Cell interaction

Sphingolipids (SPLs) are important structural components of membranes in some types of cells and are involved in numerous signaling processes. Sphingomyelin (SPM) and dihydrosphingomyelin (DHS) are the two major SPLs in membranes. Very little is known about the molecular species and role of DHS in biological systems. In this work, we employed high performance liquid chromatography with detection by atmospheric pressure chemical ionization and electrospray ionization (ESI) mass spectrometry to elucidate the SPL composition in biological extracts. No common dietary source of DHS is known to exist. A novel analytical method developed to analyze complex SPL mixtures was used to show that bovine milk contained substantial amounts of DHS. Also, the human lens is the only known system in which DHS is the most abundant SPL. The molecular species of DHS in cataractous lenses has never been reported. It was shown that there was a preference for monounsaturated species of DHS and SPM in all ages and in cataractous lenses. It was also discovered that SPLs were the primary PLs remaining in cataractous lenses. Finally, the formation of sodium adducts and dimers in the ESI source of the ion trap mass spectrometer prevented the accurate quantitative analysis of PLs. A new method was developed to eliminate these undesirable ions.

Soluble form of Junctional adhesion molecule C (sJAM-C) has been identified to cause angiogenesis as well as chemotaxis in endothelial cells. However, the role of sJAM-C in the context of cancer has not been elucidated. Our atomic force microscopy (AFM) stiffness measurements of normal mammary epithelial cells (MCF 10A) have shown a two-fold decrease in cell's stiffness in response to sJAM-C. Changes in cell stiffness are indicative of modulations in a cell's mechanical properties. Our results indicated that sJAM-C increased the MCF 10A cell migration about two-fold and also promoted a three-fold increase in chemotaxis. Additionally, sJAM-C treatment resulted in considerable filamentous-actin loss and peripheral actin ring breakage. We also found activation of Rho signaling pathway to be the main mechanism behind sJAM-C mediated alterations in MCF 10A cell cytoskeleton and motility. Our data present for the first time that sJAM-C is a pro metastatic mediator for normal mammary epithelial cells.

Talin is a ubiquitous, high-molecular-weight, flexible protein that plays a critical role in focal adhesions by activating, as well as connecting, integrins to the actin cytoskeleton. Talin's inactive auto-inhibitory state is speculated to be one of its modes of regulation inside the cell and is achieved through its head-tail interactions. In order to decipher the stability of this interaction, the head domain (residues 206-405) was cloned into a modified pET28m vector while the tail domains (residues 1654-2344 and 2225-2344) were cloned into the pET32a vector to obtain octa-histidine tagged and un-tagged peptide, respectively. Neither co-expression nor pull-down using the His-tagged head domain was successful in purifying a stable head-tail complex. Our results indicate rather weak interactions between the talin head and rod domains and hence, under our experimental conditions, do not lead to a stable auto-inhibitory complex that can be purified for further studies.