The Aspergillus nidulans IQGAP orthologue SepG is required for constriction of the contractile actomyosin ring

https://doi.org/10.1016/j.fgb.2020.103439Get rights and content

Highlights

  • We localize the Aspergillus nidulans sepG1 mutation to a gene encoding an orthologue of IQGAP1.

  • The mutation blocks contractile actomyosin ring (CAR) constriction but not SepG targeting.

  • SepG colocalizes with the CAR but is not necessary for its formation.

  • Filamentous actin, but not MyoB, is required for SepG to localize to the CAR.

Abstract

In this research we report that the sepG1 mutation in Aspergillus nidulans resides in gene AN9463, which is predicted to encode an IQGAP orthologue. The genetic lesion is predicted to result in a G-to-R substitution at residue 1637 of the 1737-residue protein in a highly conserved region of the RasGAP-C-terminal (RGCT) domain. When grown at restrictive temperature, strains expressing the sepGG1637R (sepG1) allele are aseptate, with reduced colony growth and aberrantly formed conidiophores. The aseptate condition can be replicated by deletion of AN9463 or by downregulating its expression via introduced promoters. The mutation does not prevent assembly of a cortical contractile actomyosin ring (CAR) at putative septation sites, but tight compaction of the rings is impaired and the rings fail to constrict. Both GFP::SepG wild type and the GFP-tagged product of the sepG1 allele localize to the CAR at both permissive and restrictive temperatures. Downregulation of myoB (encoding the A. nidulans type-II myosin heavy chain) does not prevent formation of SepG rings at septation sites, but filamentous actin is required for CAR localization of SepG and MyoB. We identify fourteen probable IQ-motifs (EF-hand protein binding sites) in the predicted SepG sequence. Two of the A. nidulans EF-hand proteins, myosin essential light chain (AnCdc4) and myosin regulatory light chain (MrlC), colocalize with SepG and MyoB at all stages of CAR formation and constriction. However, calmodulin (CamA) appears at septation sites only after the CAR has become fully compacted. When expression of sepG is downregulated, leaving MyoB as the sole IQ-motif protein in the pre-compaction CAR, both MrlC and AnCdc4 continue to associate with the forming CAR. When myoB expression is downregulated, leaving SepG as the sole IQ-motif protein in the CAR, AnCdc4 association with the forming CAR continues but MrlC fails to associate. This supports a model in which the IQ motifs of MyoB bind both MrlC and AnCdc4, while the IQ motifs of SepG bind only AnCdc4. Downregulation of either mrlC or Ancdc4 results in an aseptate phenotype, but has no effect on association of either SepG or MyoB with the CAR.

Introduction

The first observable stage of cytokinesis in the fungi is the formation of a plasma membrane-linked contractile actomyosin ring (CAR) at pre-selected sites around the cell cortex (Mouriño-Pérez, 2013). In filamentous fungi like Neurospora crassa and Aspergillus nidulans, the CAR assembles by degrees, appearing initially as a set of cables termed a septal actomyosin “tangle” (Delgado-Álvarez et al., 2014) or actomyosin “strings” (Hill et al., 2015; Taheri-Talesh et al., 2012) dispersed throughout the cytoplasm in the area of future septation. The strings then coalesce into a set of circumferential cortical cables that proceed to gather into a single tightly compacted cortical ring. Following compaction, the CAR constricts centripetally, while the plasma membrane invaginates and new wall material is synthesized to form a cross-wall termed the septum (Steinberg et al., 2017). The cytokinetic mechanisms operating in the fungi share much in common with those found in the metazoans (Pollard and O'Shaughnessy, 2019), with the most significant difference lying in the requirement for synthesis of new cell wall material in the fungi.

Within the fungi, the best understood cytokinetic mechanisms are those in the model yeasts Saccharomyces cerevisiae (e.g., Bhavsar-Jog and Bi, 2017) and Schizosaccharomyces pombe (e.g., Rincon and Paoletti, 2016). In S. pombe it is estimated that at least 130 different proteins are involved in cell division (Pollard and Wu, 2010), amongst which are proteins playing structural roles such as components of the CAR and ones playing regulatory roles such as involvement in the processes of site selection and coordination with the nuclear cycle. Among the proteins playing important structural roles in the CAR are myosin II, actin, actin-associated proteins like alpha actinin, and tropomyosin, as well as scaffolding proteins such as IQGAPs, anillins, and paxillins (Laplante, 2018).

In comparison to yeasts, the process of cytokinesis in filamentous fungi is much less well understood (Riquelme et al., 2018). Genomes of filamentous fungi contain orthologues of all or nearly all of the central cytokinesis proteins found in model yeasts (e.g., Harris et al., 2009), and studies have tied a number of these orthologues to roles in cytokinesis. While it is clear that cytokinetic mechanisms in yeasts and filamentous fungi share much in common, still it does not necessarily follow that the roles of cytokinetic proteins in filamentous fungi must map exactly onto those they play in the well-studied model yeasts (Harris and Momany, 2004; Seiler and Justa-Schuch, 2010). For example, disassembly of actin filaments via latrunculin B prevents localization of type II myosin to septation sites in Aspergillus nidulans (Hill et al., 2015) but not in Saccharomyces cerevisiae (Bi et al., 1998).

