Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling

Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin''s high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method''s sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.     

Fine-mapping of the BaMMV, BaYMV-1 and BaYMV-2 resistance of barley (Hordeum vulgare) accession PI1963

Barley yellow mosaic disease caused by the bymoviruses barley mild mosaic virus (BaMMV) and barley yellow mosaic virus (BaYMV) is one of the economically most important diseases of winter barley in Europe. In European barley breeding programmes, resistance is currently due to only two genes—rym4, which is effective against viruses BaMMV and BaYMV-1, and rym5, which is effective against BaYMV-2. Diversification of resistance is therefore an important task. Because the accession PI1963 confers immunity against all European strains of barley yellow mosaic disease and is not allelic to rym5, we have attempted to develop closely linked markers in order to facilitate the efficient introgression of this resistance into adapted germplasm. By means of restriction fragment length polymorphism analysis, we located a gene locus for resistance to BaMMV, BaYMV-1 and BaYMV-2 of PI1963 on chromosome 4HL using a mapping population (W757) comprising 57 doubled haploid (DH) lines. Subsequent tests for allelism indicated that the BaMMV resistance gene in PI1963 is allelic to rym11. Two DH populations, IPK1 and IPK2, comprising 191 and 161 DH lines, respectively, were derived from the initial mapping population W757 and used for further analysis. As random amplified polymorphic DNA development did not facilitate the identification of more closely linked markers, simple sequence repeat (SSR) analyses were conducted. For population IPK1, the closest SSRs detected were Bmac181 and Bmag353, which flank the gene at 2.1 cM and 2.7 cM, respectively. For the IPK2 population, the SSR markers HVM3 and Bmag353 are located proximally at 2.5 cM and distally at 8.2 cM, respectively. In order to develop markers more tightly linked to rym11, a targeted amplified fragment length polymorphism (AFLP) marker identification approach was adopted using bulks comprising lines carrying recombination events proximal and distal to the target interval. Using this approach we identified six AFLP markers closely linked to rym11, with the two markers, E56M32 and E49M33, co-segregating with rym11 in both populations. The SSRs and AFLPs identified in this study represent useful tools for marker-assisted selection.     

Hardening by partial dehydration and ABA increase desiccation tolerance in the cyanobacterial lichen Peltigera polydactylon

* BACKGROUND AND AIMS: The ability of partial dehydration and abscisic acid pretreatments to increase desiccation tolerance in the cyanobacterial lichen Peltigera polydactylon was tested. * METHODS: Net photosynthesis and respiration were measured using infrared gas analysis during a drying and rehydration cycle. At the same time, the efficiency of photosystem two was measured using chlorophyll fluorescence, and the concentrations of chlorophyll a were spectrophotometrically assayed. Heat production was also measured during a shorter drying and rehydration cycle using differential dark microcalorimetry. * KEY RESULTS: Pretreating lichens by dehydrating them to a relative water content of approx. 0.65 for 3 d, followed by storing thalli hydrated for 1 d in the light, significantly improved their ability to recover net photosynthesis during rehydration after desiccation for 15 but not 30 d. Abscisic acid pretreatment could substitute for partial dehydration. The improved rates of photosynthesis during the rehydration of pretreated material were not accompanied by preservation of photosystem two activity or chlorophyll a concentrations compared with untreated lichens. Partial dehydration and ABA pretreatments appeared to have little direct effect on the desiccation tolerance of the mycobiont, because the bursts of respiration and heat production that occurred during rehydration were similar in control and pretreated lichens. * CONCLUSIONS: Results indicate that the photobiont of P. polydactylon possesses inducible tolerance mechanisms that reduce desiccation-induced damage to carbon fixation, and will therefore improve the supply of carbohydrates to the whole thallus following stress. In this lichen, ABA is involved in signal transduction pathways that increase tolerance of the photobiont.     

