Myosin II-dependent cortical movement is required for centrosome separation and positioning during mitotic spindle assembly

The role of myosin II in mitosis is generally thought to be restricted to cytokinesis. We present surprising new evidence that cortical myosin II is also required for spindle assembly in cells. Drug- or RNAi-mediated disruption of myosin II in cells interferes with normal spindle assembly and positioning. Time-lapse movies reveal that these treatments block the separation and positioning of duplicated centrosomes after nuclear envelope breakdown (NEBD), thereby preventing the migration of the microtubule asters to opposite sides of chromosomes. Immobilization of cortical movement with tetravalent lectins produces similar spindle defects to myosin II disruption and suggests that myosin II activity is required within the cortex. Latex beads bound to the cell surface move in a myosin II-dependent manner in the direction of the separating asters. We propose that after NEBD, completion of centrosome separation and positioning around chromosomes depends on astral microtubule connections to a moving cell cortex.     

Initial diameter of the polar body contractile ring is minimized by the centralspindlin complex

Polar body formation is an essential step in forming haploid eggs from diploid oocytes. This process involves completion of a highly asymmetric cytokinesis that results in a large egg and two small polar bodies. Unlike mitotic contractile rings, polar body contractile rings assemble over one spindle pole so that the spindle must move through the contractile ring before cytokinesis. During time-lapse imaging of C. elegans meiosis, the contractile ring moved downward along the length of the spindle and completed scission at the midpoint of the spindle, even when spindle length or rate of ring movement was increased. Patches of myosin heavy chain and dynamic furrowing of the plasma membrane over the entire embryo suggested that global cortical contraction forces the meiotic spindle and overlying membrane out through the contractile ring center. Consistent with this model, depletion of myosin phosphatase increased the velocity of ring movement along the length of the spindle. Global dynamic furrowing, which was restricted to anaphase I and II, was dependent on myosin II, the anaphase promoting complex and separase, but did not require cortical contact by the spindle. Large cortical patches of myosin during metaphase I and II indicated that myosin was already in the active form before activation of separase. To identify the signal at the midpoint of the anaphase spindle that induces scission, we depleted two proteins that mark the exact midpoint of the spindle during late anaphase, CYK-4 and ZEN-4. Depletion of either protein resulted in the unexpected phenotype of initial ingression of a polar body ring with twice the diameter of wild type. This phenotype revealed a novel mechanism for minimizing polar body size. Proteins at the spindle midpoint are required for initial ring ingression to occur close to the membrane-proximal spindle pole.     

Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.     

Rho-kinase controls cell shape changes during cytokinesis

BACKGROUND: Animal cell cytokinesis is characterized by a sequence of dramatic cortical rearrangements. How these are coordinated and coupled with mitosis is largely unknown. To explore the initiation of cytokinesis, we focused on the earliest cell shape change, cell elongation, which occurs during anaphase B and prior to cytokinetic furrowing. RESULTS: Using RNAi and live video microscopy in Drosophila S2 cells, we implicate Rho-kinase (Rok) and myosin II in anaphase cell elongation. rok RNAi decreased equatorial myosin II recruitment, prevented cell elongation, and caused a remarkable spindle defect where the spindle poles collided with an unyielding cell cortex and the interpolar microtubules buckled outward as they continued to extend. Disruption of the actin cytoskeleton with Latrunculin A, which abolishes cortical rigidity, suppressed the spindle defect. rok RNAi also affected furrowing, which was delayed and slowed, sometimes distorted, and in severe cases blocked altogether. Codepletion of the myosin binding subunit (Mbs) of myosin phosphatase, an antagonist of myosin II activation, only partially suppressed the cell-elongation defect and the furrowing delay, but prevented cytokinesis failures induced by prolonged rok RNAi. The marked sensitivity of cell elongation to Rok depletion was highlighted by RNAi to other genes in the Rho pathway, such as pebble, racGAP50C, and diaphanous, which had profound effects on furrowing but lesser effects on elongation. CONCLUSIONS: We show that cortical changes underlying cell elongation are more sensitive to depletion of Rok and myosin II, in comparison to other regulators of cytokinesis, and suggest that a distinct regulatory pathway promotes cell elongation.     

