Overexpressed monomeric human acetylcholinesterase induces subtle ultrastructural modifications in developing neuromuscular junctions of Xenopus laevis embryos.
Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle-specific proteins, among them the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DNA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos of Xenopus laevis. Recombinant human AChE (rHAChE) produced in Xenopus was biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over-expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenous Xenopus AChE displayed complex oligomeric assembly. Nonetheless, cell-associated rHAChE accumulated in myotomes of 2- and 3-day-old embryos within the same subcellular compartments as native Xenopus AChE. NMJs from 3-day-old DNA-injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vertebrate synaptogenesis.
Seidman
S
S
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Aziz-Aloya
R B
RB
Timberg
R
R
Loewenstein
Y
Y
Velan
B
B
Shafferman
A
A
Liao
J
J
Norgaard-Pedersen
B
B
Brodbeck
U
U
Soreq
H
H
D000110:Acetylcholinesterase / Q000096:biosynthesis / Q000378:metabolism*; D000818:Animals; D004625:Embryo, Nonmammalian / Q000502:physiology / Q000648:ultrastructure; D005260:Female; D005306:Fertilization; D006801:Humans; D008845:Microinjections; D008854:Microscopy, Electron; D009469:Neuromuscular Junction / Q000196:embryology / Q000648:ultrastructure*; D009865:Oocytes / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D014158:Transcription, Genetic; D014982:Xenopus laevis
D012333:RNA, Messenger; D000110:Acetylcholinesterase