Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved maps kinase cascade that includes NRC-1, MEK-2 & MAK-2. During chemotrophic growth in germlings, the maps kinase cascade members localize to lớn conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to lớn the tip: SOFT, a recently identified scaffold for the MAK-1 maps kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the map kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to lớn identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown khổng lồ be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 và MAK-2 and oscillated with identical dynamics from the cytoplasm to cat tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized khổng lồ punctate complexes that did not oscillate, but still localized khổng lồ the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize lớn puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to lớn physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the map kinase cascade members for oscillation và chemotropic interactions during germling & hyphal fusion in N. Crassa. The identification of HAM-5 as a scaffold-like protein will help to links the activation of MAK-2 cascade to upstream factors và proteins involved in this intriguing process of fungal communication.
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Cell fusion between genetically identical cells of the fungus Neurospora crassa occurs when germinating asexual cells (conidia) sense each other"s proximity và redirect their growth. Chemotropic growth is dependent upon the assembly of a MAPK cascade (NRC-1/MEK-2/MAK-2) at the cell cortex (conidial anastomosis tubes; CATs), followed by disassembly over an ∼8 min cycle. A second protein required for fusion, SO, also assembles and disassembles at cat tips during chemotropic growth, but with perfectly opposite dynamics khổng lồ the MAK-2 complex. This process of germling chemotropism, oscillation and cell fusion is regulated by many genes and is poorly understood. Via a phosphoproteomics approach, we identify HAM-5, which functions as a scaffold for the MAK-2 signal transduction complex. HAM-5 is required for assembly/disassembly & oscillation of the MAK-2 complex during chemotropic growth. Our data supports a model whereby regulated modification of HAM-5 controls the disassembly of the MAK-2 MAPK complex & is essential for modulating the tempo of oscillation during chemotropic interactions.
Citation: Jonkers W, Leeder AC, Ansong C, Wang Y, Yang F, Starr TL, et al. (2014) HAM-5 Functions As a bản đồ Kinase Scaffold during Cell Fusion in Neurospora crassa. PLo
S Genet 10(11): e1004783. Https://doi.org/10.1371/journal.pgen.1004783
Editor: Joseph Heitman, Duke University Medical Center, United States of America
Received: July 3, 2014; Accepted: September 26, 2014; Published: November 20, 2014
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper và its Supporting Information files.
Funding: The work in this study was funded by a National Science Foundation grant khổng lồ NLG (MCB 1121311). Portions of this work were supported by the US DOE office of Biological và Environmental Research, và also NIH grant P41 GM103493-11 (RDS). Proteomics work was performed in the EMSL, a DOE-BER national scientific user facility PNNL. PNNL is a multi-program national laboratory operated by Battelle Memorial Institute for the DOE under contract DE-AC05-76RLO 1830. The funders had no role in study design, data collection and analysis, decision lớn publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Fusion between genetically identical cells occurs in many different organisms và plays pivotal roles in different developmental processes, such as myoblast fusion during muscle formation, macrophage fusion involved in tissue remodeling & fusion of trophoblasts during placental development <1>, <2>. Cell fusion is also important for the formation of the interconnected mycelial network that is the hallmark of filamentous fungal growth <3>, <4>, <5>. In addition to lớn hyphal fusion, fusion can also occur between genetically identical germinating asexual spores (conidia) of filamentous fungi <6>, <7>, <8>. Both hyphal và germling fusion are integral to the formation of an interconnected hyphal network và impart fitness benefits, as well as mediating genetic mixing and the sharing of resources <3>, <4>, <9>, <10>, <11>, <12>, <13>.
In the filamentous ascomycete fungus, Neurospora crassa, germinated conidia (germlings) in close proximity fuse via specialized conidial anastomosis tubes (CATs) that size at germ tube tips or between conidia <14>. In a fungal colony, hyphal fusion is observed in the central parts of the colony, in contrast lớn the peripheral parts where hyphae avoid each other. A large number of genes have been identified in N. Crassa that are important for the process of sensing, chemotropic interactions & CAT fusion and have contributed khổng lồ an understanding of this complex developmental system in filamentous ascomycete fungi <7>, <15>, <16>, <17>. An essential part of chemotropic interactions in N. Crassa is the oscillatory recruitment of three kinases of a MAPK cascade (NRC-1, MEK-2 và MAK-2) and of a protein of unknown function, SOFT (SO), to cát tips <18>, <19>. In strains carrying loss-of-function mutations in these genes, oscillatory recruitment of NRC-1/MEK-2/MAK-2 or SO, chemotropic interactions và fusion vày not occur <8>, <18>, <19>, <20>. It was proposed that the alternating oscillation of MAK-2 và SO to mèo tips may function lớn establish two distinct physiological states in interacting germlings to lớn enable chemotropism to persist, avoid self-stimulation & assure a rapid & efficient cell fusion <19>, <21>. Recently, it has been shown that an ortholog of SOFT in the related filamentous ascomycete species, Sordaria macrospora, PRO40, is a scaffold protein for the cell wall integrity maps kinase pathway (MAK-1) <22>; Δmak-1 mutants in both N. Crassa và S. Macrospora are fusion mutants <22>, <23>.
Previously, it was shown that kinase activity of MAK-2 is required to lớn maintain oscillatory recruitment of both MAK-2 và SO; addition of ATP-analog 1NM-PP1 to a mutant containing an inhibitable MAK-2 protein encoded by a mak-2Q100G allele, disrupted the oscillation of both MAK-2 và SO in communicating germlings & stalled chemotropic interactions and the fusion process <19>. The use of mak-2Q100G strain also contributed khổng lồ the identification of downstream genes whose expression levels depend on functional MAK-2 <24>. However, MAK-2 kinase targets involved in the oscillation process have not been identified và the characterization of such potential targets could help unravel molecular mechanisms associated with this highly regulated & complex process.