An important early contribution to our understanding of the process of cytokinesis in the filamentous fungi was the identification of temperature-sensitive, septation-deficient strains generated in A. nidulans through random mutagenesis (Harris et al., 1994; Morris, 1975). With the advent of molecular methods of analysis, a number of these mutant genes have been identified, including some whose products play regulatory roles in cytokinesis such as sepH (AN4385), which encodes a protein kinase in the Septation Initiation Network (Bruno et al., 2001), and others playing structural roles, such as the formin-encoding sepA (AN6523; Harris et al., 1997). One conspicuous “hold-out” amongst the early septation mutations has been sepG1, which was identified by Harris et al., (1994) as a class III sep mutation: i.e., one affecting septation alone without parallel involvement in apical growth or the nuclear cycle. Despite the absence of information on its mode of action, the mutation has proven useful in several subsequent studies as a means to experimentally block septum formation (e.g., Dynesen and Nielsen, 2003; Westfall and Momany, 2002).

In this study we report that the sepG1 mutation resides in a locus encoding the A. nidulans orthologue of the Homo sapiens IQGAP1 protein. IQGAPs are categorized as a family of scaffolding proteins (i.e., proteins whose principal role is to tether together other proteins in a manner facilitating protein-protein interactions) found in fungi and metazoans, but lacking in plants (Shannon, 2012). These are multi-domain proteins, playing roles in a diverse range of functions by binding to multiple partners in a domain-specific manner (White et al., 2012). Of particular interest to the current study is the regulation of actin-dependent functions in cell shape and motility (Brandt and Grosse, 2007). The domain complement of the canonical member of the family, H. sapiens IQGAP1, comprises (reading from the N-terminus) a calponin homology (CH) domain, a coiled-coil region, a tryptophan repeat motif (WW), a region containing four IQ motifs, a GAP-related domain (GRD), and finally a RasGAP carboxy terminal domain (RGCT or RasGAP_C) which is unique to IQGAPs (Shannon, 2012). In IQGAP1, the principal binding partner of the CH domain is F-actin (Abel et al., 2015), the GRD binds to Rho GTPases Cdc42 and Rac1 (Nouri et al., 2017), and common interactors of the IQ motifs are EF-hand proteins such as calmodulin and myosin essential light chain (Atcheson et al., 2011). IQ motifs are also found in the neck regions of myosin heavy chains, where they serve as binding sites for a variety of myosin light chains (Heissler and Sellers, 2014).

Although the RGCT domain of IQGAP1 has been associated with binding to cell surface adhesion proteins such as E-cadherin and β-catenin (reviewed in Abel et al., 2015), the domain plays a perhaps more important function as part of the auto-repression/activation mechanism in IQGAP1. As reviewed by Choi and Anderson (2016), IQGAP1 folds into an inactive conformation via intramolecular interactions involving the RGCT. Phosphorylation of residue S1443 residing between the GRD and RGCT domains by protein kinase C partially relieves these intramolecular attractions, making the protein open to binding by Rho GTPases, which then leads to the adoption of a fully open (and fully functional) conformation.

Orthologues of human IQGAP1 have been described in the yeasts S. pombe (Rng2p; Eng et al., 1998), S. cerevisiae (Iqg1p/Cyk1p; Epp and Chant, 1997; Lippincott and Li, 1998), and Candida albicans (CaIqg1; Li et al., 2008), as well as in the related saccharomycete Eremothecium (Ashbya) gossypii (AgCYK1; Wendland and Philippsen, 2002). In each of these organisms, the IQGAP orthologue plays an essential role in cytokinesis, and no other functions have been reported. As reviewed by Shannon (2012) the fungal orthologues all contain a CHD domain, an IQ region, a GRD domain, and an RGCT domain arranged in the same linear order seen in IQGAP1; but they lack the coil-coil region and WW motif that lie between the CHD and IQ region of IQGAP1. The number of IQ motifs reported within each orthologue’s IQ region varies considerably.

In this research we describe an essential role for the sole IQGAP orthologue in the filamentous fungus Aspergillus nidulans, which we designate SepG based on the published temperature-sensitive sepG1 mutation (Harris et al., 1994) which we demonstrate occurs at this locus. We show that SepG is a component of the CAR, whose function is not required for CAR assembly, but is required for CAR constriction. In addition, we present evidence to support a model in which SepG binds to the EF-hand protein AnCdc4, but not to EF-hand proteins CamA or MrlC. Finally, we also will describe a conserved feature of the SepG protein sequence, which suggests likely mechanisms of regulation involving phosphorylation via protein kinase C.

Section snippets

Media and basic culture methods

Complete medium (CM) consisted of 1% glucose, 0.2% peptone, 0.1% yeast extract, 0.1% casamino acids, 5% nitrate salts, 1% trace elements, 0.1% vitamin mix, 1.2 mM L-arginine, and 50 mg ml−1 ampicillin. Vitamin mix and nitrate salts are described in the appendix of Kafer (1977). Trace element solution is described in Hill & Kafer (2001). Minimal medium (MM) consisted of 1% glucose, 5% nitrate salts, 1% trace elements, 0.001% thiamine hydrochloride, 25 ng biotin ml−1, and 50 mg ml−1 ampicillin.

