Shigehiko Yumura

Myosin II dynamics and cortical flow during contractile ring formation in Dictyostelium cells

Myosin II is a major component of a contractile ring. To examine if myosin II turns over in contractile rings, fluorescence of GFP–myosin II expressed in Dictyostelium cells was bleached locally by laser illumination, and the recovery was monitored. The fluorescence recovered with a half time of 7.01 ± 2.62 s. This recovery was not caused by lateral movement of myosin II from the nonbleached area, but by an exchange with endoplasmic myosin II. Similar experiments were performed in cells expressing GFP–3ALA myosin II, of which three phosphorylatable threonine residues were replaced with alanine residues. In this case, recovery was not detected within a comparable time range. These results indicate that myosin II in the contractile ring performs dynamic turnover via its heavy chain phosphorylation. Because GFP–3ALA myosin II did not show the recovery, it served as a useful marker of myosin II movement, which enabled us to demonstrate cortical flow of myosin II toward the equator for the first time. Thus, cortical flow accompanies the dynamic exchange of myosin II during the formation of contractile rings.

Stretching actin filaments within cells enhances their affinity for the myosin ii motor domain

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli.

Recruitment of a myosin heavy chain kinase to actin-rich protrusions in Dictyostelium.

Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives the disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin-dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading-edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion.

Actin-based propulsive forces and myosin-II-based contractile forces in migrating Dictyostelium cells

It has been suggested that myosin II exerts traction forces at the posterior ends and retracting pseudopodia of migrating cells, but there is no direct evidence. Here, using a combination of total internal reflection fluorescence (TIRF) microscopy and force microscopy with a high spatial resolution of ∼400 nm, we simultaneously recorded GFP-myosin II dynamics and traction forces under migrating Dictyostelium cells. Accumulation of filamentous myosin II and a subsequent increase in traction forces were detected in pseudopodia just before retraction. In the case of motorless myosin II, traction forces did not increase after accumulation, suggesting that the source of the retraction force is the motor activity of accumulated myosin II. Simultaneous recording of F-actin and traction forces revealed that traction forces were exerted under spot-like regions where F-actin accumulated. Cells migrated in a direction counter to the sum of the force vectors exerted at each spot, suggesting that the stress spots act as scaffolds to transmit the propulsive forces at the leading edge generated by actin polymerization.

Myosin II can be localized to the cleavage furrow and to the posterior region of Dictyostelium amoebae without control by phosphorylation of myosin heavy and light chains

To elucidate the role of phosphorylation in regulation of intracellular distribution of myosin II, we have characterized mutant Dictyostelium cells expressing myosin II that could not be regulated by the phosphorylation on the mapped heavy chain sites, the light chain site, or both sites. Immunofluorescence microscopy demonstrated that all three mutant myosin IIs were localized in the furrow region of dividing cells and in the tail region of migrating cells, similar to wild-type cells. Thus, regulation by phosphorylation is not required to direct myosin II toward the furrow region and the tail region in Dictyostelium. However, myosins that were deficient in heavy chain phosphorylation were distributed only in the cortical region of interphase cells, whereas some myosin IIs were present throughout the endoplasm in wild-type cells. Video microscopy showed that the rate of cell migration was significantly lower in cells that were deficient in heavy chain phosphorylation- than in light chain phosphorylation-deficient cells, myosin null cells and wild-type cells. Chemotactic behavior of cells that were deficient in heavy chain phosphorylation was also retarded. These results suggest that loss of regulation by heavy chain phosphorylation results in excessive myosin in the cortex, which leads to retarded motility.

PTEN is a mechanosensing signal transducer for myosin II localization in Dictyostelium cells

To investigate the role of PTEN in regulation of cortical motile activity, especially in myosin II localization, eGFP–PTEN and mRFP–myosin II were simultaneously expressed in Dictyostelium cells. PTEN and myosin II co‐localized at the posterior of migrating cells and furrow region of dividing cells. In suspension culture, PTEN knockout (pten−) cells became multinucleated, and myosin II significantly decreased in amount at the furrow. During pseudopod retraction and cell aspiration by microcapillary, PTEN accumulated at the tips of pseudopods and aspirated lobes prior to the accumulation of myosin II. In pten− cells, only a small amount of myosin II accumulated at the retracting pseudopods and aspirated cell lobes. PTEN accumulated at the retracting pseudopods and aspirated lobes even in myosin II null cells and latrunculin B‐treated cells though in reduced amounts, indicating that PTEN accumulates partially depending on myosin II and cortical actin. Accumulation of PTEN prior to myosin II suggests that PTEN is an upstream component in signaling pathway to localize myosin II, possibly with mechanosensing signaling loop where actomyosin‐driven contraction further augments accumulation of PTEN and myosin II by a positive feedback mechanism.

