Functional Analysis of the Aspergillus nidulans Kinome

The filamentous fungi are an ecologically important group of organisms which also have important industrial applications but devastating effects as pathogens and agents of food spoilage. Protein kinases have been implicated in the regulation of virtually all biological processes but how they regulate filamentous fungal specific processes is not understood. The filamentous fungus Aspergillus nidulans has long been utilized as a powerful molecular genetic system and recent technical advances have made systematic approaches to study large gene sets possible. To enhance A. nidulans functional genomics we have created gene deletion constructs for 9851 genes representing 93.3% of the encoding genome. To illustrate the utility of these constructs, and advance the understanding of fungal kinases, we have systematically generated deletion strains for 128 A. nidulans kinases including expanded groups of 15 histidine kinases, 7 SRPK (serine-arginine protein kinases) kinases and an interesting group of 11 filamentous fungal specific kinases. We defined the terminal phenotype of 23 of the 25 essential kinases by heterokaryon rescue and identified phenotypes for 43 of the 103 non-essential kinases. Uncovered phenotypes ranged from almost no growth for a small number of essential kinases implicated in processes such as ribosomal biosynthesis, to conditional defects in response to cellular stresses. The data provide experimental evidence that previously uncharacterized kinases function in the septation initiation network, the cell wall integrity and the morphogenesis Orb6 kinase signaling pathways, as well as in pathways regulating vesicular trafficking, sexual development and secondary metabolism. Finally, we identify ChkC as a third effector kinase functioning in the cellular response to genotoxic stress. The identification of many previously unknown functions for kinases through the functional analysis of the A. nidulans kinome illustrates the utility of the A. nidulans gene deletion constructs

<i>Δsrc1</i> nuclei undergo architectural modifications from G1 that are typical of mitosis.

<p>(A) <i>Δsrc1</i> spores were germinated in the presence of 10 mM HU to arrest them in interphase. NLS-DsRed was transported within the nucleus in <i>Δsrc1</i> cells and Nup49 located around their nuclear periphery as typical of interphase Wt cells. (B-C) The localization of Nup2, NLS-DsRed, Bop1, and Nup49 were monitored in the first mitosis of <i>Δsrc1</i> cells after release from G2 arrest imposed by the <i>nimT23</i>^ts allele. (B) As occurs during normal mitosis, Nup2-GFP translocates to condensed chromatin and NLS-DsRed escapes from the nucleus during mitosis in Δ<i>src1</i> cells. However, soon after the two G1 nuclei are established and accumulate NLS-DsRed, with Nup2 now around their nuclear periphery, the nucleus indicated by the white arrowhead becomes mitotic-like with Nup2 locating to chromatin and losing its nuclear transport capacity. These effects were then largely reversed at the 21’ time point with the nucleus re-importing NLS-DsRed again. (C) As occurs during normal mitosis, Nup49 disperses from NPCs and returns to NPCs of the two new G1 nuclei generated during mitosis (0–8’). The nucleolus, marked by Bop1-GFP, then disassembles with the released nucleolar proteins being reimported into the new G1 nuclei. This process occurs normally in the absence of Src1 and is completed apparently normally (time point 17’). However the G1 nuclei are not normal as the nucleus to the right undergoes transitions typical of mitosis including the movement of Nup49 into the nucleus, presumably onto chromatin, and the disassembly of Bop1 after it is apparently expelled to the cytoplasm (indicated by a white arrowhead). This nucleolus then disassembles and Bop1 is imported into the transport competent daughter nucleus.</p

Src1 concentrates to chromatin during mitotic exit.

<p>(A) Protein domain structure of human MAN1, budding yeast Src1 (also named Heh1) compared to <i>A</i>. <i>nidulans</i> Src1 indicting the relative positions of the N-terminal LEM domain PF12949, the internal Man1-Src1p-C-terminal (MSC) domain PF09402, and the predicted transmembrane domains. (B-D) Images from live cell spinning disc confocal microscopy of mitosis in cells expressing the indicated tagged proteins. (B) Src1 associates around mitotic chromatin (marked by histone H1-chRFP) in a punctate pattern and also to two foci (white arrowheads 3’ 45”) at sites corresponding to NE abscissions during anaphase-telophase. (C) Src1 locates preferentially to the chromatin of reforming daughter nuclei in a punctate pattern and not around the nucleolus as marked by the nucleolar protein Bop1-chRFP. (D) Two nuclei shown at G2, M, and G1. The ER/NE marker Erg24 locates around both forming daughter nuclei and the nucleolus during anaphase-telophase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132489#pone.0132489.ref017" target="_blank">17</a>]. However, at this stage of mitosis Src1 preferentially locates around the reforming daughter nuclei in a punctate pattern and not around the nucleolus (merged mitotic panel). Src1 also concentrates at the two NE abscission points indicated by the pairs of arrowheads. Scale bar 5μm.</p

Src1 preferentially locates around chromatin during mitotic exit in the absence of mitotic spindle function.

