Gabrielle Larocque,1 Daniel J. Moore,1 Méghane Sittewelle,1 Cansu Kuey,1 Joseph H.R. Hetmanski,2 Penelope J. La-Borde,1 Beverley J. Wilson,2 Nicholas I. Clarke,1 Patrick T. Caswell,2 and Stephen J. Royle1
1 Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry, UK,
2 Wellcome Trust Centre for Cell-Matrix Research, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK,
Correspondence to Stephen J. Royle: [email protected]
G. Larocque’s present address is Cellular Signalling and Cytoskeletal Function Laboratory, The Francis Crick Institute, London, UK.
Medical Research, Molecular Biology
Membrane traffic is an important regulator of cell migration through the endocytosis and recycling of cell surface receptors such as integrin heterodimers. Intracellular nanovesicles (INVs) are transport vesicles that are involved in multiple membrane trafficking steps, including the recycling pathway. The only known marker for INVs is tumor protein D54 (TPD54/TPD52L2), a member of the TPD52-like protein family. Overexpression of TPD52-like family proteins in cancer has been linked to poor prognosis and an aggressive metastatic phenotype, which suggests cell migration may be altered under these conditions. Here, we show that TPD54 directly binds membrane and associates with INVs via a conserved positively charged motif in its C terminus. We describe how other TPD52-like proteins are also associated with INVs, and we document the Rab GTPase complement of all INVs. Depletion of TPD52-like proteins inhibits cell migration and invasion, while their overexpression boosts motility. We show that inhibition of migration is likely due to altered recycling of α5β1 integrins in INVs.
Cells in CDM were imaged for 16 h using an Eclipse Ti inverted microscope (Nikon) with a 20×/0.45 SPlan Fluar objective and the Nikon filter sets for bright field and a pE-300 LED (CoolLED) fluorescent light source with imaging software NIS Elements AR.5.20.02.
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