PgmNr W437: A transient, pre-cuticular apical extracellular matrix defines tiny tube diameter.

Authors:
J. D. Cohen; H. K. Gill (co first author); R. Forman-Rubinsky; M. V. Sundaram


Institutes
University of Pennsylvania SOM, Philadelphia, PA.


Keyword: Extracellular matrix

Abstract:

Apical extracellular matrix (aECM) shapes apical domains of many epithelia, but the mechanism by which it does so is still poorly understood. The aECM contains glycosaminoglycans and various glycoproteins, including mucins and zona pellucida domain (ZPD) proteins. Many ZPD proteins form fibrils via the ZPD, but how fibril formation is controlled and how fibrils influence cell shape is not clear.  Loss of ZPD proteins causes a number of human diseases, including chronic kidney disease, vascular disease, and deafness. We are interested in the role of the aECM and ZPD proteins in stabilizing seamless tubes, a specialized cell type found in capillary beds of the mammalian vascular system in which a single cell encloses a lumen without junction along its length. By forward genetic screening, we identified 85 alleles required for maintaining the duct cell, a seamless tube of the C. elegans excretory system. Two of these alleles disrupt let-653, which encodes a secreted ZPD protein that also contains mucin-like and PAN-Apple domains (putative carbohydrate or protein interaction domains also found in plasminogen and other blood coagulation proteins). LET-653 is present within the developing duct lumen as the duct elongates from a simple cylinder to a highly elongated and asymmetric shape, but then disappears prior to the bulk of cuticle secretion. In let-653 mutants, duct lumen diameter becomes irregular during elongation and the lumen and cell eventually fragment, showing that LET-653 is essential for tube integrity. The LET-653 ZPD, but not the N-terminal PAN domains, is sufficient to rescue these duct cell defects. To address the mechanism by which LET-653 defines lumen diameter, we turned to the much larger vulva lumen, whose shape LET-653 subtly influences. Tagged LET-653 was found in two distinct pools within this lumen: at the apical membrane, where it was highly stable as assessed by FRAP, and in the center of the vulva, where it was mobile. Structure-function analyses revealed that LET-653’s ZPD was necessary and sufficient for the apical localization, while the PAN domains localize the protein to the center of the vulva lumen.  Ectopic expression of full-length LET-653, but not the PAN domains alone, could expand lumen diameter in the developing gut tube, where LET-653 is not normally expressed. Together, these data support a model in which the PAN domains regulate the ability of the LET-653 ZPD to interact with other factors at the apical membrane, and this ZPD interaction promotes expansion of lumen diameter. We are currently testing if other genes identified in our screen might encode the apical factor(s) that interact with LET-653 for this mechanism.



Wormbase Genetic Index
1. let-653