Apical projections are integral functional units of epithelial cells. Microvilli and stereocilia are cylindrical apical projections that are formed of bundled actin.
Microridges on the other hand, extend laterally, forming labyrinthine patterns on surfaces of various kinds of squamous epithelial cells. So far, the structural organization and functions of microridges have remained elusive.
Our analyses have unraveled the F-actin organization supporting the most abundant and evolutionarily conserved apical projection, which functions in glycan organization. Epithelial tissues cover the outer surface of metazoans as well as line the lumen and outer surfaces of organs.
Over the course of evolution, epithelia have undergone diversification and specialization allowing them to adapt to various conditions and perform not only the barrier function but also additional functions such as absorption, secretion and mechanosensation 1 , 2. These functions require special adaptions of their apical surfaces 3 , 4 , which include diverse apical membrane protrusions. Additionally, the apical surface is kept hydrated and protected from pathogens and physical injury by the overlying glycan layer 3 , 5 , 6.
Thus, the apical zone of epithelial tissues is comprised of three components, namely, the apical plasma membrane with its projections, the cytoskeleton supporting these projections and the outer glycan layer 3. In vertebrates, epithelial cells exhibit membrane protrusions of various shapes and sizes. Columnar epithelia possess actin-based cylindrical microvilli or their derivatives such as stereocilia 3 , 7 , 8. On the contrary, the apical domain of several of the non-cornified squamous epithelia display long laterally arranged protrusions called microplicae or microridges 3 , 9.
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While previous studies have significantly enhanced our understanding of the actin organization in microvilli 7 , 10 , how actin is organized to build microridges remains controversial. They are shown to be present on several non-cornified epithelia in various species across vertebrate phyla, making them one of the more widespread actin protrusions 9 , 11 , 17 — Microridges are thought to have important roles in mucus retention, abrasion resistance and increasing the tensile strength of the apical domain 9 , 14 , 27 , They have also been proposed to function as a rapidly deployable store of F-actin and membrane during wound healing 9 , The nature of the protrusion is determined by the organization and dynamics of F-actin within it.
While actin within microridges of guppy epidermal cells is reported as bundled 11 , microridges on carp oral mucosal cells, cat epithelial cells, and guppy cells in culture are shown to contain a network of actin 11 , 20 , Furthermore, based on scanning ion-conductance microscopy in A6 cells, it has been proposed that microvilli can merge to form ridges 33 , suggesting that microridges form from microvilli containing F-actin bundled cores.
The zebrafish periderm offers a genetically and microscopically tractable model to study the formation, structure and function of microridges It has been recently shown that the cell polarity regulator atypical Protein Kinase C aPKC plays a role in restricting the elongation of microridges by controlling levels of Lethal giant larvae Lgl and nonmuscle Myosin II at the apical domain of peridermal cells However, the ultrastructure and function of these protrusions have not been characterized well.
Here, we have performed molecular as well as structural analysis of microridges in the developing zebrafish epidermis and probed for their functional importance in the context of the glycan layer. Electron tomography ET followed by segmentation analysis reveals that the actin is organized in the form of a network.
Lastly, our analyses also indicate that microridges are important for the organization of the glycan layer. The ultrastructural characterization of the entire apical zone including the microridge and its surroundings has not been carried out so far in the developing zebrafish periderm.
S1b , as measured from TEM. Using SEM Fig. To observe the organization of the glycan layer, we fixed samples in the presence of alcian blue and lysine, which has been shown to preserve the glycan layer better 35 — SEM of heads of animals fixed in such a way showed that the peridermal surface was covered by a thick glycan layer, which completely obscures the underlying microridges on the head epidermis Fig.
TEM analysis of such a sample revealed glycan enrichment around the microridges Fig.
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EM tomography of untreated samples and SEM on detergent extracted samples reveals the ultrastructural organization of the actin microridge, sub-protrusive zone and cortex. 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.
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. 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.
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 samples were fixed at 48 hpf. Since conventional TEM did not reveal the F-actin organization within the microridge due to the dense packing of actin Fig. Samples fixed using osmium and GA Fig. Aside from a few filaments parallel to the microridge membrane Fig.
In order to obtain a more detailed view of the actin organization, we used an algorithm see methods to segment filaments in a tomogram of microridges Fig.
