Recruitment of ectodermal attachment cells via an EGFR-dependent mechanism during the organogenesis of Drosophila proprioceptors

Drosophila proprioceptors (chordotonal organs) are structured as a linear array of four lineage-related cells: a neuron, a glial cell, and two accessory cells, called cap and ligament, between which the neuron is stretched. To function properly as stretch receptors, chordotonal organs must be stably anchored at both edges. The cap cells are anchored to the cuticle through specialized lineage-related attachment cells. However, the mechanism by which the ligament cells at the other edge of the organ attach is not known. Here, we report the identification of specialized attachment cells that anchor the ligament cells of pentascolopidial chordotonal organs (lch5) to the cuticle. The ligament attachment cells are recruited by the approaching ligament cells upon reaching their attachment site, through an EGFR-dependent mechanism. Molecular characterization of lch5 attachment cells demonstrated that they share significant properties with Drosophila tendon cells and with mammalian proprioceptive organs.     

Delta-catenin at the synaptic-adherens junction

Delta-catenin belongs to the p120-catenin (p120(ctn)) protein family, which is characterized by ten, characteristically spaced Armadillo repeats that bind to the juxtamembrane segment of the classical cadherins. Delta-catenin is the only member of this family that is expressed specifically in neurons, where it binds to PDZ domain proteins in the post-synaptic compartment. As a component of both adherens and synaptic junctions, delta-catenin can link the adherens junction to the synapse and, thereby, coordinate synaptic input with changes in the adherens junction. By virtue of its restriction to the post-synaptic area, delta-catenin creates an asymmetric adherens junction in the region of the synapse. The crucial nature of the specialized function of delta-catenin in neurons is demonstrated by a targeted gene mutation, which causes deficits in learning and in synaptic plasticity. Taken together, recent evidence indicates that delta-catenin is a sensor of synaptic activity and implements activity-related morphological changes at the synapse.     

Approaching the CAPRI challenge with an efficient geometry-based docking

The last 3 rounds (3-5) of CAPRI included a wide range of docking targets. Several targets were especially challenging, since they involved large-scale movements and symmetric rearrangement, while others were based on homology models. We have approached the targets with a variety of geometry-based docking algorithms that include rigid docking, symmetric docking, and flexible docking with symmetry constraints. For all but 1 docking target, we were able to submit at least 1 acceptable quality prediction. Here, we detail for each target the prediction methods used and the specific biological data employed, and supply a retrospective analysis of the results. We highlight the advantages of our techniques, which efficiently exploit the geometric shape complementarity properties of the interaction. These enable them to run only few minutes on a standard PC even for flexible docking, thus proving their scalability toward computational genomic scale experiments. We also outline the major required enhancements, such as the introduction of side-chain position refinement and the introduction of flexibility for both docking partners.     

Activated membrane patches guide chemotactic cell motility

Many eukaryotic cells are able to crawl on surfaces and guide their motility based on environmental cues. These cues are interpreted by signaling systems which couple to cell mechanics; indeed membrane protrusions in crawling cells are often accompanied by activated membrane patches, which are localized areas of increased concentration of one or more signaling components. To determine how these patches are related to cell motion, we examine the spatial localization of RasGTP in chemotaxing Dictyostelium discoideum cells under conditions where the vertical extent of the cell was restricted. Quantitative analyses of the data reveal a high degree of spatial correlation between patches of activated Ras and membrane protrusions. Based on these findings, we formulate a model for amoeboid cell motion that consists of two coupled modules. The first module utilizes a recently developed two-component reaction diffusion model that generates transient and localized areas of elevated concentration of one of the components along the membrane. The activated patches determine the location of membrane protrusions (and overall cell motion) that are computed in the second module, which also takes into account the cortical tension and the availability of protrusion resources. We show that our model is able to produce realistic amoeboid-like motion and that our numerical results are consistent with experimentally observed pseudopod dynamics. Specifically, we show that the commonly observed splitting of pseudopods can result directly from the dynamics of the signaling patches.     

Dynamic combinatorial libraries of artificial repeat proteins

Repeat proteins are found in almost all cellular systems, where they are involved in diverse molecular recognition processes. Recent studies have suggested that de novo designed repeat proteins may serve as universal binders, and might potentially be used as practical alternative to antibodies. We describe here a novel chemical methodology for producing small libraries of repeat proteins, and screening in parallel the ligand binding of library members. The first stage of this research involved the total synthesis of a consensus-based three-repeat tetratricopeptide (TPR) protein (～14 kDa), via sequential attachment of the respective peptides. Despite the effectiveness of the synthesis and ligation steps, this method was found to be too demanding for the production of proteins containing variable number of repeats. Additionally, the analysis of binding of the individual proteins was time consuming. Therefore, we designed and prepared novel dynamic combinatorial libraries (DCLs), and show that their equilibration can facilitate the formation of TPR proteins containing up to eight repeating units. Interestingly, equilibration of the library building blocks in the presence of the biologically relevant ligands, Hsp90 and Hsp70, induced their oligomerization into forming more of the proteins with large recognition surfaces. We suggest that this work presents a novel simple and rapid tool for the simultaneous screening of protein mixtures with variable binding surfaces, and for identifying new binders for ligands of interest.     

Sugars enhance the expression of gibberellin-induced genes in developing petunia flowers

Sugar is essential for the development of detached Petunia hybrida flowers. We have shown that sucrose (Suc) and gibberellic acid (GA₃) are required for anthocyanin accumulation and the expression of various genes in developing petunia corollas. The effect of GA₃ on the expression of the gibberellin-induced gene and chalcone synthase gene, in detached corollas, was promoted by metabolic sugars such as Suc, glucose (Glc) and fructose, but not by the nonmetabolized 3- O -methylglucose and the sugar alcohol, mannitol. Several pieces of evidence support sugars' signaling role in the corollas and the possible involvement of hexokinase as the sugar sensor. Mannose, which is inefficiently metabolized but is phosphorylated by hexokinase at efficiency similar to Glc, was as effective as Glc in promoting gene expression and pigmentation. 2-Deoxyglucose, which is a substrate for hexokinase but is not metabolized in glycolysis, also promoted gene expression. On the other hand, mannoheptulose, a competitive inhibitor of hexokinase, completely abolished the promotive effect of Glc. We suggest that sugar-phosphorylation-related signal transduction interacts with the gibberellin signal to induce gene expression and anthocyanin accumulation in developing petunia corollas.