UNC-11, a Caenorhabditis elegans AP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second,unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.

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Roles of BLOC-1 and Adaptor Protein-3 Complexes in Cargo Sorting to Synaptic Vesicles

Molecular Biology of the Cell ◽

10.1091/mbc.e08-05-0456 ◽

2009 ◽

Vol 20 (5) ◽

pp. 1441-1453 ◽

Cited By ~ 61

Author(s):

Karen Newell-Litwa ◽

Gloria Salazar ◽

Yoland Smith ◽

Victor Faundez

Keyword(s):

Membrane Proteins ◽

Synaptic Vesicle ◽

Synaptic Vesicles ◽

Adaptor Protein ◽

Protein Composition ◽

Organelle Biogenesis ◽

Vesicle Membrane ◽

Early Endosomes ◽

Lysosomal Sorting ◽

Lysosome Related Organelles

Neuronal lysosomes and their biogenesis mechanisms are primarily thought to clear metabolites and proteins whose abnormal accumulation leads to neurodegenerative disease pathology. However, it remains unknown whether lysosomal sorting mechanisms regulate the levels of membrane proteins within synaptic vesicles. Using high-resolution deconvolution microscopy, we identified early endosomal compartments where both selected synaptic vesicle and lysosomal membrane proteins coexist with the adaptor protein complex 3 (AP-3) in neuronal cells. From these early endosomes, both synaptic vesicle membrane proteins and characteristic AP-3 lysosomal cargoes can be similarly sorted to brain synaptic vesicles and PC12 synaptic-like microvesicles. Mouse knockouts for two Hermansky–Pudlak complexes involved in lysosomal biogenesis from early endosomes, the ubiquitous isoform of AP-3 (Ap3b1−/−) and muted, defective in the biogenesis of lysosome-related organelles complex 1 (BLOC-1), increased the content of characteristic synaptic vesicle proteins and known AP-3 lysosomal proteins in isolated synaptic vesicle fractions. These phenotypes contrast with those of the mouse knockout for the neuronal AP-3 isoform involved in synaptic vesicle biogenesis (Ap3b2−/−), in which the content of select proteins was reduced in synaptic vesicles. Our results demonstrate that lysosomal and lysosome-related organelle biogenesis mechanisms regulate steady-state synaptic vesicle protein composition from shared early endosomes.

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AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis

eLife ◽

10.7554/elife.00190 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 39

Author(s):

Mingyu Gu ◽

Qiang Liu ◽

Shigeki Watanabe ◽

Lin Sun ◽

Gunther Hollopeter ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Synaptic Vesicle ◽

Synaptic Vesicles ◽

Nematode Caenorhabditis Elegans ◽

Α Subunit ◽

Clathrin Adaptor ◽

Simultaneous Loss

The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes.

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The AP2 clathrin adaptor protein complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e14-06-1048 ◽

2015 ◽

Vol 26 (10) ◽

pp. 1887-1900 ◽

Cited By ~ 5

Author(s):

Steven D. Garafalo ◽

Eric S. Luth ◽

Benjamin J. Moss ◽

Michael I. Monteiro ◽

Emily Malkin ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Nerve Cord ◽

Ventral Nerve Cord ◽

Secretory Pathway ◽

Adaptor Protein ◽

Intracellular Compartments ◽

Clathrin Adaptor ◽

Strength And Plasticity

Regulation of glutamate receptor (GluR) abundance at synapses by clathrin-mediated endocytosis can control synaptic strength and plasticity. We take advantage of viable, null mutations in subunits of the clathrin adaptor protein 2 (AP2) complex in Caenorhabditis elegans to characterize the in vivo role of AP2 in GluR trafficking. In contrast to our predictions for an endocytic adaptor, we found that levels of the GluR GLR-1 are decreased at synapses in the ventral nerve cord (VNC) of animals with mutations in the AP2 subunits APM-2/μ2, APA-2/α, or APS-2/σ2. Rescue experiments indicate that APM-2/μ2 functions in glr-1–expressing interneurons and the mature nervous system to promote GLR-1 levels in the VNC. Genetic analyses suggest that APM-2/μ2 acts upstream of GLR-1 endocytosis in the VNC. Consistent with this, GLR-1 accumulates in cell bodies of apm-2 mutants. However, GLR-1 does not appear to accumulate at the plasma membrane of the cell body as expected, but instead accumulates in intracellular compartments including Syntaxin-13– and RAB-14–labeled endosomes. This study reveals a novel role for the AP2 clathrin adaptor in promoting the abundance of GluRs at synapses in vivo, and implicates AP2 in the regulation of GluR trafficking at an early step in the secretory pathway.

