Metastasising Fibroblasts Show an HDAC6-Dependent Increase in Migration Speed and Loss of Directionality Linked to Major Changes in the Vimentin Interactome

Caroline A Evans,Annica K B Gad,Franziska Lautenschläger,Sarah C Macfarlane,Poppy I A Nowicki,Lei Xu,Hyejeong Rosemary Kim,Carsten Baltes,Bernard M Corfe,Jerker Widengren

doi:10.3390/ijms23041961

Abstract

Metastasising cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumours in the clinic. We aimed to clarify how vimentin regulates the motility of metastasising fibroblasts. STED super-resolution microscopy, live-cell imaging and quantitative proteomics revealed that oncogene-expressing and metastasising fibroblasts show a less-elongated cell shape, reduced cell spreading, increased cell migration speed, reduced directionality, and stronger coupling between these migration parameters compared to normal control cells. In total, we identified and compared 555 proteins in the vimentin interactome. In metastasising cells, the levels of keratin 18 and Rab5C were increased, while those of actin and collagen were decreased. Inhibition of HDAC6 reversed the shape, spreading and migration phenotypes of metastasising cells back to normal. Inhibition of HDAC6 also decreased the levels of talin 1, tropomyosin, Rab GDI β, collagen and emilin 1 in the vimentin interactome, and partially reversed the nanoscale vimentin organisation in oncogene-expressing cells. These findings describe the changes in the vimentin interactome and nanoscale distribution that accompany the defective cell shape, spreading and migration of metastasising cells. These results support the hypothesis that oncogenes can act through HDAC6 to regulate the vimentin binding of the cytoskeletal and cell–extracellular matrix adhesion components that contribute to the defective motility of metastasising cells.

Highlights

Metastasising tumour cells show increased levels of the intermediate filament protein vimentin, which has, been used to diagnose invasive tumours in the clinic for decades
To determine how the three filamentous systems change during the transformation from primary fibroblasts to metastasising cells, we analysed the spatial distributions of actin microfilaments, microtubules and vimentin intermediate filaments in an isogenically matched stepwise cell model of four human cell variants that represent the transformation from primary fibroblasts to metastasising cells
We investigated whether the reversion of the cell shape, size and motility of metastasising cells to the control phenotype upon inhibition of HDAC6 was accompanied by a reversion of the vimentin interactome to that of the control cells

Summary

Introduction

Metastasising tumour cells show increased levels of the intermediate filament protein vimentin, which has, been used to diagnose invasive tumours in the clinic for decades. Vimentin is a canonical marker of epithelial-to-mesenchymal transition (EMT), a process that occurs during embryogenesis, wound healing and cancer in which stationary cells change from a round to an elongated cell shape and become migratory. Vimentin is required for the mechanical and adhesion properties of cells, and the metastatic spread of lung cancers [1,7,8,9]. This might be due, in part, to the promotion of single cell migration by vimentin, which regulates the functions of the actin microfilament and microtubule filamentous systems [9,10]

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