In recent years, highly sensitive liquid chromatography-mass spectrometry (LC-MS) based quantitative phosphoproteomic techniques have contributed lớn our understanding of kinase pathway function in eukaryotic cells <25>, <26>, <27>, <28>. Lớn identify MAK-2 kinase targets in N. Crassa, we took a global approach by identifying phosphopeptides in mak-2Q100G germlings, treated or not with 1NM-PP1. From the phosphoproteomic screen, a number of candidate MAK-2 target proteins were identified, one of which encodes a protein previously identified as being essential for germling/hyphal fusion, HAM-5 <23>, <29>. We show that HAM-5 oscillates during chemotropic interactions with components of the MAK-2 pathway, physically interacts with NRC-1, MEK-2 và MAK-2 and was required for localization of MAK-2 & MEK-2 lớn puncta. Our data supports the hypothesis that HAM-5 functions as a scaffold protein by binding to và co-localizing to cat tips with all three kinases in the MAK-2 cascade during chemotropic growth in germlings as well as in hyphae undergoing fusion events. These studies shed new light on the mechanisms of oscillation during communication và chemotropic interactions between genetically identical cells và which may be important for function of this conserved MAPK pathway in other filamentous ascomycete fungi.
Identification of MAK-2 targets in Δmak-2Q100G germlings using a phosphoproteomic approach
To better understand the role of MAK-2 during chemotropic interactions, we mix out lớn identify putative kinase targets using a global quantitative phosphoproteomics approach using a strain carrying an inhibitable mak-2Q100G allele. Altering a specific amino acid in the ATP binding site (glutamine for glycine) renders MAK-2 sensitive lớn inhibition to the ATP analogue 1NM-PP1, but does not affect MAK-2 kinase activity in the absence of inhibitor <19>. In the absence of the inhibitor 1NM-PP1, strains và germlings containing the mak-2Q100G allele (his3::mak-2Q100G; Δmak-2) showed wild-type growth và fused normally, while in the presence of inhibitor, the mak-2Q100G strain showed a mutant mak-2 phenotype; chemotropic interactions và fusion were not observed, consistent with inactivation of MAK-2 kinase activity <19>. For identifying MAK-2-dependent phosphorylation events, 4.5-hr old mak-2Q100G germlings were treated with DMSO (two samples và two biological replicates) or 1NM-PP1 (dissolved in DMSO; two samples và two biological replicates) for 10 min. Proteins were extracted, digested, and enriched phosphopeptides identified & quantified using isobaric peptide tags for relative và absolute quantification (i
TRAQ)-based LC-MS/MS analyses. Although the same peptides across experimental conditions are labeled with different i
TRAQ reagents and indistinguishable by mass, different masses will be generated in the tandem MS by releasing the reporter ions for the 4-plex i
TRAQ method (Figure S1). We performed the full experiment twice, resulting in a total of eight samples, which were analyzed for phosphopeptide identity and abundance.
From these experiments a total of 3200 unique phosphopeptides were identified. These 3200 chất lượng phosphopeptides originated from 1164 proteins (Dataset S1). A small percentage of the identified peptides have multiple phosphorylation sites (12%, Figure 1B), as compared to lớn peptides where only a single phosphorylation site was identified (88%, Figure 1B). Phosphorylation sites were predominantly identified on serine residues (75%), và to a lesser extent on threonine (22%) và tyrosine (3%) residues (Figure 1C). Fun
Cat analysis <30> showed the mix of identified phosphorylated proteins in germlings originated from a wide spectrum of functional categories (Figure 1A), including, not unexpectedly, proteins involved in metabolism, energy, cell cycle and DNA processing, protein synthesis and transcription. However, proteins within the functional categories of cellular communication/signal transduction, cell defense, và interaction with the environment were also identified, suggesting germlings are poised lớn respond khổng lồ variations in their environment (Dataset S1).
(A) Overview of Fun
Cat categories of proteins harboring the identified phosphopeptides. The upper bar shows the categories of all the proteins with identified phosphopeptides (3200 phosphopeptides, 1164 proteins), the middle bar shows functional categories of proteins with phosphopeptides that showed higher abundance after inhibition (33 phosphopeptides, 27 proteins) & the lower bar shows Fun
Cat analysis of the proteins with phosphopeptides that showed lower abundance in mak-2Q100G germlings after treatment with 1NM-PP1 (96 phosphopeptides, 67 proteins). (B) Pie chart showing the relative percentages & absolute numbers of single, double, triple và quadruple phosphosites per peptide. (C) Pie chart showing the relative percentages and absolute numbers of phosphorylated serine, threonine và tyrosine in all peptides found.
https://doi.org/10.1371/journal.pgen.1004783.g001
Of the 3200 phosphopeptides, 96 quality phosphopeptides from 67 proteins were>1.5 times less abundant (p
Q100G germlings relative lớn the DMSO-treated control cells (Table S1). Functional category analyses <30> of this phối of proteins showed enrichment for genes involved in metabolism, energy, cell cycle and DNA processing, transcription, protein fate, regulation of metabolism & protein function, cellular communication/signal transduction, interaction with the environment, cell fate & cell type differentiation (p
Figure 1A; Dataset S1). Three proteins in this group had previously been shown to be required for hyphal fusion, including HAM-9, HAM-11 và MAK-1 <23>, <24>, <31>, <32>. In addition to lớn the bản đồ kinase involved in the osmotic response signaling (OS2) <32>, CUT-1, which is implicated in the osmotic ức chế response <33>, <34> as well as the transcription factor that is a target of the OS-2 pathway (ASL-1/ATF-1, NCU01345) <35> were identified (Table 1; Table S1). Of these 3200 chất lượng phosphopeptides, 33 phosphopeptides (from 27 proteins) were identified with an increased abundance of at least 1.5 fold (p<30> of this mix of 27 proteins showed over-representation for genes/proteins involved in phosphate metabolism (perhaps as a response to 1NM-PP1 exposure), protein synthesis, protein fate and protein with binding function or cofactor requirements (Dataset S1).
Table 1. Phosphopeptides with predicted MAPK site that showed decreased abundance in 1NM-PP1-treated mak-2Q100G cells.
