Synchronization of oscillatory growth prepares fungal hyphae for fusion
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eLife assessment
This valuable study combines live cell imaging and mathematical modeling to show how an emerging model fungus engages in an oscillatory chemical dialogue to prepare for cell-cell fusion. Cell data and modeling are in compelling agreement but leave many open questions as to the nature of coordination between cells and the significance of oscillations, rendering the strength of evidence in support of the authors' inferences incomplete.
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Abstract
Communication is crucial for organismic interactions, from bacteria, to fungi, to humans. Humans may use the visual sense to monitor the environment before starting acoustic interactions. In comparison, fungi, lacking a visual system, rely on a cell-to-cell dialogue based on secreted signaling molecules to coordinate cell fusion and establish hyphal networks. Within this dialogue, hyphae alternate between sending and receiving signals. This pattern can be visualized via the putative signaling protein Soft (SofT), and the mitogen-activated protein kinase MAK-2 (MakB) which are recruited in an alternating oscillatory manner to the respective cytoplasmic membrane or nuclei of interacting hyphae. Here, we show that signal oscillations already occur in single hyphae of Arthrobotrys flagrans in the absence of potential fusion partners (cell monologue). They were in the same phase as growth oscillations. In contrast to the anti-phasic oscillations observed during the cell dialogue, SofT and MakB displayed synchronized oscillations in phase during the monologue. Once two fusion partners came into each other’s vicinity, their oscillation frequencies slowed down (entrainment phase) and transit into anti-phasic synchronization of the two cells’ oscillations with frequencies of 104±28 s and 117±19 s, respectively. Single-cell oscillations, transient entrainment, and anti-phasic oscillations were reproduced by a mathematical model where nearby hyphae can absorb and secrete a limited molecular signaling component into a shared extracellular space. We show that intracellular Ca 2+ concentrations oscillate in two approaching hyphae, and depletion of Ca 2+ from the medium affected vesicle-driven extension of the hyphal tip, abolished the cell monologue and the anti-phasic synchronization of two hyphae. Our results suggest that single hyphae engage in a ‘monologue’ that may be used for exploration of the environment and can dynamically shift their extracellular signaling systems into a ‘dialogue’ to initiate hyphal fusion.
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Author Response
Reviewer #3 (Public Review):
Wernet et al. show that there are intrinsic protein oscillations at the hyphal tips of A. flagrans, a nematode trapping fungus, that become coordinated when two hyphae become close. They create a mathematical model of this synchronization phenomenon, and then go on to show that calcium is critical to the functioning of these oscillations and hyphal fusion. The concept of interhyphal communication through signal synchronization is fascinating, and the visual matching of the output of the model to the data is compelling. However, given that the authors already showed synchronized oscillations in the SofT protein in A. flagrans in Hammadeh et al. 2022 (Figure 4), this diminishes the novelty of the findings in this study. Additionally, as it also has been established that calcium drives other …
Author Response
Reviewer #3 (Public Review):
Wernet et al. show that there are intrinsic protein oscillations at the hyphal tips of A. flagrans, a nematode trapping fungus, that become coordinated when two hyphae become close. They create a mathematical model of this synchronization phenomenon, and then go on to show that calcium is critical to the functioning of these oscillations and hyphal fusion. The concept of interhyphal communication through signal synchronization is fascinating, and the visual matching of the output of the model to the data is compelling. However, given that the authors already showed synchronized oscillations in the SofT protein in A. flagrans in Hammadeh et al. 2022 (Figure 4), this diminishes the novelty of the findings in this study. Additionally, as it also has been established that calcium drives other oscillatory communications, the characterization of calcium dependence is not especially novel or bringing new insights into the problem especially since it is unclear if the chelation is having effects due to loss of intracellular supplies and/or because it is the key signal in the dialogue. Right now the mathematical model feels a bit vague with discussion of hypothetical molecules, so the paper would be greatly strengthened if any key regulatory molecules that promote desychronization could be identified or there were some manipulations of the core known proteins that examined consequences of altering the oscillations. As it is, the reader is left intrigued but there are few concrete conceptual advancements.
We thank Reviewer #3 for the thoughtful comments on our manuscript! We would like to emphasize that the main finding of this paper is the discovery of a monologue of individual hyphae before fusion and the transition into a dialogue. This had not been shown in any fungus, and it explains nicely the onset of the communication. During the revision process, we performed co-localization of SofT-GFP and MakB-mCherry in the same hyphae and observed that both proteins were oscillating in the same phase without other hyphae in vicinity, which is the opposite of the so far observed anti-phasic oscillations observed during the cell dialogue. Additionally, we observed that decoupling of the oscillations into the anti-phasic cell dialogue occurred during the transitory phase. We included our results in (L167) and updated the figures to create a new figure 3 and supplementary figure Fig. SS.
We agree that it would be great to isolate the signaling molecule. However, this has been tried by several groups, so far without success. Therefore, we think that this one main finding is exactly the scope of short reports for eLife.
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eLife assessment
This valuable study combines live cell imaging and mathematical modeling to show how an emerging model fungus engages in an oscillatory chemical dialogue to prepare for cell-cell fusion. Cell data and modeling are in compelling agreement but leave many open questions as to the nature of coordination between cells and the significance of oscillations, rendering the strength of evidence in support of the authors' inferences incomplete.
