A Haspin-ARHGAP11A axis regulates epithelial morphogenesis through Rho-ROCK dependent modulation of LIMK1-Cofilin

Abstract

Throughout mitosis, a plethora of processes must be efficiently concerted to ensure cell proliferation and tissue functionality. The mitotic spindle does not only mediate chromosome segregation, but also defines the axis of cellular division, thus determining tissue morphology. Functional spindle orientation relies on precise actin dynamics, shaped in mitosis by the LIMK1-Cofilin axis. The kinase Haspin acts as a guardian of faithful chromosome segregation that ensures amphitelic chromosome attachment and prevents unscheduled cohesin cleavage. Here, we report an unprecedented role for Haspin in the determination of spindle orientation in mitosis. We show that, during mitosis, Haspin regulates Rho-ROCK activity through ARHGAP11A, a poorly characterized GAP, and that ROCK is in turn responsible for the mitotic activation of LIMK1 and stabilization of the actin cytoskeleton, thus supporting a functional spindle orientation. By exploiting 3D cell cultures, we show that this pathway is pivotal for the establishment of a morphologically functional tissue.

Keywords: Cell biology; Molecular biology.

Graphical abstract

Figure 1

Haspin regulates the actin cytoskeleton to sustain functional spindle orientation (A) HeLa cells were seeded on given substrates and transfected with either control or haspin-targeting siRNAs. Cells were then synchronized in mitosis either by nocodazole or RO-3306 block followed by 1 h release, before being processed to visualize DNA and microtubules. Spindles were counted as misaligned when the two MTOCs (white arrowhead) were on different focal planes. Images were acquired at a confocal microscope, single XY stack and resliced XZ maximum projection are shown; arrowheads point at centrosomes; scale bar: 5μm. The angle between the spindle and the substrate is shown. (B) Cells treated as in A were acquired in confocal microscopy, taking images every 0.5μm, to measure the angle of the spindle compared to the substrate, as shown in Figure S1A. (C) HeLa cells were treated as in panel A, staining the actin cytoskeleton with phalloidin. Arrowheads point at sites of aberrant (increased) actin distribution. Quantification is shown on the right; scale bar: 10μm. Experiments were performed at least three times. Error bars in graphs represent standard deviation, statistical analysis: T-test, significancy: n.s.: not significant; ∗p.value < 0.05; ∗∗p.value < 0.01; ∗∗∗p.value < 0.005; ∗∗∗∗p.value < 0.001.

Figure 2

Haspin stimulates LIMK1 activity to inhibit Cofilin Protein levels were analyzed by western blotting with specific antibodies as indicated. HeLa cells were seeded on fibronectin-coated plates, synchronized by nocodazole treatment and: (A) transfected with either Luc- or Haspin-targeting siRNAs (48 h before sample collection); (B and C) inhibited for Haspin by incubation with either 10nM 5-Itu or 50nM CHR-6494 for 1 h before sample collection (see Figure S2A for cell-cycle analyses) or (D) transfected with GFP or Haspin-Venus coding plasmids 24 h before nocodazole treatment (for cell synchronization controls, refer to Figure S2B). For all the experiments, isolation of mitotic cells was achieved by mitotic shake off at the end of nocodazole treatment. Experiments were performed three times (A and C), five times (B) or two times (D). Cofilin-Ser3p/Cofilin (panels A, B, and D) or LIMK1-Thr508p/LIMK1 (panels C and D) ratios are reported.

Figure 3

A Haspin-ARHGAP11A-Rho-ROCK axis modulates mitotic Cofilin phosphorylation Protein levels were analyzed by western blotting with specific antibodies as indicated. HeLa cells seeded on fibronectin-coated plates were treated as follows. (A and B) Cells were synchronized at the G2/M transition by RO-3306 treatment and released for 1 h in the absence or presence of 10μM ROCK inhibitor Y-27632. Cells were then processed to visualize actin cytoskeleton (by Alexa Fluor-phalloidin) and quantitate cells with defective actin organization (A; scale bar: 10μm) or monitor the phosphorylation status of Cofilin (B). Arrowheads in A point at sites of aberrant (increased) actin distribution. (C–E) isolation of mitotic cells was achieved by nocodazole treatment followed by mitotic shake off; silencing of given proteins was achieved by transfecting siRNAs 24 h before nocodazole treatment. Chemical inhibition of ROCK (Y-27632) and LIMK1 (BMS4) in D was performed adding the drugs 1 h before sample collection. Experiments were performed three times (A,C,E) or two times (B,D). Error bars in graphs represent standard deviation, statistical analysis: T-test, significancy: n.s.: not significant; ∗p.value < 0.05; ∗∗p.value < 0.01; ∗∗∗p.value < 0.005; ∗∗∗∗p.value < 0.001. Cofilin-Ser3p/Cofilin ratio is reported in panels B–E.

Figure 4

Impairments in Haspin activity cause tissue alterations and development issues (A) 3D Caco2 cultures grown in a Matrigel-collagen matrix were incubated for 12 days in the presence of either DMSO, 10nM 5-Itu or 50nM CHR-6494, adding 0.1 mg/ml cholera toxin for the last 12 h, before being stained with Hoechst 33342 and analyzed by fluorescent and brightfield microscopy. Panel A shows representative examples, green and red arrowheads point to single lumen or multilumen cysts, respectively; scale bar: 100μm. (B) Plot shows the percentage of multilumen cysts in control or Haspin-inhibited conditions. Experiment was performed two times. Error bars in graphs represent standard deviation, statistical analysis: T-test, significancy: n.s.: not significant; ∗p.value < 0.05; ∗∗p.value < 0.01; ∗∗∗p.value < 0.005; ∗∗∗∗p.value < 0.001.

Figure 5

Haspin activity is required for spindle orientation and a successful mitosis In mitosis, Haspin negatively regulates ARGHAP11A, preventing its excessive accumulation (blue text). This results in a buildup of Rho activity that, through the ROCK-LIMK1 axis promotes the inhibitory phosphorylation of Cofilin, stabilizing the cortical actin cytoskeleton and supporting a functional orientation of the mitotic spindle. Together with its established in monitoring amphitelic attachment of the chromosomes and in preventing unscheduled cohesin cleavage (gray text), these findings depict Haspin as a central player in mammalian cell mitosis to orchestrate not only an even distribution of the genetic material between daughters, but also maintaining a functional tissue organization.