To identify potential direct MAK-2 targets, we inspected the 96 phosphopeptides that showed reduced abundance in the 1NM-PP1 treated mak-2Q100G germlings for MAPK consensus phosphorylation sites (P-X-S*/T*-P) <36>, <37>. Nine proteins were identified, of which five were annotated as hypothetical proteins. One of the proteins was ASL-1, the transcription factor that is a target of the OS-2 pathway & which shows an ascospore-lethal phenotype <35>; Δmak-2 mutants also show an ascospore-lethal phenotype <8>, <38>. The remaining proteins included a predicted 6-phosphofructo-2-kinase (NCU01728), a predicted trehalose phosphatase (NCU05041) & a protein previously reported lớn be required for hyphal fusion, HAM-5 <23>, <29> (Table 1). In addition to lớn a predicted MAPK phosphorylation site, a predicted MAPK docking motif (R/K-R/K-(X)1-5-I/L-X-I/L) <39> was predicted in NCU07868 (hypothetical protein) & HAM-5 (Table 1). Of the nine genes encoding potential MAK-2 phosphorylation targets, five showed a reduction in expression levels in either a Δpp-1 mutant (transcription factor that is a target of the MAK-2 pathway) or in a mak-2Q100G germlings treated with 1NM-PP1 <24>, including ham-5 (Table 1).
To determine whether the putative direct or indirect MAK-2 phosphorylation targets were involved in germling fusion, strains carrying individual deletions of 8 out of the 9 genes whose proteins contained a MAPK consensus phosphorylation site and showed decreased abundance in the 1NM-PP1-treated mak-2Q100G germlings (including strains carrying deletions in all five hypothetical proteins) (Table 1), plus an additional 31 deletion mutants selected from a subset of proteins that showed decreased abundance in 1NM-PP1-treated mak-2Q100G germlings, but which lacked a predicted MAPK consensus phosphorylation site (Table S1), were evaluated for the ability to lớn undergo chemotropic interactions and cell fusion. Of these, Δham-5 in the first set, và Δham-9, Δham-11, and Δmak-1 strains in the second set, were fusion defective.
Putative MAK-2 target HAM-5-GFP localizes in an oscillatory manner to lớn cell tips during chemotrophic interactions
To further characterize putative MAK-2 targets that are required for germling fusion, we GFP-tagged proteins where localization during chemotropic interactions/cell fusion had not been determined. This effort included HAM-5. The ham-5 mutant was identified in a forward screen for mutants that failed to khung a heterokaryon <29> and encodes a large protein of 1686 amino acids (aa) with seven putative WD40 repeats at the N-terminus (aa 14-313, grey boxes; Figure 2A). At the C-terminus, an unstructured region of low complexity was identified (shaded trắng boxes; Figure 2A) that contains stretches of predominately proline and glutamine residues. Two coiled coil domains were also predicted in HAM-5 (aa 1168-1190 and 1257-1286, red boxes; Figure 2A). In total, 16 phosphorylation sites were identified, located mainly at the middle section of HAM-5 (Figure 2A). Three phosphorylation sites were identified from our phosphoproteomics analysis, one of which was a putative MAPK site at amino acid residue 506 (serine) (Figure 2A; Table 1). Thirteen additional HAM-5 phosphopeptide sites were identified in a recent phosphoproteomics study of N. Crassa hyphal cultures exposed to different carbon sources <40>. A putative MAPK docking site was also identified between residues 1128-1136 (RRKPPALDL) in the C-terminus of HAM-5 (yellow bar; Figure 2A).
(A) Schematic overview of HAM-5 protein structure. The predicted WD40 domains are shown in grey & the putative coiled coil domains are shown as red bars. The two disordered regions with low complexity are depicted by shaded trắng boxes. The MAPK phosphorylation site (aa 506) is marked by a blue star, the other two sites showing decreased abundance in treated cells (aa 1288 and 1604) are marked by green stars, và other 13 identified phosphorylation sites (S14, S414, S792, S818, S833, T838, T969, S1085, S1199, T1201, S1202, T1353, S1608) <40> are marked by black stars. The putative MAPK docking site is marked by a yellow line. (B) Localization of HAM-5-GFP lớn puncta localized to mèo tips during chemotropic interactions between genetically identical cells. HAM-5-GFP showed dynamic localization to cat tips of germlings with an oscillation of every four min (arrow). HAM-5-GFP also localized to lớn puncta within germlings and near nuclear compartments devoid of HAM-5-GFP (asterisks). The image left is a bright field image. Scale bar = 10 µM. (C) HAM-5-GFP localized to lớn the sites of contact during germling fusion (arrow). (D) Western blots of WT, WT (ham-5-gfp) và Δmak-2 (ham-5-gfp) germlings with immunoprecipitated HAM-5-GFP probed with anti-GFP antibodies (right panel shows longer run showing higher mobility of HAM-5-GFP in wild type germlings) specifically detecting HAM-5-GFP (210 k
D; Figure S4D). Lower panel shows a Western blot with identical samples probed with anti-phospho antibodies that specifically detect phosphorylated serine or threonine residues followed by a proline. (E) Localization of HAM-5-GFP lớn puncta in Δmak-2 germlings. Some puncta showed localization to lớn germling tips, but which did not oscillate during growth (white arrows). Scale bar = 10 µM.
https://doi.org/10.1371/journal.pgen.1004783.g002
In conidia, germlings và in hyphae, HAM-5-GFP fluorescence was observed in small, cytoplasmically localized puncta (white arrows, Figure S2). In mature hyphae, localization khổng lồ septa was also observed (red arrow, Figure S2A). During chemotropic interactions between germlings, HAM-5-GFP driven by the tef-1 promoter was observed as small puncta in interacting germlings và also at the tip (Figure 2B). Importantly, HAM-5 oscillated to cát tips in germling pairs during chemotropic interactions, with localization dynamics similar lớn MAK-2 or SO: HAM-5-GFP appeared at the cat tip of one germling, but was absent from the mèo tip in the partner germling, while 4 min later, HAM-5-GFP was present at the mèo tip of the second partner germling, but was absent from the mèo tip of the first germling (Movie S1). During the fusion process, a HAM-5-GFP signal was also observed in puncta that localized either to lớn the cell periphery or khổng lồ points close to nuclear compartments, which were devoid of HAM-5-GFP (Figure 2B, asterisk). When HAM-5-GFP localized khổng lồ the cat tips, the number of cytoplasmically localized puncta in the partner germling was reduced & the GFP signal was more dispersed in the cytoplasm (Figure 2B). HAM-5-GFP was detected at the site of germling liên hệ and remained there until the cytoplasm of the two germlings mixed (Figure 2C).