Long astral microtubules uncouple mitotic spindles from the cytokinetic furrow

Astral microtubules (MTs) are known to be important for cleavage furrow induction and spindle positioning, and loss of astral MTs has been reported to increase cortical contractility. To investigate the effect of excess astral MT activity, we depleted the MT depolymerizer mitotic centromere-associated kinesin (MCAK) from HeLa cells to produce ultra-long, astral MTs during mitosis. MCAK depletion promoted dramatic spindle rocking in early anaphase, wherein the entire mitotic spindle oscillated along the spindle axis from one proto-daughter cell to the other, driven by oscillations of cortical nonmuscle myosin II. The effect was phenocopied by taxol treatment. Live imaging revealed that cortical actin partially vacates the polar cortex in favor of the equatorial cortex during anaphase. We propose that this renders the polar actin cortex vulnerable to rupture during normal contractile activity and that long astral MTs enlarge the blebs. Excessively large blebs displace mitotic spindle position by cytoplasmic flow, triggering the oscillations as the blebs resolve.     

The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes

The molecular basis for asymmetric meiotic divisions in mammalian oocytes that give rise to mature eggs and polar bodies remains poorly understood. Previous studies demonstrated that the asymmetrically positioned meiotic chromosomes provide the cue for cortical polarity in mouse oocytes. Here we show that the chromatin-induced cortical response can be fully reconstituted by injecting DNA-coated beads into metaphase II-arrested eggs. The injected DNA beads induce a cortical actin cap, surrounded by a myosin II ring, in a manner that depends on the number of beads and their distance from the cortex. The Ran GTPase plays a critical role in this process, because dominant-negative and constitutively active Ran mutants disrupt DNA-induced cortical polarization. The Ran-mediated signaling to the cortex is independent of the spindle but requires cortical myosin II assembly. We hypothesize that a Ran(GTP) gradient serves as a molecular ruler to interpret the asymmetric position of the meiotic chromatin.     

Antagonists of myosin light chain kinase and of myosin II inhibit specific events of egg activation in fertilized mouse eggs

Although recent studies have demonstrated the importance of calcium/calmodulin (Ca(2+)/CAM) signaling in mammalian fertilization, many targets of Ca(2+)/CAM have not been investigated and represent potentially important regulatory pathways to transduce the Ca2+ signal that is responsible for most events of egg activation. A well-established Ca(2+)/CAM-dependent enzyme is myosin light chain kinase (MYLK2), the downstream target of which is myosin II, an isoform of myosin known to be important in cytokinesis. In fertilized mouse eggs, established inhibitors of MYLK2 and myosin II were investigated for their effects on events of egg activation. The MYLK2 antagonist, ML-7, did not decrease the activity of Ca(2+)/CAM protein kinase II or the elevation of intracellular Ca2+, and it did not delay the onset of Ca2+ oscillations. In contrast, ML-7 inhibited second polar body (PB) formation in a dose-dependent manner and reduced cortical granule (CG) exocytosis by a mean of approximately 50%. The myosin II isoform-specific inhibitor, blebbistatin, had similar inhibitory effects. Although both antagonists had no effect on anaphase onset, they inhibited second PB formation by preventing spindle rotation before telophase II and normal contractile ring constriction. To our knowledge, this is the first report that MYLK2 and myosin II are involved in regulating the position of the meiotic spindle, formation of the second PB, and CG exocytosis. The present results suggest that MYLK2 is one of a family of CAM-dependent proteins that act as multifunctional regulators and transduce the Ca2+ signal at fertilization.     

