[Proteins and Cell Regulation] Myosins Volume 7 || Non-Muscle Myosin II

CHAPTER 7

NON-MUSCLE MYOSIN II

MARY ANNE CONTI, SACHIYO KAWAMOTO,AND ROBERT S. ADELSTEINNational Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Dr MSC 1762,Building 10, Room 8N202, Bethesda, MD 20892-1762, USA

Abstract: In mammals, three different isoforms of nonmuscle myosin II, II-A, II-B and II-C,are widely distributed throughout the entire organism. While a few cells contain asingle isoform, most contain more than one, including isoforms generated by alternativesplicing. In humans, these isoforms are encoded by three different genes, MYH9 (II-A), MYH10 (II-B) and MYH14 (II-C), present on three different chromosomes. Theseproteins play a role in many fundamental cellular and developmental processes suchas cell-cell adhesion, cell migration and cytokinesis. Although all three isoforms sharea number of biochemical and structural properties, there are also important differ-ences among them that are being investigated at both the cellular level as well as inwhole animals. Small interfering RNAs specific for each of the isoforms as well asthe relatively specific inhibitor of myosin MgATPase activity, blebbistatin, are twoimportant new tools helping to elucidate the function of nonmuscle myosin II. Theuse of homologous recombination to generate mice that have been ablated for, or havemarkedly decreased amounts of each isoform, or have point mutations in the variousisoforms, is also providing new information about the role of these proteins in vivo. Thepurpose of the present chapter is to review the properties of nonmuscle myosin II andits signaling pathways and to provide examples of its function in cells from a numberof species, as well as intact animals

Keywords: biochemical and structural properties cell-cell adhesion; cell migration; cytokinesis;genetically altered mice; nonmuscle myosin II

7.1. INTRODUCTION

Nonmuscle myosin II (NM II) is a class II myosin that is distributed ubiquitouslythroughout nature. It plays a role in a variety of cellular processes including cellmigration, cytokinesis, cell adhesion and cell shape change both during developmentand in the adult organism. The purpose of the present chapter is to review thefunction, regulation and role of NM II in both cells and intact animals. Although it

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is clear that NM II is present from protists through the animal kingdom, this chapterwill emphasize the various roles played by the three different isoforms found inmammalian cells. With respect to animal models, we will emphasize what is knownfrom studies with mammalian cells and cell lines as well as mice and humans. Forrecent reviews on this subject including those not emphasizing mammalian myosins,see: Nikolaou et al., 2006 (nematode); Bosgraaf and Haastert, 2006 (Dictyostelium);Landsverk and Epstein, 2005; Matsumura, 2005; Somlyo and Somlyo, 2003; andYumura and Uyeda, 2003.

The chapter is divided into three sections: First, we will review the basic structuraland biochemical properties of NM II. In the second section, we will emphasizethree of the critical functions of NM II: its role in cell adhesion, cell motilityand cytokinesis. Recent work in this area has begun to highlight the emergingimportance of the three different mammalian NM IIs. Finally, we will discuss theroles of NM II during mammalian development and the roles that the differentisoforms play in the developing and adult organism.

It has become common to refer to each of the three different isoforms of NMII by the name designated for the heavy chain: NM II-A, NM II-B and NM II-C;however, the nomenclature pertaining to these myosins is still not uniform withsome authors using Roman numerals (II-A, II-B and II-C) and some using Arabicnumerals to designate the class isoform. The gene designation for the nonmusclemyosin heavy chains (NMHCs) in humans is MYH9 (II-A), MYH10 (II-B) andMYH14 (II-C).

The name NM II is used to designate the class of myosin as well as to differentiateit from the sarcomeric myosins of the same class. Phylogenetically, the NM IIsare closer to smooth-muscle myosin than they are to the sarcomeric myosins.However, the name ‘nonmuscle myosin’ remains a misnomer in that these myosin IIisoforms are also present in all muscle cells (skeletal, cardiac and smooth), thoughin significantly smaller quantities than the muscle myosin IIs.

7.2. STRUCTURAL, BIOCHEMICAL AND REGULATORYPROPERTIES OF NONMUSCLE MYOSIN II

7.2.1. Structural Properties

As noted above, mammals contain three different isoforms of the NMHC encoded bythree different genes in humans: MYH9, 10 and 14 (Simons et al., 1991; reviewed inBerg et al., 2001; Golomb et al., 2004). Each NMHC (230 kDa) forms a homodimerand binds two pairs of light chains, one commonly referred to as the regulatorylight chain (20 kDa MLC, MLC20) and the other as the essential light chain (17kDa MLC, MLC17; see Figure 7.1A). There is no evidence that heterodimers ofthe heavy chains exist and immunoprecipitation using isoform-specific antibodiesreveals only homodimeric NMHCs (Golomb et al., 2004). Although there are severalisoforms for each MLC, at present, it is not known whether there is any specificityto a particular set of MLC isoforms binding to a particular set of heavy chains.