To assess whether the HAM-5-GFP-tagged versions were biologically functional, we crossed wild type (WT) strains carrying ham-5-gfp driven by either the native, tef-1 or ccg-1 promoter into the Δham-5 mutant. Progeny bearing both ham-5-gfp and Δham-5 showed a fusion phenotype and frequency similar to lớn WT, indicating that ham-5-gfp driven either by the native, ccg-1 or tef-1 promoter was functional. Localization of HAM-5-GFP driven by either ccg-1 or tef-1 in the Δham-5 strain was similar to HAM-5-GFP in the WT strain, & showed localization to puncta & oscillation to mèo tips during chemotropic interactions. ham-5-gfp driven by its native promoter in the Δham-5 strain also showed localization to lớn puncta (Figure S3). However, due lớn low expression levels of HAM-5-GFP in this strain, localization during chemotropic interactions could not be fully assessed. In the following sections, HAM-5-GFP is shown driven by the tef-1 promoter unless stated otherwise. A heterokaryon of a strain carrying ham-5-gfp driven by tef-1 & a strain carrying histone H1-ds
RED (a nuclear marker), showed non-overlapping fluorescence (Figure S2), indicating that HAM-5-GFP was excluded from the nucleus. This localization pattern is similar lớn SO-GFP <19>, but different than MAK-2-GFP, which localizes lớn the cytoplasm and to nuclei (Figure S2) <19>.
MAK-2 regulates HAM-5 dynamics
HAM-5 is predicted lớn be a highly phosphorylated protein, with 16 predicted phosphorylation sites (Figure 2A). Lớn assess whether HAM-5 is a phosphorylation target of MAK-2, as indicated by the phosphoproteomics data, we introduced ham-5-GFP into a Δmak-2 strain and determined its phosphorylation status during germling fusion. HAM-5-GFP was immunoprecipitated from WT (ham-5-gfp) and Δmak-2 (ham-5-gfp) 5 hr-old germlings using anti-GFP antibodies và assayed for phosphorylation status using Western blot analysis with anti-phosphoserine/threonine antibodies that specifically recognize phosphoserine or phosphothreonine sites followed by a proline residue (MAPK phosphorylation sites). The results showed that HAM-5-GFP from both WT và Δmak-2 cells was specifically phosphorylated (Figure 2D). The HAM-5-GFP protein band from WT germlings showed a slight smear upwards và was slightly larger than that observed in Δmak-2 (ham-5-gfp) germlings (Figure 2D). These data support the phosphoproteomics data, which suggested a MAK-2-dependent modification of HAM-5 during germling fusion (Table 1). However, it is clear that HAM-5-GFP was also phosphorylated in the Δmak-2 mutant, suggested possible additional regulatory inputs into HAM-5 via phosphorylation by other proteins during conidial germination/germling fusion.
To assess whether the identified phosphosite in HAM-5 (serine 506) was required for HAM-5 function during chemotropic interactions & germling fusion, strains carrying site-directed mutations whereby serine 506 was altered khổng lồ an alanine (phosphorylation impaired) or a glutamate (phosphorylation mimic) residue were evaluated: chemotropic behavior và cell fusion in germlings carrying the ham-5S506A or ham-5S506E mutations driven by the ccg-1 promoter were indistinguishable from wild type germlings và fully complemented the growth phenotype of the Δham-5 strain (Figure S4A). Lớn assess whether mutations in the predicted MAPK docking site affected HAM-5 function, a strain was constructed where the first three amino acids of the MAPK docking site were changed to alanine (RRKPPALDL to AAAPPALDL). However, germlings bearing this ham-5 allele (ham-5RRK1128AAA) also showed a similar communication & fusion phenotype khổng lồ WT germlings resulting in similar growth phenotype lớn WT và complemented fully the growth phenotype of the Δham-5 strain (Figure S4A). Thus, the predicted MAPK docking site in HAM-5 might not be functional or additional MAPK docking sites are present making this site redundant for function.
We predicted that HAM-5-GFP would show altered localization when introduced into Δmak-2 germlings, due to lớn the inability of these cells khổng lồ undergo chemotropic interactions. However, HAM-5-GFP localized to lớn puncta in Δmak-2; ham-5-gfp germlings, as observed in wild type cells (Figure 2E). However, no oscillation of HAM-5-GFP puncta to cell tips was observed, consistent with the lack of fusion & chemotropic interactions in Δmak-2 germlings. HAM-5-GFP puncta were localized randomly within the cell, with some puncta close lớn the nuclear periphery và membrane, but cells also contained at least one HAM-5-GFP puncta localized to the cell tip (Figure 2E). The stable, tip-anchored localization of HAM-5-GFP (Figure 2E, arrows) was not observed in WT germlings in the absence of chemotropic interactions (in isolated germlings), and was unique to Δmak-2; ham-5-gfp cells.