Actomyosin-dependent cortical dynamics contributes to the prophase force-balance in the early Drosophila embryo

Background

The assembly of the Drosophila embryo mitotic spindle during prophase depends upon a balance of outward forces generated by cortical dynein and inward forces generated by kinesin-14 and nuclear elasticity. Myosin II is known to contribute to the dynamics of the cell cortex but how this influences the prophase force-balance is unclear.

Principal Findings

Here we investigated this question by injecting the myosin II inhibitor, Y27632, into early Drosophila embryos. We observed a significant increase in both the area of the dense cortical actin caps and in the spacing of the spindle poles. Tracking of microtubule plus ends marked by EB1-GFP and of actin at the cortex revealed that astral microtubules can interact with all regions of these expanded caps, presumably via their interaction with cortical dynein. In Scrambled mutants displaying abnormally small actin caps but normal prophase spindle length in late prophase, myosin II inhibition produced very short spindles.

Conclusions

These results suggest that two complementary outward forces are exerted on the prophase spindle by the overlying cortex. Specifically, dynein localized on the mechanically firm actin caps and the actomyosin-driven contraction of the deformable soft patches of the actin cortex, cooperate to pull astral microtubules outward. Thus, myosin II controls the size and dynamic properties of the actin-based cortex to influence the spacing of the poles of the underlying spindle during prophase.     

Supervillin binding to myosin II and synergism with anillin are required for cytokinesis

Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II–binding proteins anillin and supervillin, act earlier. Anillin''s role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831–1281, which bind central spindle proteins, and residues 1–170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis.     

Localization of myosin II to chromosome arms and spindle fibers in PtK1 cells: a possible role for an actomyosin system in mitosis

Summary. The enzymes of importance in moving chromosomes are called motor proteins and include dynein, kinesin, and possibly myosin II. These three molecules are all included in the category of ATPases, in that they have the ability to convert chemical energy into mechanical energy. Both dynein and kinesin have been documented as molecules that “walk” along microtubules in the mitotic spindle, carrying cargo such as chromosomes. Myosin II, analogous to the muscle contraction system, transiently interacts along actin filaments and associates with kinetochore microtubules. In this paper we present evidence that a third ATPase, myosin II, may act as a “thruster” to propel chromosomes during the mitotic process. Double-label immunocytochemistry to actin and myosin II shows that myosin II is localized on chromosome arms at the beginning of mitosis and remains localized to the chromosomes throughout mitosis. Specific staining of myosin II is relegated to the outside of chromosomes with the highest density of staining occurring between the spindle poles and the chromosomes. This specific localization could account for the movement of chromosomes during mitosis, since they segregate towards the spindle poles, along kinetochore microtubules containing actin filaments, after aligning at the equatorial region of the cell at metaphase. We conclude from this study that there is an actomyosin system present in the mitotic spindle and that myosin is attached to chromosome arms and may act as a thruster in moving chromosomes during the mitotic process. Correspondence and reprints: Department of Biological Sciences, University of Denver, 2190 E Iliff Avenue, Denver, CO 80208, U.S.A.     

Astral signals spatially bias cortical myosin recruitment to break symmetry and promote cytokinesis

BACKGROUND: After anaphase, the segregated chromosomes are sequestered by cytokinesis into two separate daughter cells by a cleavage furrow formed by the actomyosin-based contractile ring. The failure to properly position the contractile ring between the segregated chromosomes can result in aneuploidy. In both C. elegans embryos and human cells, the central spindle regulates division-plane positioning in parallel with a second pathway that involves astral microtubules. RESULTS: We combined genetic and pharmacological manipulations with live cell imaging to spatially separate the two division cues in a single cell. We demonstrate that the two pathways for furrow formation are mechanistically and genetically distinct. By following the distribution of green fluorescent protein (GFP)-tagged nonmuscle myosin, we have found that the astral pathway for furrow formation involves the negative regulation of cortical myosin recruitment. An asymmetrically positioned spindle induces the asymmetric cortical accumulation of myosin. This cortical myosin behaves as a coherent contractile network. If the cortical network is nonuniform over the cell, the cortical contractile elements coalesce into a single furrow. This coalescence requires interconnections among contractile elements. CONCLUSIONS: We conclude that the two pathways of cleavage-furrow formation are mechanistically distinct. In particular, we conclude that the astral pathway for cleavage-furrow formation involves the negative regulation of myosin distribution by astral cues.     

