Insights into the Utility of the Focal Adhesion Scaffolding Proteins in the Anaerobic Fungus Orpinomyces sp. C1A.

Shelby Calkins,Noha H Youssef

doi:10.1371/journal.pone.0163553

Abstract

Focal adhesions (FAs) are large eukaryotic multiprotein complexes that are present in all metazoan cells and function as stable sites of tight adhesion between the extracellular matrix (ECM) and the cell’s cytoskeleton. FAs consist of anchor membrane protein (integrins), scaffolding proteins (e.g. α-actinin, talin, paxillin, and vinculin), signaling proteins of the IPP complex (e.g. integrin-linked kinase, α-parvin, and PINCH), and signaling kinases (e.g. focal adhesion kinase (FAK) and Src kinase). While genes encoding complete focal adhesion machineries are present in genomes of all multicellular Metazoa; incomplete machineries were identified in the genomes of multiple non-metazoan unicellular Holozoa, basal fungal lineages, and amoebozoan representatives. Since a complete FA machinery is required for functioning, the putative role, if any, of these incomplete FA machineries is currently unclear. We sought to examine the expression patterns of FA-associated genes in the anaerobic basal fungal isolate Orpinomyces sp. strain C1A under different growth conditions and at different developmental stages. Strain C1A lacks clear homologues of integrin, and the two signaling kinases FAK and Src, but encodes for all scaffolding proteins, and the IPP complex proteins. We developed a protocol for synchronizing growth of C1A cultures, allowing for the collection and mRNA extraction from flagellated spores, encysted germinating spores, active zoosporangia, and late inactive sporangia of strain C1A. We demonstrate that the genes encoding the FA scaffolding proteins α-actinin, talin, paxillin, and vinculin are indeed transcribed under all growth conditions, and at all developmental stages of growth. Further, analysis of the observed transcriptional patterns suggests the putative involvement of these components in alternative non-adhesion-specific functions, such as hyphal tip growth during germination and flagellar assembly during zoosporogenesis. Based on these results, we propose putative alternative functions for such proteins in the anaerobic gut fungi. Our results highlight the presumed diverse functionalities of FA scaffolding proteins in basal fungi.

Highlights

In eukaryotes, focal adhesions are sites of stable contacts with the extracellular matrix (ECM) and subsequent polymerization of the cell’s cytoskeleton
Focal adhesions (FAs) are comprised of large multiprotein complexes that are mediated by integrins, heterodimeric membrane proteins that act as the point of matrix-cytoskeleton connection [1]
The integrin-cytoskeleton link is further stabilized by the recruitment of the IPP complex, comprising integrin-linked kinase (ILK), parvin, and PINCH, to promote cytoskeleton linkage and integrin signaling

Summary

Introduction

Focal adhesions are sites of stable contacts with the ECM and subsequent polymerization of the cell’s cytoskeleton They mediate interaction between the ECM and the cell interior by promoting cell anchorage and mechanical adhesion to the ECM, as well as act as signaling milieu where signaling proteins are concentrated at sites of integrin binding and connect the cell’s cytoskeleton to the ECM. The process is initiated in the presence of an ECM protein ligand, e.g. fibronectin that binds to the ECM receptor integrin. This integrin-ECM bond recruits the scaffolding protein talin to the focal adhesion site, which in turn binds actin microfilaments and functions to strengthen the integrin-ECM bond. Actin crosslinking occurs via α-actinin, which orchestrates the elongation and growth of focal adhesions

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