In Vivo Imaging and Characterization of Actin Microridges

Abstract

Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo.

Microridges in Cyprinus carpio scale epidermis

“…The two "classical fingerprint" patterns are overall similar to microridge patterns described in many studies (e.g., Hawkes, 1974;Anderson 1976, Fishelson, 1984. A recent study of microridge dynamics in zebrafish embryos (Lam et al, 2015) showed that prior to and during cytokinesis, the microridges disassemble into actin-based puncta or small ridges which later reform into microridges after completion of cell division. The second classical fingerprint pattern (classical II) is reminiscent of the first but consists of more tightly packed, virtually parallel ridges but with no obvious cross-bridging structures (Figure 2b), and was infrequently found.…”

“…Phosphorylation of tyrosine is a hallmark of signal transduction via protein tyrosine kinases (PTKs) and phosphatases (PTPs). EGFP-VASP localized to the microridges of embryonic zebrafish epidermis along with tyrosine-phosphorylated proteins (Lam et al, 2015), the latter which were present at constitutively high enough levels to be detected without PTP inhibition. The accumulation of phosphotyrosine antibody after PTP inhibition indicates PTKs and PTPs are active components of microridges under native conditions.…”

“…Alpha-actinin can bundle actin filaments or facilitate actin meshwork formation (Meyer & Aebi, 1990;Sjöblom, Salmazo, & Djinovic-Carugo, 2008), and its presence in microridges suggests it has a role in maintaining or assembling the microvillous cores as well as actin meshworks in the ridges. Cortactin was localized in a more continuous or smooth staining pattern along the microridges of embryonic zebrafish superficial epidermis in a recent study (Lam, Mangos, Green, Reiser, & Huttenlocher, 2015). Cortactin functions in assembling F-actin meshworks but has also been ascribed actin-bundling activity and can be localized to focal contacts where stress fibre filament bundles terminate (Cowieson et al, 2008;Wu & Parsons, 1993;Yamada, Takeda, Michiue, Abe, & Takei, 2016).…”

Comparison of microridges in juvenile and adult sunfish, Lepomis gibbosus

“…However some species had only microvillous structures on the body surface in earlier stages, indicating a progression to the microridge structures of later embryos. Previous studies have indicated that microvillous structures are assembled into the mature ridges (Gorelik et al, ; Lam et al, ). The former study in particular showed microridge elongation occurred through the lateral association of newly formed actin‐based microvilli with the microvillous cores of pre‐existing ridges.…”

“…Differences in the patterns of microridges of juvenile and adult sea bass (Dicentrachus labrax) were described by El–Bakry (), where a more complex, twisting arrangement of ridges was found in the adult. Lam et al () noted a progression from simple to more complex microridge patterns in developing zebrafish embryo epidermis, suggesting there is a relationship between developmental stage and extent of ridge pattern complexity. Two other studies dedicated to microridges during early teleost development are that of Foscarini () and Panhuis, Fris, Tuhela, and Kwan (), both of which carried out extensive SEM surveys of epithelial surface structures and described a transition from microvillous surface projections to microridge formations.…”