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The larval musculature of Drosophila arises by fusion of two types of myoblasts: the founder cells (FCs), which determine the identity of every individual muscle, and fusion competent myoblasts (FCMs). Cell–cell recognition and adhesion is mediated by the Ig class of transmembrane proteins. They form an adhesion ring/belt at the contact sites of FCMs and FCs/growing myotubes to establish a Fusion Restricted Myogenic Adhesive Structure (FuRMAS). FuRMAS are postulated to trigger myoblast fusion, with the formation, and dissolution of F-actin foci/plugs at the sites of cell–cell contact. Electron-dense vesicles accumulate at opposing membranes of FCMs and FCs/growing muscles, and form a pre-fusion complex (1 μm²). This is hypothesised to take place in the centre of the FuRMAS. The vesicles are thought to be exocytosed, followed by membrane vesiculation and removal of membrane remnants to achieve cytoplasmic continuity over an area of 12 μm². The FCM can then be integrated into the growing myotube. This last step depends on Arp2/3 mediated F-actin reorganisation. The data on cell adhesion, signalling and actin regulation in zebrafish, C2C12 cells and mice strongly indicate conserved factors and principles between Drosophila and vertebrate myoblast fusion. [ABSTRACT FROM AUTHOR]

Because virtually all cellular processes are based on proteins, detailed knowledge of the mitochondrial proteome represents an integral part of understanding mitochondrial function. The analysis of very complex protein mixtures such as entire cell organelles makes high demands on analysis techniques in order to ensure integrity of the obtained data set. The use of Saccharomyces cerevisiae as a model system allows the isolation of mitochondria of utmost purity in large amounts. Various approaches can be accomplished in the subsequent analysis to achieve the most complete overview possible. Combinations of orthogonal proteomics techniques include two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS-PAGE), and nano-LC-MS/MS (nano-liquid chromatography with tandem mass spectrometry) as well as multidimensional high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The inherent limitations of the individual methods can be countervailed by parallel application of these approaches. [ABSTRACT FROM AUTHOR]

With the introduction of soft ionization techniques such as Matrix Assisted Laser Desorption Ionization (MALDI), and Electrospray Ionization (ESI), proteins have become accessible to mass spectrometric analyses. Since then, mass spectrometry has become the method of choice for sensitive, reliable and inexpensive protein and peptide identification. With the increasing number of full genome sequences for a variety of organisms and the numerous protein databases constructed thereof, all the tools necessary for the high-throughput protein identification with mass spectrometry are in place. This chapter highlights the different mass spectrometric techniques currently applied in proteome research by giving a brief overview of methods for identification of posttranslational modifications and discussing their suitability of strategies for automated data analysis. [ABSTRACT FROM AUTHOR]

The Dictyostelium dicoideum centrosome consists of a box-shaped, layered core structure surrounded by dense nodules embedded in amorphous material, which make up the so-called corona. Thus, it differs markedly from centriole-containing centrosomes in animal cells or the plaque structure of yeast spindle pole bodies. For a long time, purification of Dictyostelium centrosomes was hampered by its extraordinarily tight linkage to the nucleus, which resisted all attempts to dissociate centrosomes and nuclei without destruction of the centrosome itself. Fortunately, we were able to solve this problem, and have already published a centrosome isolation protocol that is based on treatment of nucleus/centrosome complexes with sodium pyrophosphate and shear forces, followed by centrosome isolation through sedimentation and filtration techniques. However, isolated centrosomes prepared according to this protocol still contained too many impurities to allow mass spectrometrical analyses. Here, we present an improved protocol for the isolation of Dictyostelium centrosomes that contain considerably less contaminations with cytosolic and nuclear proteins. [ABSTRACT FROM AUTHOR]