DataSheet1_Regulation of cellular contractile force, shape and migration of fibroblasts by oncogenes and Histone deacetylase 6.docx

The capacity of cells to adhere to, exert forces upon and migrate through their surrounding environment governs tissue regeneration and cancer metastasis. The role of the physical contractile forces that cells exert in this process, and the underlying molecular mechanisms are not fully understood. We, therefore, aimed to clarify if the extracellular forces that cells exert on their environment and/or the intracellular forces that deform the cell nucleus, and the link between these forces, are defective in transformed and invasive fibroblasts, and to indicate the underlying molecular mechanism of control. Confocal, Epifluorescence and Traction force microscopy, followed by computational analysis, showed an increased maximum contractile force that cells apply on their environment and a decreased intracellular force on the cell nucleus in the invasive fibroblasts, as compared to normal control cells. Loss of HDAC6 activity by tubacin-treatment and siRNA-mediated HDAC6 knockdown also reversed the reduced size and more circular shape and defective migration of the transformed and invasive cells to normal. However, only tubacin-mediated, and not siRNA knockdown reversed the increased force of the invasive cells on their surrounding environment to normal, with no effects on nuclear forces. We observed that the forces on the environment and the nucleus were weakly positively correlated, with the exception of HDAC6 siRNA-treated cells, in which the correlation was weakly negative. The transformed and invasive fibroblasts showed an increased number and smaller cell-matrix adhesions than control, and neither tubacin-treatment, nor HDAC6 knockdown reversed this phenotype to normal, but instead increased it further. This highlights the possibility that the control of contractile force requires separate functions of HDAC6, than the control of cell adhesions, spreading and shape. These data are consistent with the possibility that defective force-transduction from the extracellular environment to the nucleus contributes to metastasis, via a mechanism that depends upon HDAC6. To our knowledge, our findings present the first correlation between the cellular forces that deforms the surrounding environment and the nucleus in fibroblasts, and it expands our understanding of how cells generate contractile forces that contribute to cell invasion and metastasisis.

细胞与其周围环境之间的粘附能力、施加的力以及迁移能力，决定了组织的再生与癌症的转移。细胞在此过程中施加的物理收缩力及其背后的分子机制尚未得到充分理解。因此，本研究旨在阐明细胞对其环境施加的细胞外力以及/或者变形细胞核的细胞内力，以及这些力之间的联系是否在转化和侵袭性成纤维细胞中存在缺陷，并指出其背后的调控分子机制。共聚焦、正置荧光和牵引力显微镜，随后进行计算分析，表明侵袭性成纤维细胞相较于正常对照组细胞，其施加于环境的最大收缩力增加，而细胞核的细胞内力减少。通过tubacin处理和siRNA介导的HDAC6敲低导致HDAC6活性的丧失，逆转了转化和侵袭细胞的缩小尺寸、更圆的形状以及缺陷性迁移，恢复至正常状态。然而，只有tubacin介导的，而非siRNA敲低能够逆转侵袭细胞对其周围环境的增加力至正常水平，对核力无影响。我们观察到，环境力和核力呈弱正相关，除了HDAC6 siRNA处理的细胞，其中相关性呈弱负相关。转化和侵袭性成纤维细胞相较于对照组，表现出更多的细胞-基质粘附以及更小的细胞，而tubacin处理和HDAC6敲低并未将这一表型恢复至正常，反而进一步增加了粘附。这突显了控制收缩力可能需要HDAC6在控制细胞粘附、扩散和形状之外的功能。这些数据与缺陷性力传递从细胞外环境到细胞核，通过依赖于HDAC6的机制促进转移的可能性一致。据我们所知，我们的发现首次揭示了成纤维细胞中细胞力对周围环境和细胞核变形之间的相关性，并扩展了我们对于细胞如何产生有助于细胞侵袭和转移的收缩力的理解。