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Updated: Sep 29, 2026
Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi
Published on: November 28, 2019
Live-cell imaging of vegetative hyphal fusion in Neurospora crassa
Patrick C Hickey1, David Jacobson, Nick D Read
1Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, Rutherford Building, Edinburgh, UK.
Abstract:
The process of hyphal fusion (anastomosis) in growing colonies of Neurospora crassa, stained with the membrane-selective dyes FM1-43 and FM4-64, was visualized by confocal microscopy. Time-lapse, live-cell imaging illustrated the dynamics of hyphal growth and anastomosis during its pre-contact, contact and post-contact, and post-fusion stages. Fusion-competent hyphae were morphologically distinct and exhibited remote sensing, resulting in branch initiation and/or re-direction of growth to facilitate contact between participating hyphae. A stained Spitzenkörper was often observed where fusion-competent hyphae met. It is suggested that this structure contains secretory vesicles responsible for the delivery of cell adhesion molecules at the point of contact, cell wall synthesizing enzymes for the swelling growth of fused hyphal tips, and digestive enzymes required for fusion pore formation. Dramatic changes in cytoplasmic flow frequently occurred between the participating hyphae following fusion. After anastomosis has taken place, septa commonly formed close to the fusion site. The live-cell imaging reported here has clearly shown the complexity of the hyphal homing and fusion process. The control and consequences of repeated anastomoses within a mycelium must be as complex as the process itself.
Insights
Neurospora crassa hyphae exhibit complex behaviors, including "remote sensing" and directed growth, to achieve fusion (anastomosis). This process involves specialized structures and dynamic cytoplasmic changes for successful cell joining.
Area of Science:
- Mycology
- Cell Biology
- Microbial Genetics
Background:
- Hyphal fusion, or anastomosis, is a critical process in filamentous fungi like Neurospora crassa.
- Understanding the dynamics of hyphal fusion is essential for comprehending fungal colony development and interactions.
Purpose of the Study:
- To visualize and elucidate the dynamic process of hyphal fusion in Neurospora crassa using live-cell imaging.
- To identify key morphological and cellular events preceding, during, and following anastomosis.
Main Methods:
- Confocal microscopy was employed to observe Neurospora crassa colonies stained with membrane-selective dyes FM1-43 and FM4-64.
- Time-lapse, live-cell imaging captured the dynamic stages of hyphal growth and fusion.
Main Results:
- Fusion-competent hyphae displayed distinct morphology and "remote sensing" behavior, directing growth towards each other.
- A specialized apical structure, the Spitzenkörper, was observed at hyphal contact points, potentially involved in secreting adhesion and cell wall synthesis molecules.
- Significant changes in cytoplasmic flow and septum formation near the fusion site were observed post-anastomosis.
Conclusions:
- Live-cell imaging revealed the intricate nature of hyphal homing and fusion in Neurospora crassa.
- The study suggests the Spitzenkörper plays a crucial role in mediating hyphal fusion through the delivery of specific molecules.
- The complexity of repeated anastomoses within a mycelium warrants further investigation.
