The image shows a live Drosophila melanogaster haemocyte during developmental dispersal. The actin (green) and microtubule (purple) cytoskeletons were labelled with vital probes to highlight their dynamics and coordination during motility (white reveals actin–microtubule colocalization). Image courtesy of Dr. Brian Stramer, Randall Division of Cell and Molecular Biophysics, King's College London and Department of Biochemistry and Department of Physiology and Pharmacology, University of Bristol, UK

It is over half a century since Abercrombie and Heaysman first described contact repulsion in fibroblasts, yet our molecular understanding of the regulation of this process remains limited. Now, though, in vivo imaging experiments using fluorescent probes show the dynamic interplay between actin and microtubule networks in migrating haemocytes from Drosophila melanogaster. The findings, reported in The Journal of Cell Biology, reveal a role for an 'arm'-like bundle of microtubules in polarized migration and contact repulsion.

Stramer et al. visualized microtubule dynamics in migrating D. melanogaster haemocytes during development by expressing a fusion protein comprising the microtubule-binding domain of CLIP170 and a fluorescent probe. Within the migrating cells, the microtubules arranged themselves around the cell body; some extended into the lamellae, from where they were driven back (by actin retrograde flow), converging to form a bundle — the so-called 'arm'. Migration occurred in the direction of the arm and conferred directional persistence. In response to laser wounds to embryos, haemocytes rapidly migrated towards the wound, reorganizing their microtubules to generate an arm directed towards the site of damage. Formation of the arm occurred before lamellar polarization and migration, indicating that the arm directs lamellar polarization.

When migrating haemocytes collide, they stop, repolarize and then move away from each other. Stramer et al. used time-lapse imaging to show that, following lamellar interaction and alignment of actin filaments, contacting cells transiently aligned their microtubule arms; the arms then collapsed and the cells repolarized and moved away. However, in the presence of Spastin, a microtubule-severing protein, a normal microtubule cytoskeleton could not be formed — haemocytes contained fragments of microtubules instead of bundles. Live-imaging experiments showed that Spastin-expressing haemocytes could not polarize and failed to undergo cell–cell repulsion, indicating an essential function for microtubule arms in these processes.

The microtubule plus-end-binding and -stabilizing protein Orbit/Clasp is involved in mediating repulsion in neuronal growth cones in response to the chemorepellent Slit during D. melanogaster embryogenesis. Because the microtubule architecture of haemocytes resembles that of neuronal growth cones, Stramer et al. opted to study haemocyte migration in orbit² mutant embryos to assess a potential role for Orbit in mediating cell–cell repulsion. The results seen in orbit² mutant haemocytes were strikingly similar to those of Spastin-expressing haemocytes, highlighting a role for Orbit in contact repulsion. By expressing the fluorescently-tagged CLIP170 protein, the authors showed that microtubule polymerization in orbit² mutant haemocytes was unpolarized; no bundles were formed. This defective microtubule architecture was rescued by the expression of a fluorescently tagged Orbit fusion protein, which consequently restored contact repulsion. Orbit, therefore, has an important function in regulating the formation of microtubule bundles.

Among the burning issues that remain to be resolved are how microtubules are induced to disassemble, how disassembly causes cell repulsion, and how repolarization occurs. One possibility is that when growing microtubules in opposing haemocytes collide, they generate enough force to depolymerize, followed by stochastic cellular repolarization. Alternatively, the microtubules might actively provide a signal to break up the contacting cells. Doubtless, though, this in vivo imaging approach will provide further invaluable insights into the molecular mechanisms.

Author: Katrin Legg