Figure 1.

EM tomography of untreated samples and SEM on detergent extracted samples reveals the ultrastructural organization of the actin microridge, sub-protrusive zone and cortex. TEM through a head peridermal cell revealed that peridermal cells comprise of a microridge/glycan zone, sub-protrusive zone and organelle zone (a). SEM of an Alcian blue and lysine fixed head periderm sample (b) reveals a thick glycan layer, which is chipped off on the left revealing the underlying microridges. Tomograms of Gluteraldehyde + OsO₄ fixed samples through the length of the microridge (c) and perpendicular to the cell-boundary microridge (i). Illustrations of the sectioning angle (c’, i’) with the microridge (in purple) and the sectioning plane (in green) for c-h and for i-k, respectively. Zoomed in areas of tomograms in c and i are shown in (d–h) and (j,k), respectively. Each image in c-h and i-k represents a 0.7 and 0.55 nm thick slice of the tomogram in the Z axis respectively. SEM of a detergent extracted sample (l). Magenta and green arrowheads indicate actin and keratin filaments, respectively. In the inset in (d), the arrowheads point to a filament with branch points. In (e) the arrowheads point to a keratin filament entering the microridge from the sub-protrusive zone whereas the white dashed line demarcates the boundary between the microridge above from the sub-protrusive zone below. Small filaments that appear to be parallel to each other (f) are shown by the arrowheads and inset in f. The arrowheads in (g) point to branch points in keratin filaments in the sub-protrusive zone. In (h) the arrowheads point to a filament that is parallel to the plasma membrane. The cytoskeleton from the top of the microridge to its base (indicated by white dashed line in ‘i’) and the sub-protrusive zone till the base of the tight junction (indicated by the black dashed line) is denser and made up largely of actin. In (j) smaller actin membrane crosslinks (white arrowhead) and longer actin membrane crosslinks (black arrowhead) are shown. The cortex between ridges (k) is made up largely of F-actin, which are thinner filaments (magenta arrowhead), distinctly different from the thicker keratin filament below (green arrowhead). Coloured arrowheads in (l) indicate filaments or filament bundles extending from the cortex into the microridge. All TEM images are taken on top of the head, while the SEM is from on top of the yolk. All samples were fixed at 48 hpf. The scale bars in a,b are 2 µm, in c,f,i,l are equal to 200 nm and those in d,e,g,h,j,k are 20 nm.