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Figure 7.1. (A) Diagram of the nonmuscle myosin II molecule showing the heavy chain dimer and twopairs of light chains. (B) A diagrammatic depiction of a bipolar NM II filament (modified from Craigand Woodhead, 2006). (C) Diagram showing location of the B1/C1 and B2/C2 inserted amino acids inthe head region of the NMHC. HMM is the two-headed myosin fragment and S-1 is the single-headedmyosin fragment used in kinetic studies. NHT is the non-helical tail

When the two-headed NMHC fragment, heavy meromyosin (HMM, seeFigure 7.1A) is expressed using the baculovirus system, the same set of MLCsis often co-expressed with each NMHC isoform and many of the kinetic valuesreported in the literature are based on these expressed proteins (Pato et al., 1996;Hu et al., 2002; Golomb et al., 2004; Kim et al., 2005). Using the single NM IIfrom Drosophila melanogaster, Franke et al. (2006) characterized the properties andassociation of each of the two different light chains with the MHCs. Interestingly,the MLC17, but not the MLC20, can bind to four different classes of MHCs (II, V,VI and VIIA), as well as to a microtubule-associated protein.

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Figure 7.1A is a diagram of the NM II molecule showing the globular headregion at the amino-terminal end and the coiled-coil rod portion terminating in anon-helical tail (NHT) at the carboxyl-terminus. Two of the three NMHCs (II-Band II-C) undergo alternative splicing of the pre-mRNA resulting in the insertionof a cassette of nucleotides encoding amino acids (see Figure 7.1C). The sites ofinsertion are conserved between NMHC II-B and II-C with the B1 and C1 siteslocated in loop 1 near the myosin ATP-binding region and the B2 and C2 siteslocated in loop 2 of the MHC, in the actin-binding region (Takahashi et al., 1992;Itoh and Adelstein, 1995; Golomb et al., 2004). The smooth-muscle MHC alsoundergoes alternative splicing at a site homologous to that of the B1 and C1 sites,but not at the second site (Kelley et al., 1993). Unlike the smooth-muscle MHC,the carboxyl-terminal region of the NMHC does not undergo alternative splicingnor has any alternative splicing been found for NMHC II-A in any region of themolecule.

The myosin molecule is bi-functional in containing two amino-terminal globularmotor domains, which can bind to actin and hydrolyze MgATP; and a rod domain,which is responsible for filament formation. The two heads are separated from thecoiled-coil, �-helical domain by a neck-like region, which binds the two pairs ofMLCs. Similar to other members of the myosin II family, the carboxyl-terminal,rod-like portion of the molecule forms bi-polar filaments which, in the case of NMII, are considerably smaller than those formed by skeletal and cardiac myosin (seeFigure 7.1A and 7.1B; Niederman and Pollard, 1975; Verkhovsky et al., 1995).

Structural studies from both invertebrate striated muscle and vertebrate smoothmuscle, which are regulated by phosphorylation, have shed light on the mecha-nisms underlying some of the regulatory properties of NM II. EM studies withsmooth-muscle myosin suggest that the ‘off state’ state of dephosphorylated myosinis achieved through an asymmetric intramolecular interaction between the actin-binding region of one head and the converter domain of the second head (Wendtet al., 2001; Liu et al., 2003; Tama et al., 2005). The converter domain is locatedbetween the motor domain and MLC binding regions on the MHC. These ideas wereextended using native invertebrate filaments and cryo-EM. The three-dimensionalreconstructions and atomic fitting studies demonstrate that the interacting heads arealso present in the filaments and may explain the relaxed state of thick filamentsin smooth muscle, nonmuscle cells and phosphorylation-regulated insect striatedmuscles (Woodhead et al., 2005; reviewed in Craig and Woodhead, 2006).

As can be seen from Figure 7.1A, the two globular heads are followed by therod fragment, which has the configuration of a coiled-coil, where two �-helicalchains wrap around each other. The amino-acid sequences in the rod are charac-terized by a seven-residue repeat with hydrophobic interactions occurring betweenthe two juxtaposed �-helices. Straussman et al. (2007) introduced mutations intoone of these juxtaposed regions and shows how the introduction of Asp, butnot Glu or Leu, causes flexible kinks in the coiled-coil rod at positions corre-sponding to the mutation site. The coiled-coil is followed by a non-helical tailregion (NHT, Figure 7.1A) in all three NM II isoforms. This region is of interest


because it varies in its amino-acid sequence among the three isoforms and thusprovides unique epitopes for the generation of specific antibodies that can distin-guish among the NM II isoforms. The non-helical tail also contains a number ofsites for NMHC phosphorylation by protein kinase C (PKC) and casein kinase II(see Section 7.2.2).

Nakasawa et al. (2005) has studied the regions of the myosin rod required forassembly of NM II-B. They found that two domains, a 35-amino-acid domainbetween Asp1729 and Thr1763 and a second domain extending from Ala1875 to Ala1913

are critical. Fragments lacking either region are incapable of forming filaments andare soluble at any NaCl concentration. The interactions between the positive- andnegative-charge clusters of these domains are thought to initiate filament assembly.This work is extended to explore the domains that play a role in the formation ofhomo-filaments. In Sato et al. (2007), the authors specify two additional regionsof the NM II-B rod, amino acids 1672–1728 and 1914–1976 that are involved inself-recognition when NM II-B assembles into filaments.

