Multimerization and Retention of the Scavenger Receptor SR-B1 in the Plasma Membrane.

Scavenger receptor B1 (SR-B1), the main receptor for high-density lipoprotein (HDL), is key in preventing atherosclerosis. It removes cholesterol from HDL, returning the lipid-poor lipoprotein to the circulation. To study the mechanisms controlling SR-B1 dynamics at the plasma membrane and its internalization rate, we developed a single-chain variable fragment (ScFv) antibody to image the receptor in live cells and track the behavior of single SR-B1 molecules. Unlike transferrin receptors, cholera-toxin-binding gangliosides, and bulk membrane markers, SR-B1 was internalized only marginally over hours. Plasmalemmal retention was not attributable to its C-terminal PDZ-binding domain or to attachment to the cortical cytoskeleton. Instead, SR-B1 undergoes multimerization into large metastable clusters that, despite being mobile in the membrane, fail to enter endocytic pathways. SR-B1 multimerization was impaired by mutating its C-terminal leucine zipper and by disrupting actin polymerization, causing rapid receptor internalization. Multimerization and plasmalemmal retention are critical for SR-B1 function.

An optimized method for accurate quantification of cell migration using human small intestine cells.

Quantifying the ability of a compound to modulate cell migration rate is a crucial part of many studies including those on chemotaxis, wound healing and cancer metastasis. Existing migration assays all have their strengths and weaknesses. The "scratch" assay is the most widely used because it seems appealingly simple and inexpensive. However, the scratch assay has some important limitations, as the tool introducing the "wound" might injure/stress the boundary cells and/or harm underlying matrix coatings, which in both cases will affect cell migration. This described method is a Cell Exclusion Zone Assay, in which cell-free areas are created by growing cells around removable silicone stoppers. Upon appropriate staining with fluorescent dyes and microscopically visualizing the monolayers, the migration rate is then quantified by counting the cells (nuclei) intruding the void area left by the silicone insert. In the current study human small intestine epithelial cells were seeded on a physiological substrate matrix to produce collectively migrating monolayers. Different substrates were tested to determine the optimal surface for enterocyte adherence and migration and morphological changes monitored. Recombinant human epidermal growth factor and osteopontin purified from urine were tested to see if the established migration assay produces accurate and reliable migration data with human small intestine cells. The obtained data accurately confirmed that the two bioactive proteins modulate cellular migration in a dose-dependent manner. The presented assay can likely be converted for use with other adherent cell lines or substrate matrices and allows for high throughput, while cost is kept low and versatility high. Co-staining can be applied in order to assay for cell death, different cell types, cell stress and others allowing intricate analysis of migration rate of mixed populations and correction for cell viability.

Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells.

Secretory phospholipase A2 (sPLA2) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2's do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50-60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2.