HAM-5-GFP oscillates with the bản đồ kinase cascade members to cat tips
The observation that HAM-5-GFP showed oscillation during germling fusion, but still localized to lớn puncta in Δmak-2 germlings, suggested that either HAM-5 interacted with SO or with other proteins in the MAK-2 signal transduction pathway. Previously, it was shown that the components of the MAK-2 pathway, the MAPKKK (NRC-1) & the MAPKK (MEK-2), oscillate with MAK-2 during chemotropic interactions in germlings <18>. Lớn determine if HAM-5 oscillated with the components of the MAK-2 complex or with SO, we used heterokaryons of strains carrying ham-5-gfp & either m
Cherry-tagged mak-2, mek-2, nrc-1 or so alleles & examined co-localization of these proteins during chemotropic interactions in germlings. As shown in Figure 3, HAM-5-GFP + MAK-2-m
Cherry, or HAM-5-GFP + MEK-2-m
Cherry or HAM-5-GFP + NRC-1-m
Cherry were co-recruited to lớn the cat tips during chemotropic interactions. In homokaryotic strains carrying both HAM-5-GFP and MAK-2-m
Cherry, the dynamics of the two proteins were identical and showed simultaneous oscillatory recruitment to mèo tips (Movie S2). HAM-5-GFP + MAK-2-m
Cherry & HAM-5-GFP + MEK-2-m
Cherry also co-localized khổng lồ cytoplasmic puncta during chemotropic interactions (Figure 3). As observed in <18>, the NRC-1-m
Cherry signal was weak, and recruitment of HAM-5-GFP + NRC-1-m
Cherry to cytoplasmic puncta could not be assessed. In contrast to lớn MAK-2/MEK-2/NRC-1, SO-m
Cherry + HAM-5-GFP always appeared at opposite cat tips (Figure 3D) & with exactly opposite dynamics during chemotropic interactions (Movie S3).
(A) Co-localization of HAM-5-GFP and MAK-2-m
Cherry during germling communication (arrows). (B) Co-localization of HAM-5-GFP & MEK-2-m
Cherry during germling communication (arrows). (C) Co-localization of HAM-5-GFP và NRC-1-m
Cherry during germling communication. NRC-1-m
Cherry strains show low fluorescence <18>. (D) HAM-5-GFP and SO-m
Cherry vày not co-localize during chemotropic interactions, but instead show opposite localization to cát tips in communicating germlings (arrows). The images on the left are bright field images, fluorescent images on the right. Scale bar = 10 µM. (E) Co-immunoprecipitation experiments showing an interaction between HAM-5-GFP (210 k
D; Figure S4D) & MEK-2-m
Cherry (82.9 k
D; Figure S4C) and NRC-1-m
Cherry (128 k
D; Figure S4C). Input đầu vào panels show Western blots of immunoprecipitated protein samples from 5 hr-old germlings probed with either anti-GFP (free GFP = 27 k
D; Figure S4D) or anti-m
Cherry antibodies. The output panel is a Western blot of proteins immunoprecipitated by anti-GFP antibodies (and thus HAM-5-GFP) and probed with anti-m
Cherry antibodies (detecting MEK-2-m
Cherry or NRC-1-m
Cherry).
https://doi.org/10.1371/journal.pgen.1004783.g003
The localization of HAM-5 during chemotropic interactions suggested that HAM-5 physically interacts with MAK-2, MEK-2 and/or NRC-1. To test this hypothesis, we performed co-immunoprecipitation experiments using strains carrying HAM-5-GFP + MAK-2-m
Cherry, HAM-5-GFP + MEK-2-m
Cherry or HAM-5-GFP + NRC-1-m
Cherry. As controls, we used strains carrying MAK-2-m
Cherry, MEK-2-m
Cherry or NRC-1-m
Cherry-tagged proteins with GFP driven by the ccg-1 promoter; GFP in these strains showed only cytoplasmic localization và was never observed in puncta. A specific interaction between HAM-5-GFP và MEK-2-m
Cherry & HAM-5-GFP & NRC-1-m
Cherry was detected when HAM-5-GFP was immunoprecipitated using anti-GFP antibodies from 5 hr-old germlings và subsequently re-probed using anti-m
Cherry antibodies (Figure 3E), consistent with the co-localization of these proteins observed by confocal microscopy (Figure 3A–C). No interaction between the m
Cherry-tagged proteins and cytoplasmic GFP was observed (Figure 3E; Figure S4C). An interaction between MAK-2-m
Cherry & HAM-5 could not be assessed, as MAK-2-m
Cherry showed non-specific binding. However, using phospho-specific anti-P42/P44 (Erk1/Erk2) antibodies that recognize phosphorylated MAK-2 <8>, an interaction between HAM-5-GFP và MAK-2 was detected via co-immunoprecipitation (Figure 4D). By contrast, no interaction was detected between HAM-5-GFP và SO-m
Cherry (Figure 3E).
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Figure 4. The WD40 tên miền of HAM-5 interacts with MAK-2, while the C-terminus interacts with MEK-2.
(A) Localization of HAM-51-351-GFP (WD40 domain name only) in WT germlings localized to the cytoplasm, the nucleus và the puncta at the cell periphery & at the tip during chemotropic interactions & oscillation (arrow). (B) HAM-51-351-GFP in Δham-5 germlings localized to the cytoplasm and the nucleus; no puncta were observed. The images on the left are bright field images. Scale bar = 10 µM. (C) Western blots showing a specific interaction between MEK-2-m
Cherry (82.9 k
D) with full length HAM-5-GFP (210 k
D) or HAM-5Δ67-348-GFP (180 k
D), but not with miễn phí GFP (27 k
D; Figure S4C, D). An interaction between MEK-2-m
Cherry và HAM-51-351-GFP (65.3 k
D) was not detected. Input đầu vào panels show Western blot of immunoprecipitated proteins isolated from 5 hr-old germlings probed with either anti-m
Cherry antibodies or anti-GFP antibodies. Output panel shows anti-m
Cherry immunoprecipitated proteins (MEK-2-m
Cherry) probed with anti-GFP antibodies (HAM-5-GFP). (D) Western blots showing a specific interaction between full length HAM-5-GFP or HAM-51-351-GFP and phosphorylated MAK-2. Input panels show Western blot of proteins isolated from 5 hr-old germlings probed with anti-p42/44 antibodies (which recognize phosphorylated MAK-2 (40.8 k
D; Figure S4D, E) <8>) or immunoprecipitated proteins probed with anti-GFP antibodies. The đầu ra panel shows anti-GFP immunoprecipitated protein sample probed with anti-p42/44 antibodies, showing interaction between HAM-5-GFP or HAM-51-351-GFP và phosphorylated MAK-2. (E) Schematic showing regions of interaction between HAM-5 và MAK-2 or MEK-2 based on co-immunoprecipitation experiments.
https://doi.org/10.1371/journal.pgen.1004783.g004
The HAM-5 WD40 domain is required for function và stability & binds to MAK-2 but not to lớn MEK-2
HAM-5 is a large protein with predicted protein-protein interaction domains including seven WD40 repeats, which are predicted to size β-propeller structures that have been implicated in coordinating protein assemblages in other systems <41>. To thử nghiệm the hypothesis that the WD40 domain name is involved in HAM-5-MAK/MEK-2/NRC-1 interactions, we constructed two mutant ham-5 alleles: one in which the WD40 motifs (aa 67-348) were removed (HAM-5Δ67-348) và one in which only the first 351 aa including the WD40 motifs of HAM-5 were retained (HAM-51-351). Both alleles were tagged with gfp, & function & localization were assessed in both WT and Δham-5 mutant strains.
