The epidermis is a stratified squamous epithelium forming the barrier that excludes harmful microbes and retains body fluids. To perform these functions, proliferative basal cells in the innermost layer periodically detach from an underlying basement membrane of extracellular matrix, move outward and eventually die. Once suprabasal, cells stop dividing and enter a differentiation programme to form the barrier 1 . The mechanism of stratification is poorly understood. Although studies in vitro have led to the view that stratification occurs through the delamination and subsequent movement of epidermal cells 2-4 , most culture conditions favour keratinocytes that lack the polarity and cuboidal morphology of basal keratinocytes in tissue. These features could be important in considering an alternative mechanism, that stratification occurs through asymmetric cell divisions in which the mitotic spindle orients perpendicularly to the basement membrane 5-7 . Here we show that basal epidermal cells use their polarity to divide asymmetrically, generating a committed suprabasal cell and a proliferative basal cell. We further demonstrate that integrins and cadherins are essential for the apical localization of atypical protein kinase C, the Par3-LGN-Inscuteable complex and NuMAdynactin to align the spindle.We first addressed whether oriented cell divisions participate in stratification during epidermal development. At embryonic day 12.5 (E12.5), most of the epidermis was single-layered, and most divisions occurred laterally, within the plane of the epithelium. Unexpectedly, however, a few mitotic cells seemed to be dividing perpendicularly to the basement membrane, as judged by staining with 4′,6-diamidino-2-phenylindole (DAPI) and with anti-tubulin (arrow in Fig. 1a, b). Closer inspection revealed the presence of some suprabasal cells within the E12.5 singlelayered epithelium. In these regions, the occasional suprabasal cell was often positioned directly over a basal cell, as would be expected for a perpendicular division.To facilitate quantification, we engineered mice expressing keratin 14 (K14)-centrin coupled to green fluorescent protein (GFP) and examined embryos at later stages of development, as the epidermis became multi-layered. It was now easy to distinguish parallel from perpendicular spindle alignments (Fig. 1c, d). In predominantly single-layered areas of E12.5 epithelium, 92% of divisions occurred parallel to the basement membrane. By contrast, in E12.5 areas that showed early signs of stratification, most mitotic cells had an alignment that was clearly perpendicular. At E12.5, perpendicularly aligned spindles accounted for about 22% of all mitoses, but as stratification progressed across the epithelium, this number rose markedly (Fig. 1e). From E15.5 onwards through postnatal development, more than 70% of spindles were oriented perpendicularly to the basement membrane.Author Information Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare n...
The generation of cortical actin filaments is necessary for processes such as cell motility and cell polarization. Several recent studies have demonstrated that Wiskott-Aldrich syndrome protein (WASP) family proteins and the actin-related protein (Arp) 2/3 complex are key factors in the nucleation of actin filaments in diverse eukaryotic organisms. To identify other factors involved in this process, we have isolated proteins that bind to Bee1p/Las17p, the yeast WASP-like protein, by affinity chromatography and mass spectroscopic analysis. The yeast type I myosins, Myo3p and Myo5p, have both been identified as Bee1p-interacting proteins. Like Bee1p, these myosins are essential for cortical actin assembly as assayed by in vitro reconstitution of actin nucleation sites in permeabilized yeast cells. Analysis using this assay further demonstrated that the motor activity of these myosins is required for the polymerization step, and that actin polymerization depends on phosphorylation of myosin motor domain by p21-activated kinases (PAKs), downstream effectors of the small guanosine triphosphatase, Cdc42p. The type I myosins also interact with the Arp2/3 complex through a sequence at the end of the tail domain homologous to the Arp2/3-activating region of WASP-like proteins. Combined deletions of the Arp2/3-interacting domains of Bee1p and the type I myosins abolish actin nucleation sites at the cortex, suggesting that these proteins function redundantly in the activation of the Arp2/3 complex.
Despite their importance in cell shape and polarity generation, the organization of microtubules in differentiated cells and tissues remains relatively unexplored in mammals. We generated transgenic mice in which the epidermis expresses a fluorescently labeled microtubule-binding protein and show that in epidermis and in cultured keratinocytes, microtubules stereotypically reorganize as they differentiate. In basal cells, microtubules form a cytoplasmic network emanating from an apical centrosome. In suprabasal cells, microtubules concentrate at cell–cell junctions. The centrosome retains its ability to nucleate microtubules in differentiated cells, but no longer anchors them. During epidermal differentiation, ninein, which is a centrosomal protein required for microtubule anchoring (Dammermann, A., and A. Merdes. 2002. J. Cell Biol. 159:255–266; Delgehyr, N., J. Sillibourne, and M. Bornens. 2005. J. Cell Sci. 118:1565–1575; Mogensen, M.M., A. Malik, M. Piel, V. Bouckson-Castaing, and M. Bornens. 2000. J. Cell Sci. 113:3013–3023), is lost from the centrosome and is recruited to desmosomes by desmoplakin (DP). Loss of DP prevents accumulation of cortical microtubules in vivo and in vitro. Our work uncovers a differentiation-specific rearrangement of the microtubule cytoskeleton in epidermis, and defines an essential role for DP in the process.
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