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

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 wheat-germ 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 marker-free 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.