Faculty Bibliography

Growth factors induce massive actin cytoskeletal remodeling in cells. These reorganization events underlie various cellular responses such as cell migration and morphological changes. One major form of actin reorganization is the formation and disassembly of dorsal ruffles (also named waves, dorsal rings, or circular ruffles). Dorsal ruffles are involved in physiological functions including cell migration, invasion, macropinocytosis, plasma membrane recycling, and others. Growth factors initiate rapid formation (within 5 min) of circular membrane ruffles, and these ruffles move along the dorsal side of the cells, constrict, close, and eventually disassemble ( approximately 20 min). Considerable attention has been devoted to the mechanism by which growth factors induce the formation of dorsal ruffles. However, little is known of the mechanism by which these ruffles are disassembled. Here we have shown that G proteins G(12) and G(13) control the rate of disassembly of dorsal ruffles. In Galpha(12)(-/-)Galpha(13)(-/-) fibroblast cells, dorsal ruffles induced by growth factor treatment remain visible substantially longer ( approximately 60 min) than in wild-type cells, whereas the rate of formation of these ruffles was the same with or without Galpha(12) and Galpha(13). Thus, Galpha(12)/Galpha(13) critically regulate dorsal ruffle turnover.

Heterotrimeric G proteins are critical cellular signal transducers. They are known to directly relay signals from seven-transmembrane G protein-coupled receptors (GPCRs) to downstream effectors. On the other hand, receptor tyrosine kinases (RTKs), a different family of membrane receptors, signal through docking sites in their carboxy-terminal tails created by autophosphorylated tyrosine residues. Here we show that a heterotrimeric G protein, G alpha(13), is essential for RTK-induced migration of mouse fibroblast and endothelial cells. G alpha(13) activity in cell migration is retained in a C-terminal mutant that is defective in GPCR coupling, suggesting that the migration function is independent of GPCR signaling. Thus, G alpha(13) appears to be a critical signal transducer for RTKs as well as GPCRs. This broader role of G alpha(13) in cell migration initiated by two types of receptors could provide a molecular basis for the vascular system defects exhibited by G alpha(13) knockout mice.

Purification of the prion protein (PrP) is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. A simple and efficient method for purification of recombinant bovine normal prion protein containing residues 104-242, PrP(104-242) expressed in Escherichia coli by high performance hydrophobic interaction chromatography (HPHIC) was presented in this work. The solution containing denatured and reduced protein in 8.0 mol/L urea extracted from the inclusion body was directly injected into the HPHIC column, aggregates were prevented by the interaction between the denatured PrP(104-242) molecules and the stationary phase during the chromatographic process, the soluble form of PrP(104-242) in aqueous solution was obtained after desorbed from the column. Several factors, including pH value, types of stationary phase and salt, and gradient mode, influencing the purification results were investigated. Optimal conditions were obtained for the purification of PrP(104-242) by HPHIC. This procedure yield PrP(104-242) of a purity of 96% with a recovery of 87%, respectively, for a single step purification of 40 min.

The total DNA was isolated from lymphocyte of peripheral blood of Chinese cattle. The PrPC gene was amplified by polymerase chain reaction, using two primers. It was then cloned into pGEM-T Easy Vector. The result of DNA sequencing was indicated that the 795 bp amplified fragment contains the entire PrPC coding sequence, which has no intron and is same as the published gene sequence.