ear (ep) and pearl (pe) mice, bearing mutations in the biogenesis of lysosomal organelles complex 3 subunit 1 (Hps1) and adaptor-related protein complex 3, beta 1 subunit (Ap3b1) genes respectively, are mouse models of human HPS Type 1 (HPS1) and Type 2 (HPS2) respectively. In the present study we investigated and compared the reduced fertilities of ep and pe male mice. Both ep and pe males exhibited lower abilities to impregnate C57BL/6J (B6) females, and B6 females mated with ep males produced smaller litters than those mated with pe males. Delayed testis development, reduced sperm count and lower testosterone concentrations were observed in the pe but not ep male mice. However, the reduction in sperm motility was greater in ep than pe males, likely due to the mitochondrial and fibrous sheath abnormalities observed by electron microscopy in the sperm tails of ep males. Together, the results indicate that the Hps1 and Ap3b1 genes play distinct roles in male reproductive system development and spermatogenesis in mice, even though ep and pe males share common phenotypes, including reduced lysosomes in Sertoli cells and dislocated Zn2+ in sperm heads.


Adaptor Protein Complex 3/metabolism , Adaptor Protein Complex beta Subunits/metabolism , Fertility/physiology , Lysosomes/metabolism , Membrane Proteins/metabolism , Spermatogenesis/physiology , Adaptor Protein Complex 3/genetics , Adaptor Protein Complex beta Subunits/genetics , Animals , Disease Models, Animal , Female , Litter Size , Male , Membrane Proteins/genetics , Mice , Mitochondria/metabolism , Sertoli Cells/metabolism , Spermatozoa/metabolism , Testis/metabolism , Testosterone/blood , Zinc/metabolism
15.
Haematologica ; 104(10): 2091-2099, 2019 10.
Article En | MEDLINE | ID: mdl-30630984

Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the adaptor protein complex 3 ß1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade body maturation in endothelial cells. An ex vivo patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1 Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1-/- endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2+-mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 ß1, also the µ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8-/-endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8.


Adaptor Protein Complex 3 , Adaptor Protein Complex beta Subunits , Endothelial Cells , Exocytosis , Hermanski-Pudlak Syndrome , R-SNARE Proteins/metabolism , Weibel-Palade Bodies , Adaptor Protein Complex 3/genetics , Adaptor Protein Complex 3/metabolism , Adaptor Protein Complex beta Subunits/genetics , Adaptor Protein Complex beta Subunits/metabolism , Calcium Signaling , Cells, Cultured , Endothelial Cells/metabolism , Endothelial Cells/pathology , Hermanski-Pudlak Syndrome/genetics , Hermanski-Pudlak Syndrome/metabolism , Hermanski-Pudlak Syndrome/pathology , Humans , Mutation , Protein Transport , R-SNARE Proteins/genetics , Weibel-Palade Bodies/genetics , Weibel-Palade Bodies/metabolism , Weibel-Palade Bodies/pathology
16.
Biochem Biophys Res Commun ; 507(1-4): 519-525, 2018 12 09.
Article En | MEDLINE | ID: mdl-30458990

The regulated secretory pathway is a specialized form of protein secretion found in endocrine and neuroendocrine cell types. Pro-opiomelanocortin (POMC) is a pro-hormone that utilizes this pathway to be trafficked to dense core secretory granules (DCSGs). Within this organelle, POMC is processed to multiple bioactive hormones that play key roles in cellular physiology. However, the complete set of cellular membrane trafficking proteins that mediate the correct sorting of POMC to DCSGs remain unknown. Here, we report the roles of the phosphofurin acidic cluster sorting protein - 1 (PACS-1) and the clathrin adaptor protein 1 (AP-1) in the targeting of POMC to DCSGs. Upon knockdown of PACS-1 and AP-1, POMC is readily secreted into the extracellular milieu and fails to be targeted to DCSGs.


