Abstract
Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP 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 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 to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and 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 to punctate complexes that did not oscillate, but still localized to 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 to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to 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 and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of fungal communication.
Highlights
Fusion between genetically identical cells occurs in many different organisms and plays pivotal roles in different developmental processes, such as myoblast fusion during muscle formation, macrophage fusion involved in tissue remodeling and fusion of trophoblasts during placental development [1,2]
Chemotropic growth is dependent upon the assembly of a MAPK cascade (NRC-1/MEK-2/ MAK-2) at the cell cortex, followed by disassembly over an,8 min cycle
A second protein required for fusion, SO, assembles and disassembles at conidial anastomosis tube (CAT) tips during chemotropic growth, but with perfectly opposite dynamics to the MAK-2 complex
Summary
Fusion between genetically identical cells occurs in many different organisms and plays pivotal roles in different developmental processes, such as myoblast fusion during muscle formation, macrophage fusion involved in tissue remodeling and fusion of trophoblasts during placental development [1,2]. In addition to hyphal fusion, fusion can occur between genetically identical germinating asexual spores (conidia) of filamentous fungi [6,7,8]. Both hyphal and germling fusion are integral to the formation of an interconnected hyphal network and impart fitness benefits, as well as mediating genetic mixing and the sharing of resources [3,4,9,10,11,12,13]. An essential part of chemotropic interactions in N. crassa is the oscillatory recruitment of three kinases of a MAPK cascade (NRC-1, MEK-2 and MAK-2) and of a protein of unknown function, SOFT (SO), to CAT tips [18,19]. It was proposed that the alternating oscillation of MAK-2 and SO to CAT tips may function to establish two distinct physiological states in interacting
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