Mitosis-specific mechanosensing and contractile-protein redistribution control cell shape

Abstract

Because cell-division failure is deleterious, promoting tumorigenesis in mammals, cells utilize numerous mechanisms to control their cell-cycle progression. Though cell division is considered a well-ordered sequence of biochemical events, cytokinesis, an inherently mechanical process, must also be mechanically controlled to ensure that two equivalent daughter cells are produced with high fidelity. Given that cells respond to their mechanical environment, we hypothesized that cells utilize mechanosensing and mechanical feedback to sense and correct shape asymmetries during cytokinesis. Because the mitotic spindle and myosin II are vital to cell division, we explored their roles in responding to shape perturbations during cell division. We demonstrate that the contractile proteins myosin II and cortexillin I redistribute in response to intrinsic and externally induced shape asymmetries. In early cytokinesis, mechanical load overrides spindle cues and slows cytokinesis progression while contractile proteins accumulate and correct shape asymmetries. In late cytokinesis, mechanical perturbation also directs contractile proteins but without apparently disrupting cytokinesis. Significantly, this response only occurs during anaphase through cytokinesis, does not require microtubules, and is independent of spindle orientation, but is dependent on myosin II. Our data provide evidence for a mechanosensory system that directs contractile proteins to regulate cell shape during mitosis.

Fig. 1. Progression of a mitotic cell through the stereotypical shape changes of cytokinesis without mechanical load

A. Symmetrical GFP-myosin-II is observed in 57% of unloaded dividing cells. The cell rounded up and the mitotic spindle was centrally positioned (0 s). GFP-myosin-II enriched in the cleavage furrow and the mitotic spindle elongated, positioning each daughter nucleus at opposite poles (58 s). The cleavage furrow constricted (149 s) until a bridge was formed (239 s), which finally severed (357 s). Line scans show the magnitude of GFP-myosin-II; insets show the line position. This sequence corresponds to Sup. Movie 1. B. Asymmetric GFP-myosin-II distribution in early cytokinesis is found in 43% of dividers. Initially, the cell is elongated with the mitotic spindle asymmetrically positioned within the cell (0 s, arrowheads). GFP-myosin-II localized to the polar cortex (arrows; 176 s) furthest from the spindle. As the spindle elongated (189 s and 263 s), myosin-II reoriented to the cleavage furrow, and the cell progressed through symmetric shape changes of cytokinesis (375 s). Line scans revealed the magnitude of the GFP-myosin-II response; insets show the line position. The line scan of the 263 s frame shows the asymmetry of myosin-II in the cleavage furrow cortex (compares to Fig. 1A 149 s). Scale bars, 10 µm. This sequence corresponds to Sup. Movie 2.

Fig. 2. GFP-myosin-II localized in response to mechanical load in cells undergoing cytokinesis

A. A mitotic cell in early cytokinesis (0 s) was captured with the micropipette aspirator (102 s) using a pressure of 0.34 nN/µm². Although a contractile ring had been initially formed (0s), the cell recruited GFP-myosin-II to the pipette (169 s). Under constant pressure, the cell escaped the pipette (249 s). The cell reoriented myosin-II to the equator, reestablished the correct spindle position, and underwent symmetric cytokinesis (1073 s, 1353 s). Line scans show the magnitude of the GFP-myosin-II response; insets show the line position. The 169 s panel can be compared to the 189 s panel in Fig. 1B, which also shows asymmetric myosin-II. This image sequence corresponds to Sup. Movie 3. B. A cell aspirated late in cytokinesis accumulated GFP-myosin-II both to the pipette and furrow. Even under continuous load, the cell divided. Line scan shows the magnitude of the GFP-myosin-II response; insets show the line position. This cell can be contrasted to the 239 s panel in Fig. 1A where there is no polar enrichment of myosin-II. C. An interphase cell was aspirated with a pressure of 0.3 nN/µm² for 26 minutes. GFP-myosin-II was not recruited to the site of the pipette, indicating that GFP-myosin-II recruitment is mitosis-specific. Line scans show the magnitude of the GFP-myosin-II response; insets show the line position. Scale bars in A–C, 10 µm. D–F. Frequency histograms of the distribution of magnitudes of Early (D) and Late (E) cytokinesis and Interphase (F) responses. Responders (Sup. Methods) are shaded dark gray and non-responders are shaded light gray. Both Early and Late distributions were significantly greater than the Interphase responses (Student’s t-test: P < 0.0001). As a note, we have yet to detect a clear correlation between the magnitudes of the applied loads and responses.

Fig. 3. Contractile proteins control cell shape during cytokinesis and redistribute in response to load independent of microtubules

A. The myoII null cell fails to divide under pressure (0.16 nN/µm²). Sup. Movie 4 shows a different myosin-II mutant cell failing cytokinesis under load. B. The myoII null cell divides under pressure (ranging from 0.1–0.2 nN/µm²), producing two grossly asymmetric daughter cells. Scale bar, 10 µm; applies to all panels in A, B. C. Microtubules were inhibited with nocodazole (added at 21 s). Aspiration pressure (0.35 nN/µm²) was applied after the microtubules disappeared (337 s). GFP-myosin-II localized to the pipette without microtubules (arrow, 429 s). This image sequence corresponds to Sup. Movie 6. D. With the pipettes aligned parallel to the spindle axis, crescents (arrows) of myosin-II assembled at each polar cortex. Applied pressure, 0.45 nN/µm². E. Crescents (arrows) of myosin-II assembled on opposing sides of the spindle with two pipettes oriented perpendicularly to the mitotic spindle but placed near one centrosome. Applied pressure, 0.35 nN/µm².

Fig. 4. Mechanical force triggers the re-distribution of contractile proteins

A. Symmetrical vs. asymmetrical shape changes of cytokinesis. The left column shows the symmetrical shape changes of cytokinesis. The right column depicts the asymmetrical shape changes that occur either naturally or that are induced by aspirating the cell. Cells aspirated early during anaphase recruit contractile proteins to the site of aspiration in conjunction with spindle elongation. After escaping the pipette, the cell repositions the spindle centrally and reorients myosin-II and cortexillin-I to the cleavage furrow, progressing through symmetrical cytokinesis. Cells aspirated late in cytokinesis recruited myosin-II both to the furrow and aspirated polar region. Under continuous load, these cells typically complete symmetric division. B. The diagram outlines a proposed mechanical feedback system. In the unloaded (traditional) pathway (shaded gray), spindle signals initiate the process of cytokinesis, recruiting contractile proteins (CP) to the cleavage furrow. These contractile proteins generate force, driving cell shape changes that produce cytokinesis. By applying a mechanical perturbation (load), a mechanical feedback is suggested. Mechanosensors may measure mechanical perturbations directly by measuring molecular scale strain (upper pathway) or indirectly by monitoring cell shape (lower pathway). The essential differences between these two types of mechanosensors are the length-scales and magnitudes of the strains that they detect. The feedback system then leads to the recruitment of contractile proteins, which correct for shape asymmetries.