Hierarchically-structured metalloprotein composite coatings biofabricated from co-existing condensed liquid phases

Abstract

Complex hierarchical structure governs emergent properties in biopolymeric materials; yet, the material processing involved remains poorly understood. Here, we investigated the multi-scale structure and composition of the mussel byssus cuticle before, during and after formation to gain insight into the processing of this hard, yet extensible metal cross-linked protein composite. Our findings reveal that the granular substructure crucial to the cuticle's function as a wear-resistant coating of an extensible polymer fiber is pre-organized in condensed liquid phase secretory vesicles. These are phase-separated into DOPA-rich proto-granules enveloped in a sulfur-rich proto-matrix which fuses during secretion, forming the sub-structure of the cuticle. Metal ions are added subsequently in a site-specific way, with iron contained in the sulfur-rich matrix and vanadium coordinated by DOPA-catechol in the granule. We posit that this hierarchical structure self-organizes via phase separation of specific amphiphilic proteins within secretory vesicles, resulting in a meso-scale structuring that governs cuticle function.

Fig. 1. Overview of byssus cuticle formation and structure.

a Marine mussels (Mytilus edulis) synthesize byssal threads using an organ known as the foot. b CT image of the distal region of a mussel foot highlighting the foot groove, in which the thread forms. c Schematic of a foot transverse cross-section from a region of the foot indicated by white dashed line in c showing location of specific glands in which thread-forming proteins are stockpiled. d Trichrome stained transverse section of foot gland tissue showing the core (blue) and cuticle (red) secretory vesicles. Scale bar = 10 µm. e Trichrome stained longitudinal thread section captured during induced formation showing the core and formation of the cuticle. Clusters of cuticle secretory vesicles coalesce and are partially spread over the core surface creating the cuticle. Scale bar = 4 µm. f Trichrome stained longitudinal section of a native distal byssal thread fixed on a glass slide. Scale bar = 4 µm. g SEM image of a native distal thread surface with false coloring to differentiate the cuticle (red) and exposed core (blue). Scale bar = 1 µm. h TEM image of a thin osmium stained transverse cross-section of a native distal byssal thread with false coloring to indicate the cuticle and fibrous core. Scale bar = 500 nm. i The cuticle is known to be partially comprised of a protein called mefp-1, with an extended domain made of decapeptide repeats containing 3,4-dihydroxyphenylalanine (DOPA), which is believed to be coordinated to metal ions including vanadium and iron. Panels b, d–f are adapted from ref. under the Creative Commons License.

Fig. 2. TEM and FIB-SEM imaging of cuticle secretory vesicles in mussel foot tissue.

a TEM image of osmium stained cuticle secretory vesicle from region similar to Fig. 1d. Contrast is achieved by different osmium staining with the proto-matrix (pm) staining the heaviest, the crescent phase (cp) staining the lightest and the proto-granules (pg) staining in between. Scale bar = 200 nm. b FIB-SEM image of osmium stained region of cuticle gland similar to Fig. 1d. Contrast is inverted compared to TEM, but pg, pm and cp can clearly be differentiated. Scale bar = 1000 nm. c Using electron density contrast, secretory vesicles and their inner structure were reconstructed in 3D from an image stack consisting of 392 images. d Magnified and cropped 3D image of a cuticle secretory vesicle showing all three phases. e STEM-EDS compositional analysis of native cuticle granule and matrix regions, showing distribution of nitrogen and sulfur in the region in the STEM-HAADF image in the left panel. Scale bar = 500 nm. f Relative sulfur wt% (not calibrated) collected from a transect across four secretory vesicles in dotted box in e reveals that pg has approximately one half the amount of sulfur as pm.

Fig. 3. Cuticle formation in the mussel foot groove via coalescence of cuticle secretory vesicle contents.

a Schematic overview of transverse section of mussel foot tissue showing anatomy of byssal glands. Cuticle secretory vesicles (in red) are lined up at the edge of the cuticle gland, ready to be secreted into the groove, aided by cilia. During cuticle assembly, the secretory vesicle contents cluster and coalesce into the groove spreading over the formed core surface. b TEM image from a stained section of an induced foot in the region indicated in a, showing coalesced cuticle secretory vesicles about to be released into the groove. Notably, the proto-matrix (pm) of several vesicles merges to form a continuous matrix, while the proto-granules (pg) retain their brain-like structure. Scale bar = 500 nm. c TEM image from stained section of an induced foot in the region indicated in a, showing a formed induced thread with intact cuticle. Scale bar = 500 nm. d Magnified region from c showing details of the induced cuticle including granule and matrix. Scale bar = 200 nm.

Fig. 4. FIB-SEM 3D-reconstruction of intragranular nanostructure in native thread cuticle.

a FIB-SEM image of osmium stained region of native distal thread showing substructure of the core and cuticle, including granule and matrix in the cuticle and previously described voids in the core. Scale bar = 500 nm. b Higher magnification FIB-SEM image of a single granule. Contrast arises from heavily staining (hs) and lightly staining (ls) regions within a single granule. n.b. contrast is inverted compared to TEM, with elements of higher atomic mass appearing brighter. Scale bar = 200 nm. c 3D reconstruction of FIB-SEM image stack using the contrast of the lightly staining (ls) phase of the granules and the core voids (which are parallel to the fiber axis). d Granule cropped using clipping planes parallel to the one indicated in c revealing internal structure of the ls phase, consisting of bicontinuous flattened layers with a thickness of ~20 nm. The transparent region between layers constitutes the heavily staining (hs) phase from b. Scale bar = 80 nm. e STEM-EDS compositional analysis of native cuticle granule and matrix regions, showing distribution of nitrogen, sulfur, iron, and vanadium in the region in the STEM-HAADF image on the left. Scale bar = 100 nm.

Fig. 5. Schematic model of cuticle assembly via co-existing condensed liquid phase vesicles.

To achieve the high degree of compositional, structural and mechanical hierarchy observed in the native byssus cuticle, mussels store the protein precursors as a LLPS of two co-existing phases. The main protein component mfp-1 and the Cys-rich proteins (mfp-16–19) are immiscible due to the amphiphilic nature of mfp-1, leading to phase separation into a bicontinuous structure that characterizes the proto-granule. During secretion and cuticle assembly, the proto-matrix of nearby secretory vesicles fuses forming the continuous matrix of the cuticle, possibly cross-linked via cysteine residues. The intricate nanostructure of the proto-granule is maintained in the newly formed cuticle and contains a much higher local concentration of DOPA than the surrounding matrix. Thus, when metals are added to the thread in a secondary curing, the metal ions that have the highest stability complex with DOPA (i.e., vanadium) diffuse into the granule where they become concentrated relative to iron. This results in different viscoelastic behavior of the granule and matrix, which is likely an adaptive function to life on the highly dynamic rocky seashore. Cysteine may play a secondary, but equally important role as a reducing agent that counteracts the spontaneous oxidation of DOPA to DOPA-quinone under basic conditions, which enables strong metal coordination cross-link formation.