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Altered endocytosis and vesicular trafficking are major players during tumorigenesis. Flotillin overexpression, a feature observed in many invasive tumors and identified as a marker of poor prognosis, induces a deregulated endocytic and trafficking pathway called upregulated flotillin-induced trafficking (UFIT). Here, we found that in non-tumoral mammary epithelial cells, induction of the UFIT pathway promotes epithelial-to-mesenchymal transition (EMT) and accelerates the endocytosis of several transmembrane receptors, including AXL, in flotillin-positive late endosomes. AXL overexpression, frequently observed in cancer cells, is linked to EMT and metastasis formation. In flotillin-overexpressing non-tumoral mammary epithelial cells and in invasive breast carcinoma cells, we found that the UFIT pathway-mediated AXL endocytosis allows its stabilization and depends on sphingosine kinase 2, a lipid kinase recruited in flotillin-rich plasma membrane domains and endosomes. Thus, the deregulation of vesicular trafficking following flotillin upregulation, and through sphingosine kinase 2, emerges as a new mechanism of AXL overexpression and EMT-inducing signaling pathway activation., This work was supported by the Fondation pour la Recherche Médicale (DEQ20161136700) and the Fondation ARC pour la recherche sur le cancer (ARCPJA32020060002078). Collection of breast tumor samples was supported by the Russian Science Foundation (19-75-30016). C.G.-R. was supported by the Institut National de la Santé et de la Recherche Médicale.

Molecular motors are pivotal for intracellular transport as well as cell motility and have great potential to be put to use outside cells. Here, we exploit engineered motor proteins in combination with self-assembly of actin filaments to actively pull lipid nanotubes from giant unilamellar vesicles (GUVs). In particular, actin filaments are bound to the outer GUV membrane and the GUVs are seeded on a heavy meromyosin-coated substrate. Upon addition of ATP, hollow lipid nanotubes with a length of tens of micrometer are pulled from single GUVs due to the motor activity. We employ the same mechanism to pull lipid nanotubes from different types of cells. We find that the length and number of nanotubes critically depends on the cell type, whereby suspension cells form bigger networks than adherent cells. This suggests that molecular machines can be used to exert forces on living cells to probe membrane-to-cortex attachment.

The binding strength between epithelial cells is crucial for tissue integrity, signal transduction and collective cell dynamics. However, there is no experimental approach to precisely modulate cell–cell adhesion strength at the cellular and molecular level. Here, we establish DNA nanotechnology as a tool to control cell–cell adhesion of epithelial cells. We designed a DNA-E-cadherin hybrid system consisting of complementary DNA strands covalently bound to a truncated E-cadherin with a modified extracellular domain. DNA sequence design allows to tune the DNA-E-cadherin hybrid molecular binding strength, while retaining its cytosolic interactions and downstream signaling capabilities. The DNA-E-cadherin hybrid facilitates strong and reversible cell–cell adhesion in E-cadherin deficient cells by forming mechanotransducive adherens junctions. We assess the direct influence of cell–cell adhesion strength on intracellular signaling and collective cell dynamics. This highlights the scope of DNA nanotechnology as a precision technology to study and engineer cell collectives.

Due to its versatility and programmability, DNA nanotechnology has greatly expanded the experimental toolbox for biomedical research. Recent advances allow reliable and efficient functionalization of cellular plasma membranes with a variety of synthetic DNA constructs, ranging from single strands to complex 3D DNA origami. The scope for applications, which probe biophysical parameters or equip cells with novel functions, is rapidly increasing. These applications extend from programmed cellular connectivity and tissue engineering to molecular force measurements, controlled receptor-ligand interactions, membrane-anchored biosensors, and artificial transmembrane structures. Here, we give guidance on different strategies to functionalize cellular membranes with DNA nanotechnology and summarize current trends employing membrane-anchored DNA as a tool in biophysics, cell biology, and synthetic biology.

Altered endocytosis and vesicular trafficking are major players during tumorigenesis. Flotillin overexpression, a feature observed in many invasive tumors, and identified as a marker of poor prognosis, induces a deregulated endocytic and trafficking pathway called Upregulated Flotillin-Induced Trafficking (UFIT). Here, we found that, in non tumoral mammary epithelial cells, induction of the UFIT pathway promotes epithelial-to-mesenchymal transition (EMT) and accelerates the endocytosis of several transmembrane receptors, including AXL, in flotillin-positive late endosomes. AXL overexpression, frequently observed in cancer cells, is linked to EMT and metastasis formation. In flotillin-overexpressing non-tumoral mammary epithelial cells and in invasive breast carcinoma cells, we found that the UFIT-pathway-mediated AXL endocytosis allows its stabilization and depends on sphingosine-kinase 2, a lipid kinase recruited in flotillin-rich plasma membrane-domains and endosomes.Thus, the deregulation of vesicular trafficking following flotillin upregulation, and through sphingosine kinase 2, emerges as a new mechanism of AXL overexpression and EMT-inducing signaling pathway activation.