<p>Cells expressing the indicated pairs of tagged protein were treated with the microtubule poison benomyl and imaged during spindle independent mitotic exit (SIME) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132489#pone.0132489.ref017" target="_blank">17</a>]. (A) Time-lapse images of Src1-GFP with the DNA marker H1-chRFP during the transition from a SAC imposed mitotic arrest and SIME, the start of which is indicated as time 0. Src1 can be observed to retract from the nuclear region to the right and locate preferentially around the chromatin located in the left side of the nucleus. When this process is completed (time 7’30”) the chromatin begins to undergo decondensation. (B) A similar time course as A but following the nucleolar marker Bop1 and Src1 to show that it is the nucleolus from which Src1 retracts when it preferentially locates to mitotic chromatin during mitotic exit. The arrow at time 7’ 30” indicates completion of the removal of Src1 from around the nucleolus. Scale bar 5μm.</p

Src1 is essential.

<p>(A) Conidia (asexual spores) from wildtype control (<i>pyrG</i>⁺) <i>or</i> Δ<i>src1</i>::<i>pyrG</i> deletion primary transformants were replica streaked onto nonselective (+UU) and selective (-UU) plates and grown for 40 hours for photography. Conidia that are not able to grow on–UU media are from primary Δ<i>src1</i>::<i>pyrG</i> / <i>src1 pyrG</i>89 heterokaryon transformants indicating <i>src1</i> to be essential. (B) Bright field image of the limited growth of conidia from <i>Δsrc1</i>::<i>pyrG</i> / <i>src1 pyrG</i>89 heterokaryons on selective media for 24 and 72 h. Scale bar 10 μm. (C) Diagnostic PCR using DNA isolated from a wild-type strain (Wt.), from a putative heterokaryon grown in +UU media (stops selection for <i>Δsrc1</i>), and from a putative heterokaryon growing in-UU selective media. The deletion allele carried in the heterokaryotic transformant is rapidly lost upon propagation without selection indicating the transformant is not a diploid but a heterokaryon. (D) The degree of growth of both wildtype and <i>Δsrc1</i> cells were compared at 12, 15, and 18 hours at 23.5°C by measuring the average length of germlings. (E) <i>Δsrc1</i> germlings fixed and stained with DAPI to reveal nuclear defects, including condensed DNA, odd numbers of nuclei (indicated by white arrowheads), and an unusual pattern of nuclear transport evidenced by only one nucleus being able to accumulate NLS-DsRed (indicated by arrow). Scale bar 5 μm.</p

Nuclei oscillate between mitotic-like and interphase-like states in Δ<i>src1</i> cells.

<p>(A) Time course imaging over 34 minutes of NLS-DsRed in a <i>Δsrc1</i> cell indicates its nuclei oscillate between nuclear transport active and inactive states. (B) To more clearly track individual nuclei within <i>Δsrc1</i> cells the distribution of nuclear NLS-DsRed was monitored in benomyl treated cells to stop nuclear movements. Nuclei can be seen to oscillate between transport active and inactive states through the kymograph representing the intensity of NLS-DsRed across the yellow line with time; as also plotted as nuclear fluorescence intensity. The vertical red line indicates the first ten minutes of the live cell imaging. (C) Analysis of the data revealed nuclei spent on average an equal time between nuclear transport active and inactive states with no discernable pattern between each state between nuclei within individual cells.</p

The Inner Nuclear Membrane Protein Src1 Is Required for Stable Post-Mitotic Progression into G1 in <i>Aspergillus nidulans</i>

<div><p>How membranes and associated proteins of the nuclear envelope (NE) are assembled specifically and inclusively around segregated genomes during exit from mitosis is incompletely understood. Inner nuclear membrane (INM) proteins play key roles by providing links between DNA and the NE. In this study we have investigated the highly conserved INM protein Src1 in <i>Aspergillus nidulans</i> and have uncovered a novel cell cycle response during post mitotic formation of G1 nuclei. Live cell imaging indicates Src1 could have roles during mitotic exit as it preferentially locates to the NE abscission points during nucleokinesis and to the NE surrounding forming daughter G1 nuclei. Deletion analysis further supported this idea revealing that although Src1 is not required for interphase progression or mitosis it is required for stable post-mitotic G1 nuclear formation. This conclusion is based upon the observation that in the absence of Src1 newly formed G1 nuclei are structurally unstable and immediately undergo architectural modifications typical of mitosis. These changes include NPC modifications that stop nuclear transport as well as disassembly of nucleoli. More intriguingly, the newly generated G1 nuclei then cycle between mitotic- and interphase-like states. The findings indicate that defects in post-mitotic G1 nuclear formation caused by lack of Src1 promote repeated failed attempts to generate stable G1 nuclei. To explain this unexpected phenotype we suggest a type of regulation that promotes repetition of defective cell cycle transitions rather than preventing progression past the defective cell cycle transition. We suggest the term “reboot regulation” to define this mode of cell cycle regulation. The findings are discussed in relationship to recent studies showing the Cdk1 master oscillator can entrain subservient oscillators that when uncoupled cause cell cycle transitions to be repeated.</p></div