Microtubule Assists Actomyosin to Regulate Cell Nuclear Mechanics and Chromatin Accessibility

Abstract

Cellular behaviors and functions can be regulated by mechanical cues from microenvironments, which are transmitted to nucleus through the physical connections of cytoskeletons in the cells. How these physical connections determine transcriptional activity were not clearly known. The actomyosin, which generates intracellular traction force, has been recognized to control the nuclear morphology. Here, we have revealed that microtubule, the stiffest cytoskeleton, is also involved in the process of nuclear morphology alteration. The microtubule negatively regulates the actomyosin-induced nuclear invaginations but not the nuclear wrinkles. Moreover, these nuclear shape changes are proven to mediate the chromatin remodeling, which essentially mediates cell gene expression and phenotype determination. The actomyosin disruption leads to the loss of chromatin accessibility, which can be partly recovered by microtubule interference through nuclear shape control. This finding answers the question of how mechanical cues regulate chromatin accessibility and cell behaviors. It also provides new insights into cell mechanotransduction and nuclear mechanics.

Fig. 1.

Actomyosin and microtubule synergize to regulate nuclear invagination. (A) Scheme of nuclear invagination and wrinkle. (B) Representative immunofluorescence images for lamin A/C, microtubule, and filamentous actin (F-actin) of the cells treated in shown conditions. (C and D) The percentage of the invaginated and wrinkle nucleus, respectively (n = 50, 3 technical replicates). (E) Scheme of the microtubule disruption for regulating nuclear morphology after actomyosin inhibition. The green represents actomyosin, the red represents microtubule, and the yellow represents nuclei. When the actomyosin-based traction force was inhibited by either blebbistatin or soft hydrogels, obvious invaginations and wrinkles appeared on the nuclei. After disrupting microtubule or disconnecting the link between microtubule and nucleus, the nuclear invagination, but not the wrinkle, got recovered. ns, not significant.

Fig. 2.

Actomyosin but not microtubule regulates nuclear wrinkle. (A) Representative immunofluorescence images for lamin A/C, microtubule, and F-actin of the cells treated in shown conditions. (B and C) The percentage of the invaginated and wrinkle nucleus, respectively (n = 50, 3 technical replicates). (D) Scheme of the microtubule disruption for regulating nuclear morphology after actomyosin–nucleus linkage disruption. The green represents actomyosin, the red represents microtubule, and the yellow represents nuclei. When the actomyosin–nucleus linkage was inhibited, the wrinkles appeared on the nuclear surface independent of microtubule disruption.

Fig. 3.

Intracellular traction force. (A) Representative immunofluorescence images and the (B) fluorescent intensity for the phosphorylation of myosin IIa at Ser1943 of the cells treated in shown conditions (n = 50, 3 technical replicates). (C) The representative traction fields and (D) the mean traction force of a single cell treated in shown conditions on the hydrogels with embedded fluorescent beads (n = 15, 3 technical replicates).

Fig. 4.

Cytoskeleton-induced nuclear deformation mediates chromatin accessibility. (A) Representative heatmaps of the DAPI intensity and the chromatin condensation parameter (CCP) maps estimated by corresponding edge detection method. (B) The average CCP of the cells (n = 15, 3 technical replicates). (C) The summary of the ATAC-seq results of the cells as indicated by the count of narrow peak, the fraction of reads in peaks (FRiP), and the count of summits per group. (D) The chromatin accessibility over the transcription start site (TSS) of the cells. (E) The TF motif enrichment of the cells. (F) The chromatin accessibility for ARHGAP6 in the cells with different treatment.

Fig. 5.

Microtubule regulates the early markers of cell osteogenic differentiation, apoptosis, and proliferation. The ALP staining was performed after cell culturing for 7 d in growth media with shown treatment. Other immuno-fluorescent images were acquired after cell culturing for 1 d. (A) Representative immunofluorescence images and the (B) fluorescent intensity for osterix of the cells treated in shown conditions. (C) Representative images for ALP staining and (D) the percentage of ALP-positive cells after cell culturing for 7 d in growth media with shown treatment. (E) Representative immunofluorescence images and (F) the fluorescent intensity for p53 of the cells treated in shown conditions. (G) The activity of apoptotic downstream enzyme caspase 3 of the cells treated in shown conditions. (H) Representative immunofluorescence images for Ki67 of the cells treated in shown conditions (enlarged images are shown in Fig. S6). (I) The percentage of Ki67-positive cells treated in shown conditions. (J) The cell counting kit 8 (CCK8) analysis of the cells treated in shown conditions. Each statistical result includes more than 50 cells, 3 technical replicates.

Fig. 6.

Schematic summary of how actomyosin and microtubule synergistically regulates nuclear deformation and chromatin remodeling. Microtubule negatively regulates the actomyosin-induced nuclear invaginations but not the nuclear wrinkles. The actomyosin disruption leads to the loss of chromatin accessibility, which can be partly recovered by microtubule disruption without affecting cell force.