Abstract
Flotillin-1 and flotillin-2 are two homologous, membrane raft associated proteins. Although it has been reported that flotillins are involved in cell adhesion processes and play a role during breast cancer progression, thus making them interesting future therapeutic targets, their precise function has not been well elucidated. The present study investigates the function of these proteins in cell-cell adhesion in non-malignant cells. We have used the non-malignant epithelial MCF10A cells to study the interaction network of flotillins within cell-cell adhesion complexes. RNA interference was used to examine the effect of flotillins on the structure of adherens junctions and on the association of core proteins, such as E-cadherin, with membrane rafts. We here show that the cadherin proteins of the adherens junction associate with flotillin-2 in MCF10A cells and in various human cell lines. In vitro, flotillin-1 and flotillin-2 directly interact with γ-catenin which is so far the only protein known to be present both in the adherens junction and the desmosome. Mapping of the interaction domain within the γ-catenin sequence identified the Armadillo domains 6–8, especially ARM domain 7, to be important for the association with flotillins. Furthermore, depletion of flotillins significantly influenced the morphology of the adherens junction in human epithelial MCF10A cells and altered the association of E-cadherin and γ-catenin with membrane rafts. Taken together, these observations suggest a functional role for flotillins, especially flotillin-2, in cell-cell adhesion in non-malignant epithelial cells.
Highlights
Cell-cell adhesion is based on various cellular junctions and ensures a tight contact between neighboring cells, enabling interactive exchanges necessary for morphological and functional differentiation and maintaining the homeostasis of healthy tissue organization
We have here characterized the molecular function of flotillin-1 and flotillin-2 in epithelial cell-cell adhesion using the human, non-tumorigenic epithelial MCF10A cell line
Staining of subconfluent MCF10A cells (Figure S1, upper row) showed that flotillins are mainly localized in intracellular vesicular compartments, whereas in confluent cells, they preferentially reside at the plasma membrane with only little intracellular staining (Figure S1, lower row)
Summary
Cell-cell adhesion is based on various cellular junctions and ensures a tight contact between neighboring cells, enabling interactive exchanges necessary for morphological and functional differentiation and maintaining the homeostasis of healthy tissue organization (reviewed in [1]). The molecules of the cadherin superfamily of cell-cell adhesion receptors include among other members the classical cadherins (e.g. E-cadherin and Ncadherin) and the desmosomal cadherins (e.g. desmoglein-3 and desmocollin-3). These proteins are single-span transmembrane proteins which all possess extracellular cadherin (EC) repeats. These EC repeats are capable of calcium binding and mediate the interaction capacity of the extracellular domain [5,6,7]. The differences in the structures of the cytoplasmic domains of the desmosomal and classical cadherins enable interactions with specific intracellular binding partners of the catenin protein family [8,9,10]. Classical cadherins usually associate with the catenin family members bcatenin and p120catenin, whereas desmosomal cadherins preferentially bind to c-catenin and plakophilins [10,15]
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