The Drosophila FHOD1-like formin Knittrig acts through Rok to promote stress fiber formation and directed macrophage migration during the cellular immune response

Abstract

A tight spatiotemporal control of actin polymerization is important for many cellular processes that shape cells into a multicellular organism. The formation of unbranched F-actin is induced by several members of the formin family. Drosophila encodes six formin genes, representing six of the seven known mammalian subclasses. Knittrig, the Drosophila homolog of mammalian FHOD1, is specifically expressed in the developing central nervous system midline glia, the trachea, the wing and in macrophages. knittrig mutants exhibit mild tracheal defects but survive until late pupal stages and mainly die as pharate adult flies. knittrig mutant macrophages are smaller and show reduced cell spreading and cell migration in in vivo wounding experiments. Rescue experiments further demonstrate a cellautonomous function of Knittrig in regulating actin dynamics and cell migration. Knittrig localizes at the rear of migrating macrophages in vivo, suggesting a cellular requirement of Knittrig in the retraction of the trailing edge. Supporting this notion, we found that Knittrig is a target of the Rho-dependent kinase Rok. Co-expression with Rok or expression of an activated form of Knittrig induces actin stress fibers in macrophages and in epithelial tissues. Thus, we propose a model in which Rok-induced phosphorylation of residues within the basic region mediates the activation of Knittrig in controlling macrophage migration.

Cooperative functions of the two F-BAR proteins Cip4 and Nostrin in regulating E-cadherin in epithelial morphogenesis

“…Cell culture, cell transfection and immunofluorescene microscopy Drosophila S2R+ cells were cultured and transfected as described previously (Lammel et al, 2014). For spinning disk confocal microscopy, cells were re-plated on chambered cover glass (Lab-Tek) that had not been treated or had been pre-treated with concavalin A (Sigma).…”

“…S2R+ cells were transfected as described previously (Lammel et al, 2014). Reverse transcriptase (RT)-PCR was performed with embryonic RNA as template to amplify the full-length Nostrin cDNA (short isoform).…”

Drosophila WASH is required for integrin-mediated cell adhesion, cell motility and lysosomal neutralization

“…5I; Movie 8). However, we found a reduced number of macrophages that reached the wound after 30 min by measuring the histogram-based macrophage migration score (HMMS) that we have previously established (Lammel et al, 2014; quantification in Fig. 5J).…”

Functional and Genetic Analysis of Spectraplakins in Drosophila

“…S2 cells are extremely easy to keep in Schneider's Drosophila Medium (Life Technologies), with passages being required every 3-4 days. With an average diameter of 10 μm, S2 cells are far smaller than fibroblasts but they present many helpful cytoskeletal readouts that can be used for studies of Shot: they contain acentrosomal microtubule arrays (Nye, Buster, & Rogers, 2014) and have actin-rich lamellipodia in the periphery but no stress fibers (although these can be artificially induced when manipulating formins; Lammel et al, 2014). Transfection of S2 cells is easy (Armknecht et al, 2005).…”