CD4+ T cells from lupus-prone mice avoid antigen-specific tolerance induction in vivo

Activated T cells in spontaneous lupus presumably bypass normal tolerance mechanisms in the periphery, since thymic tolerance appears intact. To determine whether such T cells indeed avoid in vivo peripheral tolerance mechanisms, we assessed their activation and recall responses after in vivo Ag stimulation in the absence of exogenously supplied costimulatory signals. Naive CD4(+) AND (transgenic mice bearing rearranged TCR specific for pigeon cytochrome c, peptides 88-104) TCR-transgenic T cells, specific for pigeon cytochrome c, from lupus-prone Fas-intact MRL/Mp+(Fas-lpr) and from H-2(k)-matched control CBA/CaJ and B10.BR mice (MRL.AND, CBA.AND, and B10.AND, respectively) were adoptively transferred into (MRL x CBA)F(1) or (MRL x B10)F(1) recipients transgenically expressing membrane-bound pigeon cytochrome c as a self-Ag. MRL.AND and control CBA.AND and B10.AND-transgenic T cells were activated and divided after transfer, indicating encounter with their cognate Ag; however, T cells from CBA.AND and B10.AND mice were impaired in their ability to proliferate and produce IL-2 after challenge with pigeon cytochrome c in ex vivo recall assays, a typical phenotype of anergized cells. By contrast, MRL.AND T cells proliferated more, and a significantly higher percentage of such cells produced IL-2, compared with control T cells. This observation that MRL T cells avoided anergy induction in vivo was confirmed in an in vitro system where the cells were stimulated with an anti-CD3 in the absence of a costimulatory signal. These experiments provide direct evidence that CD4(+) T cells from Fas-intact lupus-prone MRL mice are more resistant than nonautoimmune control cells to anergy induction. Anergy avoidance in the periphery might contribute to the characteristic finding in lupus of inappropriate T cell activation in response to ubiquitous self-Ags.     

The binding of synthetic analogs of the upstream,terminal residue of the O-polysaccharides (O-PS) of Vibrio cholerae O:1 serotypes Ogawa and Inaba to two murine monoclonal antibodies (MAbs) specific for the Ogawa lipopolysaccharide (LPS)

The binding of nineteen analogues of the upstream, terminal, monosaccharide residue of each of the O-polysaccharide (O-PS) of Vibrio cholerae O:1, serotype Ogawa and Inaba, with two murine monoclonal IgG antibodies both specific for the Ogawa LPS were measured using fluorescence spectroscopy. The use of the deoxy and the deoxyfluoro analogs allowed further refinement of the hydrogen-bonding pattern involved in the binding. Based on the binding characteristics observed for some of the ligands in the Inaba series, the binding of the monosaccharide that represents the upstream, terminal unit of the O-PS of V. cholerae O:1 serotype Inaba was redefined. We show for the first time that the upstream, terminal monosaccharide of the Inaba O-PS shows weak binding with these two anti-Ogawa antibodies. The results obtained allow further rationalization of the structural basis for the binding of V. cholerae O:1 antigens to their homologous antibodies.     

The apoplastic oxidative burst in response to biotic stress in plants: a three-component system

The oxidative burst, the generation of reactive oxygen species (ROS) in response to microbial pathogen attack, is a ubiquitous early part of the resistance mechanisms of plant cells. It has also become apparent from the study of a number of plant-pathogen interactions and those modelled by elicitor treatment of cultured cells that there may be more than one mechanism operating. However, one mechanism may be dominant in any given species. NADPH oxidases have been implicated in a number of systems and have been cloned and characterized. However, the enzyme system which is the major source of ROS in French bean (Phaseolus vulgaris) cells treated with a cell wall elicitor from Colletotrichum lindemuthianum, appears to be dependent on an exocellular peroxidase. The second component, the extracellular alkalinization, occurs as a result of the Ca(2+) and proton influxes and the K(+) efflux common to most elicitation systems as one of the earliest responses. The third component, the actual reductant/substrate, has remained elusive. The low molecular weight compound composition of apoplastic fluid was compared before and after elicitation. The substrate only becomes available some min after elicitation and can be extracted, so that by comparing the profiles by LC-MS it has been possible to identify possible substrates. The mechanism has proved to be complex and may involve a number of low molecular weight components. Stimulation of H(2)O(2) production was observed with saturated fatty acids such as palmitate and stearate without concomitant oxylipin production. This biochemical evidence is supported by immunolocalization studies on papillae forming at bacterial infection sites that show the peroxidase isoform present at sites of H(2)O(2) production revealed by cerium chloride staining together with the cross-linked wall proteins and callose and callose synthase. The peroxidase has been cloned and expressed in Pichia pastoris and has been shown to catalyse the oxidation reaction with the same kinetics as the purified enzyme. Furthermore, Arabidopsis plants transformed heterologously using the French bean peroxidase in antisense orientation have proved to be highly susceptible to bacterial and fungal pathogens. Thus it is possible that Arabidopsis is another species with the potential to mount an apoplastic oxidative burst and these transformed plant lines may be useful to identify the peroxidase that is responsible.     