Mutant phenotype

Septation is impaired in a temperature-sensitive manner in strains bearing the sepG1 mutation. In cells grown at 30 °C (permissive temperature; Fig. 1A), septa are abundant and are indistinguishable from CFW-stained septa of wild type cells grown under identical conditions (Fig. 1B). At 42 °C, wild type hyphae septate normally (Fig. 1C), but neither complete nor partial septa were observed in the sepG1 strain (Fig. 1D). At permissive temperature, the mutant colony growth rate is

Discussion

The A. nidulans IQGAP orthologue SepG conforms to the structural pattern shown in other fungal IQGAP orthologues in containing all of the domains found in the canonical IQGAP1 of H. sapiens except for the coil-coil region and WW region. The sole role that we have observed for SepG is to be an essential component of the CAR in cytokinesis. The aberrations observed in conidiophore morphology can be considered secondary consequences of the fundamental defect of impaired septation (Adams, 1995).

Author statement

No human subjects were employed in this research.

Acknowledgments

Support for this work was provided by National Science Foundation Grants RUI-0742907 and RUI-1615192, and by funds from Rhodes College in support of undergraduate research.

References (69)

  • S.D. Harris et al.

    Polarity in filamentous fungi: moving beyond the yeast paradigm

    Fung. Genet. Biol.

    (2004)
  • S.D. Harris et al.

    Morphology and development in Aspergillus nidulans: a complex puzzle

    Fungal Genet. Biol.

    (2009)
  • T.W. Hill et al.

    A mutation in the converter subdomain of Aspergillus nidulans MyoB blocks constriction of the actomyosin ring in cytokinesis

    Fungal Genet. Biol.

    (2015)
  • E. Kafer

    Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations

    Adv. Genet.

    (1977)
  • Z. Li et al.

    IQGAP1 promotes neurite outgrowth in a phosphorylation-dependent manner

    J. Biol. Chem.

    (2005)
  • R.R. Mouriño-Pérez

    Septum development in filamentous ascomycetes

    Fungal Biol. Rev.

    (2013)
  • K. Nouri et al.

    IQGAP1 interaction with RHO family proteins revisited. Kinetic and equilibrium evidence for multiple distinct binding sites

    J. Biol. Chem.

    (2016)
  • S.A. Rincon et al.

    Molecular control of fission yeast cytokinesis

    Semin. Cell Dev. Biol.

    (2016)
  • B. Romero et al.

    The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen

    Fungal Genet. Biol.

    (2003)
  • J.R. Sellers

    Myosins: a diverse superfamily

    BBA-Mol. Cell. Res

    (2000)
  • K.B. Shannon et al.

    A myosin light chain mediates the localization of the budding yeast IQGAP-like protein during contractile ring formation

    Curr. Biol.

    (2000)
  • J.M. Smith et al.

    IQGAPs choreograph cellular signaling from the membrane to the nucleus

    Trends Cell Biol.

    (2015)
  • A.G. Teepe et al.

    The protein kinase C orthologue PkcA plays a role in cell wall integrity and polarized growth in Aspergillus nidulans

    Fungal Genet. Biol.

    (2007)
  • J. Wendland et al.

    An IQGAP-related protein, encoded by AgCYK1, is required for septation in the filamentous fungus Ashbya gossypii

    Fungal Genet. Biol.

    (2002)
  • C.D. White et al.

    IQGAP1 and its binding proteins control diverse biological functions

    Cell. Signal.

    (2012)
  • J.-Q. Wu et al.

    Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis

    Dev. Cell

    (2003)
  • T.H. Adams

    Asexual sporulation in higher fungi

  • E. Atcheson et al.

    IQ-motif selectivity in human IQGAP2 and IQGAP3: binding of calmodulin and myosin essential light chain

    Biosci. Rep.

    (2011)
  • E. Bi et al.

    Involvement of an actomyosin contractile ring in Saccharomyces cerevisiae cytokinesis

    J. Cell Biol.

    (1998)
  • D.T. Brandt et al.

    Get to grips: steering local actin dynamics with IQGAPs

    EMBO Rep.

    (2007)
  • K.S. Bruno et al.

    SEPH, a Cdc7p orthologue from Aspergillus nidulans functions upstream of actin ring formation during cytokinesis

    Mol. Microbiol.

    (2001)
  • G.C. Cerqueira et al.

    The Aspergillus Genome Database: multispecies curation and incorporation of RNA-Seq data to improve structural gene annotations

    Nucl. Acids Res.

    (2014)
  • D.L. Delgado-Álvarez et al.

    Septum development in Neurospora crassa: the septal actomyosin tangle

    PLoS ONE

    (2014)
  • X. Fang et al.

    Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin II

    J. Cell Biol.

    (2010)
  • Cited by (0)

    1

    Contributions of undergraduate co-authors are listed in Supplementary Document 1.

    View full text