This allowed reconstruction of the filaments within the microridge as shown, within a small region of the tomogram Fig. These corroborated the fact that filaments are organized in a network like fashion Fig.
Segmentation of an electron tomogram reveals the arrangement of actin as a network within the microridge. Higher eigenvalue 2D matrix outlines the actin structures within the microridges shown in b.
A cubic section of Image binarization in blue outlines the actin structures c , shown at 3 different depths. Depth is indicated by colorbar.
An example of actin filament rendering in magenta f and corresponding skeleton image g from an interconnected microridge. Branch points green and endpoints black are highlighted on the 3D skeleton images showing branch lengths in nm and angle between neighboring branches. The EM tomogram analyzed here a — c was a part of the tomogram shown in Fig. To validate the performance of the segmentation algorithm, we generated artificial images Fig.
S3 , see methods for details with similar characteristics as that of EM microridge filaments and segmented these using the parameters used in Fig. This analysis revealed that our segmentation method has a sensitivity of Our ET analysis further revealed that the sub-protrusive zone contained cortical actin and a network of thick keratin filaments with discernible branching points Fig.
Some keratin filaments —discernable by their thickness as compared to actin Fig. To confirm such a contribution from the cortex, we used detergent extraction to remove apical membranes of peridermal cells and performed SEM.
In these preparations, actin filaments in the cortex were clearly visible Fig. Interestingly, we found that actin filaments and filament bundles extended a considerable distance within the cortex and into the microridge Fig. This analysis suggests that the absence of bundled actin in microridges is unlikely to be a consequence of improper fixation.
To conclude, the apical zone of zebrafish peridermal cells consists of a glycan layer, an apical domain consisting of short projections called microridges and the cytoskeleton that supports them. Within the microridge, F-actin is organized in the form of a network, supported by filaments arising from the sub-protrusive zone or terminal web and cortex.
The interior of the microridge space is accessible to vesicles indicating that the microridge is not an isolated system.
As compared to microvilli 7 , 10 , the microridge is strikingly different in both morphology and F-actin organization. This prompted us to look at the molecular composition of microridges. Thus far, only a few actin-binding proteins are known to localize to the microridge.
Since actin-binding proteins regulate and organize actin filaments into specific arrangements such as parallel or antiparallel bundles and branched networks 30 , 40 , we analyzed the localization of additional regulators of the actin cytoskeleton to microridges. We utilized two candidate-based strategies — a antibody stainings to identify proteins endogenously present at the microridge and b localization of plasmid encoded expression of tagged proteins.
Localization of actin binding proteins at the microridge at 48 hpf. Respective merges are shown in c , f , i. Plasmids were injected at the 1 cell stage and expressed in a clonal fashion. While all other images are obtained from the head epidermis, Eplin images are taken on the flank.
S5a , which later give rise to the periderm To check if these punctae were associated with protrusions, we preformed SEM.
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While ArpC2 Fig. Since keratins contribute to the terminal web, we also investigated if a temporal link exists between the formation of the keratin cytoskeleton and microridges.
Immuno-localization of actin binding proteins at the apical domain prior to microridge elongation. In all cases, inhibition resulted in a breakdown of microridges or a decrease in phalloidin punctae relative to controls. Images from 19 and 48 hpf are acquired from the head epidermis. Although Filamin and Eplin are of exogenous origin, their localization to microridges indicates their ability to bind to F-actin underlying the microridges.
Therefore, microridges form from actin punctae, presumably having branched configuration, and not from bundle based microvilli. The developing zebrafish embryo is covered by a glycan layer as early as the epiboly stages 5 , making it an excellent model to study the role of microridges in maintaining the organization of the glycan layer. In order to visualize the glycan layer with a confocal microscope, we used a fluorescently tagged wheat germ agglutinin WGA lectin This approach revealed that the glycan layer was arranged around microridges Fig.
To assess if microridges are important for the organization of the glycan layer, we used chemical inhibitors to disrupt the actin cytoskeleton and consequently the microridge pattern. While in control embryos, the WGA fluorescence followed the microridge pattern Fig. SEM analysis on CK treated samples did not reveal a major change in glycan organization, again pointing to the mild effect of CK on the glycan layer Fig. Similar to previous observations 13 , 34 , we achieved a severe perturbation of microridges using the actin monomer binding drug Latrunculin A Lat A Coue et al.
LatA treatment resulted in the decreased density and length of microridges Fig.