Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel electrophoresis and nano-LC-MS/MS analysis

The halotolerant alga Dunaliella salina is a recognized model photosynthetic organism for studying plant adaptation to high salinity. The adaptation mechanisms involve major changes in the proteome composition associated with energy metabolism and carbon and iron acquisition. To clarify the molecular basis for the remarkable resistance to high salt, we performed a comprehensive proteomics analysis of the plasma membrane. Plasma membrane proteins were recognized by tagging intact cells with a membrane-impermeable biotin derivative. Proteins were resolved by two-dimensional blue native/SDS-PAGE and identified by nano-LC-MS/MS. Of 55 identified proteins, about 60% were integral membrane or membrane-associated proteins. We identified novel surface coat proteins, lipid-metabolizing enzymes, a new family of membrane proteins of unknown function, ion transporters, small GTP-binding proteins, and heat shock proteins. The abundance of 20 protein spots increased and that of two protein spots decreased under high salt. The major salt-regulated proteins were implicated in protein and membrane structure stabilization and within signal transduction pathways. The migration profiles of native protein complexes on blue native gels revealed oligomerization or co-migration of major surface-exposed proteins, which may indicate mechanisms of stabilization at high salinity.     

ITS2 sequences as barcodes for identifying and analyzing spider mites (Acari: Tetranychidae)

The use of DNA barcodes, short DNA sequences from a standardized region of the genome, has recently been proposed as a tool to facilitate species identification and discovery. Here we show that second internal transcribed spacer of nuclear ribosomal DNA (rDNA-ITS2) barcodes effectively discriminate among 16 species of spider mites (Acari: Tetranychidae) from Israel. The barcode sequences of each species were unambiguously distinguishable from all other species and formed distinct, nonoverlapping monophyletic groups in the maximum-parsimony tree. Sequence divergences were generally much greater between species than within them. Using a 0.02 (2%) threshold for species diagnosis in our data set, 14 out of 16 species recognized by morphological criteria would be accurately identified. The only exceptions involved the low divergence, 0.011–0.015 (1.1–1.5%), between Tetranychus urticae and Tetranychus turkestani, where speciation may have occurred only recently. Still, these species had fixed alternative rDNA-ITS2 variants, with five diagnostic nucleotide substitutions. As a result, we tentatively conclude that rDNA-ITS2 sequence barcodes may serve as an effective tool for the identification of spider mite species and can be applicable as a diagnostic tool for quarantine and other pest management activities and decision-making. We predict that our work, together with similar efforts, will provide in the future the platform for a uniform, accurate, practical and easy-to-use method of spider mite species identification.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

A Pro-Nerve Growth Factor (proNGF) and NGF Binding Protein, α2-Macroglobulin,Differentially Regulates p75 and TrkA Receptors and Is Relevant to Neurodegeneration Ex Vivo and In Vivo

Nerve growth factor (NGF) is generated from a precursor, proNGF, that is proteolytically processed. NGF preferentially binds a trophic tyrosine kinase receptor, TrkA, while proNGF binds a neurotrophin receptor (NTR), p75^NTR, that can have neurotoxic activity. Previously, we along with others showed that the soluble protein α₂-macroglobulin (α₂M) is neurotoxic. Toxicity is due in part to α₂M binding to NGF and inhibiting trophic activity, presumably by preventing NGF binding to TrkA. However, the mechanisms remained unclear. Here, we show ex vivo and in vivo three mechanisms for α₂M neurotoxicity. First, unexpectedly the α₂M-NGF complexes do bind TrkA receptors but do not induce TrkA dimerization or activation, resulting in deficient trophic support. Second, α₂M makes stable complexes with proNGF, conveying resistance to proteolysis that results in more proNGF and less NGF. Third, α₂M-proNGF complexes bind p75^NTR and are more potent agonists than free proNGF, inducing tumor necrosis factor alpha (TNF-α) production. Hence, α₂M regulates proNGF/p75^NTR positively and mature NGF/TrkA negatively, causing neuronal death ex vivo. These three mechanisms are operative in vivo, and α₂M causes neurodegeneration in a p75^NTR- and proNGF-dependent manner. α₂M could be exploited as a therapeutic target, or as a modifier of neurotrophin signals.     