Myosin light chain kinases and phosphatase in mitosis and cytokinesis

At mitosis, cells undergo drastic alterations in morphology and cytoskeletal organization including cell rounding during prophase, mitotic spindle assembly during prometaphase and metaphase, chromatid segregation in anaphase, and cytokinesis during telophase. It is well established that myosin II is a motor responsible for cytokinesis. Recent reports have indicated that myosin II is also involved in spindle assembly and karyokinesis. In this review, we summarize current understanding of the functions of myosin II in mitosis and cytokinesis of higher eukaryotes, and discuss the roles of possible upstream molecules that control myosin II in these mitotic events.     

Inhibition of Chromosomal Separation Provides Insights into Cleavage Furrow Stimulation in Cultured Epithelial Cells

While astral microtubules are believed to be primarily responsible for the stimulation of cytokinesis in Echinoderm embryos, it has been suggested that a signal emanating from the chromosomal region and mediated by the interzonal microtubules stimulates cytokinesis in cultured mammalian cells. To test this hypothesis, we examined cytokinesis in normal rat kidney cells treated with an inhibitor of topoisomerase II, (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane, which prevents the separation of sister chromatids and the formation of a spindle interzone. The majority of treated cells showed various degrees of abnormality in cytokinesis. Furrows frequently deviated from the equatorial plane, twisting daughter cells into irregular shapes. Some cells developed furrows in regions outside the equator or far away from the spindle. In addition, F-actin and myosin II accumulated at the lateral ingressing margins but did not form a continuous band along the equator as in control cells. Imaging of microinjected 5- (and 6-) carboxymtetramethylrhodamine-tubulin revealed that a unique set of microtubules projected out from the chromosomal vicinity upon anaphase onset. These microtubules emanated toward the lateral cortex, where they delineated sites of microtubule bundle formation, cortical ingression, and F-actin and myosin II accumulation. As centrosome integrity and astral microtubules appeared unperturbed by (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane treatment, the present observations cannot be easily explained by the conventional model involving astral microtubules. We suggest that in cultured epithelial cells the organization of the chromosomes dictates the organization of midzone microtubules, which in turn determines and maintains the cleavage activity.     

Myosin II transport, organization, and phosphorylation: evidence for cortical flow/solation-contraction coupling during cytokinesis and cell locomotion.

The mechanism of cytokinesis has been difficult to define because of the short duration and the temporal-spatial dynamics involved in the formation, activation, force production, and disappearance of the cleavage furrow. We have investigated the structural and chemical dynamics of myosin II in living Swiss 3T3 cells from prometaphase through the separation and migration of daughter cells. The structural and chemical dynamics of myosin II have been defined using the semiautomated, multimode light microscope, together with a fluorescent analogue of myosin II and a fluorescent biosensor of myosin II regulatory light chain (RLC) phosphorylation at serine 19. The correlation of image data from live cells using different modes of light microscopy allowed interpretations not possible from single-mode investigations. Myosin II transported toward the equatorial plane from adjacent regions, forming three-dimensional fibers that spanned the volume of the equator during anaphase and telophase. A global phosphorylation of myosin II at serine 19 of the RLC was initiated at anaphase when cortical myosin II transport started. The phosphorylation of myosin II remained high near the equatorial plane through telophase and into cytokinesis, whereas the phosphorylation of myosin II at serine 19 of the RLC decreased at the poles. The timing and pattern of phosphorylation was the same as the shortening of myosin II-based fibers in the cleavage furrow. Myosin II-based fibers shortened and transported out of the cleavage furrow into the tails of the two daughter cells late in cytokinesis. The patterns of myosin II transport, phosphorylation, and shortening of fibers in the migrating daughter cells were similar to that previously defined for cells migrating in a wound in vitro. The temporal-spatial patterns and dynamics of myosin II transport, phosphorylation at serine 19 of the RLC, and the shortening and disappearance of myosin II-based fibers support the proposal that a combination of the cortical flow hypothesis and the solation-contraction coupling hypothesis explain key aspects of cytokinesis and polarized cell locomotion.     

