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Coiling and Maturation of a High-Performance Fibre in Hagfish Slime Gland Thread Cells

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Coiling and Maturation of a High-Performance Fibre in Hagfish Slime Gland Thread Cells

Timothy Winegard et al. Nat Commun.

Abstract

The defensive slime of hagfishes contains thousands of intermediate filament protein threads that are manufactured within specialized gland thread cells. The material properties of these threads rival those of spider dragline silks, which makes them an ideal model for biomimetic efforts to produce sustainable protein materials, yet how the thread is produced and organized within the cell is not well understood. Here we show how changes in nuclear morphology, size and position can explain the three-dimensional pattern of thread coiling in gland thread cells, and how the ultrastructure of the thread changes as very young thread cells develop into large cells with fully mature coiled threads. Our model provides an explanation for the complex process of thread assembly and organization that has fascinated and perplexed biologists for over a century, and provides valuable insights for the quest to manufacture high-performance biomimetic protein materials.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Hagfish slime gland, gland thread cells, and thread skeins
a, H&E stained cross-section of slime gland showing: gland thread cells (GTC), gland mucus cells (GMC), gland pore (p), striated muscle around gland (m), and skin (s). Insets show an immature GTC near the gland epithelium, and a mature GTC within the gland lumen. b, SEM of coiled thread from a mature GTC that has broken open, revealing the organization of staggered loops (partially highlighted in red), which form layers of conical loop arrangements that spiral around the skein (one layer highlighted in purple). c, Developmental progression of M. glutinosa GTCs revealed with TEM illustrating dramatic changes in nuclear size and shape. Immature GTCs lacking a slime thread can be identified by their prominent nuclei and nucleoli. As the slime thread increases in length and diameter, the nucleus becomes more spindle like, eventually receding to the basal end of the cell in mature cells. Scale bars are 5 μm.
Figure 2
Figure 2. 3D reconstruction of thread coiling within an immature GTC from FIB-SEM data
a, Stacks of images from FIB-SEM were assembled into a 3D volume using Mimics software. This image was created by combining axial and transverse sections of the rendered volume with an SEM image of a separate complete thread skein. b, Segmentation of a dozen continuous loops within a developing GTC revealed the precise 3D pattern of thread coiling (single loop in green). Segmentation of the nucleus (light blue), nucleolus (dark blue), and mitochondria (maroon) shows the relative position of these structures to the developing thread. The loops shown are not the most recent ones laid down on the nucleus (these were too small to follow), but they do reflect nuclear shape at the time of synthesis. Inset shows the position of the rendered structures within the cell. c, Based on the 3D structure of a single loop and its relationship to adjacent loops, our model (built in Maya 2013) reproduces the spiraling nature of the conical loops, the nesting of these conical structures, as well as the cabled appearance of the skein where the thread runs circumferentially along the skein surface.
Figure 3
Figure 3. Temporal and spatial models of thread assembly and coiling in GTCs
GTC growth and maturation is characterized by dramatic changes in nuclear size and morphology (left), which correspond with the shape of conical loop arrangements laid down during successive stages of thread production (right). Regular staggered loops are laid down in the space defined by previous loops and the apical surface of the nucleus, and their morphology changes as the nucleus becomes more spindle-shaped and retreats toward the basal end of the cell. The result of manufacturing the skein in the manner depicted is a mature, ovoid skein that can be ejected through the gland pore and unravel to its full extended length of ~150 mm without tangling.
Figure 4
Figure 4. Developmental series of thread ultrastructure
a, TEM sections of slime threads from very immature to fully mature GTCs. The thread depicted in (1) consists of a bundle of about ten, 12-nm IF. Threads increase in girth by the addition of more IF, and eventually by the addition of MT (2). In the next stage (3), 12-nm IF become packed more tightly, which creates electron lucent halos (asterisk) around the MT. Further IF compaction is accompanied by the appearance of a fluffy rind (arrowheads) on the thread surface (4), which likely corresponds to the direct addition of IF subunits or proteins to the thread rather than the bundling of mature IF. In the final stage, IF proteins compact further, MT are lost, the spaces they occupied are filled in, and the fluffy rind disappears. In fully mature GTCs, the thread takes up the vast majority of the cell volume and adjacent threads are packed so tightly that they conform to each other (5). Scale bars are all 200 nm. b, Models of thread development corresponding to each of the stages depicted in (a). The second panel illustrates the 12-nm diameter filament (arrows) that wraps around the thread at this stage in development c, TEM of thread longitudinal section depicting MT within a developing thread. d, Cross-section of a thread from stage (4) showing a MT penetrating from the side (3). e, Young thread from stage (2) showing the wrapping filament, which is evident as regularly spaced circular sections and corresponding lines that cross the thread at an average angle of about 86°. Scale bars in c, d, and e are 500 nm.

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