7.2.2. Biochemical Properties

The biochemical properties of the three different mammalian NM IIs show consid-erable differences. In order to study their kinetics, most laboratories have usedbaculovirus expression of both HMM and the single-headed subfragment-one (S-1)fragments (see Figure 7.1A). This avoids the problems of insolubility at the low ionicstrength required for kinetic measurements that are encountered with expression ofthe whole molecule. The HMM fragment is readily soluble for all three isoforms aswell as the alternatively spliced isoforms as long as the final ionic strength is keptin the 25–100 �M range. As noted above, the different NMHC isoforms are usuallyco-expressed with the same MLCs. To date, the only direct comparison between anendogenous NM II-derived HMM and a baculovirus-expressed HMM comes fromhuman platelet HMM, which is pure II-A. The values are in reasonable agreement(Hu et al., 2002; Sellers et al., 1988).

Table 7.1 compares the Vmax, KATPase (concentration of actin at one-half Vmax)and rate of actin-filament propulsion (in vitro motility, IVM) for the three isoforms,for some of the point mutant isoforms generated to date, and for the alternatively-spliced B1 and C1 inserted isoforms. Most of the mutations introduced into theglobular domain decrease the actin-activated MgATPase activity as well as theIVM with the one exception of the Arg730Ser mutation in HMM II-C, which has noeffect on the former. Interestingly, this mutation in NM II-C is reported to cause adefect in hearing in humans (Donaudy et al., 2004).

Studies with the S-1 II-B have shown that it has a significantly higher duty-ratio(0.2–0.4 out of 1.0, portion of time that it is bound to actin) than most other myosinIIs with values between those of skeletal-muscle myosin II (0.02–0.04) and thoseof the processive motor myosins V and VI (0.7–0.8; Rosenfeld et al., 2003; Wanget al., 2003). This prolonged duty cycle is not seen for NM II-A, which spendsa smaller fraction (0.1) of the MgATPase cycle in the strongly actin-bound state

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Table 7.1. Summary of Actin-activated MgATPase activity and in vitro motility of HMMs

HMMs Species Temperature Vmax KATPase IVM (30�C)

�Ca s−1 �M �m/s

HMM II-A (1) Human 25WT 0�45±0�03 9±2

HMM II-A (2) Human 35

WT 0�92±0�15 8�4±1�5 0.28N93K 0�03±0�002 1�7±0�2 NSMb

R702C 0�20±0�05 3�0±0�1 0.14

HMM II-B (3) Human 25

WT 0�17±0�04 3�4±1�8 0�17±0�03N97K 0�12±0�04 1�8±0�6 0�06±0�004R709C 0�05±0�003 < 0�5 NSM

HMM II-B (4) Chicken 35

B1 0�37±0�09 15�1±4�5 0�092±0�013B0 0�28±0�08 12�7±4�1 0�077±0�014

HMM II-C (3) Mouse 37

C1 0�85±0�18 26�1±6�2 0�08±0�003C0 0�29±0�04 9�9±6�0 0�03±0�005

HMM II-C (3) Mouse 25

C1 0�26±0�002 5�7±2�9 0�08±0�01C0 0�18±0�04 4�2±1�1 0�03±0�006C1-R730C 0�08±0�007 1�6±0�2 NSMC0-R722C 0�03±0�001 0�4±0�02 NSMC1-R730S 0�28±0�04 3�5±1�2 0�02±0�003

The values for Vmax, KATPase, and IVM (in vitro motility) are the mean and S.D. from two to four proteinpreparations. The numbers in parentheses refer to references below. B0, C0, noninserted NMHC II; B1,C1, inserted NMHC II containing an amino acid insert. All assays measured following phosphorylationby MLCK.a Reaction temperature for actin-activated MgATPase activity assay.b No significant movement.1. Kovacs et al., 2004a; 2. Hu et al., 2002; 3. Kim et al., 2005; 4. Pato et al., 1996.

(Kovacs et al., 2003). These important differences in their kinetic properties mayhelp to explain why NM II-A can rescue some, but not all, of the defects in NMII-B-ablated mice when NMHC II-A is expressed under control of the NM II-Bendogenous promoter (see Section 7.4.1).

Blebbistatin, a small molecule that blocks cell blebbing, has been shown to be arelatively specific inhibitor of NM IIs (Straight et al., 2003; Limouze et al., 2004;see Shu et al., 2005 for nonspecific effects). The drug has been shown to inhibit theMgATPase of S-1 II-B by blocking entry into the strong actin-binding state. It alsoreduces the rate of ADP release (Kovacs et al., 2004b; Ramamurthy et al., 2004).The three-dimensional structure of the binding of blebbistatin to the motor domainof Dictyostelium NM II has been reported (Allingham et al., 2005). Although it is


now being employed routinely, along with isoform-specific small interfering RNA(siRNA) to study all three isoforms of NM II, it is of particular use in dissectingthe steps of the cell cycle because its effect is rapid (minutes) and reversible. Ithas been shown to inhibit the contraction of the cleavage furrow without disruptingmitosis or contractile ring assembly (Straight et al., 2003).

