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Identification and characterization of agrin in "Caenorhabditis elegans"

Hrus, Ana. Identification and characterization of agrin in "Caenorhabditis elegans". 2006, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_7699

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Abstract

Agrin is a large basement membrane (BM) proteoglycan expressed in many tissues in vertebrates, with particularly important function at the neuromuscular junction (NMJ) where it clusters acetylcholine receptors (AChRs) and maintains structural stability of postsynaptic specializations. In order to cluster the receptors it has to activate muscle-specific kinase (MuSK) through an indirect interaction via an unidentified myotube-associated specificity component (MASC). Agrin has also been implicated in providing structural integrity to different tissues by connecting the extracellular matrix (ECM) to α-dystroglycan (α-DG) which is part of a large supramolecular dystrophyn-associated glycoprotein complex (DGC) spanning the cell membrane and binding the actin cytoskeleton.
Since an agrin orthologue was identified in the C. elegans genome, we decided to experimentally confirm its expression and characterize the protein. Based on the predicted sequences, we cloned the agr-1 cDNA and assembled the ORF of 4422 bp from overlapping fragments. The putative protein domain architecture shared high similarity to the vertebrate agrin, except for missing one laminin G (lamG) domain, serine/threonine-rich regions and the SEA module. Since in vertebrates agrin exists in two main isoforms varying at the amino (N)-terminal side, it was surprising to identify only one isoform in C. elegans. Likewise, additional alternative splicing that occurs at conserved sites in the vertebrate agrin orthologues having strong impact on the AChRs clustering activity, was not identified in AGR-1. Reporter constructs revealed agr-1 expression in the buccal epithelium of the pharynx, in four IL1 sensory neurons in the head, and the distal tip cell (DTC) of the gonad, but surprisingly no expression was found in the muscles or the motoneurons innervating them. The specific anti-AGR-1 antibodies detected the protein in the basement membrane of the pharynx.
We analyzed several agr-1 mutant strains and performed many different assays with the goal to identify its function. No defects related to the NMJ could be found and some indications suggested that it might be implicated in the gonad migrations through genetic interaction with other factors. Based on the expression pattern in the head neurons and pharynx, we expected a sensory or feeding-related function but did not see clear defects. AGR-1 probably acts in parallel with several other proteins in a redundant fashion.
This is the first characterization of an invertebrate agrin orthologue which sets a substantial basis for further research.
Advisors:Chiquet-Ehrismann, Ruth
Committee Members:Rüegg, Markus A.
Faculties and Departments:09 Associated Institutions > Friedrich Miescher Institut FMI
UniBasel Contributors:Rüegg, Markus A.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:7699
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:120
Language:English
Identification Number:
Last Modified:22 Apr 2018 04:30
Deposited On:13 Feb 2009 15:50

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