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Recent Breakthroughs in Neurotransmitter Release: Paradigm for Regulated Exocytosis?

Physiology ◽

10.1152/physiologyonline.1995.10.1.42 ◽

1995 ◽

Vol 10 (1) ◽

pp. 42-46

Author(s):

G Thiel

Keyword(s):

Synaptic Vesicle ◽

Brain Function ◽

Neurotransmitter Release ◽

Synaptic Vesicles ◽

Vesicle Trafficking ◽

Regulated Exocytosis ◽

Intracellular Vesicle ◽

Vesicle Cycle ◽

Synaptic Vesicle Cycle

Synaptic vesicles play a fundamental role in brain function by mediating the release of neurotransmitters. Neurons do not use an entirely unique secretion apparatus but rather a modification of the general secretion machinery. Moreover, the synaptic vesicle cycle has many similarities with intracellular vesicle trafficking pathways.

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Neurotransmitter Release

10.1093/med/9780190237493.003.0011 ◽

2017 ◽

Author(s):

Peggy Mason

Keyword(s):

Plasma Membrane ◽

Synaptic Vesicle ◽

Active Zone ◽

Neurotransmitter Release ◽

Synaptic Vesicles ◽

Synaptic Cleft ◽

Fusion Pore ◽

Synaptic Membrane ◽

Specialized Region ◽

Synaptic Vesicle Fusion

The biochemical and physiological processes of neurotransmitter release from an active zone, a specialized region of synaptic membrane, are examined. Synaptic vesicles containing neurotransmitters are docked at the active zone and then primed for release by SNARE complexes that bring them into extreme proximity to the plasma membrane. Entry of calcium ions through voltage-gated calcium channels triggers synaptic vesicle fusion with the synaptic terminal membrane and the consequent diffusion of neurotransmitter into the synaptic cleft. Release results when the fusion pore bridging the synaptic vesicle and plasma membrane widens and neurotransmitter from the inside of the synaptic vesicle diffuses into the synaptic cleft. Membrane from the active zone membrane is endocytosed, and synaptic vesicle proteins are then reassembled into recycled synaptic vesicles, allowing for more rounds of neurotransmitter release.

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Botulinum Neurotoxin a Blocks Synaptic Vesicle Exocytosis but Not Endocytosis at the Nerve Terminal

The Journal of Cell Biology ◽

10.1083/jcb.147.6.1249 ◽

1999 ◽

Vol 147 (6) ◽

pp. 1249-1260 ◽

Cited By ~ 59

Author(s):

Elaine A. Neale ◽

Linda M. Bowers ◽

Min Jia ◽

Karen E. Bateman ◽

Lura C. Williamson

Keyword(s):

Synaptic Vesicle ◽

Nerve Terminal ◽

Neurotransmitter Release ◽

Synaptic Vesicles ◽

Botulinum Neurotoxin ◽

Vesicle Membrane ◽

Botulinum Neurotoxin A ◽

Synaptic Vesicle Exocytosis ◽

Vesicle Exocytosis ◽

Botulinum Neurotoxins

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K+-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K+ depolarization, in the presence of Ca2+, triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A–blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca2+ is required for synaptic vesicle membrane retrieval.

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μ2 adaptin facilitates but is not essential for synaptic vesicle recycling in Caenorhabditis elegans

The Journal of Cell Biology ◽

10.1083/jcb.200806088 ◽

2008 ◽

Vol 183 (5) ◽

pp. 881-892 ◽

Cited By ~ 30

Author(s):

Mingyu Gu ◽

Kim Schuske ◽

Shigeki Watanabe ◽

Qiang Liu ◽

Paul Baum ◽

...

