Differential labeling of the cell surface of single ciliary ganglion neurons in vitro.

Abstract

Cationic ferritin binds in a time and concentration dependent manner to all surfaces of ciliary ganglion neurons in culture except "mounds" and "veils". In chase experiments, bound ferritin clears from the cell surfaces and forms larger and larger patches, even at low temperatures. Binding of cationic ferritin is inhibited by poly-I-lysine, potentiated by poly-L-glutamate, and not affected by neuraminidase (acylneuraminyl in HBSS, and then exposed to cationic ferritin. The cells were then washed and placed in 2.5% glutaraldehyde for 30 min. Alternatively, a 6 min chase intervened before the second glutaraldehyde fixation. For "cold" (0-4°) treatments (explained in text), all solutions (except glutaraldehyde) were kept on ice, pipettes were chilled, and dishes treated or chased in the cold were put directly on cracked ice with several pieces of ice on top of the dish cover. Neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) (Boeringer Mannheim, Clostridial, 250,ug/ml in HBSS with 75 ,tg/ml of bovine serum albumin; pH 7.0-7.1; tests for activity by M. R. Bernfield and S. Banarjee to be reported) was applied to washed, living cells for 10 or 20 min followed by washing of cells in HBSS, exposure to cationic ferritin (500 ,g/ml, 30 or 60 sec), HBSS washing, and fixation. Alternatively, cells were first labeled with ferritin (500 gtg/ml, 30 sec), washed and then exposed to the enzyme for 10 or 20 min. Control dishes run concurrently received the same series of washes and HBSS or HBSS plus albumin for equivalent times. In one experiment, cells were prefixed with 2.5% glutaraldehyde, washed in HBSS, placed in 0.1 M NH4Cl in HBSS for 4.5 hr, washed again, and then treated with neuraminidase in HBSS at pH 5.0 for 10 min. After repeated washing in HBSS, cationic ferritin (500 ,ug/ml, 30 sec) was applied. The cells were then washed and fixed again in glutaraldehyde.Hyaluronidase (hyaluronoglucosidase, hyaluronate 4-glycanhydrolase, EC 3.2.1.35; bovine testicular VI, Sigma; 3 ,tg/ml in Ca++-and Mg++-free HBSS at pH 7.1) or chondroitin ABC Iyase (EC 4.2.2.4) (Miles Laboratory; used at 1.0 or 0.2 units/ml in Ca++-and Mg++-free HBSS at pH 7.1) was applied to cells for 20 min, followed by washing, ferritin treatment, washing, and fixation. These enzymes normally remove extracellular materials from living cells or tissues (10, 11).Some cells were washed, exposed to poly-L-lysine.HBr (Miles; 500 ,ug/ml in HBSS for 5 min), washed, treated with ferritin (500 ,tg/ml, 30 sec), washed, and fixed. Others were pretreated with poly-L-glutamate-HBr (Sigma; 500,Ag/ml in HBSS for 60 sec) and treated similarly. In these and all other special proce-4100

Synthesis, insertion into the plasma membrane, and turnover of alpha-bungarotoxin receptors in chick sympathetic neurons.

“…synthesized and inserted into the surface primarily at the cell body, resulting in a gradient of membrane particles that decreases from the cell body to the tip of the growing axon. If there are sites for insertion of membrane proteins along axons as well as at the cell body as suggested in ultrastrucrural studies by Wessels et al (36), then this model fits our data for the a-bungarotoxin receptor. The intriguing possibility remains that, in the case of neurite extension, new lipid bilayers, relatively deficient in membrane proteins, are inserted into the growth cone plasma membrane.…”

Lectin labeling of sprouting neurons. II. Relative movement and appearance of glycoconjugates during plasmalemmal expansion.

“…Chase with complete medium is likely to further enhance the formation and eventual shedding from the cell surface of (glyco)protein-cationized ferritin aggregates in the experiments of Wessells et al . (50), even in the cold . Con A, especially if used at low concentration as in Letourneau's experiments (24), may indeed cause patching of membrane components as we suspect from our own observations and, therefore, may not be comparable to WGA and RCA I .…”

“…To shed some light on the mechanisms involved in plasmalemmal expansion in the neuron, we have carried out a series of surface-label pulse-chase studies' with carbohydrate-specific ligands, plant lectins (cf. references 1 and 10, and the studies with cationized ferritin by Wessells et al [50]). The lectins have been applied either in unconjugated or in ferritin-conjugated form .…”

Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin.

“…In electron microscope analysis of surface charge using glutaraldehyde-fixed cells, the effect of the fixation procedure has been minimized by the use of free amines to inactivate unreacted aldehyde groups (25,61). The effect of glutaraldehyde fixation on membrane protein surface charge has been assumed to be only that of adding free aldehydes.…”