Identification of proteins in the postsynaptic density fraction by mass spectrometry

Our understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs. In this study, we have completed the identification of most major proteins in the PSD fraction with the use of an analytical method based on mass spectrometry coupled with searching of the protein sequence databases. At least one protein in each of 26 prominent protein bands from the PSD fraction has now been identified. We found 7 proteins not previously known to be constituents of the PSD fraction and 24 that had previously been associated with the PSD by other methods. The newly identified proteins include the heavy chain of myosin-Va (dilute myosin), a motor protein thought to be involved in vesicle trafficking, and the mammalian homolog of the yeast septin protein cdc10, which is important for bud formation in yeast. Both myosin-Va and cdc10 are threefold to fivefold enriched in the PSD fraction over brain homogenates. Immunocytochemical localization of myosin-Va in cultured hippocampal neurons shows that it partially colocalizes with PSD-95 at synapses and is also diffusely localized in cell bodies, dendrites, and axons. Cdc10 has a punctate distribution in cell bodies and dendrites, with some of the puncta colocalizing with PSD-95. The results support a role for myosin-Va in transport of materials into spines and for septins in the formation or maintenance of spines

Myosin-Vb functions as a dynamic tether for peripheral endocytic compartments during transferrin trafficking

<p>Abstract</p> <p>Background</p> <p>Myosin-Vb has been shown to be involved in the recycling of diverse proteins in multiple cell types. Studies on transferrin trafficking in HeLa cells using a dominant-negative myosin-Vb tail fragment suggested that myosin-Vb was required for recycling from perinuclear compartments to the plasma membrane. However, chemical-genetic, dominant-negative experiments, in which myosin-Vb was specifically induced to bind to actin, suggested that the initial hypothesis was incorrect both in its site and mode of myosin-Vb action. Instead, the chemical-genetic data suggested that myosin-Vb functions in the actin-rich periphery as a dynamic tether on peripheral endosomes, retarding transferrin transport to perinuclear compartments.</p> <p>Results</p> <p>In this study, we employed both approaches, with the addition of overexpression of full-length wild-type myosin-Vb and switching the order of myosin-Vb inhibition and transferrin loading, to distinguish between these hypotheses. Overexpression of full-length myosin-Vb produced large peripheral endosomes. Chemical-genetic inhibition of myosin-Vb after loading with transferrin did not prevent movement of transferrin from perinuclear compartments; however, virtually all myosin-Vb-decorated particles, including those moving on microtubules, were halted by the inhibition. Overexpression of the myosin-Vb tail caused a less-peripheral distribution of early endosome antigen-1 (EEA1).</p> <p>Conclusion</p> <p>All results favored the peripheral dynamic tethering hypothesis.</p

Myosin5a tail associates directly with Rab3A-containing compartments in neurons

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of American Society for Biochemistry and Molecular Biology. The definitive version was published in Journal of Biological Chemistry, 286 (2011): 14352-14361, doi:10.1074/jbc.M110.187286.Myosin-Va (Myo5a) is a motor protein associated with synaptic vesicles (SVs) but the mechanism by which it interacts has not yet been identified. A potential class of binding partners are Rab GTPases and Rab3A is known to associate with SVs and is involved in SV trafficking. We performed experiments to determine whether Rab3A interacts with Myo5a and whether it is required for transport of neuronal vesicles. In vitro motility assays performed with axoplasm from the squid giant axon showed a requirement for a Rab GTPase in Myo5a-dependent vesicle transport. Furthermore, mouse recombinant Myo5a tail revealed that it associated with Rab3A in rat brain synaptosomal preparations in vitro and the association was confirmed by immunofluorescence imaging of primary neurons isolated from the frontal cortex of mouse brains. Synaptosomal Rab3A was retained on recombinant GST-tagged Myo5a tail affinity columns in a GTP-dependent manner. Finally, the direct interaction of Myo5a and Rab3A was determined by sedimentation v e l o c i t y analytical ultracentrifugation using recombinant mouse Myo5a tail and human Rab3A. When both proteins were incubated in the presence of 1 mM GTPγS, Myo5a tail and Rab3A formed a complex and a direct interaction was observed. Further analysis revealed that GTP-bound Rab3A interacts with both the monomeric and dimeric species of the Myo5a tail. However, the interaction between Myo5a tail and nucleotidefree Rab3A did not occur. Thus, our results show that Myo5a and Rab3A are direct binding partners and interact on SVs and that the Myo5a/Rab3A complex is involved in transport of neuronal vesicles

Separation of Volatile Metabolites from the Leaf-Derived Essential Oil of <i>Piper mollicomum</i> Kunth (Piperaceae) by High-Speed Countercurrent Chromatography

The technique of high-speed countercurrent chromatography was applied to the isolation of compounds in essential oil derived from the leaves of Piper mollicomum species. Plant leaves (200.0 g) were submitted to hydrodistillation in a modified Clevenger apparatus. The resulting crude leaf essential oil was analyzed by gas chromatography with flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) to determine the profile of the components. The purified fractions were composed of monoterpenes and sesquiterpenes such as camphor (85.0 mg at 98.5% purity), (E)-nerolidol (100.0 mg at 92.8% purity), and camphene (150.0 mg at 82.0% purity). A minor component of the essential oil, bornyl acetate (16.2 mg at 91.2% purity) was also isolated in the one-step separation protocol in 2 h. The countercurrent chromatography technique proved to be a fast and efficient method for the separation of volatile metabolites that conserved the solvent while delivering various fractions of high purity

Physical exercise stimulates salivary secretion of cystatins.

Physical exercise is known to activate the sympathetic nervous system, which influences the production of saliva from salivary glands. Our examination of saliva collected from highly trained athletes before and after a number of physical competititions showed an increase in the secretion of S-type cystatins and cystatin C as a subacute response to aerobic and anaerobic exercise. The elevation in salivary cystatins was transient and the recovery time course differed from that of amylase and other salivary proteins. An in vitro assay was developed based on a cell line from a human submandibular gland (HSG) that differentiated into acinus-like structures. Treatments with the β-adrenergic agonist isoproterenol caused a shift in the intracellular distribution of S-type cystatins and cystatin C, promoting their accumulation at the outer regions of the acinus prior to release and suggesting the activation of a directional transport involving co-migration of both molecules. In another treatment using non-differentiated HSG cells, it was evident that both expression and secretion of cystatin C increased upon addition of the β-adrenergic agonist, and these effects were essentially eliminated by the antagonist propranolol. The HSG cell line appears to have potential as a model for exploring the mechanism of cystatin secretion, particularly the S-type cystatins that originate primarily in the submandibular glands

Chemical-genetic inhibition of a sensitized mutant myosin Vb demonstrates a role in peripheral-pericentriolar membrane traffic

Selective, in situ inhibition of individual unconventional myosins is a powerful approach to determine their specific physiological functions. Here, we report the engineering of a myosin Vb mutant that still hydrolyzes ATP, yet is selectively sensitized to an N(6)-substituted ADP analog that inhibits its activity, causing it to remain tightly bound to actin. Inhibition of the sensitized mutant causes inhibition of accumulation of transferrin in the cytoplasm and increases levels of plasma-membrane transferrin receptor, suggesting that myosin Vb functions in traffic between peripheral and pericentrosomal compartments