Abstract
BackgroundThe WNT/planar-cell-polarity (PCP) pathway is a key regulator of cell polarity and directional cell movements. Core PCP proteins such as Van Gogh-like2 (VANGL2) are evolutionarily highly conserved; however, the mammalian PCP machinery is still poorly understood mainly due to lack of suitable models and quantitative methodology. WNT/PCP has been implicated in many human diseases with the most distinguished positive role in the metastatic process, which accounts for more than 90% of cancer related deaths, and presents therefore an attractive target for pharmacological interventions. However, cellular assays for the assessment of PCP signaling, which would allow a more detailed mechanistic analysis of PCP function and possibly also high throughput screening for chemical compounds targeting mammalian PCP signaling, are still missing.ResultsHere we describe a mammalian cell culture model, which correlates B lymphocyte migration of patient-derived MEC1 cells and asymmetric localization of fluorescently-tagged VANGL2. We show by live cell imaging that PCP proteins are polarized in MEC1 cells and that VANGL2 polarization is controlled by the same mechanism as in tissues i.e. it is dependent on casein kinase 1 activity. In addition, destruction of the actin cytoskeleton leads to migratory arrest and cell rounding while VANGL2-EGFP remains polarized suggesting that active PCP signaling visualized by polarized distribution of VANGL2 is a cause for and not a consequence of the asymmetric shape of a migrating cell.ConclusionsThe presented imaging-based methodology allows overcoming limitations of earlier approaches to study the mammalian WNT/PCP pathway, which required in vivo models and analysis of complex tissues. Our system investigating PCP-like signaling on a single-cell level thus opens new possibilities for screening of compounds, which control asymmetric distribution of proteins in the PCP pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0079-1) contains supplementary material, which is available to authorized users.
Highlights
The WNT/planar-cell-polarity (PCP) pathway is a key regulator of cell polarity and directional cell movements
MEC1 cells – a robust model for in vitro imaging of B cell chemotaxis Our group has recently shown that the WNT/PCP pathway drives the pathogenesis of chronic lymphocytic leukemia (CLL) [10]
Migrating MEC1 cells show a polarized distribution of planar cell polarity proteins We have shown earlier that migration of MEC1 cells depend on the PCP pathway and that cellular migration can be blocked e.g. by inhibition of casein kinase 1 (CK1) or by downregulation of DVL2 [10]
Summary
The WNT/planar-cell-polarity (PCP) pathway is a key regulator of cell polarity and directional cell movements. While the establishment of well-ordered ommatidia in the compound eye or cells in the wing epithelium in the fruitfly as well as convergent extension movements in Xenopus laevis gastrulation serve as important models of PCP signaling, assessment of PCP signaling in mammals is more difficult. In general it requires analysis of embryogenesis of mutant mouse strains where regular arrangement of sensory hair cells in the inner ear and neural tube closure phenotypes are the most commonly used readouts for PCP-like signaling in mammals [7,8]. Cellular assays for the assessment of PCP signaling, which would allow a more detailed mechanistic analysis of PCP function and possibly high throughput screening for chemical compounds targeting mammalian PCP signaling, are still missing
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