Thirty-seventh Annual Meeting February 14–18, 1993 Washington Convention Center Washington, D.C

1993 Biophysical Journal  
The effect of La3+ on LLC-PK1 (porcine kidney epithelium derived) cells was investigated by ion microscopy, a quantitative imaging technique based on mass spectrometry. Cells were incubated with LaCI3 for 10 min. (1 mM) or 30 min (0.1 mM). and intracellular calcium distributions were imaged by ion microscopy. Compared to control cells, a 10 min. kDa band shifs mobility slighdy upon digestion with Endoglycosidase FINglycosidase F (Endo F), yielding a polypeptide of -137 kDa after N-linked
more » ... drate removal. A second, minor, wheat germ agglutinin-binding form of the receptor is also present in HIT cells. This protein, with an appaent MW of 150 kDa, photolabels with low efficiency. Incubation of this form of the receptor with Endo F also yields apolypeptideof-137 kDa, suggesting the two forms of the receptor contain similar polypeptides, but differ in carbohydrate content. This idea is suppored by experimets in which each receptor is digested with Endo P, fobSowedWby V8 protease. The resultant peptides from each receptor have identical clectrophoretic mobilities. Two radiolabeled peptides from the 140 kDa protein, of 49 and 66 kDa, are glycosylated at residue 9, the same position as in the intact polypeptide. This suggests that the N-terminus is extracellular, and that the sulfonylurea binding site is in the first one-third of the receptor protein. M-Pod347 THE KINETICS OF RECEPTOR-MEDIATED CELL ADHESION UNDER FLUID FLOW. ((L.A. Tempelman, D. A. Hammer)) The physiological function of many cells is dependent on their ability to adhere via receptors to Ugand-coated surfaces under fluid flow. For example, an early step in the leukocyte-mediated inflammatory response is the adhesion of neutrophils to endothelial cells under fluid stresses of 1 -5 dynes/cm2. We haVe developed an experimental system to measure the kdnetics of cell adhesion as a function of cell and surface chemistry and fluid flow. Using a paralld-plate flow chamber, we measure the binding of rat basophillc leukemia (RBL) cells prencubated with anti-DNP IgE to polyacrylamide gels to which 2,4 dinitrophenol (DNP) is covalentiy bound. We obseve the spatial pattern of cell binding as cells travel fom the DNP-free to the DNP-coated section of the chamber, a well as the total number of cells bound. Tbere Is a rather narrow range of DNP densities, IgE coverages and shear rates for which the percentage of cells which bind changes. For example, with 4 x 104 binding sites on the cells and a ligand density of approximately 1010 sites/cm2, adhesion ranged from 99 to 2.4 % for shear rates from 30 to 120 sec-1. The spacial patterns of adhesion indicate that adhesion is a probabilistic process. Results for adhesion as a fnction of receptor number and ligand density will also be presented. Finally, we measured the adhesion of RBL cells to DNP-coated gels which were preincubated with and-DNP IgE. Adhesion in this systan results from the binding of the Fce receptor to the Fc portion of IgE , a higher affinity interaction with a hundred fold lower forward reaction rate. Adhesion under flow In this system Is very low, although static adhesion occurs readily. This demonstrates that the forward rate of reaction of the receptor-ligand pair Is more important than Its affinity In the regulation of adhesion under fluid flow. MEMBRANE RECEPTORS 1 Pediat'i & Cell Biobgy and 2Mroblgy, NY. Human parainuenza virus type 3 (HPF3), a member of the paramyxovirus group of viruses, Is an important agernt of respiratory disease In children. HPF3 establishes persistent Infection in cell culture following high multiplicity Infection. The persistently infected (pi) cells do not fuse with each other, yet rapidly fuse when seeded with uninfected cells. We have shown that the failure of the pi cells to fuse with each other is due to a lack of a receptor on these cells for the viral hemagglutinin-neurarminidase (HN) glycoprotein, and established that both the fusion (F) and HN proteins are needed for cel fusion mediated by HPF3. We now use this infomation to investigate the mechanism of membrane fusion mediated by HPF3. Low mukicity Infection with HPF3 normally reuts In extensive cell fusion. However, syncytium formation is prevented by the addition of neuraminidase to remove slaic acd, the receptorfor the viral HN glypotein. Whe the Infected cells do not fuse, the virus still spreads throughout the culture, and the cells become persistently Infected. The finding that neuraminidase treatment of cells infected at a iow moi alows spread of the virus without cell fusion suggests that there are different slallc acid recptor requirents for a viru to infect a ell (by fusing with the plasma membrane) than for fusion between cells. To Investigate this, cels Infected with low muipllcity were treated with several different amounts of bacterial neuraminidase. Wih low level neuraminidase treatment, there are sufficient sialic acid receptors for virus to Infect a cell but not enough to allow cell fusion, while with higher level neuraminidase treatment there are insufficient sialic acid receptors even for virus Infecton. Our Interptaon of these data is that there are different neuraminic acid requirements for a virus partice to infect a oell by fusion of the viral envelpe with the piasma membrane than forfusion of an infected cell with an uninfected cell. These findings offer insight into virus-induced membrane fusion, the fundamental mechanism of pathogenesis by HPF3. N%Pod35O CHEMICAL CROSS-UNKING OF IgE-RECEPTOR COMPLEXES IN RBL-2H3 CELLS ((S.-Y. Mao and H. Metzger)) NIAMS, NIH, Bethesda, MD 20892 (Spon. by D.H.H. Tsao) Aggregation of IgE-receptor complexes on mast cells by multivalent ligands activates a signal transduction pathway that leads to degranulatbon. The phosphorylatlon of aggregated receptors and cellular proteins Is thought to play a role in intracellular signaling. Aggregated IgE-receptor complexes become associated with the Triton X-100 insoiubie cytoskeleton or plasma membrane skeleton. This association Is dependent on the degree of aggregation. We Investigated whether cellular components Interact preferentially with aggregated receptors and become temporarily trapped under conditions where the receptor-cytoskeleton Interactions are promoted. Gel electrophoresis and western blot by anti-phosphotyrosine antibody showed that both the phosphorylated P and y receptor subunits as well as a 85-kDa peptide (p85) become trapped In the detergent-insoluble skeleton. A crosslinking protocol was deveoped to stabilize the interaction between the receptor and proteins physically proximal to it. RBL celis were permeabilized with tetanolysin and proteins were cross-linked with the water-soluble chemical cross-linker, DTSSP. These experiments allowed oonfirmation of the interaction between the receptor, p45, and several additional proteins.
doi:10.1016/s0006-3495(93)81403-6 fatcat:aghpptcx7jh55dha23w336z7cu