Axolemmal repair requires proteins that mediate synaptic vesicle fusion.
A damaged cell membrane is repaired by a seal that restricts entry or exit of molecules and ions to that of the level passing through an undamaged membrane. Seal formation requires elevation of intracellular Ca(2+) and, very likely, protein-mediated fusion of membranes. Ca(2+) also regulates the interaction between synaptotagmin (Syt) and syntaxin (Syx), which is thought to mediate fusion of synaptic vesicles with the axolemma, allowing transmitter release at synapses. To determine whether synaptic proteins have a role in sealing axolemmal damage, we injected squid and crayfish giant axons with an antibody that inhibits squid Syt from binding Ca(2+), or with another antibody that inhibits the Ca(2+)-dependent interaction of squid Syx with the Ca(2+)-binding domain of Syt. Axons injected with antibody to Syt did not seal, as assessed at axonal cut ends by the exclusion of extracellular hydrophilic fluorescent dye using confocal microscopy, and by the decay of extracellular injury current compared to levels measured in uninjured axons using a vibrating probe technique. In contrast, axons injected with either denatured antibody to Syt or preimmune IgG did seal. Similarly, axons injected with antibody to Syx did not seal, but did seal when injected with either denatured antibody to Syx or preimmune IgG. These results indicate an essential involvement of Syt and Syx in the repair (sealing) of severed axons. We suggest that vesicles, which accumulate and interact at the injury site, re-establish axolemmal continuity by Ca(2+)-induced fusions mediated by proteins such as those involved in neurotransmitter release.
Detrait
E
E
Department of Physiology & Biophysics, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0641, USA.
Eddleman
C S
CS
Yoo
S
S
Fukuda
M
M
Nguyen
M P
MP
Bittner
G D
GD
Fishman
H M
HM
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000918:Antibody Specificity; D003400:Astacoidea; D001369:Axons / Q000378:metabolism* / Q000648:ultrastructure; D019771:Axotomy; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D049832:Decapodiformes; D005456:Fluorescent Dyes; D015151:Immunoblotting; D066298:In Vitro Techniques; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D050765:Qa-SNARE Proteins; D013572:Synaptic Vesicles / Q000378:metabolism*; D050857:Synaptotagmins
D000906:Antibodies; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050857:Synaptotagmins