The sponge pump: the role of current induced flow in the design of the sponge body plan

Sponges are suspension feeders that use flagellated collar-cells (choanocytes) to actively filter a volume of water equivalent to many times their body volume each hour. Flow through sponges is thought to be enhanced by ambient current, which induces a pressure gradient across the sponge wall, but the underlying mechanism is still unknown. Studies of sponge filtration have estimated the energetic cost of pumping to be <1% of its total metabolism implying there is little adaptive value to reducing the cost of pumping by using "passive" flow induced by the ambient current. We quantified the pumping activity and respiration of the glass sponge Aphrocallistes vastus at a 150 m deep reef in situ and in a flow flume; we also modeled the glass sponge filtration system from measurements of the aquiferous system. Excurrent flow from the sponge osculum measured in situ and in the flume were positively correlated (r>0.75) with the ambient current velocity. During short bursts of high ambient current the sponges filtered two-thirds of the total volume of water they processed daily. Our model indicates that the head loss across the sponge collar filter is 10 times higher than previously estimated. The difference is due to the resistance created by a fine protein mesh that lines the collar, which demosponges also have, but was so far overlooked. Applying our model to the in situ measurements indicates that even modest pumping rates require an energetic expenditure of at least 28% of the total in situ respiration. We suggest that due to the high cost of pumping, current-induced flow is highly beneficial but may occur only in thin walled sponges living in high flow environments. Our results call for a new look at the mechanisms underlying current-induced flow and for reevaluation of the cost of biological pumping and its evolutionary role, especially in sponges.     

Possible role of factor XIII subunit A in Fcgamma and complement receptor-mediated phagocytosis

Besides its traditional role in hemostasis, factor XIII subunit A (FXIII-A) is supposed to function as a cellular transglutaminase and to be involved in certain intracellular processes, including cytoskeletal remodeling. To investigate its intracellular role, the aim of the present study was to follow changes in FXIII-A production in combination with the receptor-mediated phagocytic activities of monocytes/macrophages and to examine the phagocytic functions of monocytes in patients with FXIII-A deficiency. Human blood monocytes were isolated from the buffy coats of healthy volunteers and cultured for 4 days. The FcgammaR-mediated phagocytosis of sensitized erythrocytes (EA) and the complement receptor (CR)-mediated phagocytosis of complement-coated yeast particles were studied during monocyte/macrophage differentiation. Changes in the gene expression of FXIII-A were detected by real-time quantitative RT-PCR. FXIII-A protein production was investigated with fluorescent image analysis at single cell level and Western immunoblot analysis. Both the FcgammaR and CR-mediated phagocytosis increased during culturing, which peaked on day 3. The phagocytic activity of the cells could be markedly inhibited with monodansylcadaverine, an inhibitor of the transglutaminase-induced crosslinking of proteins. The phagocytosis of EA, complement-coated and uncoated yeast particles was found to be strongly diminished in monocytes of FXIII-A deficient patients. The phagocytic functions of cultured cells showed a change in parallel with the alterations in FXIII-A mRNA expression, as well as with that in FXIII-A in protein synthesis detected by image and Western immunoblot analyses in concert. Our results suggest that FXIII-A plays a role in the Fcgamma and complement receptor-mediated phagocytic activities of monocytes/macrophages.     

Structural and functional analysis of tomosyn identifies domains important in exocytotic regulation

Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 and -2, occur in mammals and produce seven different isoforms via alternative splicing. Here, we map the structural differences between the yeast homologue of m-tomosyn-1, Sro7, and tomosyn genes/isoforms to identify domains critical to the regulation of exocytotic activity to tomosyn that are outside the soluble N-ethylmaleimide-sensitive attachment receptor motif. Homology modeling of m-tomosyn-1 based on the known structure of yeast Sro7 revealed a highly conserved functional conformation but with tomosyn containing three additional loop domains that emanate from a β-propeller core. Notably, deletion of loops 1 and 3 eliminates tomosyn inhibitory activity on secretion without altering its soluble N-ethylmaleimide-sensitive attachment receptor pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region, did not reduce the ability of tomosyn to inhibit regulated secretion. However, exon variation within the hypervariable splice region resulted in significant differences in protein accumulation of tomosyn-2 isoforms. Functional analysis of s-tomosyn-1, m-tomosyn-1, m-tomosyn-2, and xb-tomosyn-2 demonstrated that they exert similar inhibitory effects on elevated K(+)-induced secretion in PC12 cells, although m-tomosyn-2 was novel in strongly augmenting basal secretion. Finally, we report that m-tomosyn-1 is a target substrate for SUMO 2/3 conjugation and that mutation of this small ubiquitin-related modifier target site (Lys-730) enhances m-tomosyn-1 inhibition of secretion without altering interaction with syntaxin1A. Together these results suggest that multiple domains outside the R-SNARE of tomosyn are critical to the efficacy of inhibition by tomosyn on exocytotic secretion.