Identification of Interphase Functions for the NIMA Kinase Involving Microtubules and the ESCRT Pathway

Abstract

The Never in Mitosis A (NIMA) kinase (the founding member of the Nek family of kinases) has been considered a mitotic specific kinase with nuclear restricted roles in the model fungus Aspergillus nidulans. By extending to A. nidulans the results of a synthetic lethal screen performed in Saccharomyces cerevisiae using the NIMA ortholog KIN3, we identified a conserved genetic interaction between nimA and genes encoding proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) pathway. Absence of ESCRT pathway functions in combination with partial NIMA function causes enhanced cell growth defects, including an inability to maintain a single polarized dominant cell tip. These genetic insights suggest NIMA potentially has interphase functions in addition to its established mitotic functions at nuclei. We therefore generated endogenously GFP-tagged NIMA (NIMA-GFP) which was fully functional to follow its interphase locations using live cell spinning disc 4D confocal microscopy. During interphase some NIMA-GFP locates to the tips of rapidly growing cells and, when expressed ectopically, also locates to the tips of cytoplasmic microtubules, suggestive of non-nuclear interphase functions. In support of this, perturbation of NIMA function either by ectopic overexpression or through partial inactivation results in marked cell tip growth defects with excess NIMA-GFP promoting multiple growing cell tips. Ectopic NIMA-GFP was found to locate to the plus ends of microtubules in an EB1 dependent manner, while impairing NIMA function altered the dynamic localization of EB1 and the cytoplasmic microtubule network. Together, our genetic and cell biological analyses reveal novel non-nuclear interphase functions for NIMA involving microtubules and the ESCRT pathway for normal polarized fungal cell tip growth. These insights extend the roles of NIMA both spatially and temporally and indicate that this conserved protein kinase could help integrate cell cycle progression with polarized cell growth.

Conserved Ankyrin Repeat Proteins and Their NIMA Kinase Partners Regulate Extracellular Matrix Remodeling and Intracellular Trafficking inCaenorhabditis elegans

“…For example, three Arabidopsis thaliana NEKs (related to NEKL-2) have been implicated in epidermal root tip growth through the possible regulation of microtubules (Sakai et al 2008;Motose et al 2011Motose et al , 2012. Perhaps most notable was the recent report that Aspergillus nidulans nimA, which was originally identified for its role in mitosis, also controls polarized cell growth and localizes to microtubule plus ends in an EB1-dependent manner during interphase (Govindaraghavan et al 2014). This study also showed that mutations in both the A. nidulans and Saccharomyces cerevisiae NIMA orthologs (nimA and kin3) are synthetically lethal with mutations in genes encoding ESCRT complex components, which promote membrane remodeling and endosome maturation (McCullough et al 2013;Govindaraghavan et al 2014).…”

“…Perhaps most notable was the recent report that Aspergillus nidulans nimA, which was originally identified for its role in mitosis, also controls polarized cell growth and localizes to microtubule plus ends in an EB1-dependent manner during interphase (Govindaraghavan et al 2014). This study also showed that mutations in both the A. nidulans and Saccharomyces cerevisiae NIMA orthologs (nimA and kin3) are synthetically lethal with mutations in genes encoding ESCRT complex components, which promote membrane remodeling and endosome maturation (McCullough et al 2013;Govindaraghavan et al 2014). Although the functional connection between nimA and ESCRT proteins is unknown, this finding implicates NIMA kinases in processes linked to intracellular trafficking.…”

The Set1/COMPASS Histone H3 Methyltransferase Helps Regulate Mitosis With the CDK1 and NIMA Mitotic Kinases in Aspergillus nidulans

“…The Set1 complex is essential when the function of NIMA or NIMT (and hence mitotic CDK1) is partially impaired By extending the results of a synthetic lethal screen conducted using the nonessential S. cerevisiae NIMA ortholog Kin3 to A. nidulans (Govindaraghavan et al 2014b), a genetic interaction was identified between NIMA and Answd1 (Figure 1), the gene encoding the A. nidulans ortholog of yeast Swd1, a WD40 repeat containing subunit of the Set1 methyltransferase complex, also called the COMPASS complex (locus AN0808; see http://www.aspergillusgenome.org/ cgi-bin/locus.pl?locus=AN0808). Deletion of An-swd1 generated haploid null strains having growth and conidiation defects that displayed cold sensitivity ( Figure S1).…”

“…A synthetic lethal screen was conducted using the deletion of the nonessential Saccharomyces cerevisiae NIMA ortholog Kin3 (Govindaraghavan et al 2014b). Here we reveal that the novel genetic interaction between nimA and the gene encoding a subunit of the Set1 protein methyltransferase complex (also called the COMPASS complex for Complex Proteins Associated with Set1) (Miller et al 2001) An-swd1 (Roguev et al 2001) is conserved in A. nidulans.…”

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation

“…On the other hand, we identified two protein kinases functionally characterized but with no known functions related to glycogen metabolism regulation. More recently, this kinase was demonstrated to regulate interphase microtubules, indicating that it could integrate cell growth and development with mitotic regulation in A. nidulans [43]. This protein kinase was first identified in S. cerevisiae as required in the meiotic cell cycle, but more recent studies revealed that this kinase displays multiple cellular functions beyond meiosis [41].…”