# High-speed atomic force microscopy reveals a surface-catalyzed elongation mechanism of the fungal functional amyloid hydrophobin RolA

## TL;DR

Scientists used high-speed atomic force microscopy to discover how fungal proteins form protective rodlet films through a process called surface-catalyzed elongation.

## Contribution

The study introduces the novel concept of surface-catalyzed elongation in fungal amyloid formation.

## Key findings

- Rodlet elongation occurs at both ends and alternates between growth and pause states.
- Surface-catalyzed elongation promotes bundling and alignment of rodlets, increasing elongation rates.
- Monte Carlo simulations confirmed the role of surface-catalyzed elongation in forming oriented rodlet structures.

## Abstract

The fungal functional amyloid hydrophobin RolA self-assembles into a fibrous form called a rodlet, which functions as a protective coat, but the mechanism of rodlet formation remains largely unknown. Here, we used high-speed atomic force microscopy to observe rodlet formation at the single-fibril level in real time. We identified a phenomenon termed surface-catalyzed elongation, by which the bundling of rodlets promotes their elongation. This phenomenon increases the likelihood of rodlet alignment, resulting in the formation of a dense film similar to that observed on the actual cell surface. Our findings revealed rodlet formation kinetics driven by surface-catalyzed elongation and provide an important conceptual advance for understanding the rodlet film-formation mechanism.

Hydrophobins are functional amyloids conserved in filamentous fungi. They act as a protective coat in the fibrous form, called rodlet. Rodlets further assemble to form dense films where they are bundled and densely aligned, contributing to the hydrophobicity of the mycelium surface. The mechanism of this dense film formation is completely unknown. Here, we used high-speed atomic force microscopy to directly observe the structural dynamics of rodlet bundling and subsequent film formation by hydrophobin RolA from the industrial fungus Aspergillus oryzae at a single-fibril level, and we revealed the film-formation mechanism. Rodlet elongation occurred at both ends and was discontinuous, alternating between periods when rodlets could elongate (growth state) and could not elongate (pause state). This suggests an equilibrium of two distinct structural states at the rodlet ends. We also identified a pathway, termed “surface-catalyzed elongation,” in which elongation is promoted by lateral interactions between bundled rodlets. Surface-catalyzed elongation decreased the energy barrier of both structural switching between growth and pause states and elongation at rodlet ends, doubling the elongation rate in bundled rodlets. The rodlet surface could be considered as a catalyst for the elongation of neighboring rodlets. Surface-catalyzed elongation could contribute to rodlet bundling, whereby rodlets tend to form oriented domain structures; our Monte Carlo simulations confirmed this. The concept we propose here provides a clear explanation of the mechanism by which rodlets form a dense coat on the cell surface.