Determinants of myosin II cortical localization during cytokinesis

Abstract

Myosin II is an essential component of the contractile ring that divides the cell during cytokinesis. Previous work showed that regulatory light chain (RLC) phosphorylation is required for localization of myosin at the cellular equator. However, the molecular mechanisms that concentrate myosin at the site of furrow formation remain unclear. By analyzing the spatiotemporal dynamics of mutant myosin subunits in Drosophila S2 cells, we show that myosin accumulates at the equator through stabilization of interactions between the cortex and myosin filaments and that the motor domain is dispensable for localization. Filament stabilization is tightly controlled by RLC phosphorylation. However, we show that regulatory mechanisms other than RLC phosphorylation contribute to myosin accumulation at three different stages: (1) turnover of thick filaments throughout the cell cycle, (2) myosin heavy chain-based control of myosin assembly at the metaphase-anaphase transition, and (3) redistribution and/or activation of myosin binding sites at the equator during anaphase. Surprisingly, the third event can occur to a degree in a Rho-independent fashion, gathering preassembled filaments to the equatorial zone via cortical flow. We conclude that multiple regulatory pathways cooperate to control myosin localization during mitosis and cytokinesis to ensure that this essential biological process is as robust as possible.

Figure 1. Thick filament assembly and RLC phosphorylation, but not actin binding are necessary for myosin stabilization at the cortex

(A) Schematic representation of five versions of the myosin heavy chain protein. All deletions but the ΔC construct possess the full domain for self assembly which has been described in [9]. IQ domain consists of ELC-binding domain (ELC) and RLC-binding domain (RLC). (B) The full-length, and GFP-ΔMotor proteins showed enrichment at the equatorial cortex during anaphase (indicated by yellow arrowheads), but the GFP-ΔC, GFP-ΔN, and GFP-1350–1940 proteins did not show cortical accumulation at the equator of anaphase S2 cells. See also Figure S1A and Movie S1. Bars, 5 μm. (C) The phosphomimetic RLC[E20E21]-GFP concentrates on the cortex prior to anaphase onset but does not localize to the equator until anaphase onset.

Figure 2. Stable association of myosin to the equatorial cortex after anaphase is driven by RLC phosphorylation

(A) GFP-myosin localization from metaphase to telophase. We divided anaphase/telophase into three phases according to the ratio of cell width over length. (B) FRAP of RLC-GFP and RLC[E20E21]-GFP. We observed slower recovery rates as anaphase progresses for both constructs and at each stage the recovery of the phosphomimetic RLC was slower. Error bars represent SEM. Observed cell number is indicated for each sample. (C) Mean recovery rate of RLC-GFP and RLC[E20E21]-GFP in metaphase and early anaphase, with SEM. See also Figure S1.

Figure 3. Preassembled myosin filaments can target the equatorial cortex in the absence of Rho

(A) An S2 cell expressing RLC-GFP and mCh-tubulin was plated on a conA coated dish and then imaged from metaphase until late anaphase using TIRF microscopy. (B) Cells expressing only the RLC[E20E21]-GFP and mCh-tubulin were imaged as above. (C) Cells expressing the RLC-GFP were depleted of Rho and imaged in TIRF. In 20% of cells some transient bursts of myosin filament formation were observed. However, in 80% of cells no myosin filament formation was observed (n > 10). (D) In cells depleted of Rho and expressing exclusively the phosphomimetic RLC[E20E21]-GFP, higher levels of cortical myosin are observed prior to anaphase and the protein slowly enriches at the equatorial cortex after anaphase (indicated by yellow arrows) See also Figures S2, S3, and S4 and Movies S2, S3, and S4. Bar, 10 μm.

Figure 4. A model for myosin regulation during mitosis and cytokinesis

(A) During mitosis myosin phosphatase (via dephosphorylating the RLC, red ovals) and an unknown mechanism (via and interaction with the MHC) both work to keep myosin as single myosin molecules. At the metaphase to anaphase transition, both inhibitors of filament assembly are inactivated. Then when myosin reaches the equatorial cortex, the activity of ROCK phosphorylates the RLC (green ovals) to locally stimulate filament formation. (B) Localized activation of filament assembly and binding to the cortex and the rest of the cytokinetic machinery occurs at equatorial sites that correspond to the zones of microtubule attachment and overlap. Then after the formation of filaments occurs, cortical flow can drive any peripheral myosin molecules and filaments to the equator.