The ham-51-351 construct failed to complement the growth or fusion defects of the Δham-5 mutant (Figure S4A). When observed microscopically, the localization of the HAM-51-351-GFP in Δham-5 germlings was cytoplasmic & nuclear; no puncta were observed (Figure 4B; Figure S5A). This result is in contrast to the full length HAM-5-GFP, which localized to puncta & was excluded from the nucleus (Figure 2; Figure S2). However, in a WT background, a portion of the HAM-51-351-GFP localized to puncta và showed oscillation during chemotropic interactions between germlings (Figure 4A). These observations suggest that in WT germlings, HAM-51-351-GFP may bind the native untagged HAM-5, resulting in localization khổng lồ puncta when the complex oscillates to mèo tips during chemotropic interactions. Khổng lồ determine whether HAM-51-351-GFP (in a Δham-5 mutant) physically interacted with MAK-2 or MEK-2, we performed co-immunoprecipitation experiments using either anti-m
Cherry antibodies (for MEK-2-m
Cherry) or anti-p42/44 antibodies for MAK-2 <8>. HAM-51-351 specifically immunoprecipitated phosphorylated MAK-2 but not MEK-2-m
Cherry (Figure 4C, D; Figure S4D).
The ham-5Δ67-348 construct also failed to lớn complement the growth or fusion defects of the Δham-5 mutant (Figure S4A), consistent with an essential role for the HAM-5 WD40 domain. Cellular fluorescence of HAM-5Δ67-348-GFP in germlings or hyphae was not observed và less protein was produced than other GFP-tagged proteins (Figure S4B), suggesting that the WD40 tên miền is required for HAM-5-GFP stability. However, co-immunoprecipitation experiments revealed a specific interaction between HAM-5Δ67-348 & MEK-2-m
Cherry, but not with MAK-2 (Figure 4C; Figure S4F). These biochemical interaction studies indicated that the WD40 domain name of HAM-5 is important for interactions with MAK-2 and the C-terminus is important for interactions with MEK-2. The predicted MAPK docking site, which was not required for function by mutational analyses (see above), is located in the C-terminus of HAM-5, indicating that this site is not essential for MAK-2-HAM-5 interactions.
HAM-5 is required for assembly of the MAPK-cascade members MAK-2 & MEK-2 into puncta
Scaffold proteins such as Ste5 in Saccharomyces cerevisiae <42>, which assembles the pheromone response MAPK pathway, regulates spatial functionality of this pathway via nuclear/plasma membrane shuttling of Fus3 during mating. We hypothesized that HAM-5 was also required for the assembly of the MAK-2 cascade members in complexes & subsequent recruitment of these complexes to their correct cellular location during chemotropic interactions (e.g. The mèo tip). To demo this hypothesis, we introduced MAK-2-GFP or MEK-2-m
Cherry into a Δham-5 strain. In contrast khổng lồ WT & Δmak-2 germlings where HAM-5-GFP was localized khổng lồ puncta (Figure 2), MAK-2-GFP showed cytoplasmic and nuclear localization in the Δham-5 mutant; no puncta were observed (Figure 5A, B). In Δham-5 hyphae, MEK-2-m
Cherry localized khổng lồ the cytoplasm & to septa, but puncta were not observed as in WT hyphae (Figure 5C, D; Movie S4). We then tested whether HAM-5 was required to lớn establish a stable interaction between MEK-2 and MAK-2. When MEK-2-m
Cherry was immunoprecipitated from WT germlings, phosphorylated MAK-2 was also detected, while in Δham-5 germlings, co-immunoprecipitation of phosphorylated MAK-2 with MEK-2-m
Cherry was not detectable (Figure 5E; Figure S4E). SO-GFP was also cytoplasmically localized in Δham-5 (so-gfp) germlings (Figure S5), a localization pattern identical lớn that observed in WT (so-gfp) germlings not undergoing chemotropic interactions.
(A) MAK-2-GFP in isolated WT germlings localizes to the nucleus, cytoplasm & to puncta (white arrows). (B) In the Δham-5 mutant, MAK-2-GFP localization is cytoplasmic và nuclear; no puncta are observed. Scale bar = 10 µM. (C) In WT hyphae, MEK-2-m
Cherry localizes khổng lồ the septum (green arrow) & also to cytoplasmic puncta (white arrow). Scale bar = 10 µM. (D) In Δham-5 hyphae, septum localization of MEK-2-m
Cherry is observed (green arrow), but puncta are not. Scale bar = 10 µM. (E) Co-immunoprecipitation experiments showing an interaction between MEK-2-m
Cherry và phosphorylated MAK-2 in WT (mek-2-m
Cherry) germlings, but a significant reduction in interaction between MAK-2 và MEK-2-m
Cherry in a Δham-5 (mek-2-m
Cherry) strain. đứng đầu panel is a Western blot of protein samples probed with anti-p42/44 antibodies (MAK-2, 40.8 k
D, MAK-1, 46.7 k
D; Figure S4E). Middle panel is anti-m
Cherry immunoprecipitated proteins probed with anti-m
Cherry antibodies (MEK-2-m
Cherry, 82.9 k
D; Figure S4C, E). Bottom panel is anti-m
Cherry (MEK-2-m
Cherry) immunoprecipitated proteins probed via Western blot with anti-p42/44 antibodies that recognize phosphorylated MAK-2.
