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Results: 8

1.
FIGURE 2:

FIGURE 2:. From: A mechanosensory system governs myosin II accumulation in dividing cells.

IQGAP2 is sensitive to mechanical stress in a manner dependent on myosin II and cortexillin I. (A) Of the cortexillin I regulators rac1A, IQGAP1, IQGAP2, and cortexillin II expressed in WT or complemented mutant (iqgap1, iqgap2, cortII) strains, only IQGAP2 is recruited to the micropipette in dividing cells. The IQGAP2 response magnitude is significantly higher than that of rac1A, IQGAP1, and cortexillin II (ANOVA: p = 0.001). (B) IQGAP2 does not accumulate at the micropipette in myoII- or cortI-null cells (white arrows; ANOVA: p = 0.008). Note that cortI-mutant cells, which form a lot of blebs, showed strong accumulation of IQGAP2 at the blebs (arrowhead). Scale bars,10 μm.

Yee-Seir Kee, et al. Mol Biol Cell. 2012 April 15;23(8):1510-1523.
2.
FIGURE 5:

FIGURE 5:. From: A mechanosensory system governs myosin II accumulation in dividing cells.

Myosin II cleavage furrow accumulation is significantly reduced in unperturbed iqgap2-null cells. (A) In cells grown on surfaces (without perturbation), GFP-myosin II accumulated at the cleavage furrow at comparable levels in WT and kif12-null cells. (B) GFP-myosin II levels at the cleavage furrow were similar in incenp-rescued cells and incenp-null cells. (C) Images of GFP-myosin II in WT, cortI, iqgap1, iqgap2, and iqgap1/2 cells grown on surfaces. By pairwise analysis of each mutant as compared with the WT control, only the iqgap2 null is significantly lower (Student's t test: p = 0.008). Scale bars, 10 μm. The bar graphs show the mean furrow-to-pole intensity ratio (If/Ip ± SEM), and the sample sizes are listed on the bars.

Yee-Seir Kee, et al. Mol Biol Cell. 2012 April 15;23(8):1510-1523.
3.
FIGURE 3:

FIGURE 3:. From: A mechanosensory system governs myosin II accumulation in dividing cells.

Roles of IQGAP proteins in mechanosensitivity regulation and cortical tension. (A) GFP-myosin II showed WT levels of mechanosensitive accumulation in rac1A/C double mutants, cortII single mutants, and iqgap1/2 double mutants (Student's t test: p = 0.9, 0.1, and 0.07, respectively) but not iqgap2 and cortI single mutants (Student's t test: p < 0.0001 for both cases). GFP-myosin II responsiveness is higher in iqgap1 null cells than in WT (Student's t test: p = 0.01). Although GFP-myosin II recruitment in cortI/II is not statistically different from WT (Student's t test: p = 0.4), the recruitment behavior is not WT like (see Supplemental Figure S2). (B) Rescue of iqgap2 cells with IQGAP2 restored the myosin II mechanosensitive accumulation, whereas IQGAP1 overexpression in WT cells inhibited myosin II mechanosensitive accumulation (Student's t test: p = 0.01). (C) Cortexillin I showed a similar dependence as myosin II on IQGAP function. Cortexillin I accumulated in iqgap1 and iqgap1/2 and cortI/II mutants but not in iqgap2 single-mutant cells (ANOVA: p = 0.0003). Scale bars (A, C), 10 μm. (D) The effective cortical tension of WT, iqgap1, iqgap2, iqgap1/2, cortI, cortII, and cortI/II mutant cells. Each strain is significantly different from WT (and each other; Student's t test: p < 0.0001 for all except cortII, p = 0.006). Sample sizes are listed on the bar graph.

Yee-Seir Kee, et al. Mol Biol Cell. 2012 April 15;23(8):1510-1523.
4.
FIGURE 6:

FIGURE 6:. From: A mechanosensory system governs myosin II accumulation in dividing cells.

Mechanically challenged cells require kif12, INCENP, cortexillin I, and IQGAP2 for normal myosin II accumulation. (A) On agarose overlay, the myosin II levels at the cleavage furrow increased threefold in WT and kif12-rescued cells (as compared with unperturbed cells; Figure 5A). This amplification was lost in kif12-null cells (Student's t test: p = 0.0002). (B) INCENP was required for stress-induced amplification of myosin II accumulation at the cleavage furrow cortex. Under agarose overlay, the rescued incenp cell line showed threefold higher myosin II accumulation than the incenp-null cells had (Student's t test: p = 0.001). (C) WT cells treated with the DMSO carrier and WT cells treated with 10 μM nocodazole showed comparable accumulation of myosin II in response to agarose overlay. The spindle was confirmed to be disrupted by tracking RFP-tubulin. (D) Except for iqgap1- and iqgap2-rescued cells, all mutants showed significant differences in the GFP-myosin II If/Ip ratios as compared with WT cells. Statistical analysis was performed using an ANOVA with a Student Neuman–Keuls post hoc test. The p values are indicated in the inset, and level of significance is indicated with asterisks. Scale bars, 10 μm. The bar graphs show the mean furrow-to-pole intensity ratio (If/Ip ± SEM), and the sample sizes are listed on the bars.