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Reviewer #1 (Public Review):
Hyphal fusion is a common process in filamentous fungi that requires a tightly regulated, oscillatory cell-to-cell dialogue between the two fusion partners. While several signaling components functioning in this process have previously been identified, the actual signal(s) exchanged during the molecular dialogue between two genetically identical cells have remained a mistery. In this study, the authors show that even when growing in the absence of a fusion partner, hyphae of a nematode pathogenic fungus already undergo signal oscillations that are in phase with their growth oscillations. After detecting the presence of a fusion partner, a slowdown of the oscillation frequencies occurs (entrainment), followed by a transition to an anti-phasic synchronization of the oscillations between the two partners. Based …
Reviewer #1 (Public Review):
Hyphal fusion is a common process in filamentous fungi that requires a tightly regulated, oscillatory cell-to-cell dialogue between the two fusion partners. While several signaling components functioning in this process have previously been identified, the actual signal(s) exchanged during the molecular dialogue between two genetically identical cells have remained a mistery. In this study, the authors show that even when growing in the absence of a fusion partner, hyphae of a nematode pathogenic fungus already undergo signal oscillations that are in phase with their growth oscillations. After detecting the presence of a fusion partner, a slowdown of the oscillation frequencies occurs (entrainment), followed by a transition to an anti-phasic synchronization of the oscillations between the two partners. Based on a mathematical model the authors postulate a mechanism involving the oscillatory secretion/uptake of a signaling compound from a shared extracellular space. To experimentally validate the model, they visualize anti-phasic oscillations of intracellular Ca2+ concentrations in two approaching hyphae and find that they are anti-phasic with the recruitment of chitin synthase B. Moreover, addition of a calcium-chelating agent to the medium abolishes molecular oscillations and anti-phasic synchronization in the two hyphae. Based on these results, the authors conclude that extracellular Ca2+ is essential for the signaling mechanism during the cell-to-cell dialogue.
This is a very solid and well-performed microscopical study that provides new insights into the signaling mechanisms during hyphal fusion. Novel findings include: 1) the occurrence of signal oscillations at the tip of individual growing hyphae (monologue) that are in phase with the growth oscillations; 2) the presence of an entrainment phase involving a slowdown of the oscillation frequency upon detection of a potential fusion partner (entrainment) followed by a transition to an anti-phasic synchronization; 3) the detection of anti-phasic intracellular calcium oscillations during the molcular dialogue; 4) the establishment of a model predicting the secretion/uptake of a signaling compound (possibly calcium).
In general, the results are clearly presented and most of the conclusions are well justified by the data. I had some problems in interpreting the model based on the accompanying text, likely because of a confusion between the two different concepts of signaling component and signaling compound. Furthermore, the fluctuations of the fluorescent calcium probe R-GECO in Fig. 3d are difficult to detect for the untrained eye. Finally, the conclusion that intracellular Ca2+ oscillations are caused by uptake of extracellular Ca2+ is not fully supported by the data. These points can all be addressed by minor changes in the text and Figures.
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Reviewer #2 (Public Review):
Using live cell imaging, this article describes the oscillation of a tip localized protein, SofT, to hyphal tips during growth of a nematode-trapping fungus, independently of the oscillation of this protein during chemotropic interactions prior to cell fusion. The authors observe oscillation of SofT, which becomes entrained as opposing oscillations at hyphal tips during chemotropic interactions, a process that requires calcium signaling. The authors build on a previously developed mathematical model describing oscillation of proteins to fusion tips during chemotropic interactions with a transition period from single hyphal tip oscillation to coordinated oscillation during chemotropic interactions.
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Reviewer #3 (Public Review):
Wernet et al. show that there are intrinsic protein oscillations at the hyphal tips of A. flagrans, a nematode trapping fungus, that become coordinated when two hyphae become close. They create a mathematical model of this synchronization phenomenon, and then go on to show that calcium is critical to the functioning of these oscillations and hyphal fusion. The concept of inter-hyphal communication through signal synchronization is fascinating, and the visual matching of the output of the model to the data is compelling. However, given that the authors already showed synchronized oscillations in the SofT protein in A. flagrans in Hammadeh et al. 2022 (Figure 4), this diminishes the novelty of the findings in this study. Additionally, as it also has been established that calcium drives other oscillatory …
Reviewer #3 (Public Review):
Wernet et al. show that there are intrinsic protein oscillations at the hyphal tips of A. flagrans, a nematode trapping fungus, that become coordinated when two hyphae become close. They create a mathematical model of this synchronization phenomenon, and then go on to show that calcium is critical to the functioning of these oscillations and hyphal fusion. The concept of inter-hyphal communication through signal synchronization is fascinating, and the visual matching of the output of the model to the data is compelling. However, given that the authors already showed synchronized oscillations in the SofT protein in A. flagrans in Hammadeh et al. 2022 (Figure 4), this diminishes the novelty of the findings in this study. Additionally, as it also has been established that calcium drives other oscillatory communications, the characterization of calcium dependence is not especially novel or bringing new insights into the problem especially since it is unclear if the chelation is having effects due to loss of intracellular supplies and/or because it is the key signal in the dialogue. Right now the mathematical model feels a bit vague with discussion of hypothetical molecules, so the paper would be greatly strengthened if any key regulatory molecules that promote desychronization could be identified or there were some manipulations of the core known proteins that examined consequences of altering the oscillations. As it is, the reader is left intrigued but there are few concrete conceptual advancements.
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