The mammalian LINC complex controls mechanosensing at a genome-wide level: RNA-Seq

Mechanical cues influence the shape, growth, and function of tissues and organs and are necessary for the development of engineered tissues. Yet, how cells sense mechanical cues and transduce them into changes in gene expression is not well understood. It is known that mechanical forces transmitted to the nucleus induce chromatin remodeling, promote DNA repair, contribute to the motion of intranuclear organelles and cause direct dissociation of protein complexes inside nuclei. Yet, the extent to which such signals impact gene expression is not understood. Because mechanical forces from the cytoskeleton to the nucleus interior are transmitted by the LINC (linker of nucleoskeleton-to-cytoskeleton) complex, we disrupted the LINC complex and performed genome wide expression studies using RNA sequencing. LINC disruption altered the expression of hundreds of genes at a genome-wide scale. We asked how LINC disruption affected the mechanosensitivity of individual genes by quantifying fold changes in gene expression on soft and stiff substrates. Remarkably, LINC disruption tended to preserve gene mechanosensitivity, but to reverse its direction. LINC disruption did not cause changes in nuclear shape, nor eliminated nuclear shape sensitivity to substrate rigidity. Our results show for the first time that the LINC complex regulates mechano-sensing at a genome-wide level, and argue for a distinct mechanism that does not require changes in nuclear morphology. Overall design: mRNA profiles of NIH 3T3 TetON cells that were induced to express either SS-GFP-KDEL (control) or SS-HA-Sun1L-KDEL by the addition of doxycycline. Two (2) substrate stiffnesses were used (1 kPa and 308 kPa), Y27632 or blebbistatin was used for certain samples to inhibit myosin II activity. A total of 6x3 reps= 18 samples were analyzed.

机械信号可影响组织与器官的形态、生长及功能，亦是工程化组织发育的必要条件。然而，细胞感知机械信号并将其转导为基因表达变化的机制尚未完全阐明。已知传递至细胞核的机械力可诱导染色质重塑、促进DNA修复、参与核内细胞器的运动，并直接解离细胞核内的蛋白质复合物，但此类信号对基因表达的影响程度仍不明确。由于从细胞骨架到细胞核内部的机械力经由LINC复合物（linker of nucleoskeleton-to-cytoskeleton complex）传递，我们对该复合物进行了干扰，并通过RNA测序（RNA sequencing）开展全基因组表达分析。LINC复合物干扰可在全基因组范围内改变数百个基因的表达水平。我们通过量化软、硬底物上基因表达的倍数变化，探究了LINC复合物干扰对单个基因机械敏感性的影响。值得注意的是，LINC复合物干扰往往会保留基因的机械敏感性，但会反转其作用方向。LINC复合物干扰未引起细胞核形态的改变，也未消除细胞核形态对底物刚度的敏感性。本研究首次证实，LINC复合物可在全基因组层面调控机械感知过程，且其作用机制不依赖于细胞核形态的变化。整体实验设计：经强力霉素诱导表达SS-GFP-KDEL（对照组）或SS-HA-Sun1L-KDEL的NIH 3T3 TetON细胞的mRNA转录谱。实验采用两种底物刚度（1 kPa与308 kPa），部分样本使用Y27632或blebbistatin抑制肌球蛋白II活性。共计6组×3次重复，合计18个样本完成分析。