Adaptor Protein Complex 1/metabolism , Adrenocorticotropic Hormone/metabolism , Secretory Pathway , Vesicular Transport Proteins/metabolism , Adaptor Protein Complex 3/metabolism , Animals , Cell Line , Lysosomes/metabolism , Mice , Pro-Opiomelanocortin/metabolism , Protein Binding
17.
Mol Biol Cell ; 29(17): 2055-2068, 2018 08 15.
Article En | MEDLINE | ID: mdl-29949458

Transport of late endosomes and lysosome-related organelles (LE/LROs) in axons is essential for supplying synaptic cargoes and for removing damaged macromolecules. Defects in this system are implicated in a range of human neurodegenerative and neurodevelopmental disorders. The findings reported here identify a novel mechanism regulating LE/LRO transport based on the coordinated coupling of microtubule motors and vesicle coat proteins to the neuron-enriched, transmembrane protein calcyon (Caly). We found that the cytoplasmic C-terminus of Caly pulled down proteins involved in microtubule-dependent transport (DIC, KIF5A, p150Glued, Lis1) and organelle biogenesis (AP-1 and AP-3) from the brain. In addition, RNA interference-mediated knockdown of Caly increased the percentage of static LE/LROs labeled by LysoTracker in cultured dorsal root ganglion axons. In contrast, overexpression of Caly stimulated movement of organelles positive for LysoTracker or the AP-3 cargo GFP-PI4KIIα. However, a Caly mutant (ATEA) that does not bind AP-3 was unable to pull down motor proteins from brain, and expression of the ATEA mutant failed to increase either LE/LRO flux or levels of associated dynein. Taken together, these data support the hypothesis that Caly is a multifunctional scaffolding protein that regulates axonal transport of LE/LROs by coordinately interacting with motor and vesicle coat proteins.


Adaptor Protein Complex 3/metabolism , Axons/metabolism , Membrane Proteins/metabolism , Microtubules/metabolism , Molecular Motor Proteins/metabolism , Nerve Tissue Proteins/metabolism , Organelles/metabolism , Animals , Cell Line , Dyneins/metabolism , Endosomes/metabolism , Lysosomes/metabolism , Membrane Proteins/chemistry , Mice, Inbred C57BL , Protein Binding , Protein Interaction Domains and Motifs , Rats , rab5 GTP-Binding Proteins
18.
Plant Physiol ; 177(1): 216-225, 2018 05.
Article En | MEDLINE | ID: mdl-29523712

Pollen tube growth is an essential step for successful plant reproduction. Vacuolar trafficking and dynamic organization are important for pollen tube growth; however, the key proteins involved in these processes are not well understood. Here, we report that the ADAPTOR PROTEIN-3 (AP-3) complex and its tonoplast cargo PROTEIN S-ACYL TRANSFERASE10 (PAT10) are critical for pollen tube growth in Arabidopsis (Arabidopsis thaliana). AP-3 is a heterotetrameric protein complex consisting of four subunits, δ, ß, µ, and σ. AP-3 regulates tonoplast targeting of several cargoes, such as PAT10. We show that functional loss of any of the four AP-3 subunits reduces plant fertility. In ap-3 mutants, pollen development was normal but pollen tube growth was compromised, leading to reduced male transmission. Functional loss of PAT10 caused a similar reduction in pollen tube growth, suggesting that the tonoplast association of PAT10 mediated by AP-3 is crucial for this process. Indeed, the Ca2+ gradient during pollen tube growth was reduced significantly due to AP-3 loss of function, consistent with the abnormal targeting of CALCINUERIN B-LIKE2 (CBL2) and CBL3, whose tonoplast association depends on PAT10. Furthermore, we show that the pollen tubes of ap-3 mutants have vacuoles with simplified tubules and bulbous structures, indicating that AP-3 affects vacuolar organization. Our results demonstrate a role for AP-3 in plant reproduction and provide insights into the role of vacuoles in polarized cell growth.


