Fellows' research: 'Claudin' days in worm neuromuscular junctions: Redefining roles and responsibilities


11 Dec 2018

Fellows' research: 'Claudin' days in worm neuromuscular junctions: Redefining roles and responsibilities

 

Recently published research of Dr Kavita Babu, Intermediate Fellow, Indian Institute of Science Education and Research, Mohali

By Preethi Ravi

The notion of boundaries and junctions is especially intriguing in biological systems. For example, individual cells are encapsulated within walls called cell membranes that have tightly regulated doors, aka junctions. These junctions allow cells to exchange molecules and proteins and thus communicate with each another. One such critical interface is that between neurons and muscles, referred to as neuromuscular junctions, NMJs (similar to 'synapses', the junction between neurons for neuron-to-neuron communication). Since any muscular movement involves efficient conversations between the neurons and muscle cells, proteins involved in regulating molecular traffic across NMJs are heavily investigated.

A group of researchers from the Indian Institute of Science Education and Research (IISER) Mohali, led by Dr. Kavita Babu (in collaboration with Dr. Zhitao Hu and his research team at Queensland Brain Institute, Australia), have discovered for the first time, a novel role for a family of ‘junction’ proteins known as claudins in the NMJs of Caenorhabditis elegans (C. elegans), a nematode widely used as a model organism and commonly called the ‘worm’. Claudins cement cells and help in regulated transport of proteins within the interstitial space and across neighbouring cells. Worms have around 18 genes that make claudin and claudin-like proteins. However, two publications, one in the Journal of Neuroscience (August 2018) and another in Cell Reports (November 2018), together show that claudins are not merely structural components, as was believed earlier, but in fact, regulate cellular processes at NMJs.

Pallavi, a graduate student in Dr. Babu's laboratory, decided to use the transparent and genetically amenable C. elegans for investigating the role of claudins as patrols of NMJs. To do this, Pallavi set up a behavioural screen in which normal worms (wild-type) were exposed to aldicarb, a drug capable of inducing paralysis to a certain degree. She then treated various worms carrying mutations for genes encoding claudin (claudin-deficient worms). The hope was that if the function of any one of the claudins was required in the NMJs, their loss would disrupt the neuromuscular communication in the mutant, claudin-deficient worms and thus result in paralytic characteristics that were different from those of the aldicarb-treated, normal worms.

Particularly, Pallavi tested a population of worms that lacked the claudin-like protein, HPO-30, and found that almost 90% of the mutant worms were resistant to aldicarb exposure. In parallel, another graduate student, Vina, noticed that a claudin-like protein, HIC-1, when absent, resulted in severe paralysis of the aldicarb-treated, mutant worms compared to aldicarb-treated, normal worms. Pallavi, Vina, and their colleagues found that both these claudins, HPO-30 and HIC-1, expressed in the muscles and in acetylcholine-producing cholinergic motor neurons, respectively, regulated the expression of acetylcholine receptors at the postsynaptic muscle end. These acetylcholine receptors are activated by the chemical, acetylcholine, at the postsynaptic terminals.

Owing to the divergent observations, the team delved into the question: If both the claudins regulate acetylcholine receptors, why did their loss lead to contrasting results in mutant worms post aldicarb exposure? They found that the claudins regulated two different types of acetylcholine receptors (levamisole-sensitive and nicotine-sensitive) and modulated acetylcholine receptor expression in different directions.

HPO-30 positively moderates the levels of a levamisole-sensitive acetylcholine receptor. In addition, the team found that HPO-30 in association with another cell adhesion molecule called NLG-1 helps maintain levels of levamisole-sensitive acetylcholine receptors on the postsynaptic terminals.

HIC-1, on the other hand, negatively influences the levels of a nicotine-sensitive acetylcholine receptor. However, what was puzzling was that HIC-1, from one end of the NMJ fence (presynaptic), seemed to be influencing the expression of receptors on the opposite (post-synaptic) end of the NMJ. Claudin's protein structure provided clues to this puzzle.

Crisscrossing the cell wall like a snake, claudins have both their head and tail regions within the cell's interior—a structural feature that helps them pick internal signals. Claudins, at their tail end also harbour domains that interact with proteins involved in maintaining a cell's shape and integrity. Interestingly, imaging experiments revealed that lack of HIC-1 messes up the underlying mesh of actin filaments, a family of proteins that give structural stability to cells. Using these two clues, Vina and colleagues identified that Neurabin, an actin-interacting protein associates with HIC-1. This partnership seemed to season the synapse for the release of a veteran called Wnt, which is a signalling protein well known for its role during early animal development. The team proposes that the binding of the released Wnt molecules to their related receptors on the muscle initiates cellular signals that then influence acetylcholine receptor levels. However, the missing link of how the actin fabric of the cell could affect Wnt release remains to be investigated. 

Cell Rep. 2018 Nov 13; 25(7): 1856–1871.e6.

 

While the team returns to the lab bench to solve some missing parts of this puzzle, it's time to redefine our textbook definition of claudins.

References:

1) The Claudin-like Protein HPO-30 Is Required to Maintain LAChRs at the C. elegans Neuromuscular Junction. Journal of Neuroscience. August 2018

2) Wnt Secretion Is Regulated by the Tetraspan Protein HIC-1 through Its Interaction with Neurabin/NAB-1. Cell Reports, November 2018

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Preethi Ravi (@catchpreethir) graduated from the National University of Singapore and carried out her graduate studies at the National Centre for Biological Sciences, Bangalore. Under the guidance of Prof. Gaiti Hasan, she investigated the molecular and cellular basis of flight in the fruit fly, Drosophila melanogaster. She is currently a freelance popular science writer, pursuing her interest in science and passion for writing.