Interactions between myosin and actin crosslinkers control cytokinesis contractility dynamics and mechanics

Abstract

Introduction: Contractile networks are fundamental to many cellular functions, particularly cytokinesis and cell motility. Contractile networks depend on myosin-II mechanochemistry to generate sliding force on the actin polymers. However, to be contractile, the networks must also be crosslinked by crosslinking proteins, and to change the shape of the cell, the network must be linked to the plasma membrane. Discerning how this integrated network operates is essential for understanding cytokinesis contractility and shape control. Here, we analyzed the cytoskeletal network that drives furrow ingression in Dictyostelium.

Results: We establish that the actin polymers are assembled into a meshwork and that myosin-II does not assemble into a discrete ring in the Dictyostelium cleavage furrow of adherent cells. We show that myosin-II generates regional mechanics by increasing cleavage furrow stiffness and slows furrow ingression during late cytokinesis as compared to myoII nulls. Actin crosslinkers dynacortin and fimbrin similarly slow furrow ingression and contribute to cell mechanics in a myosin-II-dependent manner. By using FRAP, we show that the actin crosslinkers have slower kinetics in the cleavage furrow cortex than in the pole, that their kinetics differ between wild-type and myoII null cells, and that the protein dynamics of each crosslinker correlate with its impact on cortical mechanics.

Conclusions: These observations suggest that myosin-II along with actin crosslinkers establish local cortical tension and elasticity, allowing for contractility independent of a circumferential cytoskeletal array. Furthermore, myosin-II and actin crosslinkers may influence each other as they modulate the dynamics and mechanics of cell-shape change.

Figure 1

Actin filaments are organized into a meshwork and actin and myosin-II are not enriched in a uniform ring as revealed by platinum-shadowed transmission electron microscopy (Pt-TEM; panel A), 3D-electron tomography (3D-EM; panel B), 3D-deconvolution (3D-decon, panel C-E), and total internal reflection fluorescence (TIRF; panel F, lower images) microscopy. A. The actin network is observed at the furrow of a wild type cell by Pt-TEM. Scale bars, 2 μm and 500 nm. B. Rotated 3D-EM images of a model of a 0.5-μm section (derived from combining two adjacent sections) of the lower surface of a cleavage furrow reveal disordered actin filaments. Mitochondria are green, vesicles are cyan, plasma membrane is blue, and the actin filaments are yellow. The first 2 panels show the furrow model viewed from top and bottom, respectively. The third panel is the furrow viewed down the long axis of the furrow. Scale bar, 2μm, applies to all panels. The z-series of the raw EM data can be found in Sup. Movie S1. The corresponding movie of the model can be found at Sup. Movie S2. C. Non-uniform cleavage furrow cortical actin. TRITC-phalloidin staining of filamentous actin in a wild type cell. Inset shows a cross-section of the furrow actin where the actin is enriched along the lateral surface. Scale bar, 10 μm applies to all images. Equatorial localization of GFP-myosin-II and binucleation (DAPI) confirms cell was undergoing cytokinesis prior to fixation. D, E. Non-circumferential distribution of myosin-II thick filaments. Wild type cells expressing GFP-tubulin and GFP-myosin-II reveal that, like actin, myosin-II does not form a continuous ring at the cleavage furrow. D. Early stage dividing cell. E. Late stage dividing cell. The C-S images show the cross-sectional fluorescence intensities of the furrow. F. Epi-fluorescence and TIRF images indicate that myosin-II is not circumferentially-oriented at the basal region of the furrow. Note that at later stages of cytokinesis, the furrow lifts from the surface.