Distinct pathways for the early recruitment of myosin II and actin to the cytokinetic furrow

Equatorial organization of myosin II and actin has been recognized as a universal event in cytokinesis of animal cells. Current models for the formation of equatorial cortex favor either directional cortical transport toward the equator or localized de novo assembly. However, this process has never been analyzed directly in dividing mammalian cells at a high resolution. Here we applied total internal reflection fluorescence microscope (TIRF-M), coupled with spatial temporal image correlation spectroscopy (STICS) and a new analytical approach termed temporal differential microscopy (TDM), to image the dynamics of myosin II and actin during the assembly of equatorial cortex. Our results indicated distinct and at least partially independent mechanisms for the early equatorial recruitment of myosin and actin filaments. Cortical myosin showed no detectable directional flow during early cytokinesis. In addition to equatorial assembly, we showed that localized inhibition of disassembly contributed to the formation of the equatorial myosin band. In contrast to myosin, actin filaments underwent a striking flux toward the equator. Myosin motor activity was required for the actin flux, but not for actin concentration in the furrow, suggesting that there was a flux-independent, de novo mechanism for actin recruitment along the equator. Our results indicate that cytokinesis involves signals that regulate both assembly and disassembly activities and argue against mechanisms that are coupled to global cortical movements.     

A mechanosensory system governs myosin II accumulation in dividing cells

The mitotic spindle is generally considered the initiator of furrow ingression. However, recent studies suggest that furrows can form without spindles, particularly during asymmetric cell division. In Dictyostelium, the mechanoenzyme myosin II and the actin cross-linker cortexillin I form a mechanosensor that responds to mechanical stress, which could account for spindle-independent contractile protein recruitment. Here we show that the regulatory and contractility network composed of myosin II, cortexillin I, IQGAP2, kinesin-6 (kif12), and inner centromeric protein (INCENP) is a mechanical stress-responsive system. Myosin II and cortexillin I form the core mechanosensor, and mechanotransduction is mediated by IQGAP2 to kif12 and INCENP. In addition, IQGAP2 is antagonized by IQGAP1 to modulate the mechanoresponsiveness of the system, suggesting a possible mechanism for discriminating between mechanical and biochemical inputs. Furthermore, IQGAP2 is important for maintaining spindle morphology and kif12 and myosin II cleavage furrow recruitment. Cortexillin II is not directly involved in myosin II mechanosensitive accumulation, but without cortexillin I, cortexillin II's role in membrane-cortex attachment is revealed. Finally, the mitotic spindle is dispensable for the system. Overall, this mechanosensory system is structured like a control system characterized by mechanochemical feedback loops that regulate myosin II localization at sites of mechanical stress and the cleavage furrow.     

Regulation of myosin II during cytokinesis in higher eukaryotes

Cellular myosin II is the principal motor responsible for cytokinesis. In higher eukaryotes, phosphorylation of the regulatory light chain (MLC) of myosin II is a primary means of activating myosin II and is known to be crucial for the execution of cell division. Because signals transmitted by the mitotic spindle coordinate key spatial and temporal aspects of cytokinesis, such signals should ultimately function to activate myosin II. Thus, it follows that identification of regulatory factors involved in MLC phosphorylation should elucidate the nature of spindle-derived regulatory signals and lead to a model for how they control cytokinesis. However, the identity of these upstream molecules remains elusive. This review (which is part of the Cytokinesis series) summarizes current views of the regulatory pathway controlling MLC phosphorylation and features four candidate molecules that are likely immediate upstream myosin regulators. I discuss proposed functions for MLCK, ROCK, citron kinase and myosin phosphatase during cytokinesis and consider the possibility of a link between these molecules and the signals transmitted by the mitotic spindle.     

