Figure 1. EPS-8 interacts with VAB-19 and colocalizes with VAB-19 at epithelial attachment structures. (A) The VAB-19 ANK repeat domain interacts with the central domain of EPS-8. The initial interacting clone corresponds to almost full length EPS-8A. Deletion derivatives of EPS-8A were screened for interaction with VAB-19ANK in yeast. A fragment containing the central domain (EPS-8A residues 245502) is sufficient to interact with VAB-19 ANK domains, although constructs containing part of the PTB domain interact more strongly. The PTB domain alone (residues 25264) does not interact with VAB-19ANK. None of the constructs cause self-activation (pBTM116 controls). (B, C) GFP expressed under the control of a 3.3 kb
eps-8 promoter (Peps-8-GFP, juEx526) is first seen in ventral and dorsal epidermal cells at comma stage (350 min) and persists in these tissues throughout larval and adult stages. Peps-8-GFP was also expressed in sublateral neurons (arrow, C). The 3.3 kb
eps-8 promoter likely drives a subset of the
eps-8 expression pattern, as
eps-8 has been shown to be expressed in intestinal and pharyngeal cells. (D) EPS-8B::GFP (juEx700) was localized to circumferential bands in muscle-adjacent epidermis in embryos (not shown) and in larvae. We detected EPS-8B::GFP at other known sites of trans-epidermal attachments: adjacent to mechanosensory neuron processes (arrow, E), and at the uterine seam cell attachment (arrow, F). EPS-8::GFP was also expressed in the uterus and in the excretory cell (not shown). (G) In pharyngeal marginal cells, Pvab-19-EPS-8B::GFP was localized to the apical (lumenal) ends of attachment structures (arrow) and not to the basal surface (arrowhead), in contrast to VAB-19::GFP (juEx433), which is found at both basal and apical ends of pharyngeal marginal cells (arrowhead and arrow, respectively, H). The subcellular localization of EPS-8A::GFP in these tissues was indistinguishable from EPS-8B::GFP (not shown). (I, J) Localization of mCherry:: EPS-8(central) (juEx1784) to embryonic attachment structures (arrow). Truncated mCherry::EPS-8 fusion proteins also displayed aggregation (bright blobs); as these aggregates are not observed with GFP-tagged full length EPS-8 they may result from overexpression of truncated proteins. (K) Co-localization of Pdpy-7-mCherry::EPS-8(central) (juEx1784) and VAB-19::GFP (juEx433) to attachment structures in larval epidermis. Scales, 10 um.