Mussels Fabricate Porous Glues via Multiphase Liquid–Liquid Phase Separation of Multiprotein Condensates

Abstract

Mussels (Mytilus edulis) adhere to hard surfaces in intertidal marine habitats with a porous underwater glue called the byssus plaque. The plaque is an established role model for bioinspired underwater glues and comprises at least six proteins, most of which are highly cationic and enriched in the post-translationally modified amino acid 3,4-dihydroxyphenylalanine (DOPA). While much is known about the chemistry of plaque adhesion, less is understood about the natural plaque formation process. Here, we investigated plaque structure and formation using 3D electron microscopic imaging, revealing that micro- and nanopores form spontaneously during secretion of protein-filled secretory vesicles. To better understand this process, we developed a method to purify intact secretory vesicles for in vitro assembly studies. We discovered that each vesicle contains a sulfate-associated fluid condensate consisting of ∼9 histidine- and/or DOPA-rich proteins, which are presumably the required ingredients for building a plaque. Rupturing vesicles under specific buffering conditions relevant for natural assembly led to controlled multiphase liquid–liquid phase separation (LLPS) of different proteins, resulting in formation of a continuous phase with coexisting droplets. Rapid coarsening of the droplet phase was arrested through pH-dependent cross-linking of the continuous phase, producing native-like solid porous “microplaques” with droplet proteins remaining as fluid condensates within the pores. Results indicate that histidine deprotonation and sulfates figure prominently in condensate cross-linking. Distilled concepts suggest that combining phase separation with tunable cross-linking kinetics could be effective for microfabricating hierarchically porous materials via self-assembly.

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“…36,42,199 There is considerable evidence in the literature that specific purified byssus proteins, including mfp-1, mfp-3, and mfp-5 can undergo LLPS in vitro to form coacervate phases, 242,250−255 and experiments on induced plaque and isolated plaque secretory vesicles suggest that the plaque proteins are indeed stored as a protein condensate within the secretory vesicles. 36,42,202,237 The plaque gland surrounds the distal depression near the tip of the foot and contains an extensive network of microfluidic-like secretory channels known as the longitudinal ducts (LDs), which have diameters ranging from 10−100 μm (Figure 13). 42,199 Surrounding the LDs are the secretory cells of the plaque gland, which are fully packed with spherical secretory vesicles that contain a dense fluid mixture of at least 10 different plaque proteins.…”

“…DOPA, cysteine, and histidine chemistry is proposed to play a crucial roles in achieving both strong cohesion and adhesion in the plaque. FIB-SEM reproduced with permission from ref . Copyright 2022 American Chemical Society.…”

“…Confirmed proteins include DOPA-rich proteins mfp-2, mfp-3, and mfp-5, cysteine-rich protein mfp-6, and histidine-containing protein mfp-4 (Figure ). Additionally, recent studies provide compelling evidence for the presence of mfp-8 (DOPA-rich) and mfp-11 (histidine-rich) in the plaque. ,, Structural and chemical analysis has demonstrated with varying degrees of certainty that mfp-3 and mfp-5 are present at the adhesive interface, , mfp-3 and mfp-6 are present in the pores, mfp-2 is present in large amounts in the struts of the porous structure, and it has been hypothesized but not shown that mfp-4 might be present at the interface between the core fibers and the plaque porous structure (Figure ). Along these lines, each protein has been hypothesized to play a role commensurate with its biochemical composition and location in the plaque.…”

“…Purified plaque vesicles will fuse and undergo liquid–liquid phase separation (LLPS) in vitro, forming a porous microplaque material. Adapted with permission from refs and . Copyright 2021 American Association for the Advancement of Science and 2022 American Chemical Society, respectively.…”

“…The plaque is flattened disc-like structure into which the thread core is embedded analogous to the roots of a tree into the ground (Figure ). The plaque possesses a hierarchically porous morphology with two dominant pore sizes of approximately 100 nm and 1.5 μm, respectively. , The micro- and nanoporous structure has been suggested to enhance the mechanical damping behavior of the adhesive (and thus the whole byssus fiber) , but more recently has also been suggested to function as a reservoir of phase-separated cysteine-rich reducing proteins that hinder oxidation of the DOPA moieties critical for achieving surface adhesion …”