A role for fascin in preventing filopodia breakage in Drosophila tracheal cells

Abstract

Filopodia are long and thin finger-like protrusions essential for cell migration. They are formed by parallel actin bundles tightly packed by cell type and context dependent actin-bundling proteins. Our recent work analyzing the role of Fascin during tracheal development in Drosophila has shown that Singed (the Drosophila Fascin homolog) acts as a molecular link between the Branchless (FGF)/Breathless (FGFR) pathway and the actin cytoskeleton. We have reported that the lack of Singed (Sn) leads to wavy and flaccid filopodia due to the disorganization of the tracheal actin cytoskeleton. Here we describe for the first time filopodia breakage in Drosophila, and show that Fascin plays a role in this event. We propose that actin filaments in sn mutant filopodia buckle under membrane pressure due to lower bending stiffness, eventually undergoing breakage. Both Filopodia buckling and breakage would impair correct cell navigation and migration.

Keywords: FGF pathway; actin-cytoskeleton; buckling; fascin; filopodia; filopodia breakage; migration; tracheal development.

Figure 1.

For figure legend, see page 3.Figure 1 (See previous page). Filopodial breakage and re-elongation occur during tracheal tissue morphogenesis. Time-lapse images of in vivo movies of tip cells from tracheal dorsal branches (DBs) during their approach toward the dorsal midline. (A) Tip cells of a DB of a stage 15 control embryo projecting stiff and straight filopodia. We show the rare case of a filopodium breaking (green arrowheads). During the retraction phase, this long filopodium first bends almost 90° (at 02:07 min) and later breaks in 2 pieces (04:04, magnified in the inset). The freed piece eventually shrinks, the cell body-attached piece retracts and is reabsorbed (04:15-08:08). Before breakage it is possible to observe the “necking” of the filopodium (03:53, magnified in the inset) just where the filopodium will bend and break. Black arrows show the process of re-elongation. The arrows point to one filopodium that elongates (01:14 to 02:07), retracts (02:07 to 03:53) and shows a period of stasis (03:53-04:35). Subsequently, instead of being reabsorbed, the filopodium elongates again (04:35-05:50) to finally retract and reabsorb. Note the extension of the lamellipodium toward this re-elongating filopodium (04:15-04:35). Also note that this lamellipodium extension continues after the retraction of the marked filopodium, likely helped by the formation formation of more filopodia in the same direction (04:35-08:08). Images were taken every 10s624ms. (B) Tip cells of a DB of a stage 15 sn mutant embryo (sn^P1. FBal0035641) show bent filopodia and very irregular cell edges and cell shapes. We show 3 cases of filopodia breakage (red, blue and purple arrowheads). All filopodia break during the retraction phase in regions of pronounced bending. The free filopodia remnant shrinks and the attached filopodia retracts toward the cell. Images were taken every 10s627ms. In order to visualize tracheal cells and filopodia, the Src membrane protein tagged with GFP is expressed in the tracheal tissue driven by the specific tracheal driver breathless-Gal4 in both control and sn mutant embryos. Live-time imaging was performed in a TCS-SP5 Leica confocal microscope HCX PL APO lambda blue 63.0×1.40 Oil UV. Images were imported into Fiji and Photoshop software. Scale Bar 10 μm, and 2 μm in magnifications. Time is shown in min:sec.