Phospho-Regulation of the <i>Neurospora crassa</i> Septation Initiation Network

<div><p>Proper cell division is essential for growth and development of uni- and multicellular organisms. The fungal septation initiation network (SIN) functions as kinase cascade that connects cell cycle progression with the initiation of cytokinesis. Miss-regulation of the homologous Hippo pathway in animals results in excessive cell proliferation and formation of tumors, underscoring the conservation of both pathways. How SIN proteins interact and transmit signals through the cascade is only beginning to be understood. Moreover, our understanding of septum formation and its regulation in filamentous fungi, which represent the vast majority of the fungal kingdom, is highly fragmentary. We determined that a tripartite kinase cascade, consisting of CDC-7, SID-1 and DBF-2, together with their regulatory subunits CDC-14 and MOB-1, is important for septum formation in the model mold <i>Neurospora crassa</i>. DBF-2 activity and septum formation requires auto-phosphorylation at Ser499 within the activation segment and phosphorylation of Thr671 in the hydrophobic motif by SID-1. Moreover, SID-1-stimulated DBF-2 activity is further enhanced by CDC-7, supporting a stepwise activation mechanism of the tripartite SIN kinase cascade in fungi. However, in contrast to the situation described for unicellular yeasts, the localization of the entire SIN cascade to spindle pole bodies is constitutive and cell cycle independent. Moreover, all SIN proteins except CDC-7 form cortical rings prior to septum initiation and localize to constricting septa. Thus, SIN localization and activity regulation significantly differs in unicellular versus syncytial ascomycete fungi.</p> </div

<i>N. crassa</i> SIN components localize to SPBs and septa (A)

<p>Functional GFP fusion proteins of CDC-7, SID-1, CDC-14 and DBF-2 localized to spindle pole bodies (arrows) and as constricting rings at forming septa. Nuclei were labeled with histone H1-RFP, the cell wall was stained with Calcofluor White. (<b>B</b>) The localization of the three SIN kinases CDC-7, SID-1 and DBF-2 to SPBs is constitutive and cell cycle independent. The three SIN kinases associate with SPBs of interphase nuclei as well as during early and late mitotic stages (as indicated by nuclear morphology). Nuclei were labeled with histone H1-RFP. </p

Dual phosphorylation of DBF-2 is required for kinase activity and septum formation.

<p>(<b>A</b>) Functional characterization of two conserved phosphorylation sites of DBF-2. The phosphomimetic DBF-2(T671E) variant complemented Δ<i>dbf-2</i>, while substitution of Ser499 to alanine and glutamate and Thr671 to alanine did not. Cell wall and septa were labeled with Calcofluor White. (<b>B</b>) Kinase activity and MOB-1 interaction pattern of the indicated DBF-2 variants. Hydrophobic motif phosphorylation of Thr671 was required for maximal kinase activity, while either modification of Ser499 within the activation segment reduced DBF-2 activity to ca. 30% of the wild type DBF-2 control. Phospho-site double mutant analysis indicated that substitution of Thr671 to glutamate in a S499A and S499E background could only partly restore kinase activity. Precipitated DBF-2 variants were assayed <i>in </i><i>vitro</i> using the synthetic NDR kinase peptide (KKRNRRLSVA) as substrate (n = 5). Western Blot analysis indicated equal precipitation of the co-activator protein MOB-1 with DBF-2 activation segment and hydrophobic motif variants. </p

<i>N. crassa</i> SIN components are required for septum formation but display distinct mutant characteristics

<p>(<b>A</b>) Deletion strains defective in the indicated SIN components generate thin and aseptate hypha in young colonies (18 h time point). In older colonies, the septation defects were suppressed in ∆<i>sid-1</i> and ∆<i>cdc-14</i> strains (36 h time point). Cell wall and septa were labeled with Calcofluor White. (<b>B</b>) SIN mutants showed cytoplasmic leakage (magnified inserts), but, due to the fast ability to septate, ∆<i>sid-1</i> and ∆<i>cdc-14</i> generated abundant aerial mycelium and asexual spores (conidia; plate morphology). (<b>C</b>) SIN mutants displayed distinct abnormalities during sexual development. wt x ∆ crosses with ∆<i>cdc-7</i>(het) and ∆<i>dbf-2</i>(het) resulted in the frequent formation of large, banana-shaped ascospores. In contrast, wt x ∆<i>sid-1</i>(het) progeny morphology was normal, while crosses of wt x ∆<i>cdc-14</i>(het) produced no mature perithecia .</p

Model depicting network organization of the MAK-2 pathway and putative regulatory mechanisms.

<p>Ligand-induced activation of an unknown receptor may be transmitted through plasma membrane-associated STE-20, RAS-2 and CAP-1, which signal toward the NRC-1/STE-50 complex and recruit the MAPK cascade through activation and clustering of the scaffold HAM-5 at intracellular puncta. MAK-2 activation triggers nuclear gene expression through interaction with the transcription factor PP-1 and the RCO-1/RCM-1 complex and the cytosolic activation of the secretory pathway and cell polarity machineries to coordinate pulsed signal release and chemotrophic growth towards the partner cell, respectively. MAK-2 activity is also required for termination of the receiver phase, potentially through negative feedback phosphorylation of the MAPK and disassembly of the MAK-2/HAM-5 module. MAK-2 pathway function may also be regulated through the STRIPAK complex, the CK2 heterodimer, membrane lipid composition, the septum-associated septation initiation network SIN and motor protein-dependent vesicle trafficking.</p

The MAK-2 pathway elements STE-20 and RAS-2 are important for cell-cell communication.

