Spatial organization of acetylcholine receptors at the neuromuscular junction Pubblico

Abstract

The coordinated movement of humans’ everyday lives is dependent in part upon the neuromuscular junction (NMJ). The NMJ is a large synapse that connects motor neurons to skeletal muscles for precise and controlled movement. Defects in the development or function of this synapse commonly underlie many neuromuscular diseases, such as amyotrophic lateral sclerosis (ALS) and myasthenia gravis. Of all of the molecular components at the NMJ, the neurotransmitter receptor, acetylcholine receptor (AChR), plays one of the largest roles in the initiation of muscle contraction. AChRs are highly concentrated at the NMJ on the surface of the muscle fiber where they are responsible for receiving and responding to neurotransmitter released from the motor neuron terminal. This dissertation focuses on the organization of AChRs and the developmental processes that lead to such an organization. I have identified a novel organization of acetylcholine receptors at the postsynaptic membrane of the adult NMJ. The postsynaptic membrane of the NMJ contains numerous infoldings in which many key postsynaptic proteins, including AChRs, are segregated to either the top of the folds or the bottom of the infolded membrane. Using super-resolution microscopy, I found that AChRs located at the top of membrane folds are specifically concentrated in the area directly opposite that of presynaptic neurotransmitter release sites, and not distributed across the entire top of the membrane fold as previously thought. I also examined the developmental processes responsible for the spatial organization of AChRs and found that the actin cytoskeleton, including the actin binding proteins cortactin and profilin, plays a significant role in the clustering of AChRs. A highly dynamic actin cytoskeleton was found to be necessary for the clustering of AChRs during synaptogenesis. Thus, the data presented in this dissertation reveal a novel organization of AChRs, whereby the actin cytoskeleton plays a significant role in orchestrating this organization during development. 

Table of Contents

Chapter I: Introduction...........................................................................1

1.1 Significance of the neuromuscular junction .................................................. 1

1.2 NMJ synaptogenesis & development ............................................................ 3

1.3 Neurotransmission ......................................................................................... 7

1.4 Molecular composition & signaling of the NMJ ........................................... 8

1.4.1 AChR clustering ............................................................................... 9

1.4.2 AChR dispersal............................................................................... 11

1.4.3 Synaptic basal lamina ..................................................................... 12

1.5 Postsynaptic cytoskeleton....................................................................................... 15

1.5.1 Actin biology ............................................................................................ 15

1.5.2 Two pools of actin at the NMJ ................................................................. 16

1.5.3 Microtubules ............................................................................................ 19

1.5.4 Intermediate filaments .............................................................................. 20

1.5.5 Complementary roles of the postsynaptic cytoskeleton ........................... 21

1.6 Diseases of the NMJ ............................................................................................... 22

1.6.1 Amyotrophic lateral sclerosis ................................................................... 23

1.6.2 Myasthenia gravis .................................................................................... 24

1.6.3 Congenital myasthenic syndromes ........................................................... 25

1.6.4 Lambert-Eaton myasthenic syndrome ...................................................... 26

1.7 Summary................................................................................................................. 27

1.7 Figures .................................................................................................................... 29

Chapter II: The actin cytoskeleton regulates the clustering of aneural acetylcholine receptors ..... 33

2.1 Introduction ................................................................................................. 34

2.2 Results ......................................................................................................... 36

2.2.1 AChR and actin organization at aneural AChR clusters ................ 36

2.2.2 Dynamic F-actin puncta are concentrated at aneural AChR clusters .......................... 37

2.2.3 Actin binding proteins associated with aneural actin puncta.......... 39

2.2.4 Dynamic F-actin puncta are involved in the accumulation of AChRs into aneural clusters .................... 40

2.3 Discussion............................................................................................................... 43

2.4 Materials & Methods ................................................................................... 47

2.5 Figures ......................................................................................................... 53

Chapter III: Super-resolution microscopy reveals a nanoscale organization of acetylcholine receptors for trans-synaptic alignment at neuromuscular synapses.... 63

3.1 Introduction ................................................................................................. 64

3.2 Results ......................................................................................................... 66

3.3 Discussion.................................................................................................... 72

3.4 Materials & Methods ................................................................................... 76

3.5 Figures ......................................................................................................... 80

Chapter IV: Discussion: from structure to function ..........................89

4.1 Fine-tuning AChR organization at the NMJ................................................ 90

4.2 What is the role of the actin puncta in NMJ development?......................... 91

4.3 How do postsynaptic nanodomains relate to NMJ development and function?..........94

4.3.1 How do postsynaptic nanodomains contribute to NMJ function?.. 95

4.3.2 What regulates the formation of postsynaptic nanodomains? ........ 97

4.4 Outlook ........................................................................................................ 97

4.5 Summary...................................................................................................... 98

References ........................................................................................... 100 

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Biology, Cell
Parola chiave	synapse neuromuscular junction acetylcholine receptor
Committee Chair / Thesis Advisor	Zheng, James, Emory University
Committee Members	Ono, Shoichiro, Emory University Sale, Winfield, Emory University Faundez, Victor, Emory University Kowalczyk, Andrew, Emory University

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