Inter-cellular forces orchestrate contact inhibition of locomotion.

Contact inhibition of locomotion (CIL) is a multifaceted process that causes many cell types to repel each other upon collision. During development, this seemingly uncoordinated reaction is a critical driver of cellular dispersion within embryonic tissues. Here, we show that Drosophila hemocytes require a precisely orchestrated CIL response for their developmental dispersal. Hemocyte collision and subsequent repulsion involves a stereotyped sequence of kinematic stages that are modulated by global changes in cytoskeletal dynamics. Tracking actin retrograde flow within hemocytes in vivo reveals synchronous reorganization of colliding actin networks through engagement of an inter-cellular adhesion. This inter-cellular actin-clutch leads to a subsequent build-up in lamellar tension, triggering the development of a transient stress fiber, which orchestrates cellular repulsion. Our findings reveal that the physical coupling of the flowing actin networks during CIL acts as a mechanotransducer, allowing cells to haptically sense each other and coordinate their behaviors.

Cells on film - the past and future of cinemicroscopy.

Movie making is now a ubiquitous experimental tool that biologists use alongside more traditional techniques such as molecular biology and biochemistry. It is no longer just cell biologists, but scientists from many other disciplines, such as immunology and neuroscience, that utilise movies to dissect their processes of interest. When did filming become such a standard laboratory technique? Who developed the use of the movie as an experimental tool? The Wellcome Library has recently restored and digitized a number of original 16-mm films from two pioneering cinemicroscopists, Ronald Canti and Michael Abercrombie, which are now freely available to the scientific community. In light of these films, this Essay will give a brief history of the early cinemicroscopists and discuss what is driving the use of movies in the laboratory today.

Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo.

Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. Here, we report that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd's Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo.

Emergence of embryonic pattern through contact inhibition of locomotion.

The pioneering cell biologist Michael Abercrombie first described the process of contact inhibition of locomotion more than 50 years ago when migrating fibroblasts were observed to rapidly change direction and migrate away upon collision. Since then, we have gleaned little understanding of how contact inhibition is regulated and only lately observed its occurrence in vivo. We recently revealed that Drosophila macrophages (haemocytes) require contact inhibition for their uniform embryonic dispersal. Here, to investigate the role that contact inhibition plays in the patterning of haemocyte movements, we have mathematically analysed and simulated their contact repulsion dynamics. Our data reveal that the final pattern of haemocyte distribution, and the details and timing of its formation, can be explained by contact inhibition dynamics within the geometry of the Drosophila embryo. This has implications for morphogenesis in general as it suggests that patterns can emerge, irrespective of external cues, when cells interact through simple rules of contact repulsion.

Contact inhibition of locomotion in vivo controls neural crest directional migration.

Contact inhibition of locomotion was discovered by Abercrombie more than 50 years ago and describes the behaviour of fibroblast cells confronting each other in vitro, where they retract their protrusions and change direction on contact. Its failure was suggested to contribute to malignant invasion. However, the molecular basis of contact inhibition of locomotion and whether it also occurs in vivo are still unknown. Here we show that neural crest cells, a highly migratory and multipotent embryonic cell population, whose behaviour has been likened to malignant invasion, demonstrate contact inhibition of locomotion both in vivo and in vitro, and that this accounts for their directional migration. When two migrating neural crest cells meet, they stop, collapse their protrusions and change direction. In contrast, when a neural crest cell meets another cell type, it fails to display contact inhibition of locomotion; instead, it invades the other tissue, in the same manner as metastatic cancer cells. We show that inhibition of non-canonical Wnt signalling abolishes both contact inhibition of locomotion and the directionality of neural crest migration. Wnt-signalling members localize at the site of cell contact, leading to activation of RhoA in this region. These results provide the first example of contact inhibition of locomotion in vivo, provide an explanation for coherent directional migration of groups of cells and establish a previously unknown role for non-canonical Wnt signalling.

Arp2/3 complex interactions and actin network turnover in lamellipodia.

Cell migration is initiated by lamellipodia-membrane-enclosed sheets of cytoplasm containing densely packed actin filament networks. Although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for Arp2/3 complex and its activator WAVE complex. Here, we combine fluorescence recovery after photobleaching (FRAP) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly/disassembly. We show that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip. In contrast, cortactin-another prominent Arp2/3 complex regulator-and ADF/cofilin-previously implicated in driving both filament nucleation and disassembly-were rapidly exchanged throughout the lamellipodium. These results suggest that Arp2/3- and WAVE complex-driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. Network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh.

Role of vinculin in regulating focal adhesion turnover.

Although vinculin (-/-) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053-1066) and referred to as the deltaC mutation. This mutation reduced PIP2 binding to a Vt deltaC polypeptide by >90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculin deltaC mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.

PTEN couples Sema3A signalling to growth cone collapse.