7.2.3. Distribution of the NM II Isoforms

The three NMHC isoforms are 60–80% identical at the level of amino acids, but aresignificantly different, particularly at their amino-terminal and carboxyl-terminalends, so that it has been possible to raise specific antibodies to each of the threeisoforms (Golomb et al., 2004). Figure 7.2 illustrates use of these antibodies toshow the distribution of all three isoforms during mouse embryonic development.It shows that each of the isoforms is ubiquitously expressed and that, in additionto areas where they clearly overlap, there are areas where each isoform appears

Figure 7.2. Detection of NM II isoforms in mouse tissues during development. Sections ofparaformaldehyde-fixed mouse tissues were probed with antibodies to NMHC II-A, top row, NMHCII-B, middle row, and NMHC II-C, bottom row. E11.5, saggital sections from an E11.5 mouse show thatall three proteins are widely distributed throughout the embryo. Brain E11.5, enlarged area of the brainshows increased staining for II-C in the pituitary (arrow), intense staining at the pial and ventricularsurfaces for II-B, and enhanced vascular staining for II-A. Inner ear E16.5, shows staining of the mouseinner ear. II-C is particularly intense in the developing sensory cells of the cochlea, II-B is expressed inboth the mesenchymal and epithelial cells, and II-A staining is most intense in the vasculature. IntestineE16.5, shows staining of the mouse small intestine at E16.5. Both II-C and II-A are intensely stained inthe epithelial cells, but II-C is particularly concentrated at the apical border of these cells. In contrast,II-B appears more intense in the surrounding serosal cells. (Immunofluorescence microscopy by XuefeiMa, from Golomb et al., 2004.)

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to be uniquely enriched compared to the surrounding tissue (see, for example, thearrow which points to the pituitary gland at embryonic day (E) 11.5, which isenriched for NM II-C). Although the three isoforms are widely distributed andmore than one isoform can be present in a single cell, there are certain cells whichcontain a particular isoform exclusively or are enriched for a particular isoform.Cardiac myocytes are unusual in containing NM II-B and II-C, but lacking II-A.The nonmyocyte cells in the heart do contain NM II-A. Platelets, many of the bloodcells, lymph nodes, spleen and the thymus are enriched for II-A, while the brainand testes are enriched for II-B and II-C. NM II-C is not detected in the spleen andliver (although the mRNAs are present in the liver) and along with II-A, is presentin the colon and stomach, which lack II-B (Golomb et al., 2004). The distributionof the isoforms changes during development. For example, II-C is not presentearly in development and II-A is present in the cardiac myocytes, but only up toE8.5 and is absent thereafter. During embryonic development, NM II-B is presentthroughout the cardiac myocytes, but after birth II-B is markedly decreased in themature cardiac muscle cell and is mostly confined to the intercalated disks (Takedaet al., 2000). The alternatively spliced isoforms B1, B2 and C2 are confined to thebrain and spinal cord. In contrast to the other alternatively spliced isoforms, C1 ispresent in most of the tissues containing II-C with the exception of adult heart andskeletal muscle. The inserted C1 isoform is present in these last two tissues duringthe embryonic stage (Golomb et al., 2004; Ma et al., 2006; Jana et al., 2006).

The presence of the three isoforms raises another point related to distribution,which is quantification. As is well understood, antibodies, because of differentdegrees of antigenicity of their epitopes, do not directly convey quantitative data.As will be discussed below (see Section 7.4.1), the total quantity of NM II presentin a cell regardless of isoform may have a significant effect on the cell phenotype.Thus, it has become important to devise a method to quantitate the various isoformsof myosin in any given cell. One way to accomplish this has been outlined in Baoet al. (2005), which makes use of both siRNA and a pan-myosin antibody. However,other, more direct methods are urgently needed to address this critical point.

7.2.4. Regulation

NM II regulation refers to the various mechanisms controlling the actin-activatedMgATPase activity as well as the formation of filaments by myosin II molecules.The critical step in activating the activity of NM II is phosphorylation of the MLC20

on Ser19. This was initially described for NM II-A in vitro, using purified plateletNM II (Adelstein and Conti, 1975) and then by a number of authors for vertebratesmooth-muscle myosins (Sobieszek and Small, 1976; Gorecka et al., 1976; Chackoet al., 1977; Ikebe et al., 1977). The Ser19 phosphorylation is essential for actin-activated MgATPase activity, filament assembly and in vitro motility. Additionalphosphorylation on Thr18 results in a further increase in the MgATPase activityand filament assembly, but no change in the in vitro motility (Ikebe et al., 1988;Umemoto et al., 1989). Usually phosphorylation of Ser19 precedes that of Thr18.


