cytochalasin-d and Lymphoma--T-Cell

In order to investigate the role of microfilaments in the crawling movements of lymphoid cells, we have analyzed the effects of botulinum C2 toxin and of cytochalasin D (cytoD) on the actin cytoskeleton and on the motility of a BW5147 T-lymphoma-derived cell line. Actin was ADP-ribosylated by C2 toxin in the living cells, and this resulted in a time and dose-dependent disappearance of F-actin, as assessed by staining with labeled phalloidin. CytoD did not affect the amount of polymerized actin, but rather changed its distribution from a diffuse peripheral network to focal accumulations on one side of the cell. Both treatments affected the motility of the lymphoma cells in two assay systems. Fourier analysis was used to quantify shape changes performed by the cells. C2 toxin as well as CytoD caused the cessation of pseudopodal protrusion. Invasion of the lymphoma cells through a monolayer of fibroblast-like cells was also inhibited by the treatments, in a dose-dependent way. C2 toxin significantly inhibited invasion at concentrations at which only part of the actin pool had been ADP-ribosylated. We conclude that partial depolymerization, as well as disorganization, of the microfilament network impairs the active cellular deformations that are involved in the crawling movements of the lymphoma cells. From previous work, there is evidence to state that the monolayer invasion assay to some extent mimics tissue infiltration by hematopoietic cells. The present study is the first to analyze the role of actin polymerization in a model system that is relevant for the migration of lymphoid cells in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actins; Animals; Botulinum Toxins; Cell Movement; Cell Size; Cytochalasin D; Humans; Lymphoma, T-Cell; Mice; Mice, Inbred C3H; Neoplasm Invasiveness; Tumor Cells, Cultured

Studies were carried out to characterize the interaction between inositol 1,4,5-trisphosphate (IP3) receptors and the plasma membrane fraction. Extraction of the membranes with the nonionic detergents Nonidet P-40 and Triton X-100, followed by centrifugation at 100,000 g, resulted in the doubling of the IP3 receptor in the pellets, whereas no detectable binding was found in the supernatants. These data indicate that the detergents did not solubilize the receptor, that it remained associated with membrane particles, and that it is likely to be associated with the cytoskeleton. The cytoskeleton proteins actin, ankyrin, and spectrin were identified in the plasma membrane fraction. However, comparison of the amount of these proteins in different fractions of the detergent, or otherwise treated plasma membrane fractions, showed no direct correlation between the presence of any of these proteins in the plasma membrane fraction and their ability to bind [3H]IP3. This is in contrast to the brain and T-lymphoma cells in which the IP3 receptor is attached to ankyrin (L. Y. W. Bourguigon, H. Jin, N. Iida, N. R. Brandt, and S. H. Zhang. J. Biol. Chem. 268: 6477-6486, 1993; and S. K. Joseph and S. Samanta. J. Biol. Chem 268: 6477-6486, 1993). Thus the hepatic IP3 receptor, which is different from the brain receptor, might attach to the cytoskeleton by anchoring to a different protein. Because cytochalasin D treatment of livers diminishes the ability of IP3 to raise cytosolic free Ca2+ levels, the attachment of the IP3 receptor to the cytoskeleton seems to involve an association with microfilaments.

Topics: Actins; Animals; Ankyrins; Blotting, Western; Brain; Calcium Channels; Cell Membrane; Cytochalasin D; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; Gelsolin; Gluconeogenesis; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Kinetics; Liver; Lymphoma, T-Cell; Male; Oxygen Consumption; Phosphorylase a; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Tetracaine; Vasopressins