Arf-GEF localization and function at myosin-rich adherens junctions via coiled-coil heterodimerization with an adaptor protein

Abstract

Epithelial morphogenesis often depends on regulation of actomyosin networks at adherens junctions. The Arf-GEF Steppke has been implicated in a negative feedback loop that locally down-regulates junctional actomyosin. We find that coiled-coil heterodimerization with the adaptor protein Stepping stone recruits Steppke to myosin-rich adherens junctions to facilitate tissue stretching during Drosophila dorsal closure.

The Arf-GEF Steppke promotes F-actin accumulation, cell protrusions and tissue sealing during Drosophila dorsal closure

“…F-tractin-tdTomato decorated the actin bundles more strongly than the lamellar regions, whereas GFP-Step localized to the lamellar regions without enrichment at bundles (seen in 25 embryos with ectopic protrusions). The overexpressed GFP-Step also localized to cell-cell contacts, as reported previously [27,47].…”

“…Thus, we examined the spatial relationship between Step and Ena along the LE. To image Step at endogenous levels, we used a GFP-tagged allele of step expressed from the endogenous step gene locus [ 47 ]. Immunostaining of Ena in the Step-GFP embryos revealed close associations of the proteins at both LE cell-cell junctions ( Fig 4A–4A’ , boxes 1 and 2, arrows) and tri-cellular junctions of rear cells ( Fig 4A , arrowheads).…”

“…An N-terminal coiled-coil (CC) domain is needed for interaction with the adaptor protein Stepping stone and for both cortical association in the early embryo and localization to myosin-rich AJs at DC [ 42 , 47 ], and the C-terminus contains a pleckstrin homology (PH) domain that binds the cell cortex through PIP3 [ 43 , 50 , 51 ] and, via separate residues, GTP-bound Arf small G proteins [ 43 , 52 ]. A construct lacking the CC domain (GFP-Step ΔCC ) was deficient in cell-cell junction localization compared with wild-type GFP-Step ( Fig 6A ; [ 47 ]), but remarkably, it induced ectopic protrusions ( Fig 6A , arrows; penetrance quantified in Fig 6C ). These protrusions lacked actin bundles, with both F-tractin-tdTomato and GFP-Step ΔCC labelling only their lamellar structure (seen for 10 embryos with ectopic protrusions).…”

“…The construct also enhanced the lamellar structure of LE protrusions ( Fig 6B , outlined; penetrance quantified in Fig 6D ). A construct lacking the PH domain (GFP-Step ΔPH ) was effectively recruited to cell-cell junctions ( Fig 6A ; [ 47 ]), but failed to modify LE protrusions ( Fig 6B and 6D ) or to induce ectopic protrusions ( Fig 6A and 6C ). Taken together, these data demonstrate that GFP-Step can induce protrusions, and it requires both its GEF activity and its PH domain to do so.…”

Planar Cell Polarity and E-Cadherin in Tissue-Scale Shape Changes in Drosophila Embryos

“…Adherens junctions and the binding proteins could be the potential candidates. For example, Arf-GEF Steppke is recruited to the myosin-rich adherens junction via coiled-coil heterodimerization with an adaptor protein, where the complex downregulates junctional tension and facilitates tissue stretching ( West et al, 2017 ; Zheng et al, 2019 ). It is worth expanding the research focus from the amnioserosa and actin cable to the surrounding epidermis.…”