Phosphorylation of MLC20 affects the kinetic (Vmax) and structural properties ofmyosin (reviewed in Somlyo and Somlyo, 2003) and has no significant effect on theaffinity of myosin for actin (Sellers et al., 1982; Sellers, 1985). One dramatic effectof phosphorylation that has yet to be demonstrated in intact cells is an alterationof its unphosphorylated, folded configuration, with the rod portion of the moleculefolded in on the two heads, to a stretched, phosphorylated configuration that canparticipate in filament formation (Trybus and Lowey, 1984).

MLC20 can be phosphorylated on Ser19 by a number of kinases, which lead tomyosin activation, but to date, the best-described ones are the Ca2+-calmodulin-dependent myosin light chain kinase (MLCK) and the Rho-activated kinase(reviewed in Matsumara, 2005 and Zhao and Manser, 2005). Although Rho kinasecan phosphorylate a number of substrates including the myosin-binding subunit(MBS) of myosin phosphatase (Kimura et al., 1996), MLCK appears to be specificfor the MLC20 (see Section 7.3.3.3). In addition to MLCK and Rho kinase, otherkinases that have been reported to phosphorylate the MLC20 include citron kinase,p21-activated protein kinase (PAK), myotonic dystrophy protein kinase-relatedCdc42-binding kinase (MRCK) and Zip kinase.

The most significant advances in our recent understanding of the regulation ofNM II activity relate to the signaling pathways upstream of the various kinases thatcan phosphorylate the MLC20 and of these, the upstream and downstream signals ofthe Rho signaling system appear to be the most informative. A number of reviewshave appeared (Garcia-Mata and Burridge, 2007; Burridge and Wennerberg, 2004and the extensive review by Somlyo and Somlyo, 2003). Recent work has centeredon the importance of GTPase exchange factors (GEFs) and the roles that they playin regulating downstream activity. Indeed, it is the plethora of GEFs that may holdthe secret to the ability of Rho kinase to affect so many different cellular processes(Nikolaidou and Barrett, 2004; Dawes-Hoang et al., 2005).

A number of investigators (Katoh et al., 2001; Totsukawa et al., 2004; Sandquistet al., 2006) provide evidence for differences in the location of the two major kinaseswith MLCK being located in the cell’s periphery and Rho kinase being locatedmore centrally. Thus, in fibroblasts, Totsukawa et al. (2004) suggest that MLCKcan regulate membrane ruffling in the periphery, whereas Rho kinase controls focaladhesions at the cell center (see Section 7.3.2.2).

In addition to sites for phosphorylation at Ser19/Thr18 on MLC20, Ser1 and Ser2,and Thr9 have been described as sites for phosphorylation by protein kinase C(PKC) on MLC20. The effects of this phosphorylation are of interest because theyhave a negative effect on the actin-activated MgATPase activity. In vitro PKCphosphorylation decreases the affinity of MLCK for NM II and decreases theaffinity of actin for the phosphorylated myosin (Nishikawa et al., 1984; Ikebe andReardon, 1990). However, there is still a controversy as to which of the threesites that have been identified in vitro as putative phosphorylation sites (Ser1,Ser2 and Thr9� are the relevant sites affecting NM II activity in vivo. Turbedskyet al. (1997) provide evidence for Thr9, but the issue remains open. In intact cells,phosphorylation of Ser1 and Ser2, but not Thr9, is detected following stimulation of

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the cells with phorbol ester as well as physiological agonists (Kawamoto et al., 1989;Ludowyke et al., 1989). It may be that the isoform of PKC used to phosphorylatethe NM II MLC20 both in vitro and in vivo is of major importance.

Although MHC phosphorylation has been shown to play a critical role inregulating the activity of Dictyostelium and Acanthamoeba NM II (reviewed inRedowicz, 2001 and Bosgraaf and Van Haastert, 2006), the role of NMHC phospho-rylation in regulating mammalian NM II is still being elucidated. With respect toNMHC II-A, a number of laboratories report that Ser1917 is a substrate for PKCand recently, Ludowyke et al. (2006) report that this phosphorylation is mediatedby PKC �II in RBL-2H3 cells, where it plays a role in granule-mediated secretion.Ser1944 on NMHC II-A is a substrate for casein kinase II, and phosphorylation ateither Ser1917 or Ser1944 inhibits the assembly of II-A rod into filaments. Interest-ingly, PKC phosphorylation has no effect on the binding of the S100A4 metastasisfactor to NM II-A, but casein kinase II phosphorylation decreases the affinity ofNM II-A rod for the factor. The S100A4 factor, also called mts1, regulates cellmotility in HeLa cells by reducing side protrusions and favoring forward protrusions(Dulyaninova et al., 2005; Li and Bresnick, 2006).

Experiments with a prostate cancer cell line (TSU-pr1) demonstrate phosphory-lation of NM II-B on Ser1937 by atypical PKC� (Even-Faitelson and Ravid, 2006;Even-Faitelson et al., 2005). This phosphorylation is stimulated by epidermal growthfactor and involves a complex of PAK1 and PKC�. Rosenberg and Ravid (2006)also report evidence for phosphorylation of a number of serine residues in thenon-helical tail of myosin II-B by PKC�.

Having surveyed some of the general properties of NM II, we now will seek tounderstand its role in three important processes: cell adhesion, cell migration andcytokinesis. Following this section, we will consider some of the functions of NMII in vivo.