Yee-Seir Kee, et al. Mol Biol Cell. 2012 April 15;23(8):1510-1523.
5.
FIGURE 4:

FIGURE 4:. From: A mechanosensory system governs myosin II accumulation in dividing cells.

Mechanosensitive and cleavage furrow accumulation of kif12 and normal spindle morphology requires IQGAP2. (A) GFP-kif12 recruitment was deficient in iqgap2- and iqgap1/2-null cells. WT control data were redrawn from Figure 1A. The difference between the WT control and the iqgap single and double mutants was significant (ANOVA: p < 0.0001). (B) WT and iqgap1-null cells had normal spindle morphology and cleavage furrow localization of GFP-kif12 (white arrows). The iqgap2- and iqgap1/2-null cells had disrupted spindle morphology, and GFP-kif12 furrow localization was reduced or absent. Scale bars (A, B), 10 μm. (C) The quantification of the intensity ratio of GFP-kif12 at the furrow to polar cytoplasm (If/Ip cyto). The If/Ip cyto values in WT and iqgap1 cells were indistinguishable (Student's t test: p = 0.44). The intensity ratio was significantly lower in iqgap2 (Student's t test: p = 0.03) and iqgap1/2 (Student's t test: p = 0.02) cells as compared with WT cells. (D) The percentage of iqgap2 and iqgap1/2 null cells with normal spindles was much lower than that in WT and iqgap1 (comparison of proportions: p ≤ 0.0006). (E) The iqgap2 and iqgap1/2 mutants were deficient in cytokinesis, as the percentage of cells completing cytokinesis was much lower than for WT and iqgap1-null cells (comparison of proportions: p ≤ 0.006). The sample sizes for C–E are listed on the bar graphs. (F) Cartoon summarizes data from this figure and Figures 1–3. Mechanical stress detected by the myosin II/cortexillin I mechanosensor is transduced through IQGAP2 to kif12 and INCENP. IQGAP2 is also important for maintaining normal spindle morphology and is required to suppress IQGAP1, which inhibits mechanosensing.

Yee-Seir Kee, et al. Mol Biol Cell. 2012 April 15;23(8):1510-1523.
6.
FIGURE 7:

FIGURE 7:. From: A mechanosensory system governs myosin II accumulation in dividing cells.

WT myosin II lever arm determines cleavage furrow cortex concentration and furrow ingression dynamics. (A) Citrine (CIT)-labeled WT and ΔBLCBS myosin II (expressed in GFP-tubulin-labeled myoII-null cells) accumulated at the cleavage furrow under agarose overlay, but ΔBLCBS did not integrate as tightly at the lateral edges of the cleavage furrow cortex. Line scans at the line in the fluorescence images show the distribution of myosin II in the cortex vs. the central region of these furrows. Scale bar, 10 μm. A comparison by confocal microscopy is presented in Supplemental Figure S5A. (B) The bar graph shows the quantification of the mean furrow-to-pole intensity ratio of (If/Ip) of CIT-WT and CIT-ΔBLCBS myosin II (Student's t test: p = 0.77). (C) The bar graph shows the quantification of the mean ratio of lateral furrow cortex to furrow center (If/If center) of CIT-WT and ΔBLCBS myosin II (Student's t test: p = 0.0004). Sample sizes are shown on the bar graphs in B and C. (D, E) The graphs depict the furrow-thinning dynamics of myoII-null cells (black line) and myoII-null cells complemented with a CIT-labeled WT myosin II (gray line), long-lever-arm 2xELC myosin II (light gray line), and the short-lever-arm ΔBLCBS myosin II. (D) The WT and 2xELC myosin II, which had indistinguishable furrow ingression dynamics, whereas myoII cells showed accelerated furrow thinning. (E) Individual ΔBLCBS furrow ingression dynamics were similar to those of myoII-null cells, but they failed to collapse onto a single universal curve. Unaveraged myoII-null, WT, and 2xELC curves are given in Supplemental Figure S5, B–D.

Yee-Seir Kee, et al. Mol Biol Cell. 2012 April 15;23(8):1510-1523.
7.
FIGURE 8:

FIGURE 8:. From: A mechanosensory system governs myosin II accumulation in dividing cells.

