The mammalian LINC complex regulates genome transcriptional responses to substrate rigidity

Abstract

Mechanical integration of the nucleus with the extracellular matrix (ECM) is established by linkage between the cytoskeleton and the nucleus. This integration is hypothesized to mediate sensing of ECM rigidity, but parsing the function of nucleus-cytoskeleton linkage from other mechanisms has remained a central challenge. Here we took advantage of the fact that the LINC (linker of nucleoskeleton and cytoskeleton) complex is a known molecular linker of the nucleus to the cytoskeleton, and asked how it regulates the sensitivity of genome-wide transcription to substratum rigidity. We show that gene mechanosensitivity is preserved after LINC disruption, but reversed in direction. Combined with myosin inhibition studies, we identify genes that depend on nuclear tension for their regulation. We also show that LINC disruption does not attenuate nuclear shape sensitivity to substrate rigidity. Our results show for the first time that the LINC complex facilitates mechano-regulation of expression across the genome.

Figure 1. The LINC complex exerts control over the transcriptome.

(A) Inducible perturbation of the LINC-complex by SUN1L-KDEL. NIH 3T3 TetON cells were induced to express either SS-GFP-KDEL (control, KDEL) or SS-HA-SUN1L-KDEL (SUN1L) by the addition of doxycycline (+Dox). Untreated cells received no doxycycline (−Dox). When expressed, only the SUN1L but not the KDEL control (both green) led to loss of nesprin-3 (red) from the nuclear envelope. DNA is labeled with Hoechst dye (blue) in the merged image. Bar, 30 μm. (B) Volcano plots of statistical significance versus fold change between KDEL and SUN1L cells on 1-kPa (left) and 308-kPa (right) substrates; significantly differentially expressed genes are shown in red (downregulated) and green (upregulated). The horizontal line indicates the significance threshold, p = 0.05.

Figure 2. LINC disruption affects genes differently on soft versus stiff substrates.

(A) Venn diagram summarizing counts of genes that were significantly differentially expressed (p < 0.05; |fold change| >2) between KDEL and SUN1L samples on 1 and 308 kPa substrates. (B) Heat maps depicting statistically significant gene expression changes upon LINC complex disruption for selected genes for cells cultured on different substrate rigidities. (C) Results of a DAVID analysis showing selected significantly enriched biological processes for genes that were differentially expressed upon LINC complex disruption on soft and stiff substrates. (D) Upon LINC disruption, a few genes gained mechanosensitivity (ratio, blue solid circles), other genes lost mechanosensitivity (, yellow solid circles) and the majority of genes reversed the direction of mechanosensitivity (, red solid circles). Mechanosensitivity is calculated as the ratio of the expression levels (EL) between the two substrate rigidities as shown.

Figure 3. Myosin tension-dependent genes that require an intact LINC complex for their regulation.

(A) Shown are Venn diagrams summarizing counts of genes that were significantly differentially expressed (p < 0.05; fold change >2 (left) and <−2 (right)) in a similar direction upon LINC complex disruption (red), non-muscle myosin II inhibition with blebbistatin (blue), and inhibition of Rho-kinase activity with Y27632 (green). (B) Intersection of the list of interacting genes (gray) with each of the three perturbations in A: LINC disruption (red), Y27 (green) and blebbistatin (blue) treatment.

Figure 4. Nuclear shape sensitivity to substrate rigidity is weakly dependent on the LINC complex.

(A) Confocal images of fixed KDEL (top) and SUN1L (bottom) cells on 1-kPa (left) and 308-kPa (right) substrates, stained with phalloidin for F-actin (red) and Hoechst for DNA (blue), showing the cell and nuclear shapes under various conditions. LINC complex disruption caused minor nuclear shape changes and did not eliminate the nuclear shape sensitivity to substrate rigidity. Bar, 20 μm (x-y view) and 5 μm (x-z view). (B) Bar graphs of nuclear height (top) and nuclear aspect ratio (bottom) of KDEL and SUN1L cells on 1 and 308 kPa gels showing that disruption of the LINC complex does not alter nuclear shape considerably. Error bars represent standard error of the mean (s.e.m.). n ≥ 30 for each condition.