Multiple mechanisms for accumulation of myosin II filaments at the equator during cytokinesis.

Total internal reflection fluorescence microscopy revealed how individual bipolar myosin II filaments accumulate at the equatorial region in dividing Dictyostelium cells. Direct observation of individual filaments in live cells provided us with much convincing information. Myosin II filaments accumulated at the equatorial region by at least two independent mechanisms: (i) cortical flow, which is driven by myosin II motor activities and (ii) de novo association to the equatorial cortex. These two mechanisms were mutually redundant. At the same time, myosin II filaments underwent rapid turnover, repeating their association and dissociation with the actin cortex. Examination of the lifetime of mutant myosin filaments in the cortex revealed that the turnover mainly depended on heavy chain phosphorylation and that myosin motor activity accelerated the turnover. Double mutant myosin II deficient in both motor and phosphorylation still accumulated at the equatorial region, although they displayed no cortical flow and considerably slow turnover. Under this condition, the filaments stayed for a significantly longer time at the equatorial region than at the polar regions, indicating that there are still other mechanisms for myosin II accumulation such as binding partners or stabilizing activity of filaments in the equatorial cortex.

Molecular dynamics and forces of a motile cell simultaneously visualized by TIRF and force microscopies

Cells must exert traction forces onto the substratum for continuous migration. Molecular dynamics such as actin polymerization at the front of the cell and myosin II accumulation at the rear should play important roles in the exertion of forces required for migration. Therefore, it is important to reveal the relationships between the traction forces and molecular dynamics. Traction forces can be calculated from the deformation of the elastic substratum under a migrating cell. A transparent and colorless elastic substratum with a high refractive index (1.40) and a low Youngs modulus (1.0 kPa) were made from a pair of platinum-catalyzed silicones. We used this substratum to develop a new method for simultaneous recording of molecular dynamics and traction forces under a migrating cell in which total internal refractive fluorescence (TIRF) and force microscopies were combined. This new method allows the detection of the spatiotemporal distribution of traction forces produced by individual filopodia in migrating Dictyostelium cells, as well as simultaneous visualization of these traction forces and the dynamics of filamentous myosin II.

De novo formation of basal bodies in Naegleria gruberi : regulation by phosphorylation

The de novo formation of basal bodies in Naegleria gruberi was preceded by the transient formation of a microtubule (MT)-nucleating complex containing γ-tubulin, pericentrin, and myosin II complex (GPM complex). The MT-nucleating activity of GPM complexes was maximal just before the formation of visible basal bodies and then rapidly decreased. The regulation of MT-nucleating activity of GPM complexes was accomplished by a transient phosphorylation of the complex. Inhibition of dephosphorylation after the formation of basal bodies resulted in the formation of multiple flagella. 2D-gel electrophoresis and Western blotting showed a parallel relationship between the MT-nucleating activity of GPM complexes and the presence of hyperphosphorylated γ-tubulin in the complexes. These data suggest that the nucleation of MTs by GPM complexes precedes the de novo formation of basal bodies and that the regulation of MT-nucleating activity of GPM complexes is essential to the regulation of basal body number.

Cell-scale dynamic recycling and cortical flow of the actin–myosin cytoskeleton for rapid cell migration

Summary Actin and myosin II play major roles in cell migration. Whereas pseudopod extension by actin polymerization has been intensively researched, less attention has been paid to how the rest of the actin cytoskeleton such as the actin cortex contributes to cell migration. In this study, cortical actin and myosin II filaments were simultaneously observed in migrating Dictyostelium cells under total internal reflection fluorescence microscopy. The cortical actin and myosin II filaments remained stationary with respect to the substratum as the cells advanced. However, fluorescence recovery after photobleaching experiments and direct observation of filaments showed that they rapidly turned over. When the cells were detached from the substratum, the actin and myosin filaments displayed a vigorous retrograde flow. Thus, when the cells migrate on the substratum, the cortical cytoskeleton firmly holds the substratum to generate the motive force instead. The present studies also demonstrate how myosin II localizes to the rear region of the migrating cells. The observed dynamic turnover of actin and myosin II filaments contributes to the recycling of their subunits across the whole cell and enables rapid reorganization of the cytoskeleton.