7.3. FUNCTIONAL PROPERTIES OF NONMUSCLE MYOSIN II

7.3.1. Cell-cell Adhesion

7.3.1.1. Introduction

Morphogenic movements during development, tissue formation and turnover ofcells such as those lining the gut are mediated by dynamic rearrangements of cell-cell contacts. Disease processes such as metastasis of cancer cells may be facilitatedby downregulation of components of the intact cell-cell adhesion complex. Therole of actin in the apical junction complex (AJC) has been investigated in the past(reviewed in Bershadsky, 2004), but the emergence of a role for NM II has onlyrecently been appreciated. Both the structural and enzymatic properties of myosinmay be important in maintaining and dynamically re-organizing cell-cell contacts.

The AJC is the name applied to the structures near the apicolateral surface ofepithelial cells, which are responsible for cell-cell adhesion and for effecting thebarrier function of diverse tissue types. The AJC plays a key structural role in


cell sorting and organogenesis, and additionally functions as a part of intracellularsignaling pathways (reviewed in Goodwin and Yap, 2004; Erez et al., 2005). Thecomplex is composed of the adherens junctions and tight junctions (TJs). Themajor proteins of the adherens junctions are the transmembrane cadherins and thecytosolic catenins, and of the TJs, the transmembrane occludins and claudins, andthe cytosolic ZO-1.

The adherens junctions are formed by homotypic binding of transmembraneglycoproteins of the large and diverse cadherin family (reviewed in Halbleib andNelson, 2006). The extracellular portion of classical cadherins recognizes and bindsto cadherins on neighboring cells while the intracellular domains of the moleculeare responsible for signaling and linkage to the cytoskeleton. Cadherins extendextracellular domains to bind the extracellular domains of like cadherins on neigh-boring cells in a Ca2+-dependent manner. Actin polymerization and branchingmediated by Arp2/3 binding are the driving force, which brings cells into contact(Vasioukhin et al., 2000). As cell membranes contact each other, cadherins cluster,the regions of nascent cell-cell contacts extend, and the cadherins recruit cytoplasmicproteins, which reorganize and link to the actin cytoskeleton (reviewed in Adamsand Nelson, 1998; Bershadsky, 2004; Vaezi et al., 2002). In addition to providingsites for actin assembly (Kovacs et al., 2002), cadherins form a scaffold whichincludes catenins, �-actinin, vinculin, actin-binding proteins such as formin andArp2/3, and signaling molecules of the Rho family of small GTPases (Vasioukhinet al., 2000; reviewed in Yap and Kovacs, 2003). As the junctions mature, actin isorganized into a continuous apical belt or perijunctional ring of filaments parallelto the lateral cell membrane (Mooseker, 1985).

TJs maintain the barrier function of tissues by sealing the space between cells andpreventing passive diffusion. They actively regulate permeability of the cell barrierto water, ions, and other solutes notably in the lining of the gastrointestinal tractand the kidneys. The transmembrane claudins form the pores in the paracellularspace, which selectively regulate solutes based on size or charge. The TJs formnearer the apical surface of the cell than adherens junctions (reviewed in D’Atriand Citi, 2002; Turner, 2006).

It is clear that upon cell-cell adhesion, cadherins act to transmit signals in orderto assemble and regulate the network of proteins on the cytoplasmic side of thecell membrane (reviewed in Yap and Kovacs, 2003; Fukata and Kaibuchi, 2001).The Rho family of signaling proteins, RhoA, Rac1, and Cdc42, are involved incadherin signaling (Braga et al., 1997; Noren et al., 2001): E-cadherin signalingthrough Rac to Arp 2/3 regulates actin polymerization and reorganization ofthe actin cytoskeleton. RhoA acting through the actomyosin network and Cdc42through filopodia formation help initiate cell-cell contacts (reviewed in Fukata andKaibuchi, 2001; Jaffer and Chernoff, 2004).

7.3.1.2. NM II in cell-cell adhesion

In an effort to test the relationship between E-cadherin and NM II localization,Shewan et al. (2005) found an interdependence of the two proteins in the Mcf7

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breast tumor cell line and in Chinese hamster ovary cells exogenously expressingE-cadherin. E-cadherin and NM II partially co-localize at the cell-cell contactsupon homophilic binding of cadherin. This interdependence of E-cadherin andactive NM II localization is diminished in response to Rho kinase inhibition orinhibition of NM II activity. When the motor activity of NM II is inhibited withblebbistatin, E-cadherin redistributes on the cell surface and away from areas ofcontact. Y27632, the Rho kinase inhibitor, causes a loss of NM II from cell-cell adhesions accompanied by decreased levels of MLC20 phosphorylation. Bothtreatments also result in the loss of actin localization from the perijunctional belt.

