cytochalasin-d and lucifer-yellow

Mechanisms that maintain the close coupling between the formation of photoreceptor disk membranes and the displacement of disk membranes toward the pigment epithelium are poorly understood. This study was designed to determine whether the axial displacement of disk membranes requires the assembly and insertion of new disk lamellae.. Retinal detachment and treatment with cytochalasin D were employed to interrupt the normal formation of disk membranes in cultured Xenopus laevis retinas. The effect of disrupting disk initiation and assembly upon disk displacement was documented and quantified.. Isolating retinas from the retinal pigment epithelium prevented the normal morphogenesis of disks, but previously formed disks moved distally at a rate that is greater than or equal to the rate in attached retinas or in vivo. Treatment of attached retinas in eyecups with cytochalasin D blocked initiation of new disks and resulted in the formation of ectopic, disk-like membranes, but it did not stop axial displacement of previously formed disks. Rod cells in retinas that were cultured while slightly elevated from the retinal pigment epithelium sometimes formed disks of a smaller diameter than normal, even though the rate of initiation and displacement of disks was the same as in vivo.. Observations on detached retinas and or retinas treated with cytochalasin D suggest that disk displacement does not depend upon normal disk formation and that the motive mechanism does not involve filamentous actin. The formation of small diameter disks in elevated retinas suggests that disk initiation and displacement is independent of the completion of normal diameter disks.

Topics: Animals; Cytochalasin D; Fluorescent Dyes; Intracellular Membranes; Isoquinolines; Morphogenesis; Organ Culture Techniques; Retinal Detachment; Rod Cell Outer Segment; Xenopus laevis

Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm.

Topics: Actin Cytoskeleton; Actins; Animals; Antibodies, Monoclonal; Calcium; Cell Line; Cell Membrane; Coated Pits, Cell-Membrane; Cytochalasin D; Dogs; Endocytosis; Epithelial Cells; Epithelium; Fluorescent Antibody Technique; Fluorescent Dyes; Iodine Radioisotopes; Isoquinolines; Kidney; Membrane Glycoproteins; Methionine; Receptors, Transferrin; Transfection; Transferrin; Vesicular stomatitis Indiana virus; Viral Envelope Proteins

Cytochalasin D was found to reduce the endocytosis of ricin and the fluid phase markers [14C]sucrose and Lucifer Yellow in Vero cells without reducing the uptake of transferrin. The number of coated pits at the plasma membrane was not affected by the treatment. Cytochalasin D also reduced the endocytosis of ricin in cells where uptake of transferrin from coated pits was blocked by low cytosolic pH. Colchicine had a similar effect as cytochalasin D. Both drugs inhibited the exocytosis of ricin from the cells, and they reduced the rate by which ricin intoxicated the cells. Cytochalasin D had essentially no effect on the ability of the cells to bind transferrin, whereas colchicine reduced the binding to some extent. Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the endocytic uptake of ricin in A431 cells both under normal culture conditions and when the coated pit/coated vesicle pathway was blocked by acidification of the cytosol. In contrast, EGF and TPA had no stimulatory effect on the uptake of transferrin at normal cytoplasmic pH, and they did not abolish the ability of low cytoplasmic pH to inhibit endocytic uptake of transferrin. The results indicate that cytochalasin D and colchicine selectively inhibit endocytic uptake from non-clathrin-coated areas of the cell membrane whereas EGF and TPA stimulate it. The data support the view that there are different endocytic mechanisms, and they indicate that at least in some cell types the non-clathrin-coated endocytosis can be modulated.

Topics: Animals; Biological Transport; Coated Pits, Cell-Membrane; Colchicine; Cytochalasin D; Endocytosis; Endosomes; Epidermal Growth Factor; Fluorescent Dyes; Isoquinolines; Kinetics; Membrane Fluidity; Microscopy, Electron; Ricin; Sucrose; Tetradecanoylphorbol Acetate; Transferrin; Vero Cells