Fiber Formation from Liquid Crystalline Collagen Vesicles Isolated from Mussels

Abstract

Marine mussels (Mytilus edulis) fabricate byssal threads, highperformance biopolymeric fibers, which exhibit exceptional toughness and selfhealing capacity. These properties are associated with collagenous proteins in the fibrous thread core known as preCols that self-organize into a hierarchical semicrystalline structure. Threads assemble individually in a bottom-up process lasting just minutes via secretion of membrane bound vesicles filled with preCols. However, very little is understood about the details and dynamics of this assembly process. Here, we explore the hypothesis that preCols are stored within the vesicles in a liquid crystalline phase, which contributes to fiber assembly by preordering molecules. To achieve this, a protocol was developed for extracting and isolating intact preCol secretory vesicles in high yield and purity. Vesicles were characterized and were manipulated in vitro, clearly indicating the dynamic liquid crystalline nature of the proteins within. Moreover, mechanical shearing of vesicles led to formation of highly birefringent preCol fibers. These findings have relevance for efforts toward sustainable production of advanced polymeric materials, and possibly for engineering biomedical scaffolds based on collagenous proteins.

Collagen Pentablock Copolymers Form Smectic Liquid Crystals as Precursors for Mussel Byssus Fabrication

“…liquid-liquid phase separations (LLPS)) to produce a microporous underwater adhesive 12,13 , and to deploy co-existing condensed LLPS phases comprised of several functionally distinct proteins to produce a hard, yet extensible composite coating 14,15 . Of particular relevance to the current study, mussels also seem to use liquid crystalline (LC) phases to build the tough and self-healing collagenous core of the byssal threads [16][17][18] . Despite recent progress on these topics, many questions are unanswered.…”

“…Despite recent progress on these topics, many questions are unanswered. Here, we utilized advanced electron microscopic imaging to further investigate the hypothesis that mussels utilize a LC collagen precursor phase to initiate rapid assembly of the hierarchically structured self-healing core 16,18,19 .…”

“…Assembly of concentrated protein precursors has been proposed to be triggered in part by the pH transition from the presumed acidic conditions within the secretory vesicles to the basic pH of seawater 17,18 . The core gland (alternatively called white gland or collagen gland 8,30,32 ) produces and stockpiles prolate ellipsoid secretory vesicles (~2 µm long axis) that are filled with preCols in a concentrated fluid phase 16 (Fig. 1a-c).…”

“…Based on strong birefringence of the vesicles 8,31 and their characteristic layered banding pattern in TEM images 32 (Fig. 1c,d), preCols were hypothesized to be stored as a smectic LC phase 16,18,19 . The smectic phase is proposed to be a means of preorganizing the preCols in an ordered fluid state that can be rapidly mobilized and assembled into the highly organized thread hierarchical structure (Fig.…”

“…The smectic phase is proposed to be a means of preorganizing the preCols in an ordered fluid state that can be rapidly mobilized and assembled into the highly organized thread hierarchical structure (Fig. 1e, i) 8,16,18 . However, the preCol LC phase has been exceedingly challenging to characterize experimentally at the nanoscale, especially in 3D -although this information is crucial for understanding the byssus assembly process.…”