Drosophila integrin adhesion complexes are essential for hemocyte migration in vivo

Abstract

Cell migration is an important biological process which has been intensively studied in the past decades. Numerous techniques, mainly involving two-dimensional cell culture systems, have contributed to dissecting the essential mechanisms underlying this process. However, the development of three-dimensional cell culture and in vivo systems has shown some differences with what was previously believed to be well-established cell migration mechanisms, suggesting that two-dimensional cell motility would be a poor predictor of in vivo behaviour. Drosophila is a widely recognized model organism to study developmental and homeostatic processes and has been widely used to investigate cell migration. Here, we focus on the migration of small groups of pupal hemocytes that accumulate during larval stages in dorsal patches. We show that integrins, and other known nascent adhesion-related proteins such as Rhea and Fermitin 1, are crucial for this process and that their depletion does not affect polarization in response to environmental cues. We also present evidence for the importance of adhesion maturation-related proteins in hemocyte migration, namely Zyxin. Zyxin depletion in hemocytes leads to a significant increase of cell speed without affecting their response to a chemotactic cue. This is the first report of a systematic analysis using Drosophila melanogaster hemocytes to study adhesion-related proteins and their function in cell migration in vivo. Our data point to mechanisms of cell migration similar to those described in three-dimensional in vitro systems and other in vivo model organisms.

Keywords: Drosophila; Hemocyte; Integrin; Migration.

Fig. 1.. Myospheroid is required for proper hemocyte migration.

(A) Outline of the MARCM protocol in hemocytes. Cross between DEMON males and FRT19a control or mys¹ mutant virgin female flies for MARCM analysis. (B) Crosses are placed at 25°C for 24 hours. The progeny is submitted to three 1 hour heat-shocks (indicated by arrows) at 37°C before selection of 3^rd instar females containing GFP-expressing hemocytes. (C) Movement of wild-type and mys¹ GFP-expressing hemocytes in 3 to 4 hour APF flies, tracked for 12 minutes (1 min time-lapse interval). Arrowheads indicate filopodium-like protrusions produced by both wild-type and mys¹ mutant hemocytes. Scale bars: 10 µm. (D) Graph showing individual mean cell speed for wild-type and mys¹ clones (2 to 4 hours APF). (E) Graph showing average mean cell speeds for yv control and mys (valium 20) RNAi-expressing flies (between 3 to 4 hours APF). Mann–Whitney tests for non-Gaussian populations were used. Black lines indicate the samples' means; Error bars  =  standard deviation; P-values shown above tested groups.

Fig. 2.. NA and FA proteins are essential for optimal hemocyte migration.

(A) Example tracks of yv controls and mys RNAi-expressing flies. Scale bars: 10 µm. (B) Graph showing average mean cell speeds for different genetic backgrounds in 3 to 4 hour APF flies. val 10  =  TRIP valium 10; val 20  =  TRIP valium 20. Mann–Whitney tests for non-Gaussian populations were used. Both rhea RNAi lines were compared to the y¹sc¹v¹ control line. All other lines were compared to the y¹v¹ control line. Black lines indicate the samples' means; Error bars  =  standard deviation; P-values shown above tested groups.

Fig. 3.. Polarization towards a chemotactic cue does not depend on NA proteins.

(A) yv control flies immediately before and 28 minutes after wounding, respectively. Zoom of a selected cell presented at the top right hand corners, showing changes in the polarization arm. Red arrows point in the direction of the polarization arm of cells at the pictured time point. Green arrows point towards wound center (depicted by a white cross). Scale bars: 10 µm. (B) Graph showing the mean hemocyte numbers at the wound area along time for different control and RNAi flies. Dotted line under x axis depicts the chosen time interval for the polarization analysis. (C) Graph showing the mean polarization angle for individual cells before and after wounding in 3 to 4 hour APF flies. val 10  =  TRIP valium 10; val 20  =  TRIP valium 20. Black lines indicate the samples' means; Error bars  =  standard deviation; P-values shown above tested groups.