Figure 2

Wild type myosin-II mechanochemistry is required for wild type interphase mechanics, but not cytokinesis mechanics and kinetics. A. Comparison of furrow-thinning trajectories in cells expressing wild type myosin-II or myosin-II S456L to myoII null cells shows that S456L is able to fully restore the uniform furrow-thinning kinetics of wild type dividing cells. Note that myoII null cells have a faster furrow-thinning rate at later stages of division. B.-D. Expression of myosin-II S456L in myoII cells does not fully recover wild type cellular mechanics during interphase. B. MyoII cells have lower viscoelasticity (|G*|) than wild type cells as measured by LTM. C, D. MyoII cells are more deformable (panel C) and have a lower effective cortical tension (panel D) as measured by MPA. The S456L cells have cortical mechanics similar to wild type cells at longer time-scales (10 rad/s, panel B) and smaller deformations (lower L_p/R_p, panel C) but are more like myoII cells at shorter time-scales (>10² rad/s, panel B) and larger deformations (larger L_p/R_p, panel C). E. Representative micrographs showing cells aspirated at metaphase and during cytokinesis at the pole and furrow. For the polar cortex, we aspirated at angles ranging from parallel to perpendicular to the spindle axis with no detectable differences in the level of deformability. F. The degree of deformability of wild type interphase and metaphase cells was not significantly different. During anaphase, the furrow was slightly less deformable than during metaphase, while the pole was more deformable than the furrow or metaphase cortices. G. Conversely, the furrow and pole of myoII cells were not significantly different from each other and both regions were much more deformable than the polar region of wild type cells. H. S456L reduces the level of deformability of the furrow and polar regions to wild type levels. Error bars indicate standard error of the mean. Sample sizes for panel D are shown on the histograms. Samples sizes for A are provided in Table S2; sample sizes for panel B are shown in the histograms in Fig. S3, and the calculated E values and sample sizes for C and F-H are provided in Table S3.

Figure 3

Dynacortin has a greater contribution to cortical mechanics and furrow-thinning kinetics compared to fimbrin. A. Removal of dynacortin and/or myosin-II reduces the viscoelasticity (|G*|) of interphase cells as measured by LTM. B. Likewise, myosin-II and dynacortin contribute to the effective tension (T_eff) as measured by MPA. C. Removal of fimbrin reduces the viscoelasticity (|G*|) of interphase cells as measured by LTM. D. In wild type cells, fimbrin does not contribute to cortical tension as measured by MPA, but it does contribute significantly in a myoII null background. Both the fimbrin knockout strain (fimbrin) and fimbrin RNAi (fimhp) strains have the same effects. The control for fimbrin is the parental strain and the control cells for the fimhp expressing cells are wild type cells carrying the empty vector. E. In a myoII null background where fimbrin has a significant mechanical contribution on longer time-scales (MPA), reduction of fimbrin or dynacortin increases the rate of furrow ingression compared to control cytokinesis. However, dynacortin has a greater contribution to the furrow-thinning kinetics than fimbrin has. Error bars, standard error of the mean.

Figure 4

Model for cytokinesis cell shape change through the contraction of an actin meshwork: myosin-II and actin crosslinkers interact to control furrow ingression dynamics, equatorial and polar cortical tension, and crosslinker lifetimes. Here, the equatorial cortex is principally controlled by myosin-II and cortexillin. The global/polar cortex is modulated by dynacortin, fimbrin, and myosin-II. The local increased cortical tension (S_cf>S_cp) by myosin-II generates equatorial stresses (orange arrows) that help squeeze cytoplasm from the furrow region whereas the globally distributed crosslinkers generate resistive stresses (green arrows) that slow furrow ingression (this paper and [13]). The equatorial crosslinker cortexillin-I and equatorial populations of fimbrin and dynacortin (represented as red ellipses) persist much longer at the cortex, perhaps contributing to the increased tension in this region. Conversely, polar actin crosslinkers (fimbrin and dynacortin represented as blue ellipses) release from the network on fast time-scales, making the global cortex more deformable. This system of myosin-II and equatorial and global actin crosslinkers generates the stress differential that drives and controls the dynamics of cytokinesis cell shape change.