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

Abstract

To study the dynamics of membrane components during neuritic growth, we carried out a series of pulse-chase experiments with ferritin-conjugated and unconjugated lectins on sympathetic neurons sprouting in vitro. Labeling of aldehyde-prefixed cultures with wheatgerm agglutinin or with the galactose-specific lectin of Ricinus communis is consistently dense near the distal end of the neurites . By contrast, if live cultures are labeled with these lectins and chased for 3-20 min, label-free plasmalemmal areas appear in the most peripheral regions of the growth cone, on filopodia and, furthermore, over vesicle clusters (SPVs) . These markerfree areas, however, contain lectin receptors, as can be shown by relabeling the chased cultures with the same lectins after the aldehyde fixation . In a further set of experiments, cultures are labeled with a saturating concentration of native lectin, chased, aldehyde-fixed, and then relabeled with the ferritin conjugate of the same lectin . In this case, the surfaces of filopodia and of SPV clusters are selectively labeled with the ferritin conjugate, indicating the insertion of new lectin receptors into the plasma membrane in the growth cone periphery. These results indicate that plasmalemmal expansion in the neuron occurs by a mechanism of polarized growth, possibly involving SPVs as plasmalemmal precursor vesicles .Recent studies on a variety of eukaryotic cell systems have produced a considerable body of data on mechanisms and location of synthesis of membrane components (e .g., references 41 and 42). However, only in a few cases are there data that suggest how newly synthesized membrane components, lipids and proteins, may reach the plasmalemma, or where and by what mechanism insertion of these components into the plasmalemma occurs . Results relevant to these questions have been obtained from studies on the assembly of viral membranes and have suggested that the transfer to the cell surface of certain membrane components occurs in particulate, rather than soluble, form (3,40,41) . In another system, the retinal rod (e .g., reference 54), disk membrane components have been demonstrated to migrate distally into the outer segment, presumably after localized insertion near the connecting stalk (cf. references 4 and 5) . Similarly, localized insertion of new membrane components may occur during egg cleavage . In that situation, fusion of vesicles with the plasma membrane of the cleavage furrow is thought to lead to its localized expansion (2, 7) . The fusion hypothesis has also been proposed for hyphal growth in

New genes associated with schizophrenia in neurite formation: a review of cell culture experiments

“…114,116 The activation of this pathway in hippocampal cells induced membrane assembly at the axonal growth cone, a key parameter of axonal growth. [126][127][128][129][130] Whereas in PC12 cells the PI3/Akt signaling mechanism was accompanied by an increase in neurofilament protein NF68 a marker of neuronal differentiation. 114,131,132 Three studies done with PC12 cells 114,115,117 and one done with hippocampal cells 116 found that Akt activation was followed by GSK-3 phosphorylation which as a result can lead to microtubule polymerization 112 and consequently, neurite formation.…”

Localization of Synaptotagmin in the Regenerating Sprouts Emanating from the Nodes of Ranvier.

“…The Ca2+-dependent membrane fusion in growth cones has been studied by the Phenninger's group, though proteins involved in the fusion have not been identified. They have developed a cell-free membrane expansion assay using intact growth cone particles and found that high potassium depolarization and calcium-ionophore treatment cause significant increases in exposed WGA binding sites within minutes [22,23]. By morphometric analysis, the source of the newly added plasma membranes has been identified as clear vesicles (150 nm in diameter).…”

“…The source of the membrane for the rapidly growing axolemma and the mode by which the membrane is added to the axolemma have been of primary interest. Pfeninger et al have demonstrated in vitro that some clear vesicles localized in growth cones fuse with the axolemma in a Cat-dependent manner [22,23]. The molecules involved in the Cat-dependent membrane fusion in the growth cones are, however, largely unknown.…”

Lectin labeling of sprouting neurons. I. Regional distribution of surface glycoconjugates.

“…Prolonged exposure to lectin at 36°C is expected to lead to a decrease in the density of binding sites because of internalization and/or capping-like rearrangement ofthe membrane-bound ligand. The observed decrease in binding sites (20).…”

“…Thus, cellular polarity is also reflected in cell surface chemistry . This finding has to be viewed in light of the fact that neuronal plasmalemma is expanding very rapidly during neuritic growth, and that addition of certain new components to the plasma membrane occurs predominantly in the growth cone area, whereas other membrane components appear to be added primarily at the perikaryon (19,20,22 Labeling of spinal cord anterior horn neurons with F-RCA II and F-SBA. All samples were aldehyde-fixed before labeling .…”