Coordinated crosstalk between microtubules and actin by a spectraplakin regulates lumen formation and branching

Abstract

Subcellular lumen formation by single-cells involves complex cytoskeletal remodelling. We have previously shown that centrosomes are key players in the initiation of subcellular lumen formation in Drosophila melanogaster, but not much is known on the what leads to the growth of these subcellular luminal branches or makes them progress through a particular trajectory within the cytoplasm. Here, we have identified that the spectraplakin Short-stop (Shot) promotes the crosstalk between MTs and actin, which leads to the extension and guidance of the subcellular lumen within the Terminal Cell (TC) cytoplasm. Shot is enriched in cells undergoing the initial steps of subcellular branching as a direct response to FGF signalling. An excess of Shot induces ectopic acentrosomal luminal branching points in the embryonic and larval tracheal TC leading to cells with extra subcellular lumina. These data provide the first evidence for a role for spectraplakins in single-cell lumen formation and branching.

An endosome-associated actin network involved in directed apical plasma membrane growth

“…The results so far suggested an instructive role of late endosomes in tube guidance, but it was not clear how this relates mechanistically to known regulators of this process, in particular the important function of the actin cytoskeleton (Oshima et al, 2006; JayaNandanan et al, 2014; Schottenfeld-Roames et al, 2014; Gervais and Casanova, 2010; Levi et al, 2006; Okenve-Ramos and Llimargas, 2014; Ricolo and Araujo, 2020). When we stained embryonic terminal cells expressing YRab7 with markers for actin (phalloidin-ATTO647) and the subcellular lumen (CBP-Alexa546), we found actin enriched around YRab7 vesicles in the area ahead of the tube (Fig.…”

“…An alternative model is that microtubule organization and therefore transport of late endosomes is an indirect consequence of cell growth. Gervais and Casanova (2010) have proposed that the actin cytoskeleton, downstream of FGF signalling, allows polarized microtubule organization, and the distribution of Shot, an actin-microtubule crosslinker, supports this model (Ricolo and Araujo, 2020). Several studies have shown that multiple cytoskeletal regulators lie downstream of FGF signalling activation, namely, Ena (VASP family founding member), Fascin/Singed, and Shot itself (Gervais and Casanova, 2010; Okenve-Ramos and Llimargas, 2014; Ricolo and Araujo, 2020).…”

“…Gervais and Casanova (2010) have proposed that the actin cytoskeleton, downstream of FGF signalling, allows polarized microtubule organization, and the distribution of Shot, an actin-microtubule crosslinker, supports this model (Ricolo and Araujo, 2020). Several studies have shown that multiple cytoskeletal regulators lie downstream of FGF signalling activation, namely, Ena (VASP family founding member), Fascin/Singed, and Shot itself (Gervais and Casanova, 2010; Okenve-Ramos and Llimargas, 2014; Ricolo and Araujo, 2020). The actin cytoskeleton is widely used to control guidance of microtubules (Dogterom and Koenderink, 2019), so it is reasonable to assume that FGF signalling could control microtubule orientation by locally organizing F-actin at the leading edge, which in turn serves as a cue to organize microtubules via Shot.…”

“…Absence of Shortstop (Shot), a fly homolog of the F-actin-microtubule crosslinker Spectraplakin, also results in shorter subcellular tubes. Shot is thought to anchor the microtubules that surround the subcellular tube to an actin core that lies ahead of the subcellular tube, but whose function has remained elusive (Oshima et al, 2006; Ricolo and Araujo, 2020).…”

“…Furthermore, loss of either IKKe or Singed/Fascin leads to general actin disorganization (Oshima et al, 2006; Okenve-Ramos and Llimargas, 2014). While myosin is dispensable for tracheal morphogenesis (Ochoa-Espinosa et al, 2017), actin crosslinking alone is sufficient to generate tension in other contexts(Bun et al, 2018), so it is likely that the action of these molecules is able to 3 connect the actin cortices with the endosomal actin and drive tube guidance in response to signalling.Another key component involved in tube formation are microtubules, which initiate tube membrane invagination and allow further tube branching(Ricolo et al, 2016;Ricolo and Araujo, 2020). Microtubules are stiffer and less dynamic than actin, so it is likely that their role in tube formation is the stabilization of the membrane invagination as it continues to grow.…”