Epidermal Cell Surface Structure and Chitin-Protein Co-assembly Determine Fiber Architecture in the Locust Cuticle

Abstract

The geometrical similarity of helicoidal fiber arrangement in many biological fibrous extracellular matrices, such as bone, plant cell wall, or arthropod cuticle, to that of cholesteric liquid mesophases has led to the hypothesis that they may form passively through a mesophase precursor rather than by direct cellular control. In search of direct evidence to support or refute this hypothesis, here, we studied the process of cuticle formation in the tibia of the migratory locust, Locusta migratoria, where daily growth layers arise by the deposition of fiber arrangements alternating between unidirectional and helicoidal structures. Using focused ion beam/scanning electron microscopy (FIB/SEM) volume imaging and scanning X-ray scattering, we show that the epidermal cells determine an initial fiber orientation, from which the final architecture emerges by the self-organized co-assembly of chitin and proteins. Fiber orientation in the locust cuticle is therefore determined by both active and passive processes.

Keywords: chitin; extracellular matrices; liquid crystal; microvilli; protein.

Figure 1

Cuticle deposition in L. migratoria. (A) Adult locust (AIDAsign—stock.adobe.com, reprinted with permission). Inset showing a cross section of the hind tibia and the coordinate system used throughout: (z) longitudinal, (r) radial, and (t) transverse directions. Hind tibia of L. migratoria contains proximal (PRO) and distal (DIS) parts. Area marked in magenta, upper region of distal part, was sampled and analyzed in this study. (B) Confocal light scanning microscopy (CLSM) image of tibia cross section stained with Direct Yellow 96 stain showing the daily growth layers of chitin fibers in unidirectional and helicoidal fiber arrangements, leading to, respectively, alternating nonlamellate and lamellate layers. (C) CLSM tibia cross section double stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and Nile red (green) showing the epidermal cell layer adjacent to the cuticle. Orange rectangle indicates similar area of the cross section imaged using FIB/SEM in (D). (D) FIB/SEM slice showing epidermal cells between the basal membrane (Bm) and the cuticle in fifth instar. The nuclei are false colored in light blue and the pore canals are indicated with magenta arrowheads. (E) FIB/SEM slice showing the assembly zone between the cuticle (right) and the surface of the epidermal cell layer (left) in fifth instar. Magenta arrowheads point to a pore canal; cell nuclei are false colored in light blue. The orange rectangle marks an area from which the micrograph shown in (F) is taken. (F) Magnification of the assembly zone in a region indicated in (E). The micrograph is obtained from a different depth in t direction than shown in (E). The microvillar structures on the apical cell surface, which end with bright contrast at the location of the presumably chitin synthesizing plaques (light blue arrowhead), can be seen, as well as the newly deposited cuticle (yellow asterisk). Cell interior is marked with blue asterisk.

Figure 2

FIB/SEM and reconstructed 3D volume of locust cuticle and apical surface of epidermal cells. (A) FIB/SEM slices of Night and (C) Day samples of cryofixed locust tibiae obtained from adult and fifth instar specimen, respectively, 2 days after ecdysis. Reconstruction of microvilli in Night (B) and Day (D) samples. Cyan arrowheads point to plaques at the tips of the microvilli. Orange rectangles in (A) and (C) indicate the regions for which 3D volume renderings are shown in (B) and (D), respectively. White dotted lines in (D) indicate merged microvilli structures.

Figure 3

Microvilli structure and fiber deposition within the assembly zone. FIB/SEM images (A, D) obtained by reslicing the 3D datasets shown in Figure 2 along (t) (top) and (z) (bottom) directions for Night (A) and Day (D) samples. The comparison shows that the microvilli have similar dimensions in (z) and (t) directions in the Night samples ((A) top and bottom), whereas they are elongated along the (z) direction in the Day samples ((D) top). The plaques are depicted in bright blue. (B, E) (zr) and (tr) plane views of resliced volumes. Only one plaque (bright blue arrowhead) is situated at the tips of each microvillus in Night samples (B), but two or three plaques are observed in the Day samples on top of microvilli merged along the (z) direction (E). (C, F) Three-dimensional volume rendering of the chitin fibers/fiber bundles (yellow) observed in the assembly zone and the apical cell surface in Night (C) and Day (F) samples. For simplicity, only half of the assembly zone thickness is shown. White dotted lines in (F) indicate merged microvilli structures.

Figure 4

Reconstruction and quantification of 3D FIB/SEM data of apical cell surface structures obtained from chemically fixed locust tibiae. (A, B) Volume rendering of the segmented apical protrusions at the surface of the epidermal cells (cyan) and the pore canals (magenta) in Night (A) and Day (B) samples of fifth instar animals 2 days after ecdysis. (C, D) “Top-view” of the rendered volume of the segmented apical surface of the epidermal cells (cyan) and the lateral cell membrane (orange) in Night (C) and Day (D) samples. Note that the respective apical protrusion organization is continuous across multiple cells. (E, G) Three-dimensional Fourier transform (FT) of the segmented volumes representing the epidermal cell surfaces in Night (E) and Day (G) samples. (F, H) Azimuthal partial integration of the FT pattern in (E) and (F), respectively, in the meridional (light gray) and equatorial (dark gray) directions, showing increased isotropy and reduced long-range-order correlation of the apical protrusions in Night vs Day samples.

Figure 5

Fiber orientation in the assembly zone. Orientation color maps of the fibers in the assembly zone (entire volume) in Night (A) and Day (B) samples from datasets shown in Figures 2 and 3. (C, D) Fiber orientation (angle) vs assembly zone depth (d, nm) histograms showing the variation in fiber orientation as a function of their position within the assembly zone along the (r) direction from the cell surface (0 nm) to the cuticle (300 nm). The color (yellow, pink, blue, orange) arrowheads represent the respective dominant orientations in (A) and (B).

Figure 6

Apical surface of the epidermal cells in the absence of microvilli. (A) FIB/SEM slice at the cell surface showing irregular structures in a Day sample of fifth instar animal 2 days after ecdysis. (B, C) Volume rendering of the 3D reconstructed FIB/SEM data represented in (A) in the (tz) plane. In (B), the cell surface is viewed from the outside of the cell. In (C), the cell surface is viewed from within the cell outward. Lateral cell membranes are depicted in orange. Individual structures depicted in (E)–(G) are marked with a white dashed line in both (B) and (C). (D) Three-dimensional FIB/SEM data resliced to show the (tr) and (zr) plane views. (E–G) Three-dimensional volume rendering of representative structures visible in (B) and (C). (E) Multivesicular body. (F) Elongated openings at the cell surface. (G) Vesicles presumably fusing with the apical cell membrane.

Figure 7

Scanning X-ray diffraction measurements of adult locust tibia cross sections 4 days after ecdysis. (A) Intensity variation of the (020) reflection allows identification of “Day” (high I₍₀₂₀₎) and “Night” (low I₍₀₂₀₎) regions. Exo marks the region of exocuticle, D marks Day regions, and N marks Night regions. (B) Integrated intensity ratio between the chitin reflection (020) and the protein reflection (q ∼ 5.5 nm^–1). The ratio is increased in the assembly zone relative to the cuticle. (C) Peak position (q) of the (020) reflection. The q(020) position is shifted in the cuticle relative to the assembly zone.