Hair follicle bulge cultures yield class III β-tubulin-positive melanoglial cells

Class III β-tubulin (TUBB3)-positive cells from the hair follicle bulge are thought to be neuronal cells derived from a local neural crest stem cell. However, TUBB3 has recently been shown to be expressed in the melanocytic lineage. To evaluate the neural-crest-associated immunophenotype of TUBB3-positive cells from hair follicle bulge explants, we dissected hair follicle bulges out from mouse whisker pads and cultured for 1 month and assessed outgrowing cells by means of immunocytochemistry using the biomarkers TUBB3, nestin, NGFR, SOX9, TYRP1 and laminin. Large amounts of TUBB3-positive cells could be cultured that co-expressed nestin, NGFR, SOX9 and, to a lesser degree, TYRP1, matching a melanoglial phenotype. In addition, a small population of TUBB3-negative but laminin-positive cells was found, which presumably are of glial origin. It can be concluded that cells of melanoglial origin can easily be obtained from hair follicle bulge explants. These cells may be of use in experimental animal or human disease and wound healing models. Notably, the TUBB3-positive cells are of melanoglial rather than neuronal origin

Asymmetry of VANGL2 in migrating lymphocytes as a tool to monitor activity of the mammalian WNT/planar cell polarity pathway

International audienceBackground: The 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. Results: Here 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. Conclusions: The 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