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Localization of the Mutant SepG1 Protein to Sites of Cell Division in the Filamentous Fungus Aspergillus nidulans
Author(s) -
Ugwu Uchenna,
JacksonHayes Loretta,
Hill Terry William
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.533.107
Subject(s) - aspergillus nidulans , biology , mutant , cytokinesis , microbiology and biotechnology , green fluorescent protein , gene , cell division , genetics , wild type , cell
Cell division (cytokinesis) in filamentous fungi involves inward growth of the plasma membrane at sites of crosswall (septum) formation, under the guidance of a contractile actomyosin ring (CAR). The process involves the coordinated activities of multiple proteins, most of which are poorly characterized or wholly undiscovered. A forward‐genetics approach to identifying proteins with unrecognized roles in cell division has been to artificially mutagenize cells and isolate viable progeny defective in septum formation (septation), followed by identification of mutation sites at the molecular level. Prior work in this laboratory has localized a classic temperature‐sensitive septation mutation, termed sepG1 , in the model fungus Aspergillus nidulans to a gene locus predicted to encode an IQGAP homologue. Reduced expression of the wild type gene under the regulatable AlcA promoter blocks septation, and a GFP‐tagged version of the wild type protein localizes to sites where septa are forming. We have also succeeded in GFP‐tagging the protein encoded by the mutant sepG1 allele and have demonstrated that it too localizes to septation sites, even under restrictive conditions that prevent completion of septation, indicating that the sepG1 defect is not in protein targeting. Multiple attempts using Mendelian methods to create double‐mutant strains expressing both GFP‐tagged SepG1 protein plus RFP‐tagged versions of actin or myosin have not been successful, preventing investigation of whether the SepG1 protein associates normally with the CAR. Therefore, we have generated riboflavin‐auxotrophic strains containing untagged sepG1 mutant alleles along with RFP‐tagged wild‐type alleles of actin or myosin, with the goal of tagging the sepG1 allele in these strains via direct genetic transformation. We have constructed a plasmid‐based cassette containing sequences that, after integration, will allow the simultaneous expression of GFP‐tagged SepG1 protein in the same strains that are also expressing RFP‐tagged actin or myosin. Support or Funding Information National Science Foundation Grant 1615192 (Loretta Jackson‐Hayes, PI). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .