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

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.

Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungusPecoramyces ruminantiumstrain C1A

“…The amount of fungal biomass at the time of quantification was derived from the headspace pressure as previously described ( Ranganathan et al, 2017 ). The fungal biomass was vacuum filtered on 0.45 µm filters, and immediately crushed in a bath of liquid nitrogen using a mortar and pestle as described previously ( Calkins & Youssef, 2016 ). The crushed cells were then poured into 2 separate 15-mL plastic falcon tubes, and stored at −80 °C for subsequent RNA, and protein extraction, respectively.…”

“…RNA was extracted following the protocol in Epicentre ® MasterPure™ Yeast RNA Purification Kit, with few modifications as detailed previously ( Calkins & Youssef, 2016 ). RNA concentrations were measured using the Qubit ® RNA HS Assay Kit (Life Technologies ® , Carlsbad, CA, USA).…”

Characterization and rank assignment criteria for the anaerobic fungi (Neocallimastigomycota)

“…Such procedures, however, should not impede progress in wider aspects of Neocallimastigomycota biology. Indeed, the use of alphanumeric designations to identify strains has been a long-standing and widely accepted approach in the fields of biotechnology [30][31][32][33], biochemistry [34][35][36], cell biology [37,38] and beyond.…”

Peer Review #1 of "Development of an RNA interference (RNAi) gene knockdown protocol in the anaerobic gut fungus Pecoramyces ruminantium strain C1A (v0.1)"

“…Germinating spores were previously shown to be most amenable for accumulating the highest amount of exogenously added nucleic acids (23). We, therefore, reasoned that addition of chemically synthesized siRNA to the sterile anoxic flooding water at the onset of spore germination (at around 75 minutes from the onset of flooding) followed by re-incubation at 39ºC Manuscript to be reviewed previously described (46).The fungal biomass was vacuum filtered on 0.45 µm filters, and immediately crushed in a bath of liquid nitrogen using a mortar and pestle as described previously (69). The crushed cells were then poured into 2 separate 15-mL plastic falcon tubes, and stored at -80°C for subsequent RNA, and protein extraction, respectively.…”

“…RNA extraction, qRT-PCR, and RNA-seq. RNA was extracted following the protocol in Epicentre ® MasterPure TM Yeast RNA Purification Kit, with few modifications as detailed previously (69). RNA concentrations were measured using the Qubit® RNA HS Assay Kit (Life Technologies®).…”