Toxin harvested from the snake species Bungarus multicinctus that binds Acetylcholine receptors and therefore paralyzes its prey. Alpha bungarotoxin is used as a label for Acetylcholine receptors.
A 400 kD proteoglycan made by nerve and glia. Agrin is transported to the nerve terminal and synaptic cleft. Due to the phenotype of agrin knockout mice (dispersed acetylcholine receptors), agrin was believed to be the factor which organizes the aggregation of acetylcholine receptors into clusters. Later experiments in model systems in which agrin could not have been present due to the absence of the pre synaptic nerve (Homeobox 9 or HB9 knockouts) showed that Agrin was not necessary for clustering. It has since been elucidated that agrin stops the dispersion of acetylcholine receptors. Dispersion of acetylcholine receptors is caused by the receptor's own ligand, the neurotransmitter acetylcholine.
A class of glycoproteins which contain glycosaminoglycan chains
The polysaccharides which form the carbohydrate moiety of glycoproteins.
A receptor tyrosine kinase found in muscle necessary for aggregation of Acetylcholine receptors into clusters. MuSK co-localizes with Acetylcholine receptors. Its expression peaks during the formation of neuro-muscular junctions.
A 43 kD cytosolic protein necessary for proper Acetylcholine aggregation. During early stages of muscle development Rapsyn co-localizes with acetylcholine receptors. Mice without rapsyn show normal localization of MuSK but no AchR clusters.
Choline Acetyl transferase. The enzyme is responsible for the synthesis of Acetylcholine and Coenzyme A from Choline and Acetyl-CoA. In ChAT mutant mice, there is an overabundance of acetylcholine receptor clusters. Additionally, in a ChAT-/-, agrin-/- double knock-out, the agrin single knock-out defect (lack of acetylcholine receptor clustering) is rescued. This suggested that acetylcholine is the negative regulator of acetylcholine receptor clustering, thereby leading to "Paradigm lost!" and a modified agrin hypothesis model to be formed.
A protein which is a known ligand for the erbB type receptor tyrosine kinase. Originally thoungt to be essential for NMJ development, recent studies have shown that its role is probably secondary, having to do with Schwann cell development.
Lecture 4 Model Systems
A neuromuscular junction (NMJ) is the synapse or junction of the axon terminal of a motoneuron with the motor end plate, the highly-excitable region of muscle fiber plasma membrane responsible for initiation of action potentials across the muscle's surface, ultimately causing the muscle to contract. The signal passes through the neuromusclar junction via the neurotransmitter acetylcholine. Mature NMJs contain three cell types - presynaptic cell, postsynaptic cell, and Schwann cells.
Torpedo californica generates electric current. A large amount of AchR and other proteins involved in the neuromuscular junction are present at high density in the electric ray, making it a suitable model organism for study of the neuromuscular junction.
Zebrafish are a useful model organism since their fertilization process is external, which allows scientists to study all stages of development with ease. In addition, they have a large number of offspring which allows for easy analysis of general trends in offspring. Most importantly, the embryos and early stages are transparent, which makes it easy to view internal structures. It also allows for labeling and tracking of proteins or cells labeled with GFP that can be seen through the skin of the animal. Work in zebrafish showed that AchR clusters appear prior to nerve arrival with axons binding to these AchR pre-clusters.
Squid giant axon
The giant squid axon has been used to elucidate many of the most exciting problems in the history of neurobiology, such as how action potentials initiate and what ion channels contribute to the resting membrane potential. The squid giant axon is about 1 mm in diameter, making its large size easy for axons to be visualized. In particular, Hodgkin and Huxley were able to perform their work on action potentials by inserting electrodes directly into the luman of the axon. The squid giant axon has provided the highest rate in measurement accuracy of action potentials, and they are still used in contemporary studies. The squid giant axon prep likewise proved to be useful to identify components of the presynaptic apparatus, especially motor proteins.
Genetic mosaic animals
Genetic mosaic animals have cells of different genotypes. Genetic mosaics can be used to determine whether a particular cell acts autonomously based on its own genotype and not those of neighboring cells. Genetic mosaic animals proved to be useful to study competition between axons and maintenance of synaptic stability. For example, in Lecture 4, we discussed that a group of axons were ChAT+ while others were ChAT-. With ChAT+ axons occupying over 50% of the area, Ach fueled the competition and ChAT+ axons were able to outcompete ChAT- axons.
Lecture 4 Techniques
α-bungarotoxin is one of the toxic components of the venom of the elapid snake Taiwanese banded krait (Bungarus multicinctus). It binds irreversibly to the acetylcholine receptor found at the neuromuscular junction, causing paralysis, respiratory failure and death. Bungarotoxin was discovered by researchers of the National Taiwan University in 1963. α-bungarotoxin is also a selective antagonist of the α7 nicotinic acetylcholine receptor in the brain.
Yellow fluorescent protein allows scientists to visualize and monitor cellular processes in organisms using optical microscopy and confocal microscopy. YFP is made usuing molecular cloning methods by fusing the fluorophore to a diversity of proteins or enzyme targets. After the crystal structure of green fluorescent proteins was elucidated, it was found that threonine residue 203 was near the chromophore. By mutating this residue to tyrosine, the excited state of the chromophore may be manipulated and shifted to longer wavelengths, resulting in YFP or proteins of various colors.
CFP, or cyan fluorescent protein, is a mutant form of GFP (Green Fluorescent Protein) that floresceses with the color cyan instead of green. The obvious benefit is to allow for staining of different markers in the same organism. Yellow Fluorescent Protein (YFP) is also an analogue of CFP and GFP. Fluorescent proteins are used in Fluorescence resonance energy transfer (FRET) experiments, in which these proteins can be coupled to important cellular proteins to visualize cell activity in vivo easily without causing undue disturbance to the living cell. For more information, please see the entry for GFP.
Radiolabeled amino acids
Radiolabeled amino acids are made by replacing a carbon atom with 11C in a physiologic amino acid. This does not chemically change the molecule, but allows for detection through positive electron tomography. Or radiolabeled amino acids can be imaged after being infused into cells and incorporated into synthesized proteins while in culture or in vivo. Tracking where the amino acids travel and their activities serves to garner information about cellular function and can aid in imaging structures. Methionine is the most popular amino acid for PET when made into L-[methyl-11C]-methionine (MET), and it is extremely effective at diagnosing brain tumors.
GFP fusion protein
GFP has been widely used to visualize receptors and various intracellular proteins and their movements in live cells. To investigate the dynamics of protein trafficking or membrane receptor diffusion, GFP-fusion proteins may be used. GFP may be fused to the protein of interest, and using Fluorescence Recovery After Photobleaching (FRAP), the rates of diffusion or trafficking of proteins may be determined. This process is completed by repeatedly bleaching areas of the cell containing the protein under investigation, then measuring the recovery intensity of the region after bleaching.
Lethal enhancer screen