Search

MDCK cells were engineered to reversibly express mutant E-cadherin protein with a large extracellular deletion. Mutant cadherin overexpression reduced the expression of endogenous E- and K-cadherins in MDCK cells to negligible levels, resulting in decreased cell adhesion. Despite severe impairment of the cadherin adhesion system, cells overexpressing mutant E-cadherin formed fluid-filled cysts in collagen gel cultures and responded to hepatocyte growth factor/scatter factor (HGF/SF) that induced cellular extension formation with a frequency similar to that of control cysts. However, cells were shed from cyst walls into the lumen and into the collagen matrix prior to and during HGF/SF induced tubule extension. Despite the propensity for cell dissociation, MDCK cells lacking cadherin adhesion molecules were not capable of anchorage-independent growth in soft agar and cell proliferation rate was not affected. Thus, cadherin loss does not induce transformation, despite inducing an invasive phenotype, a later stage of tumor progression. These experiments are especially relevant to tumor progression in cells with altered E-cadherin expression, particularly tumor samples with identified E-cadherin extracellular domain genomic mutations.

Gelsolins are actin-binding proteins that cap, nucleate, and sever actin filaments. Microinjection of cytoplasmic or plasma gelsolin into living fibroblasts and macrophages did not affect the shape, actin distribution, deformability, or ruffling activity of the cells. Gelsolin requires calcium for activity, but the NH2-terminal half is active without calcium. Microinjection of this proteolytic fragment had marked effects: the cells rounded up, stopped ruffling, became soft, and stress fibers disappeared. These changes are similar to those seen with cytochalasin, which also caps barbed ends of actin filaments. Attempts to raise the cytoplasmic calcium concentration and thereby activate the injected gelsolin were unsuccessful, but the increases in calcium concentration were minimal or transient and may not have been sufficient. Our interpretation of these results is that at the low calcium concentrations normally found in cells, gelsolin does not express the activities observed in vitro at higher calcium concentrations. We presume that gelsolin may be active at certain times or places if the calcium concentration is elevated to a sufficient level, but we cannot exclude the existence of another molecule that inhibits gelsolin. Microinjection of a 1:1 gelsolin/actin complex had no effect on the cells. This complex is stable in the absence of calcium and has capping activity but no severing and less nucleation activity as compared with either gelsolin in calcium or the NH2-terminal fragment. The NH2-terminal fragment-actin complex also has capping and nucleating activity but no severing activity. On microinjection it had the same effects as the fragment alone. The basis for the difference between the two complexes is unknown. The native molecular weight of rabbit plasma gelsolin is 82,500, and the extinction coefficient at 280 nm is 1.68 cm2/mg. A new simple procedure for purification of plasma gelsolin is described.

Voltage-dependent sodium channels are distributed nonuniformly over the surface of nerve cells and are localized to morphologically distinct regions. Fluorescent neurotoxin probes specific for the voltage-dependent sodium channel stain the axon hillock 5-10 times more intensely than the cell body and show punctate fluorescence confined to the axon hillock which can be compared with the more diffuse and uniform labeling in the cell body. Using fluorescence photobleaching recovery (FPR) we measured the lateral mobility of voltage-dependent sodium channels over specific regions of the neuron. Nearly all sodium channels labeled with specific neurotoxins are free to diffuse within the cell body with lateral diffusion coefficients on the order of 10(-9) cm2/s. In contrast, lateral diffusion of sodium channels in the axon hillock is restricted, apparently in two different ways. Not only do sodium channels in these regions diffuse more slowly (10(-10)-10(-11) cm2/s), but also they are prevented from diffusing between axon hillock and cell body. No regionalization or differential mobilities were observed, however, for either tetramethylrhodamine-phosphatidylethanolamine, a probe of lipid diffusion, or FITC-succinyl concanavalin A, a probe for glycoproteins. During the maturation of the neuron, the plasma membrane differentiates and segregates voltage-dependent sodium channels into local compartments and maintains this localization perhaps either by direct cytoskeletal attachments or by a selective barrier to channel diffusion.

To investigate the physiologic role of gelsolin in cells, we have studied the location and mobility of gelsolin in a mouse fibroblast cell line (C3H). Gelsolin was localized by immunofluorescence of fixed and permeabilized cells and by fluorescent analog cytochemistry of living cells and cells that were fixed and/or permeabilized. Overall, the images show that in living cells gelsolin has a diffuse cytoplasmic distribution, but in fixed cells a minor fraction is associated with regions of the cell that are rich in actin filaments. The latter fraction is more prominent after permeabilization of the fixed cells because some diffuse gelsolin is not fixed and is therefore lost during permeabilization, confirmed by immunoblots. To determine quantitatively whether gelsolin is bound to actin filaments in living cells, we measured the mobility of microinjected fluorescent gelsolin by fluorescence photobleaching recovery. Gelsolin is fully mobile with a diffusion coefficient similar to that of control proteins. As a positive control, fluorescent phalloidin, which binds actin filaments, is totally immobile. These results are supported by immunoblots on cells permeabilized with detergent. All the endogenous gelsolin is extracted, and the half-time for the extraction is approximately 5 s, which is about the rate predicted for diffusion. Therefore, gelsolin is not tightly bound to actin filaments in cells. The most likely interpretation of the difference between living and fixed cells is that fixation traps a fraction of gelsolin that is associated with actin filaments in short-lived complexes.

Experiments were performed to study the feasibility of two mechanisms of acetylcholine receptor (ACHR) accumulation in chick myotubes: diffusion and trapping of previously dispersed surface receptors and localized insertion of new receptors at accumulation sites. Fluorescence photobleaching recovery (FPR) measurements indicated that the majority of diffusely distributed ACHRs in chick myotube membranes were mobile whereas nearly all receptors within high density clusters were effectively immobile. Unlike previous reports, two rates of ACHR movement characterized the mobile population. Moreover, we found that the estimated diffusion coefficient depended critically on the objective (spot size) used to assay recovery from bleaching. Implications of this finding for mechanisms of receptor immobilization are discussed. Extracts of chick brain, known to increase the number of surface receptors, did not alter receptor mobility. Extracts of Torpedo electric organ that increase the number of receptor aggregates, decreased the mobile fraction of ACHRs. Simulations of the diffusion and trapping mechanism indicated that captured receptors should congregate around the periphery of a receptor patch during the first hour after they were inserted into the membrane. However, newly inserted ACHRs were found to be located centrally within receptor patches under neurites, and this was not consistent with an exclusive diffusion-trapping mechanism. We also studied the mobility of ACHRs near points of contact made by cholinergic growth cones. The rate of receptor movement was increased in the vicinity of growth cones, but the magnitude of this effect was small.