Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.

Dynamin is a GTPase enzyme involved in membrane constriction and fission during endocytosis. Phospholipid binding via its pleckstrin homology domain maximally stimulates dynamin activity. We developed a series of surface-active small-molecule inhibitors, such as myristyl trimethyl ammonium bromide (MiTMAB) and octadecyltrimethyl ammonium bromide (OcTMAB), and we now show MiTMAB targets the dynamin-phospholipid interaction. MiT-MAB inhibited dynamin GTPase activity, with a K i of 940 Ϯ 25 nM. It potently inhibited receptor-mediated endocytosis (RME) of transferrin or epidermal growth factor (EGF) in a range of cells without blocking EGF binding, receptor number, or autophosphorylation. RME inhibition was rapidly reversed after washout. The rank order of potency for a variety of MiTMAB analogs on RME matched the rank order for dynamin inhibition, suggesting dynamin recruitment to the membrane is a primary cellular target. MiTMAB also inhibited synaptic vesicle endocytosis in rat brain nerve terminals (synaptosomes) without inducing depolarization or morphological defects. Therefore, the drug rapidly and reversibly blocks multiple forms of endocytosis with no acute cellular damage. The unique mechanism of action of MiTMAB provides an important tool to better understand dynamin-mediated membrane trafficking events in a variety of cells.

The large-scale resolution and detection of proteins from complex native mixtures is fundamental to quantitative proteomic analyses. Comprehensive analyses depend on careful tissue handling and quantitative protein extraction and assessment. To most effectively link these analyses with an understanding of underlying molecular mechanisms, it is critical that all protein types - isoforms, splice variants and those with functionally important PTMs - are quantitatively extracted with high reproducibility. Methodological details concerning protein extraction and resolution using 2DE are discussed with reference to current in-gel protein detection limits. We confirm a significant increase in total protein, and establish that extraction, resolution and detection of phospho- and glycoproteins are improved following automated frozen disruption relative to manual homogenisation. The quality of 2DE protein resolution is established using third-dimension separations and 'deep imaging'; substantially more proteins/protein species than previously realised are actually resolved by 2DE. Thus, the key issue for effective proteome analyses is most likely to be detection, not resolution. Thus, these systematic methodological and technical advances further solidify the role of 2DE in top-down proteomics. By routinely assessing as much proteomic data from a sample as possible, 2DE enables more detailed and critical insights into molecular mechanisms underlying different physiological states.

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The septins are GTPase enzymes with multiple roles in cytokinesis, cell polarity or exocytosis. The proteins from the mammalian septin genes are called Sept1-10. Most are expressed in multiple tissues, but the mRNA for Sept5 (CDCrel-1) and Sept3 (G-septin) appear to be primarily expressed in brain. Sept3 is phosphorylated by cGMP-dependent protein kinase I (PKG-I) and the cGMP/PKG pathway is involved in presynaptic plasticity. Therefore to determine whether Sept3 specifically associates with neurones and nerve terminals we investigated its distribution in rat brain and neuronal cultures. Sept3 protein was detected only in brain by immunoblot, but not in 12 other tissues examined. Levels were high in all adult brain regions, and reduced in those enriched in white matter. Expression was developmentally regulated, being absent in the early embryo, low in late embryonic rat brain and increasing after birth. Like dynamin I, Sept3 was specifically enriched in synaptosomes compared with whole brain, and was only found in a peripheral membrane extract and not in the soluble or membrane extracts. Sept3 was particularly abundant in mossy fibre nerve terminals in the hippocampus. In primary cultured hippocampal neurones Sept3 immunoreactivity was punctate in neurites and predominantly localized to presynaptic terminals, strongly colocalizing with synaptophysin and dynamin I. The specific nerve terminal localization was confirmed by immunogold electron microscopy. Together this shows that Sept3 is a neurone-specific protein highly enriched in nerve terminals which supports a secretory role in synaptic vesicle recycling. Keywords: cGMP, G-septin, PKG, protein phosphorylation, septin 3, synaptosomes. The septins are a multigene family in mammals comprising 10 genes, producing a variety of protein products by further alternative splicing. The proteins are named Sept1-10 and are GTPase enzymes of around 40-70 kDa (Macara et al. 2002;Surka et al. 2002). The GTPase domain comprises the bulk of the sequence and no other large domains are found, although a few short functional motifs are known (Saraste et al. 1990;Zhang et al. 1999). The septins have multiple cellular functions, primarily playing an essential role in cytokinesis (Kinoshita et al. 1997;Hsu et al. 1998;Xie et al. 1999;Surka et al. 2002). In non-mitotic cells septins appear to assemble into filaments and/or associate with the plasma membrane Fung and Scheller 1999;Zhang et al. 1999). Groups of different septins also assemble as filaments in vitro (Field et al. 1996;Hsu et al. 1998;Kinoshita 2003). The filaments appear to be strongly associated with either actin (Kinoshita et al. 1997;Kinoshita et al. 2002) or tubulin networks (Surka et al. 2002), or in some cases with both (Surka et al. 2002). The filaments can self-assemble into higher-order structures to form