https://doi.org/10.1371/journal.pgen.1004783.g005
Phosphorylation of MAK-2 by the upstream kinase, MEK-2, is required for fusion <18>, but is not fully dependent on functional HAM-5. In a Δham-5 mutant, phosphorylated MAK-2 is still detectable in hyphal preparations <29>. We confirmed this result in germlings, where phosphorylated MAK-2 was also observed in the Δham-5 samples (Figure 6A). MAK-2 phosphorylation is also observed in two other fusion mutant strains: Δham-7 and Δham-11 <24>, <43> (Figure 6A). To lớn investigate whether the localization of MAK-2 complexes khổng lồ puncta was dependent on HAM-5 or the ability khổng lồ undergo chemotropic interactions, we expressed HAM-5-GFP and MAK-2-m
Cherry in Δham-7 và Δham-11 cells. Although Δham-7 và Δham-11 germlings are unable to lớn undergo chemotropic interactions and cell fusion <23>, <24>, <43>, co-localization of HAM-5-GFP và MAK-2-m
Cherry to puncta was still observed (Figure 6B-D). Interestingly, as seen in the Δmak-2; ham-5-gfp strain, tip-anchored HAM-5-GFP and MAK-2-m
Cherry were present in Δham-7 & Δham-11 germlings (Figure 6, arrows).
Figure 6. HAM-5-GFP và MAK-2-m
Cherry localize to lớn puncta in Δham-7 and Δham-11 fusion-deficient germlings.
(A) Western blot of protein samples from 5 hr-old WT, Δham-5, Δham-7 và Δham-11 germlings probed with anti-p42/44 antibodies, which recognize phosphorylated MAK-1 và MAK-2 <8>. As previously shown <43>, MAK-1 phosphorylation is reduced in the Δham-7 mutant. (B) HAM-5-GFP & MAK-2-m
Cherry localization in WT germlings undergoing chemotropic interactions. Arrows show localization to lớn the mèo tip và to cytoplasmic puncta. (C) HAM-5-GFP và MAK-2-m
Cherry co-localization in Δham-7 (ham-5-gfp; mak-2-m
Cherry) germlings. Cảnh báo lack of chemotropic interactions. Arrows show puncta of HAM-5-GFP & MAK-2-m
Cherry in Δham-7 (ham-5-gfp; mak-2-m
Cherry) germlings, which are often co-localized at the germ tube tip. (D) HAM-5-GFP and MAK-2-m
Cherry co-localization in Δham-11 (ham-5-gfp; mak-2-m
Cherry) germlings. Cảnh báo lack of chemotropic interactions. HAM-5-GFP và MAK-2-m
Cherry co-localized khổng lồ both cytoplasmic và tip-localized puncta (arrows). The upper left panels are bright field images, the upper right panels show GFP fluorescence, the lower left panels show m
Cherry fluorescence. Lower right panels show merged images of GFP & m
Cherry images. Scale bars = 10 µM.
https://doi.org/10.1371/journal.pgen.1004783.g006
HAM-5-GFP oscillates with MAK-2 during hyphal fusion
Most mutants affected in germling fusion are also deficient in hyphal fusion <7>, although localization of MAK-2 or SO during hyphal interactions has not been previously reported. Whether hyphal fusion is similarly coordinated as germling fusion is unknown, as different avoidance & fusion signals may be present at the periphery và older parts of a colony <44>. Another difference between germlings and hyphae is the presence of cytoplasmic flow in hyphae <10> that may influence the oscillation of proteins to lớn sites of fusion. We therefore evaluated the localization of HAM-5-GFP during hyphal fusion in a mature colony. As shown in Figure 7, oscillation of HAM-5-GFP lớn the tips of hyphae undergoing chemotropic interactions was observed with dynamics very similar to lớn that during germling fusion (where MAK-2 has a cycling time of ∼8 min at a single hyphal tip). Similar to lớn germlings, MAK-2-m
Cherry also showed co-localization with HAM-5-GFP and oscillated with identical dynamics. MAK-2-m
Cherry was also observed in nuclei, while HAM-5-GFP was excluded (Figure S6A và Movie S5).
Figure 7. HAM-5-GFP shows oscillatory localization khổng lồ fusion points và puncta in hyphae showing chemotropism.
(A) Time course of HAM-5-GFP localization to interacting hyphae prior to cell fusion. HAM-5-GFP localized to the hyphal tip of a homing hyphae (white arrow T = 0; T = 8), followed by a disappearance and localization of HAM-5-GFP at the cell surface in the receptive hypha (white arrow; T = 4). Red arrow shows localization to lớn septa near fusion points. At T = 30, HAM-5 is observed at the site of tương tác (white arrow). Bright field image is shown in upper left panel; remaining panels show GFP fluorescence. Scale bar = 50 µM. (B) Graphical representation of relative fluorescence intensity (R.F.I.) of HAM-5-GFP localization lớn the receptive hypha và the homing hypha over the time course (panel A). × axis shows time (min). (C) Graphical representation of HAM-5-GFP fluorescence of interacting fusion hyphae shown in Figure S6B over an extended time course. Note that following the fusion sự kiện (T = 22 min), HAM-5-GFP puncta co-oscillate in both hyphae for an additional 30 minutes, see (D). y axis shows maximal fluorescence intensity (M.F.I.) while the × axis shows time (min). The time points at which the individual pictures taken at T = 4, T = 8, T = 46 & T = 59 minutes are pointed out in the graph (black arrows) (Figure S6 & Movie S5). The time of fusion at T = 22 min is marked with a black line. (D) Example of HAM-5-GFP appearing in puncta in both hyphae after fusion at T = 46.
https://doi.org/10.1371/journal.pgen.1004783.g007
In addition khổng lồ localization to lớn sites at fusion tips of hyphae, puncta containing HAM-5-GFP & MAK-2-m
Cherry within adjoining hyphal compartments also showed oscillation (Figure 7 & Figure S6). Interestingly, upon membrane merger (t = 22 min), oscillation in both fusion hyphae was completely coordinated for an additional 30 minutes (Figure 7C,D, Figure S6 and Movie S5). We further assessed how far oscillation of HAM-5-GFP puncta extended in hyphal compartments that surrounded a fusion point. In filamentous fungi like N. Crassa, hyphal compartments are delineated by septa, but septa contain a pore through which organelles, including nuclei, can move <45>. We observed coordinated oscillation of HAM-5 in over six hyphal compartments that were ∼100 µm from the point of fusion for a total distance of ∼200 µm (Figure S7). Hyphal compartments that showed different or no oscillation of HAM-5 distant from the point of fusion were delineated by septa (Movie S6). These data show that the oscillation of HAM-5-GFP in the hyphal network was coordinated over large distances surrounding a fusion point, but could be restricted by septa. Cytoplasmic flow, septal plugging and fusion events in nearby hyphae may also affect the oscillation of HAM-5 in compartments surrounding hyphal fusion points.