Myosin II isoforms promote internalization of spatially distinct clathrin-independent endocytosis cargoes through modulation of cortical tension downstream of ROCK2

Although the actomyosin cytoskeleton has been implicated in clathrin-mediated endocytosis, a clear requirement for actomyosin in clathrin-independent endocytosis (CIE) has not been demonstrated. We discovered that the Rho-associated kinase ROCK2 is required for CIE of MHCI and CD59 through promotion of myosin II activity. Myosin IIA promoted internalization of MHCI and myosin IIB drove CD59 uptake in both HeLa and polarized Caco2 intestinal epithelial cells. In Caco2 cells, myosin IIA localized to the basal cortex and apical brush border and mediated MHCI internalization from the basolateral domain, while myosin IIB localized at the basal cortex and apical cell–cell junctions and promoted CD59 uptake from the apical membrane. Atomic force microscopy demonstrated that myosin IIB mediated apical epithelial tension in Caco2 cells. Thus, specific cargoes are internalized by ROCK2-mediated activation of myosin II isoforms to mediate spatial regulation of CIE, possibly by modulation of local cortical tension.     

Rapid redistribution of myosin II in livingDictyostelium amoebae,as revealed by fluorescent probes introduced by electroporation

Summary Fluorescently labeled myosin II fromDictyostelium and fluorescently labeled antibody Fab fragments against myosin II fromDictyostellium were introduced into livingDictyostelium amoebae by electroporation. Fluorescent labeling of myosin II impairs neither actin-activated ATPase activity nor the ability to form filaments in vitro. Fluorescently labeled Fab also did not interfere with the functions of myosin II in vitro. After electroporation, introduced fluorescently labeled myosin II was distributed diffusely in the endoplasm but some of it accumulated at the tail cortical region of migrating cells. During the course of observations, intense fluorescence due to myosin II disappeared and then it appeared again instantaneously in the cortical regions during amoeboid movement. Fluorescently labeled Fab, after electroporation, bound to endogenous myosin II in amoebae and the dynamic changes in its distribution were similar to those of fluorescently labeled myosin II. The fluorescence due to myosin II also underwent dynamic redistribution during the division of cells and chemotactic stimulation. The introduction of labeled Fab and labeled myosin II did not impair the motility ofDictyostelium. During changes in direction associated with cell locomotion, myosin II accumulated at the original front region of the cell and, thereafter, the accumulation was observed at the new tail region of the cell. These results are consistent with the hypothesis that myosin II has two possible roles for cell locomotion. One is that myosin II accumulates at tail regions to produce the power required for contraction. The other is that it hinders the extension of pseudopods in directions other than the frontal direction.     

Functional divergence of human cytoplasmic myosin II: kinetic characterization of the non-muscle IIA isoform

Cytoplasmic (or non-muscle) myosin II isoforms are widely expressed molecular motors playing essential cellular roles in cytokinesis and cortical tension maintenance. Two of the three human non-muscle myosin II isoforms (IIA and IIB) have been investigated at the protein level. Transient kinetics of non-muscle myosin IIB showed that this motor has a very high actomyosin ADP affinity and slow ADP release. Here we report the kinetic characterization of the non-muscle myosin IIA isoform. Similar to non-muscle myosin IIB, non-muscle myosin IIA shows high ADP affinity and little enhancement of the ADP release rate by actin. The ADP release rate constant, however, is more than an order of magnitude higher than the steady-state ATPase rate. This implies that non-muscle myosin IIA spends only a small fraction of its ATPase cycle time in strongly actin-bound states, which is in contrast to non-muscle myosin IIB. Non-muscle myosin II isoforms thus appear to have distinct enzymatic properties that may be of importance in carrying out their cellular functions.