<p>(<b>A</b>) STE-20-GFP localizes in a stable manner to the apices of two communicating germlings and marks the site of contact. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s015" target="_blank">Video S6</a> for time course. (<b>B</b>) GFP-RAS-2 associates with the entire plasma membrane of germinating spores and localizes at the contact point of two communicating cells. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s016" target="_blank">Video S7</a> for time course. (<b>C</b>) Time course experiments to determine rates of germination and of chemotropic interactions of the indicated mutants. Note that due to delayed germination Δ<i>cr-1</i> and Δ<i>cap-1</i> strains are assayed at later time points than the other mutants. (<b>D</b>) Quantification of basal and stress-induced MAK-2 phosphorylation levels (detected with p42/44 antibodies) in cell extracts of exponentially growing liquid cultures of the indicated strains (n = 3). A representative Western blot is depicted below; tubulin was used as loading control.</p

WSC-1 and HAM-7 Are MAK-1 MAP Kinase Pathway Sensors Required for Cell Wall Integrity and Hyphal Fusion in <em>Neurospora crassa</em>

<div><p>A large number of cell wall proteins are encoded in the <em>Neurospora crassa</em> genome. Strains carrying gene deletions of 65 predicted cell wall proteins were characterized. Deletion mutations in two of these genes (<em>wsc-1</em> and <em>ham-7</em>) have easily identified morphological and inhibitor-based defects. Their phenotypic characterization indicates that HAM-7 and WSC-1 function during cell-to-cell hyphal fusion and in cell wall integrity maintenance, respectively. <em>wsc-1</em> encodes a transmembrane protein with extensive homology to the yeast Wsc family of sensor proteins. In <em>N. crassa</em>, WSC-1 (and its homolog WSC-2) activates the cell wall integrity MAK-1 MAP kinase pathway. The GPI-anchored cell wall protein HAM-7 is required for cell-to-cell fusion and the sexual stages of the <em>N. crassa</em> life cycle. Like WSC-1, HAM-7 is required for activating MAK-1. A Δ<em>wsc-1;</em>Δ<em>ham-7</em> double mutant fully phenocopies mutants lacking components of the MAK-1 MAP kinase cascade. The data identify WSC-1 and HAM-7 as the major cell wall sensors that regulate two distinct MAK-1-dependent cellular activities, cell wall integrity and hyphal anastomosis, respectively.</p> </div

STE-50 functions as regulatory subunit of NRC-1.

<p>(<b>A</b>) GFP-STE-50 localizes in a dynamic manner to opposing tips of two communicating germlings and the site of cell-cell contact. The oscillation period is approximately three to five minutes. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s010" target="_blank">Video S1</a> for time course. (<b>B</b>) GFP-STE-50 localizes in a diffuse cytosolic manner in non-communicating hyphae and is excluded from nuclei (left image). Moreover, GFP-STE-50 accumulates at septa (arrowheads) and the contact point (arrow) of communicating hyphal tips (right image). (<b>C</b>) Δ<i>ste-50</i> fully phenocopies Δ<i>mak-2</i> defects (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s003" target="_blank">Figure S3</a> for comparative characterization of mutant defects), and the mutant characteristics are complemented by expression of a constitutive active NRC-1(P488S) allele in Δ<i>ste-50</i>. The slant images represent macroscopic appearance and conidiation pattern, while the Table summarizes rates of mycelial extension, protoperithecia (PP) formation, germling communication and MAK-2 activities of the indicated strains.</p

Characterization of the <i>Neurospora crassa</i> Cell Fusion Proteins, HAM-6, HAM-7, HAM-8, HAM-9, HAM-10, AMPH-1 and WHI-2

<div><p>Intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. Cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. Approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the model filamentous fungus <i>Neurospora crassa</i>. Genetic, biochemical, and microscopic techniques were used to characterize the functions of seven previously poorly characterized cell fusion proteins. HAM-6, HAM-7 and HAM-8 share functional characteristics and are proposed to function in the same signaling network. Our data suggest that these proteins may form a sensor complex at the cell wall/plasma membrane for the MAK-1 cell wall integrity mitogen-activated protein kinase (MAPK) pathway. We also demonstrate that HAM-9, HAM-10, AMPH-1 and WHI-2 have more general functions and are required for normal growth and development. The activation status of the MAK-1 and MAK-2 MAPK pathways are altered in mutants lacking these proteins. We propose that these proteins may function to coordinate the activities of the two MAPK modules with other signaling pathways during cell fusion.</p></div

Interaction network of MAK-2 pathway components.

<p>(<b>A</b>) Proteins associated with the MAK-2 cascade were identified in affinity purification experiments coupled to mass spectrometry (AP-MS), and identified proteins were filtered against control purifications using GFP as bait (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s007" target="_blank">Table S1</a>). Only proteins identified in two biological replicates and absent from the control data set are shown. Protein coverage by peptides and the number of identified total and unique peptides identified are given for the better of the two purifications. Bold numbers indicate the GFP-fusion protein used as bait. (<b>B</b>) Physical interactions between MAK-2 pathway components were mapped in yeast two-hybrid (Y2H) tests. The indicated constructs were co-expressed in strain AH109 and yeast growth was analyzed on the indicated selective media. The reciprocal Y2H assays are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004762#pgen.1004762.s002" target="_blank">Figure S2</a>. (<b>C</b>) Summary schema of Y2H-based interactions of the indicated proteins and their domains. Color-coded lines below the protein schemas and dashed connectors indicate the used constructs and detected interactions, respectively.</p