medium-scale-preparation-drosophila-embryo-extracts-for-proteomic

Medium-scale Preparation of <em>Drosophila</em> Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

building-simple-versatile-illumination-system-for-optogenetic

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...

University of California, San Diego

Tandem affinity purification (TAP) has been widely used for the analysis of protein complexes. We investigated the parameters of the recently developed TAP method (GS-TAP) and its application in Drosophila. This new tag combination includes two Protein G modules and a streptavidin binding peptide (SBP), separated by one or two TEV protease cleavage sites. We made pMK33-based GS-TAP vectors to allow for generation of stable cell lines using hygromycin selection and inducible expression from a metallothionein promoter, as well as pUAST-based vectors that can be used for inducible expression in flies. Rescue experiments in flies demonstrated that the GS-TAP tag preserves the function of the tagged protein. We have done parallel purifications of proteins tagged with the new GS-TAP tag or with the conventional TAP tag (containing the Protein A and calmodulin binding peptide domains) at the amino terminus, using both cultured cells and embryos. A major difference between the two tags was in the levels of contaminating proteins, which were significantly lower in the GS-TAP purifications. The GS-TAP procedure also resulted in higher yield of the bait protein. Overall, GS-TAP is an improved method of protein complex purification because it provides a superior signal-to-noise ratio of the bait protein relative to contaminants in purified material.

Tandem affinity purification in Drosophila: the advantages of the GS-TAP system.

β-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. In this study, we report a new in vivo function of the Drosophila β-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. Our findings uncover a new in vivo function of β-arrestins and present a new mechanism of ERK inhibition by the Drosophila β-arrestin Krz.

β-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development.

Heparin and heparan sulfate mediated basic fibroblast growth factor (bFGF) signaling plays an important role in skeletal muscle homeostasis by maintaining a balance between proliferation and differentiation of muscle progenitor cells. In this study we investigate the role of a synthetic mimic of heparin, poly(sodium-4-styrenesulfonate) (PSS), on myogenic differentiation of C2C12 cells. Exogenous supplementation of PSS increased the differentiation of C2C12 cells in a dose-dependent manner, while the formation of multinucleated myotubes exhibited a nonmonotonic dependence with the concentration of PSS. Our results further suggest that one possible mechanism by which PSS promotes myogenic differentiation is by downregulating the mitogen activated extracellular regulated signaling kinase (MAPK/ERK) pathway. The binding ability of PSS to bFGF was found to be comparable to heparin through molecular docking calculations and by native PAGE. Such synthetic heparin mimics could offer a cost-effective alternative to heparin and also reduce the risk associated with batch-to-batch variation and contamination of heparin.

Phillip Kyriakakis

Department of Bioengineering

University of California, San Diego

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Phillip Kyriakakis

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Bioengineering

University of California, San Diego

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Department of Bioengineering