RESUMO
Amphiphysin 1 is a brain-specific protein enriched at the synapse and a major binding partner of several components of the clathrin-mediated endocytic machinery (Proc Natl Acad Sci USA 93 (1996) 331). It interacts with clathrin-coat proteins, dynamin, and membranes (Nat Cell Biol 1 (1999) 33; JBC). A role of amphiphysin in synaptic vesicle recycling is supported by both acute and chronic perturbation studies (Science 276 (1997) 259; Neuron 33 (2002) 789). Here we show that amphiphysin directly stimulates clathrin recruitment onto liposomes in an in vitro assay. Amphiphysin-dependent clathrin-coat recruitment is enhanced by the interaction of amphiphysin with dynamin. We also show that the amphiphysin SH3 domain binds full-length amphiphysin, likely via an internal poly-proline region, and that clathrin recruitment onto liposomes by amphiphysin is enhanced in the presence of the isolated amphiphysin SH3 domain. Expression of a mutant amphiphysin harboring two amino acid substitutions in the SH3 domain, and therefore unable to bind proline-containing motifs, induces an accumulation of large intracellular aggregates including amphiphysin, clathrin, AP-2, and other endocytic proteins, as well as a concomitant block of transferrin endocytosis. Thus, putative intramolecular interactions between the amphiphysin COOH-terminal SH3 domain and its internal poly-proline region may regulate clathrin recruitment onto membranes.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Endocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Encéfalo/metabolismo , Células CHO , Células COS , Chlorocebus aethiops , Vesículas Revestidas por Clatrina/genética , Vesículas Revestidas por Clatrina/metabolismo , Cricetinae , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Proteínas do Tecido Nervoso/genética , RatosRESUMO
Synaptojanin 1 is a polyphosphoinositide phosphatase implicated in synaptic vesicle recycling. We used FM1-43 imaging and electron microscopy in cultured cortical neurons from control and synaptojanin 1 knockout mice to study how the absence of this protein affects specific steps of the synaptic vesicle cycle. Exoendocytosis after a moderate stimulus was unchanged. However, during prolonged stimulation, the regeneration of fusion-competent synaptic vesicles was severely impaired. In stimulated nerve terminals, there was a persistent accumulation of clathrin-coated vesicles and a backup of newly reformed vesicles in the cytomatrix-rich area around the synaptic vesicle cluster. These findings demonstrate that synaptojanin 1 function is needed for the progression of recycling vesicles to the functional synaptic vesicle pool.
Assuntos
Proteínas do Tecido Nervoso/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Fusão Celular , Células Cultivadas , Córtex Cerebral/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Endocitose , Exocitose , Camundongos , Camundongos Knockout , Microscopia EletrônicaRESUMO
In striated muscle, the plasma membrane forms tubular invaginations (transverse tubules or T-tubules) that function in depolarization-contraction coupling. Caveolin-3 and amphiphysin were implicated in their biogenesis. Amphiphysin isoforms have a putative role in membrane deformation at endocytic sites. An isoform of amphiphysin 2 concentrated at T-tubules induced tubular plasma membrane invaginations when expressed in nonmuscle cells. This property required exon 10, a phosphoinositide-binding module. In developing myotubes, amphiphysin 2 and caveolin-3 segregated in tubular and vesicular portions of the T-tubule system, respectively. These findings support a role of the bilayer-deforming properties of amphiphysin at T-tubules and, more generally, a physiological role of amphiphysin in membrane deformation.
Assuntos
Estruturas da Membrana Celular/ultraestrutura , Desenvolvimento Muscular , Músculo Esquelético/ultraestrutura , Proteínas do Tecido Nervoso/metabolismo , Animais , Células CHO , Caveolina 3 , Caveolinas/metabolismo , Diferenciação Celular , Linhagem Celular , Membrana Celular/metabolismo , Estruturas da Membrana Celular/metabolismo , Cricetinae , Dinaminas , Éxons , GTP Fosfo-Hidrolases/metabolismo , Lipossomos/metabolismo , Camundongos , Microscopia Eletrônica , Morfogênese , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Interferente Pequeno , RNA não Traduzido/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
The function of the clathrin coat in synaptic vesicle endocytosis is assisted by a variety of accessory factors, among which amphiphysin (amphiphysin 1 and 2) is one of the best characterized. A putative endocytic function of amphiphysin was supported by dominant-negative interference studies. We have now generated amphiphysin 1 knockout mice and found that lack of amphiphysin 1 causes a parallel dramatic reduction of amphiphysin 2 selectively in brain. Cell-free assembly of endocytic protein scaffolds is defective in mutant brain extracts. Knockout mice exhibit defects in synaptic vesicle recycling that are unmasked by stimulation and suggest impairments at multiple stages of the cycle. These defects correlate with increased mortality due to rare irreversible seizures and with major learning deficits, suggesting a critical role of amphiphysin for higher brain functions.