However, MLC20 phosphorylation of NM II is not required in order for E-cadherinto accumulate at sites of cell-cell adhesion. Avizienyte et al. (2004), investigatingthe Src signaling pathways in epithelial cancer cells, shows that NM II phospho-rylated on MLC20 accumulates at the cell periphery in cells adopting a migratoryphenotype. This accumulation can be blocked by inhibitors of Src kinase, mitogen-activated protein kinase kinase (MEK), Rho kinase, and MLCK, but E-cadherinstill localizes to sites of cell-cell contact. Thus, in the initial stages of formation ofcell-cell junctions, although actin is required, myosin activation and the contractileresponse seem not to be required and may even be disruptive of the junctions.The downstream effector of RhoA signaling may act to differentially modulateactomyosin filaments: RhoA acting through Rho kinase increases myosin contrac-tility and disrupts nascent cell-cell adhesion, whereas RhoA signaling through theformin homology protein, Dia, promotes cadherin-catenin complex formation (Sahaiand Marshall, 2002).

In studying the AJC in intestinal epithelial cells, the formation of TJs, butnot adherens junctions, is accompanied by recruitment of NM II to the AJC andblocked by the NM II ATPase inhibitor, blebbistatin (Ivanov et al., 2005; Miyakeet al., 2006). NM II is responsible for cell polarity and positioning of the AJC.Rapid turnover of actin is required for the nascent junctions to form although morestable actin filaments contribute to the mature junctions. NM II phosphorylation isdetected only after assembly of apical junctions. In addition, disassembly of theAJC in calcium-depleted intestinal cells, which occurs through contractile F-actinring intermediates, is blocked by blebbistatin treatment indicating a requirement forNM II activity in disassembly (Ivanov et al., 2004).

The barrier function of TJs appears to be regulated by NM II activity in thatstudies show that a constitutively active MLCK induces reversible changes in TJpermeability, morphology and biochemical properties in differentiated epithelialmonolayers (Shen et al., 2006). Since these changes occur in the absence of alter-ations in expression of the claudins, occludins or ZO-1, they may represent fine-tuning of TJ permeability in response to physiological challenges. Upregulation ofMLCK is also observed to contribute to barrier dysfunction (Wang et al., 2005).Studies by Srinivas et al. (2006) support a role for NM II in TJ function throughhistamine-induced phosphorylation of MLC20 and inhibition of MLC20 phosphatase.Treatment with histamine disrupts the apical actin belt and decreases the barrierfunction of corneal endothelial cells in culture.


7.3.1.3. Cell-cell adhesion during embryonic development

In vivo studies of mammalian embryonic development are increasingly contributingto our understanding of the role of myosin in cell-cell adhesion. When NM II-A is deleted in embryonic stem cells and in mouse embryos, there is a loss ofcell-cell adhesion and a decrease in E-cadherin and �-catenin localization at cell-cell contacts. The embryos are disorganized and unable to progress beyond E6.5.In embryoid bodies, which lack NM II-A, the cells detach from the surface andmigrate out from the cell cluster (Conti et al., 2004; see also Section 7.4.1). Geneticdeletion of NM II-B also causes a cell-cell adhesion defect during mouse embryonicdevelopment. In this case the defect appears to be confined to the neuroepithelialcells lining the embryonic spinal canal. These cells are more vulnerable becausethe only isoform of NM II they contain is NM II-B. This defect can be rescued byexpression of NM II-B containing a point mutation, which compromises enzymaticactivity (Ma et al., 2007; see also Section 7.4.1) and indicates a structural ratherthan contractile role for NM II-B in maintenance of the AJC in these cells.

Although this chapter concentrates on mammalian NM II, there are signif-icant results from studies of Drosophila embryo germ band elongation, which areof particular interest. Similar remodeling occurs during mammalian developmentduring stages such as gastrulation and the process of neurulation. In the type oftissue remodeling which is termed ‘convergent extension’, cell layers narrow andelongate. This occurs independently of tension at tissue boundaries of the cell sheetor cell division to increase the number of cells. The elongation is mediated by inter-calation of cells along one axis, in the process breaking the linkage to adjacent cellsand reforming that linkage to new neighboring cells (reviewed in Lecuit, 2005).NM II (a single gene product in Drosophila) is enriched at disassembling cell-cell junctions where it is concentrated along the anterior-posterior axis (Zallen andWieschaus, 2004) localized at transient junctions and appears to drive the formationof new junctions by preventing reversion of junctions to the original cell orien-tation (Bertet et al., 2004). Since the Rho kinase inhibitor Y27632, which preventsphosphorylation of MLC20 and activation of NM II, also prevents recruitment ofNM II to the cell-cell junctions, NM II activation appears to be required. AlthoughE-cadherin localization is not affected, Drosophila mutants for NM II are unable tointercalate cells or elongate and the embryos appear frozen at the stage of junctionremodeling. Such experiments point towards the involvement of tension maintainedby NM II at cell-cell boundaries in the oriented disassembly of cell junctions.