Adaptor Protein Complex 3/metabolism , Arabidopsis/growth & development , Pollen Tube/growth & development , Vacuoles/metabolism , Acyltransferases/genetics , Acyltransferases/metabolism , Adaptor Protein Complex 3/genetics , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Calcium Signaling/genetics , Mutation , Plants, Genetically Modified , Pollen/genetics , Pollen/growth & development , Pollen Tube/cytology , Pollen Tube/metabolism , Seeds/genetics , Seeds/growth & development
19.
Dev Biol ; 436(1): 66-74, 2018 04 01.
Article En | MEDLINE | ID: mdl-29476722

A deficit of GABA (γ-aminobutyric acid) transmission will lead to epilepsy and other cognitive disorders. Recent evidence has shown that neuronal miRNAs affect various synapses, including GABAergic synapses. However, the miRNAs that control GABAergic synapses remain not fully understood. Here, we identified miR-51, a member of Caenorhabditis elegans miR-99/100 family, as a key regulator of GABAergic synapses. Loss of mir-51 increased PTZ (Pentylenetetrazole) and aldicarb hypersensitivities, and decreased the number of GABAergic synapses and abundance of GABAA receptors. A Rab guaninenucleotide exchange factor (GEF) GLO-4, a well-known component in lysosomal trafficking-related GLO-4/GLO-1/AP-3 (GLO/AP-3) pathway, was discovered to be the direct target of miR-51. Rescue experiments showed that GLO-4 expressed in GABAergic motor neurons functioned as a suppressor of miR-51. Disruption of glo-1 or AP-3 gene apm-3 attenuated the defects of GABAergic synapse in mir-51 mutants, suggesting miR-51 regulated GABAergic synapses through GLO/AP-3 pathway. The present study implies the essential roles of miRNAs on the nervous pathologies characterized by mis-regulated GABA signaling, such as epilepsy.


Caenorhabditis elegans Proteins/metabolism , GABAergic Neurons/metabolism , Guanine Nucleotide Exchange Factors/metabolism , MicroRNAs/metabolism , Protein Transport/genetics , Adaptor Protein Complex 3/metabolism , Animals , Caenorhabditis elegans/genetics , Cell Culture Techniques , GABAergic Neurons/physiology , Lysosomes/metabolism , Lysosomes/physiology , Protein Transport/physiology , Signal Transduction/genetics , Synapses/metabolism , Synaptic Transmission/physiology
20.
PLoS One ; 12(10): e0184629, 2017.
Article En | MEDLINE | ID: mdl-29028839

Human Respiratory Syncytial Virus (HRSV) is a leading cause of bronchopneumonia in infants and the elderly. To date, knowledge of viral and host protein interactions within HRSV is limited and are critical areas of research. Here, we show that HRSV Matrix (M) protein interacts with the cellular adaptor protein complex 3 specifically via its medium subunit (AP-3Mu3A). This novel protein-protein interaction was first detected via yeast-two hybrid screen and was further confirmed in a mammalian system by immunofluorescence colocalization and co-immunoprecipitation. This novel interaction is further substantiated by the presence of a known tyrosine-based adaptor protein MU subunit sorting signal sequence, YXXФ: where Ф is a bulky hydrophobic residue, which is conserved across the related RSV M proteins. Analysis of point-mutated HRSV M derivatives indicated that AP-3Mu3A- mediated trafficking is contingent on the presence of the tyrosine residue within the YXXL sorting sequence at amino acids 197-200 of the M protein. AP-3Mu3A is up regulated at 24 hours post-infection in infected cells versus mock-infected HEp2 cells. Together, our data suggests that the AP-3 complex plays a critical role in the trafficking of HRSV proteins specifically matrix in epithelial cells. The results of this study add new insights and targets that may lead to the development of potential antivirals and attenuating mutations suitable for candidate vaccines in the future.


Adaptor Protein Complex 3/metabolism , Respiratory Syncytial Virus, Human/metabolism , Viral Matrix Proteins/metabolism , Adaptor Protein Complex 3/chemistry , Amino Acid Motifs , Amino Acid Sequence , Conserved Sequence , HeLa Cells , Humans , Protein Binding , Protein Stability , Protein Subunits/chemistry , Protein Subunits/metabolism , Protein Transport , Respiratory Syncytial Virus, Human/physiology , Up-Regulation , Viral Matrix Proteins/chemistry , Virus Assembly
...