ABSTRACT
Collapsin-1/Sema III, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Cellular and molecular mechanisms by which collapsin-1 exerts its action are not fully understood. Collapsin-1 induces growth cone collapse via a pathway which may include neuropilin-1, a cellsurface collapsin-1 binding protein, as well as intracellular CRMP-62 and heterotrimeric G proteins. We previously identified a second action of collapsin-1, the facilitation of antero- and retrograde axoplasmic transport. This response occurs via a mechanism distinct from that causing growth cone collapse. To investigate the possible involvement of neuropilin-1 in the action of collapsin-1 on axoplasmic transport, we produced a soluble neuropilin-1 (sNP-1) lacking the transmembrane and intracellular region. sNP-1 progressively displaced the dose-response curve for collapsin-1 to induce growth cone collapse to higher concentrations. sNP-1 also inhibited collapsin-1-induced augmentation of both antero- and retrograde axoplasmic transport. Furthermore, an anti-neuropilin-1 antibody blocked the collapsin-induced axoplasmic transport. These results together indicate that neuropilin-1 mediates collapsin-1 action on axoplasmic transport. To visualize collapsin-1 binding to endogenous neuropilin-1, we used a truncated collapsin-1-alkaline phosphatase fusion protein (CAP-4). CAP-4 stains the growth cone, neurite, and cell body. However, local application of collapsin-1 to growth cone but to neither neurite nor cell body promotes axoplasmic transport. Thus, growth cone NP-1 mediates the facilitatory action of collapsin-1 on antero- and retrograde axoplasmic transport.
Subject(s)
Axonal Transport/physiology , Glycoproteins/metabolism , Growth Cones/metabolism , Nerve Tissue Proteins/metabolism , Alkaline Phosphatase/genetics , Animals , Antibodies/pharmacology , Cells, Cultured , Chick Embryo , Ganglia, Spinal/cytology , Gene Expression/physiology , Glycoproteins/genetics , Glycoproteins/immunology , Growth Cones/chemistry , Mice , Microscopy, Video , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/immunology , Neuropilin-1 , Plasmids , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Semaphorin-3A , Solubility , TransfectionABSTRACT
Chick collapsin-1, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Collapsin-1 induces growth cone collapse via a pathway which may include CRMP-62 and heterotrimeric G proteins. CRMP-62 protein is related to UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. Mutations in unc-33 affect neural microtubules, the basic cytoskeletal elements for axoplasmic transport. Using computer-assisted video-enhanced differential interference contrast microscopy, we now demonstrate that collapsin-1 potently promotes axoplasmic transport. Collapsin-1 doubles the number of antero- and retrograde-transported organelles but not their velocity. Collapsin-1 decreases the number of stationary organelles, suggesting that the fraction of time during which a particle is moving is increased. Collapsin-1-stimulated transport occurs by a mechanism distinct from that causing growth cone collapse. Pertussis toxin (PTX) but not its B oligomer blocks collapsin-induced growth cone collapse. The holotoxin does not affect collapsin-stimulated axoplasmic transport. Mastoparan and a myelin protein NI-35 induce PTX-sensitive growth cone collapse but do not stimulate axoplasmic transport. These results provide evidence that collapsin has a unique property to activate axonal vesicular transport systems. There are at least two distinct pathways through which collapsin exerts its actions in developing neurons.