Monoclonal Antibody Self-Association, Cluster Formation, and Rheology at High Concentrations

I used immunogold labeling and quick-freeze, deep-etch, rotary replication to characterize the membrane skeleton at regions with high concentrations of acetylcholine receptor domains in receptor clusters of cultured rat muscle cells. This membrane skeleton consists of a network of filaments closely applied to the cytoplasmic membrane surface. The filaments are specifically decorated by immunogold labeling with a monoclonal antibody, VIIF7, that recognizes an isoform of beta-spectrin colocalizing with acetylcholine receptors. The filaments are 32 +/- 11 nm in length and three to four filaments (average 3.1-3.3) join at each intersection to form the network. These parameters are nearly identical to those reported previously for the membrane skeleton of erythrocytes. Depending on the amount of platinum coating, filament diameters range from 9 to 11 nm in diameter, and are 1.4 nm larger on average than spectrin filaments of erythrocytes replicated at the same time. Filaments are decorated with gold particles close to one end, consistent with the location of the epitope recognized by the monoclonal antibody. Computer modeling shows that all filament intersections in the membrane skeletal network are equally capable of being labeled by the monoclonal antibody. This pattern of labeling is consistent with a network containing antiparallel homodimers of beta-spectrin.

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Thrombospondin stimulates motility of human neutrophils.

The Journal of Cell Biology ◽

10.1083/jcb.111.6.3077 ◽

1990 ◽

Vol 111 (6) ◽

pp. 3077-3086 ◽

Cited By ~ 51

Author(s):

P J Mansfield ◽

L A Boxer ◽

S J Suchard

Keyword(s):

Monoclonal Antibody ◽

Cell Types ◽

Human Neutrophils ◽

Heparin Binding ◽

Directed Movement ◽

Surface Receptors ◽

Chemotactic Peptides ◽

Heparin Binding Domain ◽

High Concentrations ◽

Priming Activity

Polymorphonuclear leukocytes (PMNs) migrate to sites of inflammation or injury in response to chemoattractants released at those sites. The presence of extracellular matrix (ECM) proteins at these sites may influence PMN accumulation at blood vessel walls and enhance their ability to move through tissue. Thrombospondin (TSP), a 450-kD ECM protein whose major proteolytic fragments are a COOH-terminal 140-kD fragment and an NH2-terminal heparin-binding domain (HBD), is secreted by platelets, endothelial cells, and smooth muscle cells. TSP binds specifically to PMN surface receptors and has been shown, in other cell types, to promote directed movement. TSP in solution at low concentrations (30-50 nM) "primed" PMNs for f-Met-Leu-Phe (fMLP)-mediated chemotaxis, increasing the response two- to fourfold. A monoclonal antibody against the HBD of TSP totally abolished this priming effect suggesting that the priming activity resides in the HBD of TSP. Purified HBD retains the priming activity of TSP thereby corroborating the antibody data. TSP alone, in solution at high concentrations (0.5-3.0 microM), stimulated chemotaxis of PMNs and required both the HBD and the 140-kD fragment of TSP. In contrast to TSP in solution, TSP bound to nitrocellulose filters in the range of 20-70 pmol stimulated random locomotion of PMNs. The number of PMNs migrating in response to bound TSP was approximately two orders of magnitude greater than the number of cells that exhibited chemotaxis in response to soluble TSP or fMLP. Monoclonal antibody C6.7, which recognizes an epitope near the carboxyl terminus of TSP, blocked migration stimulated by bound TSP, suggesting that the activity resides in this domain. Using proteolytic fragments, we demonstrated that bound 140-kD fragment, but not HBD, promoted migration of PMNs. Therefore, TSP released at injury sites, alone or in synergy with chemotactic peptides like fMLP, could play a role in directing PMN movement.

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