Cloning and sequencing of the serine dehydrogenase gene from Agrobacterium tumefaciens

The structural gene for NADP+-dependent serine dehydrogenase [EC 1.1.1.-] from Agrobacterium tumefaciens ICR 1600 was cloned into Escherichia coli cells and its complete DNA sequence was analyzed. The gene encodes a polypeptide containing 249 amino acid residues. The enzyme had high sequence similarity to short-chain alcohol dehydrogenases from bacteria and unknown proteins of Haemophilus influenzae, Escherichia coli, and Saccharomyces cerevisiae.     

Studies on the dehydrative cyclization of epimeric 4-(L-xylo and L-lyxo-tetritol-1-yl)-2-phenyl-2H-1,2,3-triazoles. Circular dichroism and NMR assignment of the formed anomeric C-nucleoside L-threofuranosyl triazole analogs

Dehydrative cyclization of epimeric 4-(L-xylo- and L-lyxo-tetritol-1-yl)-2-phenyl-2H-1,2,3-triazoles afforded the anomeric alpha and beta-L-threofuranosyl analogs. The anomeric configuration of the formed anomeric C-nucleoside analogs was determined by circular dichroism and NMR spectroscopy.     

Desmoglein 3 acting as an upstream regulator of Rho GTPases, Rac-1/Cdc42 in the regulation of actin organisation and dynamics

Desmoglein 3 (Dsg3), a member of the desmoglein sub-family, serves as an adhesion molecule in desmosomes. Our previous study showed that overexpression of human Dsg3 in several epithelial lines induces formation of membrane protrusions, a phenotype suggestive of Rho GTPase activation. Here we examined the interaction between Dsg3 and actin in detail and showed that endogenous Dsg3 colocalises and interacts with actin, particularly the junctional actin in a Rac1-dependent manner. Ablation of Rac1 activity by dominant negative Rac1 mutant (N17Rac1) or the Rac1 specific inhibitor (NSC23766) directly disrupts the interaction between Dsg3 and actin. Assembly of the junctional actin at the cell borders is accompanied with enhanced levels of Dsg3, while inhibition of Dsg3 by RNAi results in profound changes in the organisation of actin cytoskeleton. In accordance, overexpression of Dsg3 results in a remarkable increase of Rac1 and Cdc42 activities and to a lesser extent, RhoA. The enhancements in Rho GTPases are accompanied by the pronounced actin-based membrane structures such as lamellipodia and filopodia, enhanced rate of actin turnover and cell polarisation. Together, our results reveal an important novel function for Dsg3 in promoting actin dynamics through regulating Rac1 and Cdc42 activation in epithelial cells.     

The Arabian cradle: mitochondrial relicts of the first steps along the southern route out of Africa

A major unanswered question regarding the dispersal of modern humans around the world concerns the geographical site of the first human steps outside of Africa. The "southern coastal route" model predicts that the early stages of the dispersal took place when people crossed the Red Sea to southern Arabia, but genetic evidence has hitherto been tenuous. We have addressed this question by analyzing the three minor west-Eurasian haplogroups, N1, N2, and X. These lineages branch directly from the first non-African founder node, the root of haplogroup N, and coalesce to the time of the first successful movement of modern humans out of Africa, ～60 thousand years (ka) ago. We sequenced complete mtDNA genomes from 85 Southwest Asian samples carrying these haplogroups and compared them with a database of 300 European examples. The results show that these minor haplogroups have a relict distribution that suggests an ancient ancestry within the Arabian Peninsula, and they most likely spread from the Gulf Oasis region toward the Near East and Europe during the pluvial period 55-24 ka ago. This pattern suggests that Arabia was indeed the first staging post in the spread of modern humans around the world.