Preparation of intact, highly purified phagosomes from Dictyostelium.

Phagocytosis plays a fundamental role in the immune system for the defense against invading microorganisms and the clearing of apoptotic and cancerous cells. The common amoeba Dictyostelium discoideum is a recognized model for professional immune phagocytes and is now commonly used to study host-pathogen interactions. Dictyostelium is genetically and biochemically tractable and is a most versatile experimental system. The classical protocol for purifying phagosomes formed by ingestion of latex beads particles has been adapted to Dictyostelium. It was improved in yield, purity, and synchronicity, allowing isolation of milligram amounts of phagosomal proteins and lipids. This method has been used successfully to highlight membrane trafficking and phagosome maturation. Here, we present a step-by-step protocol including detailed notes necessary for ensuring access to a large number of highly synchronized phagosomes of high purity and integrity.

Phosphacan short isoform, a novel non-proteoglycan variant of phosphacan/receptor protein tyrosine phosphatase-beta, interacts with neuronal receptors and promotes neurite outgrowth.

Phosphacan, one of the principal proteoglycans in the extracellular matrix of the central nervous system, is implicated in neuron-glia interactions associated with neuronal differentiation and myelination. We report here the identification of a novel truncated form of phosphacan, phosphacan short isoform (PSI), that corresponds to the N-terminal carbonic anhydrase- and fibronectin type III-like domains and half of the spacer region. The novel cDNA transcript was isolated by screening of a neonatal brain cDNA expression library using a polyclonal antibody raised against phosphacan. Expression of this transcript in vivo was confirmed by Northern blot hybridization. Analysis of brain protein extracts reveals the presence of a 90-kDa glycosylated protein in the phosphate-buffered saline-insoluble 100000 x g fraction that reacts with antisera against both phosphacan and a recombinant PSI protein and that has the predicted N-terminal sequence. This protein is post-translationally modified with oligosaccharides, including the HNK-1 epitope, but, unlike phosphacan, it is not a proteoglycan. The expression of the PSI protein varies during central nervous system development in a fashion similar to that observed for phosphacan, being first detected around embryonic day 16 and then showing a dramatic increase in expression to plateau around the second week post-natal. Both the native and recombinant PSI protein can interact with the Ig cell adhesion molecules, F3/contactin and L1, and in neurite outgrowth assays, the PSI protein can promote outgrowth of cortical neurons when used as a coated substrate. Hence, the identification of this novel isoform of phosphacan/receptor protein tyrosine phosphatase-beta provides a new component in cell-cell and cell-extracellular matrix signaling events in which these proteins have been implicated.