Mycelium is the fabric of fungal populations: fungi produce thread-like roots called hyphae, which branch and fuse with one another to form a vast, interconnected network—the mycelium. It allows fungi to grow rapidly, transport nutrients, and even share information about the local environment over long distances. The network is also vulnerable; a wound could lead to catastrophic bleeding of protoplasm that can lead to death. While some fungal species separate their filaments into compartments with septal walls that can limit leakage, other fungi do not make any walls, and mycologists haven’t known how they respond to an injury.
Now, a team at the National University of Singapore has discovered their secret: large, mechanosensitive proteins called gellins that have not been described before. When a hyphal filament is injured, the pressurized liquid protoplasm inside the hyphae gushes out. Immediately, gellins inside the hyphae respond to the shear stress of the protoplasm flow by quickly crosslinking, unfolding, and sticking to each other and to cell membranes. The crosslinked gellins form a gel plug at the site of injury almost instantaneously, preventing the fungus from losing all of its protoplasm, evolutionary cell biologist Greg Jedd of the National University of Singapore and colleagues report in Current Biology on November 12.
“What’s super neat about the gellins is that you’re using a physical cue to drive phase separation,” says cell biologist Amy Gladfelter at the University of North Carolina, Chapel Hill who was not involved in the study. That is, shear stress of the rapid flow of protoplasm at the injured site triggers the gellins to unfold and crosslink, creating two distinct phases in the protoplasm—liquid and gel. “There aren’t too many examples of that.”
The project started when Jedd observed that injuries in Phycomyces blakesleeanus and Mucor circinelloides, two fungal species that do not make septal walls, triggered a rapid gelation response that stopped the protoplasm from leaking out. Suspecting that this response was mediated by a large polymer, the researchers ground up the cells and used protein electrophoresis to identify the large proteins inside the filaments. They called these proteins gellins—there are two kinds, gellin A and gellin B—and identified the genes that encode them. Finally, they used CRISPR to eliminate both types of gellins from Mucor circinelloides. When fungi lacking gellins were injured, they bled out into a protoplasmic puddle and died, confirming that gellins play a key role in sealing up breaks.
“This was a creative way to discover the proteins that might be responsible for this phenomenon,” says mycologist Jason Stajich at University of California, Riverside, who was not involved in the research. He praised the team’s ability to make observations of the fungal gelation response and “work backwards to identify the actual molecular basis of that.”
To find out how the gellins actually work, the researchers turned to structural biologist Daiwen Yang also of the National University of Singapore. Yang purified the gellins and used nuclear magnetic resonance to discover that gellins have 10 “beta-barrel” domains. The first beta barrel domain, which the researchers focused on, had an unusual, very hydrophobic loop that they suspected was involved in binding to cell membranes. Mechanobiologist Jie Yan then used magnetic tweezers to grab a gellin by one end and gently pull on the other end, revealing that the protein unfolded at forces consistent with what gellins would experience inside a bursting hypha.
“It’s an elegant set of experiments,” Stajich says.
The process to stop fungal bleeding has some parallels to mammalian blood clotting. For example, the von Willebrand factor (VWF) that helps blood clot is also mechanically sensitive and responds to the force of flow by changing shape and binding with other VWF proteins.
All multicellular systems have evolved transport networks, which are required to overcome the limit of diffusion as an organism gets large, Jedd explains. “Each has found ways of dealing with injury, but there is some degree of convergence.”
Spider silk, too, is made of large proteins that are extruded through the spider’s silk glands, and the shear stress from that flow induces the formation of intermolecular bonds, thus producing a thread from liquid protein. Understanding the design principles for these mechanosensitive proteins could have all kinds of bioengineering applications, such as self-healing soft robots, the researchers suggest.
T.A. Nguyen et al., “Fungal wound healing through instantaneous protoplasmic gelation,” Current Biology, doi: 10.1016/j.cub.2020.10.016, 2020.