Silencing of CD44 in Glioma Leads to Changes in Cytoskeletal Protein Expression and Cellular Biomechanical Deformation Properties as Measured by AFM Nanoindentation
CD44, a transmembrane glycoprotein receptor for extracellular matrix molecules such as hyaluronic acid and osteopontin, is involved in glioma cellular signalling, adhesion and invasion. Although a great deal is known concerning the molecular players in adhesion, migration and invasion, little is known relating to how these invasive and migratory-promoting proteins influence biomechanical properties of glioma cells. Herein, we extend previous CD44 blocking experiments to examine effects of CD44 knock-down on expression of cytoskeletal proteins and cellular stiffness. An atomic force microscope (AFM) nanoindentation method was used to measure deformation or cellular stiffness (Young’s modulus, E) in real time, at the single-cell level over nuclear and cytoplasmic regions. A glioblastoma cell line (SNB-19) was transfected with either CD44 small interfering RNA (siRNA), scrambled siRNA or a non-related gene siRNA. In SNB-19 CD44 knock-down cells, levels of microtubule, vimentin and glial fibrillary acidic protein (GFAP) proteins were lower compared to cells transfected with scrambled siRNA. Functionally, CD44 knock-down cells were less migratory compared to controls. AFM nanoindentation results show that the areas over the nuclei of both knock-down and parental control cells examined were significantly more compliant than their cytoplasmic regions (p < 0.001). The most striking difference was seen when comparing nuclear regions of parental control cells versus CD44 knock-down cells. CD44 knock-down SNB-19 cells (E = 0.56 ± 0.50 kPa) were less stiff than parental cells (E = 1.93 ± 2.86 kPa; p < 0.001). Based on these results, we hypothesise that CD44 signalling via cytoskeletal proteins such as vimentin may influence the ability of glioma cells to respond to host tumour-derived mechanical pressures.
KeywordsGlioma Migration Cytoskeleton Cell stiffness Atomic force microscopy (AFM)
We thank Drs Robert Field and Alex Winkle from JPK Instruments, Cambridge, UK, for loan of the CellHesion 200 and NanoWizard 3 AFM instruments and Dr Torsten Muller from JPK Instruments, Berlin, Germany, for initial AFM training. We also thank Brain Tumour Research and Institute for Biomedical and Biomolecular Sciences for support.
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