Abstract
One of the most important questions in cell biology concerns how cells reorganize after sensing polarity cues. In the present study, we describe the formation of an actin-rich domain on the apical surface of human primary endothelial cells adhering to the substrate and investigate its role in cell polarity. We used confocal immunofluorescence procedures to follow the redistribution of proteins required for endothelial cell polarity during spreading initiation. Activated Moesin, vascular endothelial cadherin and partitioning defective 3 were found to be localized in the apical domain, whereas podocalyxin and caveolin-1 were distributed along the microtubule cytoskeleton axis, oriented from the centrosome to the cortical actin-rich domain. Moreover, activated signaling molecules were localized in the core of the apical domain in tight association with filamentous actin. During cell attachment, loss of the apical domain by Moesin silencing or drug disruption of the actin cytoskeleton caused irregular cell spreading and mislocalization of polarity markers. In conclusion, our results suggest that the apical domain that forms during the spreading process is a structural organizer of cell polarity by regulating trafficking and activation of signaling proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-012-0965-9) contains supplementary material, which is available to authorized users.
Highlights
Cell polarity is the asymmetric organization of most of the physical structures of the cell
Consistent with previous work on melanoma cells (Estecha et al 2009), we observed that when endothelial cells contacted the substrate and began to adhere, they underwent transition from round to hemispheric shape and filamentous actin (F-actin) mainly localized to the periphery of the cells (Fig. 1a, 2 min; b, top left)
To assess whether the apical actin domain is involved in cell polarization of adhering cells, we focused on Moesin, VE-cadherin and partitioning defective 3 (Par3), which have been demonstrated to be required for endothelial apical–basal polarity in vitro and during embryonic development (Strilic et al 2009; Lampugnani et al 2010; Wang et al 2010; Zovein et al 2010)
Summary
Cell polarity is the asymmetric organization of most of the physical structures of the cell. Different cell types and organisms show cellular asymmetries, establishment, maintenance, and transduction of cell polarity are characterized by common general steps: a polarity cue, asymmetric accumulation of polarity factors, and reorganization of the cytoskeleton. The establishment of apical–basal polarity is a fundamental requirement for lumen formation in different types of vessels. Direct interaction of endothelial cells with the extracellular matrix (ECM) through b1 integrin was identified as an initial and critical cue for asymmetric distribution of regulatory proteins, among which partitioning defective 3 (Par3) plays a key role in the acquisition of apical–basal polarity (Zovein et al 2010). PODXL, which is able to generate apical-domain structures (Nielsen and McNagny 2008), connects with Moesin, a member of the Ezrin/Radixin/Moesin (ERM) family of actin-binding proteins (Strilic et al 2009; Zovein et al 2010). In spite of the molecular polarity pathways identified, it remains unclear how cortical polarity controls the reorganization of the actin and microtubule cytoskeletons and the polarization of trafficking pathways
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