Distinct changes in glycogen synthase kinase-3 (GSK-3) signalling can regulate neuronal morphogenesis including the determination and maintenance of axonal identity, and are required for neurotrophin-mediated axon elongation. In addition, we have previously shown a dependency on GSK-3 activation in the semaphorin 3A (Sema3A)-mediated growth-cone-collapse response of sensory neurons. Regulation of GSK-3 activity involves the intermediate signalling lipid phosphatidylinositol 3,4,5-trisphosphate, which can be modulated by phosphatidylinositol 3-kinase (PI3K) and the tumour suppressor PTEN. We report here the involvement of PTEN in the Sema3A-mediated growth cone collapse. Sema3A suppresses PI3K signalling concomitant with the activation of GSK-3, which depends on the phosphatase activity of PTEN. PTEN is highly enriched in the axonal compartment and the central domain of sensory growth cones during axonal extension, where it colocalises with microtubules. Following exposure to Sema3A, PTEN accumulates rapidly at the growth cone membrane suggesting a mechanism by which PTEN couples Sema3A signalling to growth cone collapse. These findings demonstrate a dependency on PTEN to regulate GSK-3 signalling in response to Sema3A and highlight the importance of subcellular distributions of PTEN to control growth cone behaviour.

Cell motility under the microscope: Vorsprung durch Technik.

The fashion today is to disparage technology-led research but our view is that cell biologists, in particular, should be proud of their 'progress through technology'. The 'cell theory' itself, arguably the oldest cornerstone in the theoretical foundations of biology, emerged because Hooke, van Leeuwenhoek and others had, more than a century earlier, pioneered the enabling technology--the microscope. We develop this theme with reference to our own field of research: the locomotion of cultured tissue cells.

Fluorescence localization after photobleaching (FLAP).

Fluorescence localization after photobleaching is a new method for localized photolabeling and subsequent tracking of specific molecules within living cells. The molecular species to be located carries two different fluorophores that can be imaged independently but simultaneously by fluorescence microscopy. For the method to work, these two fluorophores should be accurately colocalized throughout the cell so that their images are closely matched. One of the fluorophores (the target fluorophore) is then rapidly photobleached at a chosen location. The unbleached (reference) fluorophore remains colocalized with the target fluorophore; thus, the subsequent fate of the photobleached molecules can be revealed by processing simultaneously acquired digital images of the two fluorophores. Here we demonstrate the simplicity and effectiveness of the FLAP method in revealing both fast and slow molecular dynamics in living cells using a Zeiss LSM 510 laser scanning confocal microscope.

Requirement for PI 3-kinase gamma in macrophage migration to MCP-1 and CSF-1.

Phosphoinositide 3-kinases (PI3Ks) are important regulators of cell migration. The PI3K isoform gamma is primarily expressed in haematopoietic cells, and is activated by G protein-coupled receptors (GPCRs). Here, we investigate the contribution of PI3Kgamma to macrophage responses to chemoattractants, using bone marrow-derived macrophages from wild-type and PI3Kgamma-null mice. We observe that early membrane ruffling induced by MCP-1, which activates a GPCR, or by CSF-1, which activates a tyrosine kinase receptor, is unaltered in PI3Kgamma(-/-) mice, although by 30 min MCP-1-induced cell polarization was strongly reduced in PI3Kgamma(-/-) compared to wild-type macrophages. The migration behaviour of the macrophages was analysed by time-lapse microscopy in Dunn chemotaxis chambers. PI3Kgamma(-/-) macrophages showed reduced migration speed and translocation, and no chemotaxis to MCP-1. Interestingly, there was also a reduction in migration efficiency in PI3Kgamma(-/-) macrophages stimulated with CSF-1 although early CSF-1R signalling was normal. These results indicate that the initial actin reorganization induced by either a GPCR or tyrosine kinase receptor agonist is not dependent on PI3Kgamma, whereas PI3Kgamma is needed for optimal migration of macrophages to either agonist.

Rapid actin transport during cell protrusion.

Transformed rat fibroblasts expressing two variants of green fluorescent protein, each fused to beta-actin, were used to study actin dynamics during cell protrusion. The recently developed FLAP (fluorescence localization after photobleaching) method permits the tracking of one fluorophore after localized photobleaching by using the other as a colocalized reference. Here, by visualizing the ratio of bleached to total molecules, we found that actin was delivered to protruding zones of the leading edge of the cell at speeds that exceeded 5 micrometers per second. Monte Carlo modeling confirmed that this flow cannot be explained by diffusion and may involve active transport.

Restoration of podosomes and chemotaxis in Wiskott-Aldrich syndrome macrophages following induced expression of WASp.

We used a direct-viewing (Dunn) chemotaxis chamber to analyse the chemotactic responses of human normal and Wiskott-Aldrich syndrome (WAS) macrophages to the cytokine colony stimulating factor-1 (CSF-1). In five patients with classic WAS, where specialised adhesion complexes called podosomes are absent, chemotaxis of macrophages was abolished. The deficient chemotactic responses of WAS macrophages following cytokine stimulation could be correlated with abnormalities in cell polarisation and actin organisation. In a series of cell microinjection studies we found that normal chemotactic responses were restored in WASp macrophages transfected with a full-length human WAS construct. Expression of exogenous WAS protein (WASp) in these cells also restored normal polarised cell morphology and the ability to form podosomes.