Model for how a control system regulates CM recruitment at sites of mechanical stress, including the cleavage furrow. (A) The mitotic spindle (red line) elongates as the symmetry of the round, dividing cell is broken and the contractile machinery (CM) composed of cortexillin I and myosin II begins to accumulate at the mid zone, initiating furrow formation (green patch at the cleavage furrow; see C for detail view). On micropipette aspiration, CM, which constitutes the mechanosensor module, is recruited to the mechanically stressed cortex (green patch inside the micropipette; see B for a detailed view). (B) Mechanical stress—for example, by micropipette aspiration—recruits CM, which generates contractile stress to counteract the applied mechanical stress. The mechanical stress detected by the mechanosensor then leads to the accumulation of spindle signaling proteins. The mitotic spindle is dispensable for both CM mechanosensing and the mechanosensitive accumulation of spindle signaling proteins. Other mechanisms, such as diffusion, active transport, and/or cortical receptor (R) binding, also contribute to CM accumulation. (C) During cleavage furrow ingression, CM is recruited to the cleavage furrow cortex by spindle signals from spindle microtubules (MTs) and CM mechanosensing induced by mechanical stress such as from contractility, which acts against the resistive stress at the furrow. Diffusion and active transport and/or cortical receptor binding also contribute to CM accumulation. If the MTs are disrupted, myosin II can still be recruited to the cleavage furrow. The control system may also be engaged by the mechanical stress generated by the CM as it pulls against the viscoelastic cortex. (D) Schematic, depicting the control system that tunes the level of myosin II at the cleavage furrow cortex. The system may be activated by the spindle and/or mechanical stress. Cortexillin I and myosin II define the core mechanosensor. IQGAP2 antagonizes IQGAP1 to maintain an active mechanosensory module, and IQGAP2 mediates mechanosensitive localization of kif12 and INCENP. IQGAP2 also plays a role in maintaining morphology of the mitotic spindle.

Yee-Seir Kee, et al. Mol Biol Cell. 2012 April 15;23(8):1510-1523.
8.
FIGURE 1:

FIGURE 1:. From: A mechanosensory system governs myosin II accumulation in dividing cells.

Kinesin-6 (kif12) and INCENP are directed to cortical regions of high mechanical stress in a myosin II–dependent and microtubule-independent manner but are not required for myosin II mechanosensitive accumulation. (A) GFP-kif12 and GFP-INCENP, but not GFP-aurora, localized to sites of mechanical deformation induced by micropipette aspiration of dividing WT or rescued cells (arrows). The graph shows the GFP-tagged protein response (log[Ip/Io], where Ip is the intensity in the pipette and Io is the intensity of the opposite cortex) in WT and rescued cells. The distributions of GFP-kif12 and GFP-INCENP response intensity ratio in WT and rescued cells are significantly higher than in WT cells expressing either GFP or mCherry alone (Student's t test: p < 0.0001) and cells expressing GFP-aurora (Student's t test: p = 0.003). The distribution of GFP-aurora is not different from that of GFP/mCherry (Student's t test: p = 0.14). Each dot represents an aspirated cell. The numbers in the differential interference contrast images represent the start of the movie (time 0 s) and the time of the response after the pressure was applied. (B) GFP-kif12 accumulated at the micropipette independent of microtubules but dependent on myosin II. Cells were treated with 10 μM nocodazole (NOC), and the RFP-tubulin was monitored to confirm that the mitotic spindle was disrupted. The response distribution of GFP-kif12 in myoII-null cells is significantly lower than in the untreated (reproduced from A) and 10 μM nocodazole-treated cells (ANOVA: p < 0.0001). Note that the bright spherical structures shown in GFP-kif12 images are the centrosomes. (C) GFP-myosin II mechanosensitive accumulation occurred similarly in WT vs. kif12-null cells and incenp-rescued vs. incenp-null cells. The distributions are statistically indistinguishable (WT and kif12 null, Student's t test: p = 0.22; incenp rescued and incenp null, Student's t test: p = 0.53). Scale bars (A–C), 10 μm. (D) The effective cortical tension (Teff) of kif12 and incenp null cells was only slightly reduced compared with WT and GFP-INCENP–rescued controls (Student's t test: p = 0.08 and 0.67, respectively). Sample sizes are listed on the bar graph. (E) Cartoon summarizes the data in this figure. Mechanical stress directs the recruitment of myosin II and cortexillin I, which in turn are required to direct mechanosensitive accumulation (mechanotransduction) of kif12 and INCENP.

Yee-Seir Kee, et al. Mol Biol Cell. 2012 April 15;23(8):1510-1523.

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