Keyword(s):

Nervous System ◽

Caenorhabditis Elegans ◽

Synaptic Vesicle ◽

Synaptic Vesicles ◽

Evoked Responses ◽

Nematode Caenorhabditis Elegans ◽

Synaptic Vesicle Recycling ◽

Specific Loss ◽

Vesicle Recycling ◽

Cargo Proteins

Synaptic vesicles must be recycled to sustain neurotransmission, in large part via clathrin-mediated endocytosis. Clathrin is recruited to endocytic sites on the plasma membrane by the AP2 adaptor complex. The medium subunit (μ2) of AP2 binds to cargo proteins and phosphatidylinositol-4,5-bisphosphate on the cell surface. Here, we characterize the apm-2 gene (also called dpy-23), which encodes the only μ2 subunit in the nematode Caenorhabditis elegans. APM-2 is highly expressed in the nervous system and is localized to synapses; yet specific loss of APM-2 in neurons does not affect locomotion. In apm-2 mutants, clathrin is mislocalized at synapses, and synaptic vesicle numbers and evoked responses are reduced to 60 and 65%, respectively. Collectively, these data suggest AP2 μ2 facilitates but is not essential for synaptic vesicle recycling.

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The AP-3 Complex Required for Endosomal Synaptic Vesicle Biogenesis is Associated with a Casein Kinase Ια-Like Isoform

Molecular Biology of the Cell ◽

10.1091/mbc.11.8.2591 ◽

2000 ◽

Vol 11 (8) ◽

pp. 2591-2604 ◽

Cited By ~ 49

Author(s):

Victor V. Faundez ◽

Regis B. Kelly

Keyword(s):

Synaptic Vesicle ◽

Synaptic Vesicles ◽

Enzymatic Reaction ◽

Casein Kinase ◽

Small Gtpase ◽

Casein Kinase I ◽

Vesicle Membranes ◽

Vesicle Biogenesis ◽

Hinge Domain

The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes ( Faundez et al., 1998 ). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15°C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the β3 subunit of the complex by a kinase similar to casein kinase 1α. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the β3A and β3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes.

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A new life for an old pump: V-ATPase and neurotransmitter release

The Journal of Cell Biology ◽

10.1083/jcb.201403040 ◽

2014 ◽

Vol 205 (1) ◽

pp. 7-9 ◽

Cited By ~ 11

Author(s):

Stefano Vavassori ◽

Andreas Mayer

Keyword(s):

Plasma Membrane ◽

Complex Formation ◽

Membrane Fusion ◽

Synaptic Vesicle ◽

Neurotransmitter Release ◽

Synaptic Vesicles ◽

Snare Complex ◽

Spontaneous Release ◽

Synaptic Vesicle Fusion ◽

Snare Complex Formation

Neurons fire by releasing neurotransmitters via fusion of synaptic vesicles with the plasma membrane. Fusion can be evoked by an incoming signal from a preceding neuron or can occur spontaneously. Synaptic vesicle fusion requires the formation of trans complexes between SNAREs as well as Ca2+ ions. Wang et al. (2014. J. Cell Biol. http://dx.doi.org/jcb.201312109) now find that the Ca2+-binding protein Calmodulin promotes spontaneous release and SNARE complex formation via its interaction with the V0 sector of the V-ATPase.

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Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor

The Journal of Cell Biology ◽

10.1083/jcb.200405032 ◽

2004 ◽

Vol 167 (2) ◽

pp. 293-302 ◽

Cited By ~ 75

Author(s):

Fubito Nakatsu ◽

Motohiro Okada ◽

Fumiaki Mori ◽

Noriko Kumazawa ◽

Hiroto Iwasa ◽

...

Keyword(s):

Synaptic Vesicles ◽

Neuronal Excitability ◽

Critical Role ◽

Physiological Role ◽

Adaptor Protein ◽

Epileptic Seizures ◽

Gaba Release ◽

Long Term Potentiation ◽

Clathrin Adaptor ◽

And Function

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking μ3B, a subunit of AP-3B. μ3B−/− mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of γ-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in μ3B−/− mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.

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