<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

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

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 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

MST-1/POD-6 hetero-dimerization inhibits POD-6.

<p>(<b>A</b>) Yeast two-hybrid tests indicate hetero-dimerization of MST-1 and POD-6. (<b>B</b>) Co-immunoprecipitation experiments of MST-1-GFP and HA-POD-6 and of myc-COT-1 and HA-POD-6 from cell extracts co-expressing both functionally tagged proteins indicate interaction of the two kinase pairs. However, the MST-1/POD-6 interaction was more stable and withstood two washes with IP buffer, while the interaction between COT-1 and POD-6 was abolished under these conditions. (<b>C</b>) Protein displacement assay of a precipitated myc-COT-1/HA-POD-6 complex by the addition of separately purified MST-1 kinase, but not a mock-IP precipitate abolished the COT-1/POD-6 interaction. The upper panel displays a representative experiment, while three independent experiments are quantified in the lower graph. (<b>D</b>) POD-6 activity was determined <i>in vitro</i> in the presence of increasing levels of added kinase-dead MST-1(D157A) (n = 4).</p

The regulation of MST-1 and SID-1 by CDC-7 is based on distinct mechanisms.

<p>(<b>A</b>) <i>In vitro</i> DBF-2 activity assays after addition of the indicated kinases. MST-1-stimulated DBF-2 activity is inhibited by addition of CDC-7 or CDC-7(D195A), while SID-1-stimulation requires active CDC-7. Western blot analysis of the precipitated proteins was used to determine comparable kinase levels (n = 4). (<b>B</b>) A GFP fusion construct of wt CDC-7 localized to spindle pole bodies and accumulated around the mature septal pore, while a CDC7(D195A)-GFP construct exclusively localized to SPBs. Nuclei were labeled with histone H1-RFP and plasma membrane with FM4-64.</p

Kinetics of cortex association of SIN and MOR components DBF-2, MST-1 and COT-1 during septum formation.

<p>(<b>A</b>) Composite images of time series of the indicated proteins during septum formation. The start of plasma membrane invagination was monitored with FM4-64 and defined as time point 0:00 min. Arrows indicate the time point when the GFP-fusion proteins occurred first at the cell cortex and arrowhead the time point of complete CAR constriction. (<b>B</b>) Schematic representation of the chronology of emergence of the SIN and MOR at the cell cortex relative to the actin cytoskeleton.</p

SID-1 and POD-6 are pathway-specific activators of the SIN and MOR, while MST-1 regulates both NDR kinase pathways.

<p>(<b>A</b>) <i>In vitro</i> kinase assays of precipitated MST-1 and SID-1 specifically stimulated DBF-2, but not DBF-2(T671A). (<b>B</b>) COT-1 was specifically phosphorylated by the upstream GC kinases POD-6 and MST-1, but not by SID-1. Western blot analysis of the precipitated proteins was used to determine comparable kinase levels.</p

MST-1 is required for proper contractile actin ring formation.

<p>(<b>A</b>) 4D reconstruction of z-stacks in time lapse series revealed cortical, concentrically constricting BNI-1-GFP rings in wt cells, which resulted in centrally positioned septal pores. In contrast, BNI-1 formed asymmetric and frequently open BNI-1-GFP rings in Δ<i>mst-</i>1 that led to acentric CAR constriction and asymmetric septal pores. (<b>B</b>) 3D reconstruction of z-stacks illustrates BNI-1-GFP association with extensive cortical Calcofluor White-labeled spirals in Δ<i>mst-</i>1. (<b>C</b>) Comparison of actin dynamics during CAR assembly and constriction in wt and Δ<i>mst-</i>1. Lifeact-GFP labeled a dynamic meshwork of actin cables and patches around the future septation site in wt cells, which subsequently coalesced to form the CAR. The actin meshwork was mis-organized and irregularly distributed in Δ<i>mst-1</i> (<b>D</b>) 4D reconstruction of z-stacks in time lapse series visualized open actin spirals labeled by Lifeact-GFP, which were unable to constrict. Cell wall, septa and cortical spirals were labeled by Calcofluor White.</p

CDC-7 forms mutually exclusive complexes with SID-1 and MST-1.

<p>(<b>A</b>) AP-MS data from two independent biological replicates were used for identification of predicted SIN components. * denotes % protein coverage and number of unique peptides of the identified protein. Only SIN components identified in both replicate purifications and absent from a control data set obtained in purifications with GFP-expressing cells are displayed (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004306#pgen.1004306.s005" target="_blank">Table S1</a>). GFP-fusion constructs used as bait proteins are in bold type. (<b>B</b>) Yeast two-hybrid tests with the indicated SIN constructs and summary schema of the observed interactions. (<b>C</b>) Reciprocal co-immunoprecipitation experiments of CDC-7-GFP and HA-MST-1 from cell extracts co-expressing both functionally tagged proteins indicate interaction of the two kinases. (<b>D, E</b>) GC-kinase displacement assays of precipitated CDC-7-GFP/GC-kinase complexes. Addition of a second, separately purified GC kinase, but not a mock-IP precipitate to these complexes reduced the abundance of the co-purified GC kinase. The upper panel displays a representative experiment, while three independent experiments are quantified in the lower graph.</p