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 W WM), astroglial cells displayed a biphasic [Ca 2+ ] i response consisting of an initial peak followed by a sustained elevation. Suramin and pyridoxalphosphate-6-azophenyl-2P P,4P P-disulfonic acid blocked both components, albeit to a di¡erent extent. By contrast, the selective P2X7 antagonist oxidized ATP irreversibly abrogated the sustained [Ca 2+ ] i signal without a¡ecting the transient phase. Finally, astrocyte challenge with the selective P2X7 agonist 3P P-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+ in£ux through P2X7-like receptors. ß

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 requiredforthisstimulation.Acidicliposome-triggered,N-WASPdependent 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 colocalizes 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.The dynamic nature of the actin cytoskeleton is crucial for a variety of cellular events, including cell morphogenesis, cell migration, and intracellular membrane traffic (1). Actin polymerization is stimulated by a variety of actin regulatory proteins, prominent among which are WASP family proteins that function by triggering Arp2/3-mediated actin nucleation (2). Activation of WASP family proteins, in turn, is controlled by factors that bind these proteins and release an autoinhibitory intramolecular interaction that prevents their VCA domain from interacting with the Arp2/3 complex. As extensively shown for N-WASP, many such factors are proteins that bind to the N-WASP proline-rich region via the SH3 4 domain. Recently, we have found that the SH3 domain containing protein amphiphysin 1 stimulates actin polymerization during phagocytosis in testicular Sertoli cells, and this effect requires interactions of its C-terminal SH3 domain (3). Amphiphysin 1 is an endocytic adaptor present at high levels in brain at neuronal synapses but is also expressed at significant levels in Sertoli cells (4,5). In addition to a C-terminal SH3 domain, known to bind the GTPase dynamin and the phosphoinositide phosphatase synaptojanin (6), amphiphysin 1 contains an N-terminal BAR domain, a curved protein module that binds lipid bilayers and generates and senses curvature (7,8). It also contains binding motifs for clathrin and for the clathrin adaptor AP-2 (9). Hence, amphiphysin 1 was primarily studied as an endocytic protein capable of assembling at the neck of endocytic pits and of coupling clathrin-mediated budding to dynamin-mediated fission (10, 11). However, regulatory roles of amphiphysin 1 in actin cytoskeleton have also been suggested by studies of neuronal growth cones (12) and by the function of the amphiphysin homologue in yeast, . 81-86-235-7126; E-mail: kohji@md.okayama-u.ac.jp. 4 The abbrevi...

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.

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 requiredforthisstimulation.Acidicliposome-triggered,N-WASPdependent 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 colocalizes 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.The dynamic nature of the actin cytoskeleton is crucial for a variety of cellular events, including cell morphogenesis, cell migration, and intracellular membrane traffic (1). Actin polymerization is stimulated by a variety of actin regulatory proteins, prominent among which are WASP family proteins that function by triggering Arp2/3-mediated actin nucleation (2). Activation of WASP family proteins, in turn, is controlled by factors that bind these proteins and release an autoinhibitory intramolecular interaction that prevents their VCA domain from interacting with the Arp2/3 complex. As extensively shown for N-WASP, many such factors are proteins that bind to the N-WASP proline-rich region via the SH3 4 domain. Recently, we have found that the SH3 domain containing protein amphiphysin 1 stimulates actin polymerization during phagocytosis in testicular Sertoli cells, and this effect requires interactions of its C-terminal SH3 domain (3). Amphiphysin 1 is an endocytic adaptor present at high levels in brain at neuronal synapses but is also expressed at significant levels in Sertoli cells (4,5). In addition to a C-terminal SH3 domain, known to bind the GTPase dynamin and the phosphoinositide phosphatase synaptojanin (6), amphiphysin 1 contains an N-terminal BAR domain, a curved protein module that binds lipid bilayers and generates and senses curvature (7,8). It also contains binding motifs for clathrin and for the clathrin adaptor AP-2 (9). Hence, amphiphysin 1 was primarily studied as an endocytic protein capable of assembling at the neck of endocytic pits and of coupling clathrin-mediated budding to dynamin-mediated fission (10, 11). However, regulatory roles of amphiphysin 1 in actin cytoskeleton have also been suggested by studies of neuronal growth cones (12) and by the function of the amphiphysin homologue in yeast, . 81-86-235-7126; E-mail: kohji@md.okayama-u.ac.jp. 4 The abbrevi...