Acetylcholine Receptors Enable the Transport of Rapsyn from the Golgi Complex to the Plasma Membrane

Abstract

The accumulation of acetylcholine receptors (AChRs) at nerve terminals is critical for signal transmission at the neuromuscular junction, and rapsyn is essential for this process. Previous studies suggest that AChRs might direct rapsyn self-clusters to the synapse. In vivo experiments with fluorescently tagged AChR or rapsyn in zebrafish larvae revealed that rapsyn self-clusters separate from AChRs did not exist before synapse formation. Examination of rapsyn in the AChR-less mutant sofa potato revealed that rapsyn in the absence of AChR was localized in the Golgi complex. Expression of muscle-type AChR in sofa potato restored synaptic clustering of rapsyn, while neuronal type AChR had no effect. To determine if this requirement of protein interaction is reciprocal, we examined the mutant twitch once, which has a missense mutation in rapsyn. While the AChRs distributed non-synaptically on the plasma membrane in twitch once, mutant rapsyn was retained in the Golgi complex. We conclude that AChRs enable the transport of rapsyn from the Golgi complex to the plasma membrane through a molecule-specific interaction.

Midkine-a Protein Localization in the Developing and Adult Retina of the Zebrafish and Its Function During Photoreceptor Regeneration

“…The primary antibodies used were: rabbit anti-Mdka 1:200 (Zymed Laboratories-Invitrogen; see [ 49 ]), mouse anti-BrdU 1:100 (BD Biosciences 347580), mouse anti-GM130 1:200 (BD Biosciences 610822; [ 50 ]), mouse anti-Glutamine Synthetase 1:200 (Millipore MAB302), mouse anti-GFP 1:200 (Millipore MAB3580), mouse anti-Zpr1 1:200 (anti-Arrestin 3, ZIRC), mouse anti-Zpr3 1:200 (anti-Rhodopsin, ZIRC). Secondary antibodies (Invitrogen) were raised against in either mouse or rabbit antibodies and conjugated to Alexa Fluor 488 and 555 and diluted 1:500.…”

Zebrafish mutants of the neuromuscular junction: swimming in the gene pool

“…One notable feature of the zebrafish NMJ is its accessibility for optical, time-lapse studies in vivo. Ex-utero development combined with genetically introduced fluorescent molecules enable studies in which synapse formation can be observed in its entirety, both spatially and temporally [ 14 , 41 , 42 ]. Studies utilizing these unique approaches complement studies using human patients or mammalian animal models.…”

“…However a conserved leucine residue is changed to proline near the N-terminus, which blocks the assembly of pentamers. In nic and sofa potato , AChR pentamers fail to form and the remaining normal subunits are retained in the endoplasmic reticulum [ 14 ]. These null mutants are completely paralyzed until their death approximately 7 days post-fertilization (dpf), but can be rescued by introducing a normal copy of the mutant subunit [ 13 , 15 ].…”

“…Rapsyn cannot localize to the synapse correctly in the absence of AChRs [ 13 ]. In particular, analysis of rapsyn in sofa potato revealed that rapsyn cannot reach the plasma membrane without AChRs and is retained in the Golgi apparatus [ 14 ]. When normal AChRs were introduced in sofa potato , the transport of rapsyn to the plasma membrane was restored.…”

“…When normal AChRs were introduced in sofa potato , the transport of rapsyn to the plasma membrane was restored. This effect of AChR on rapsyn was highly specific: the expression of a different AChR molecule, normally expressed in the brain [ 33 ], failed to restore rapsyn localization [ 14 ].…”