Discussion
In this study, we show that HAM-5 functions as a scaffold protein for the MAK-2 maps kinase complex và is required for oscillation of this complex during chemotropic interactions during germling & hyphal fusion in N. Crassa. Our findings are complemented by the accompanying study of Dettmann et al., <46> that show physical interaction between HAM-5 và MAK-2/MEK-2/NRC-1 via mass spectrometry & yeast two hybrid, assessing both indirect & direct physical interactions of HAM-5 with the MAK-2 kinase complex. In other filamentous ascomycete species, mutations in nrc-1, mek-2, mak-2 orthologs results in strains unable to undergo vegetative cell fusion <47>, <48>, <49>, <50> as well as defects in growth, reproduction, virulence & host colonization phenotypes, indicating expanded functions for this MAPK pathway in filamentous fungi as compared to yeast <51>, <52>, <53>, <54>, <55>, <56>. ham-5 is highly conserved in the genomes of filamentous ascomycete species <51>. We predict that these ham-5 homologs will function as a scaffold in these species for mak-2/mek-2/nrc-1 orthologs, & which may be important for mediating growth, reproduction và virulence functions of this important & conserved signal transduction pathway.
The MAK-2 MAPK signal transduction pathway (MAK-2, MEK-2 và NRC-1) in filamentous fungi is orthologous to the pheromone response pathway in S. Cerevisiae (Fus3, Ste7 và Ste11). Previously, it was shown that a FUS3/KSS1 ortholog in the filamentous ascomycete species Magnaporthe grisea, (PMK1) as well as its ortholog in Aspergillus nidulans (mpk
B) complements the pheromone response/mating defect of a S. Cerevisiae fus3Δ/kss1Δ mutant <49>, <57>. In S. Cerevisiae, the Ste5 scaffold protein allosterically facilitates Ste7 phosphorylation of N. Crassa MAK-2 (called N. Cra mpk
B) <58> and A. Nidulans Mpk
B <57>, indicating conservation of regulation of these kinases by allosteric motifs within Ste5. However, although components of these two MAPK pathways are highly homologous in fungi, an ortholog of STE5 is absent in the genomes of filamentous ascomycete species. Future experiments comparing the function of these non-homologous scaffold proteins (STE5 và HAM-5) in regulating conserved signal transduction pathways will reveal how selection & evolution has shaped convergent evolution of these processes.
In S. Cerevisiae, Gβγ is involved in the recruitment of Ste5 khổng lồ the plasma membrane upon pheromone exposure, thereby recruiting the Fus3 MAPK cascade lớn the membrane. Membrane binding of Ste5 likely concentrates the bound maps kinases spatially, promoting amplification of the signal <59>, <60>. In N. Crassa, how HAM-5 và MAK-2 are recruited lớn the membrane is still elusive, but is dissimilar from Ste5 since the Gβγ ortholog in N. Crassa is not involved in germling fusion <61>, <62>. Other upstream factors shared between S. Cerevisiae and N. Crassa might regulate the activation of the MAPK pathway. One is STE50, a component in yeast that helps lớn activate Ste11. In the accompanying article, Dettmann et al., <46> identified a role for N. Crassa STE-50 as an activator of NRC-1; Δste-50 mutants were fusion deficient. Three other proteins acting upstream of NRC-1 and STE-50 were also identified: the MAP4 kinase STE-20, the small GTPase RAS-2/SMCO-7 và the capping protein of the adenylate cyclase (AC) complex, CAP-1/NCU08008. Strains carrying a deletion of any of these three genes still showed residual germling fusion, suggesting multiple and redundant inputs into the MAK-2 signal transduction pathway. In S. Cerevisiae, Bem1 interacts with Ste20, Ste5 & actin <63>. In N. Crassa, strains carrying either a deletion of bem-1, encoding a predicted scaffold for NADPH oxidase (NOX), or its regulator (NOXR), are germling/hyphal fusion deficient <23>. Activated RAC-1 also localizes to cat tips during chemotropic interactions & may also function as an upstream activator <64>; Δrac-1 mutants are also are germling fusion defective <23>.
During chemotropic interactions, HAM-5/MAK-2 complex assembles in puncta at the cát tip and in the cytoplasm, followed by disassembly of HAM-5/MAK-2 complex from puncta, not just at the cat tip, but from puncta observed throughout the germling and fusion hyphae, a cycle that repeats itself during chemotropic interactions every ∼8 min. In the absence of HAM-5, localization of MAK-2 kinase complex khổng lồ puncta was impaired, while in Δmak-2 mutants, HAM-5 puncta were still observed. These observations indicate that MAK-2 kinase activity is essential for disassembly of the HAM-5/MAK-2 complex (Figure 8) during chemotropic interactions. The formation of HAM-5/MAK-2 complexes in puncta was not disrupted in other fusion mutants, such as Δham-7 & Δham-11; cortical localization of the HAM-5/MAK-2 complexes were observed at cell tips, although oscillation was not. These data suggest that the HAM-5/MAK-2/MEK-2/NRC-1 complexes are poised to lớn signal for chemotropic interactions, but that the absence of HAM-7 or HAM-11 disrupts signaling that results in disassembly of the HAM-5/MAK-2 complexes, both within the cell và at the cell cortex.
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