The STE20/Germinal Center Kinase POD6 Interacts with the NDR Kinase COT1 and Is Involved in Polar Tip Extension inNeurospora crassa

Abstract

Members of the Ste20 and NDR protein kinase families are important for normal cell differentiation and morphogenesis in various organisms. We characterized POD6 (NCU02537.2), a novel member of the GCK family of Ste20 kinases that is essential for hyphal tip extension and coordinated branch formation in the filamentous fungus Neurospora crassa. pod-6 and the NDR kinase mutant cot-1 exhibit indistinguishable growth defects, characterized by cessation of cell elongation, hyperbranching, and altered cell-wall composition. We suggest that POD6 and COT1 act in the same genetic pathway, based on the fact that both pod-6 and cot-1 can be suppressed by 1) environmental stresses, 2) altering protein kinase A activity, and 3) common extragenic suppressors (ropy, as well as gul-1, which is characterized here as the ortholog of the budding and fission yeasts SSD1 and Sts5, respectively). Unlinked noncomplementation of cot-1/pod-6 alleles indicates a potential physical interaction between the two kinases, which is further supported by coimmunoprecipitation analyses, partial colocalization of both proteins in wild-type cells, and their common mislocalization in dynein/kinesin mutants. We conclude that POD6 acts together with COT1 and is essential for polar cell extension in a kinesin/dynein-dependent manner in N. crassa. INTRODUCTIONFactors that determine and modulate cell polarity have been the subject of extensive investigations in a variety of experimental organisms (Drubin and Nelson, 1996;Nelson, 2003), with the most progress having been made in the unicellular yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe (Bähler and Peter, 2000; Bretscher, 2000a, 2000b;Pruyne et al., 2004). The mechanisms by which polarity is established in filamentous fungi have remained largely obscure, but it is likely that the fundamental principles leading to the initial polarization of the cell are conserved among unicellular organisms (Bähler and Peter, 2000;Wendland, 2001), filamentous fungi (Galagan et al., 2003;Borkovich et al., 2004;Harris and Momany, 2004), and animals (Hall, 1998). But in contrast to baker's yeast, where growth becomes isotropic soon after bud emergence, the growth of filamentous fungi must stay highly polar to produce a tipgrowing hypha that can extend at astonishing rates of more than 1 m/s (Lopez-Franco et al., 1994;Seiler and Plamann, 2003;Harris et al., 2005). Thus, filamentous fungi present good model systems to study how this highly polar shape is maintained over long distances, the way in which new branch points are initiated and how their spatial relationship is regulated.In recent years, protein kinases of the NDR Ser/Thr protein kinase family have emerged as being important for normal cell differentiation and polar morphogenesis in various organisms, yet their specific functions are still elusive (Tamaskovic et al., 2003;Hergovich et al., 2006). In Drosophila melanogaster, the NDR kinases Tricornered and Warts are required for control of the extent and direction of cell proliferation as well as fo...

Regulation of polarised growth in fungi

“…Moreover, a pod-6 -cot-1 -double mutant shows the same phenotype as each single mutant (Seiler et al, 2006). Taken together these observations suggest that COT1 and POD6 act in the same pathway.…”

“…Cot-1 -mutations are also suppressed by ropy mutations affecting the dynein/dynactin minus-end directed microtubular motor complex. In these mutants COT1 becomes concentrated at the tips (Seiler et al, 2006). An interpretation of this result is that COT1 is transported along microtubules to the hyphal tip by plus-end directed microtubule motors and away from the tip along microtubules by the dynein/dynactin complex.…”

The Nuclear Dbf2-Related Kinase COT1 and the Mitogen-Activated Protein Kinases MAK1 and MAK2 Genetically Interact to Regulate Filamentous Growth, Hyphal Fusion and Sexual Development in Neurospora crassa

“…This efficient procedure makes N. crassa ideal for the genetic dissection of Ndr signaling. Several mutants designated ''gulliver'' that act as modifiers of the compact cot-1(ts) morphology at restrictive temperature have been described (Terenzi and Reissig 1967;Bruno et al 1996b;Seiler et al 2006). gul-4 has been mapped to nic-3 (17%) on linkage group VII (Perkins et al 2001).…”

“…Both kinases have been shown to interact, and they share common suppressors and are localized in a kinesin/dynein-dependent manner (Seiler et al 2006). We have provided evidence indicating that COT1/POD6 and PKA act in parallel pathways that regulate polarity formation in a positive or negative manner, respectively, in N. crassa (Seiler et al 2006). However, the input and outcome components of the Ndr kinase network as well as its integration into a cellular signaling context have not been described in any system.…”

More Than a Repair Enzyme:Aspergillus nidulansPhotolyase-like CryA Is a Regulator of Sexual Development

“…A 6.8-kb cryA genomic fragment was subcloned into pGEM5 (SpeI/NsiI), creating pME3182. For construction of the complementation cassette, by using primers OZG189/192, nat R cassette was amplified from pNV1 (Seiler et al, 2006), and 3Ј untranslated region (UTR) of cryA was amplified from genomic (OZG191/ 10) DNA, and these two fragments were fused by fusion PCR (Yu et al, 2004) and nat R ϩ 3Ј UTR fusion fragment was cloned in pCR-Blunt II-TOPO (Invitrogen). VspI site of this plasmid served as recipient for OZG199/200 amplified and VspI digested 5Ј UTR ϩ cryA ORF from pME3182, resulting in the complementation plasmid pME3183 from which 6.5-kb complementation fragment was released with PfIMI digest and used for complementation of cryA⌬ mutant.…”