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1.
Biochem J ; 426(1): 55-64, 2010 Jan 27.
Article in English | MEDLINE | ID: mdl-19922412

ABSTRACT

Synapsins are abundant SV (synaptic vesicle)-associated phosphoproteins that regulate synapse formation and function. The highly conserved C-terminal domain E was shown to contribute to several synapsin functions, ranging from formation of the SV reserve pool to regulation of the kinetics of exocytosis and SV cycling, although the molecular mechanisms underlying these effects are unknown. In the present study, we used a synthetic 25-mer peptide encompassing the most conserved region of domain E (Pep-E) to analyse the role of domain E in regulating the interactions between synapsin I and liposomes mimicking the phospholipid composition of SVs (SV-liposomes) and other pre-synaptic protein partners. In affinity-chromatography and cross-linking assays, Pep-E bound to endogenous and purified exogenous synapsin I and strongly inhibited synapsin dimerization, indicating a role in synapsin oligomerization. Consistently, Pep-E (but not its scrambled version) counteracted the ability of holo-synapsin I to bind and coat phospholipid membranes, as analysed by AFM (atomic force microscopy) topographical scanning, and significantly decreased the clustering of SV-liposomes induced by holo-synapsin I in FRET (Förster resonance energy transfer) assays, suggesting a causal relationship between synapsin oligomerization and vesicle clustering. Either Pep-E or a peptide derived from domain C was necessary and sufficient to inhibit both dimerization and vesicle clustering, indicating the participation of both domains in these activities of synapsin I. The results provide a molecular explanation for the effects of domain E in nerve terminal physiology and suggest that its effects on the size and integrity of SV pools are contributed by the regulation of synapsin dimerization and SV clustering.


Subject(s)
Phospholipids/metabolism , Protein Multimerization/physiology , Synapsins/chemistry , Synapsins/metabolism , Animals , Chromatography, Affinity , Fluorescence Resonance Energy Transfer , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Liposomes/metabolism , Microscopy, Atomic Force , Phospholipids/chemistry , Protein Multimerization/genetics , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Rats , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Synapsins/genetics
2.
J Biol Chem ; 284(49): 34244-56, 2009 Dec 04.
Article in English | MEDLINE | ID: mdl-19759398

ABSTRACT

Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP- and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are required for this stimulation. Acidic liposome-triggered, N-WASP-dependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 co-localizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.


Subject(s)
Actins/chemistry , Gene Expression Regulation , Nerve Tissue Proteins/metabolism , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Actins/metabolism , Animals , Brain/metabolism , Cell Membrane/metabolism , Cytosol/metabolism , Endocytosis , Fluorescence Resonance Energy Transfer , Liposomes/chemistry , Male , Mice , Mice, Knockout , Rats , Receptors, Cell Surface/metabolism , Sertoli Cells/metabolism
3.
FEBS Lett ; 538(1-3): 71-6, 2003 Mar 13.
Article in English | MEDLINE | ID: mdl-12633855

ABSTRACT

The receptor mechanisms regulating the ATP-induced free cytosolic Ca(2+) concentration ([Ca(2+)](i)) changes in cultured rat cortical type-1 astrocytes were analyzed using fura-2-based Ca(2+) imaging microscopy. Upon prolonged ATP challenge (1-100 microM), astroglial cells displayed a biphasic [Ca(2+)](i) response consisting of an initial peak followed by a sustained elevation. Suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid blocked both components, albeit to a different extent. By contrast, the selective P2X7 antagonist oxidized ATP irreversibly abrogated the sustained [Ca(2+)](i) signal without affecting the transient phase. Finally, astrocyte challenge with the selective P2X7 agonist 3'-O-(4-benzoyl)benzoyl-ATP evoked a sustained [Ca(2+)](i) elevation, which occluded that induced by ATP. We can conclude that in cultured cortical astrocytes the ATP-mediated sustained [Ca(2+)](i) rise does not implicate capacitative Ca(2+) entry but involves Ca(2+) influx through P2X7-like receptors.


Subject(s)
Adenosine Triphosphate/metabolism , Astrocytes/metabolism , Calcium Signaling , Cerebral Cortex/metabolism , Pyridoxal Phosphate/analogs & derivatives , Receptors, Purinergic P2/metabolism , Animals , Astrocytes/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Purinergic P2 Receptor Agonists , Purinergic P2 Receptor Antagonists , Pyridoxal Phosphate/pharmacology , Rats , Receptors, Purinergic P2X7 , Suramin/pharmacology
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