An apically localized actomyosin network is required for apical cell constrictionduring ventral furrow formation during Drosophila gastrulation. Studies fromseveral laboratories (Nikolaidou and Barrett, 2004; Dawes-Hoang et al., 2005; Foxand Peifer, 2007) elucidate the upstream pathways leading to cell shape changes,which form the developing embryo. In mutants of armadillo (the Drosophila �-catenin analog), which disrupt apical cell junctions, NM II mislocalizes, forminga tight contracted ball at the center of the cell, and apical constriction and cellshape change do not occur (Dawes-Hoang et al., 2005). Parallel pathways operatingthrough RhoGEF2 and Abelson kinase affect actin organization while Cta, a G�

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subunit downstream of twist and the folded gastrulation gene, acting throughRhoGEF2 (Rogers et al., 2004) affects activated apical NM II accumulation leadingto contraction of the actomyosin ring and apical cell constriction.

Wound closure by a purse string composed of actin and myosin (Bementet al., 1993) represents a particular example of cell adhesion in the movement ofepithelial sheets. In studies in Drosophila, it has been shown that NM II is thedriving force in contraction of the supracellular purse string during dorsal closure inembryonic development (Franke et al., 2005b). In mammalian MDCK cells, woundclosure requires NM II contractile activity, involves actomyosin rings at both apico-lateral tight junction sites and basal-lateral cell sites, and is inhibited by the Rhokinase inhibitor Y-27632 (Tamada et al., 2007; Bement et al., 1993). In humanintestinal epithelial cells, although assembly of an actomyosin ring is dependenton Rho kinase, wound closure itself requires active MLCK. Additionally, NM IIphosphorylation is detected around sites of injury in human colonic disease biopsyspecimens (Russo et al., 2005).

Earlier models of the connection between the transmembrane cadherin and theactomyosin networks have been modified in light of experiments, which show that�-catenin does not simply connect a stable complex of E-cadherin and �-cateninto actin (Yamada et al., 2005; Drees et al., 2005). It is possible that the connectionfrom cadherin to the actomyosin network is achieved by locally high concentrationsof �-catenin dimers, which promote actin polymerization, by linkages through thePDZ domain of �-catenin to other proteins, which bind to actin, or is mediated byother adhesion molecules such as nectin (reviewed in Weis and Nelson, 2006).

The importance of NM II as a downstream effector of cell signals initiated uponcadherin engagement is an active field of study. The elucidation of the signal cascadeas well as the precise function of NM II in maintenance and disassembly of cellcontacts is increasing our understanding of embryonic development, homeostasis,and of disease processes.

7.3.2. Cell Migration


NM II contributes to the stability of tissues through maintenance of cell-celladhesion and it is also required for cell motility both during normal developmentand disease processes. Directed cell migration is key to the early stages in devel-opment of the embryo such as gastrulation, when the newly forming cell layersmigrate and form the three-layered embryo. Growth cone motility, wound healing,angiogenesis, immune surveillance, renewal of cell layers in skin and gut epitheliumall require properly timed and spatially limited cell migration. Disease processessuch as metastases of cancer cells, tissue invasion, and tumor-directed angiogenesisare examples of cell migration gone awry.

Cell migration can be looked on as a series of coordinated steps, which movethe cell body forward in response to a stimulus. The initial step is extension ofthe leading edge of the cell by protrusion of a lamellipodium. The protrusive force


generated by the lamellipodium is due to the dendritic network of Arp2/3-boundactin filaments which extend the cell membrane by adding actin at the leading edgeand depolymerizing filaments towards the back of the lamellipodium (reviewed inPollard and Borisy, 2003; Ponti et al., 2004). The lamellipodium makes contact withthe surface and is stabilized by the formation of small, punctate focal contacts atthe base of the lamellipodium, which provide stability and traction. Focal adhesions,which form at the base of the lamellipodium, indirectly connect actomyosin containingstress fibers to transmembrane integrins, which, in turn, link to the extracellularmatrix. The cell body contracts and advances in the direction of the lamellipodiumat the front of the cell and detaches from the contact surface at the cell rear.

In order to accomplish these steps, the cell requires coordinated, transientsignaling and response networks (reviewed in Ridley et al., 2003; Xu et al., 2003;Raftopoulou and Hall, 2004). Initial signals for migration activate small GTPases ofthe Rho family, which cycle between active GTP-bound and inactive GDP-boundstates. Rac signals are important for lamellipodia formation and Cdc42 for filopodiaat the cell front. Rac and Cdc42 inhibit RhoA activation at the leading edge sothat RhoA signaling is higher at the sides and rear of the cell where it signals toactomyosin filaments.

7.3.2.2. Cell migration, front to back

NM II activation is required at the front of the cell in order to exert tension onnascent focal complexes through stress fibers. The size and density of adhesionsin cells plated on high concentrations of fibronectin decrease when NM II activityis inhibited by low concentrations of blebbistatin. When low concentrations ofcalyculin A, an inhibitor of serine/threonine phosphatases 1 and 2A, are used toinhibit NM II phosphatase and thereby increase activated NM II, the size anddensity of focal adhesions increase as does the speed of migration of the cells(Gupton and Waterman-Storer, 2006). NM II activation by MLCK is requiredfor formation of focal adhesions and stress fibers at the cell periphery based onstudies using the inhibiter ML7 (Totsukawa et al., 2004; Katoh et al., 2001).A fluorescent biosensor also detects the activated state of MLCK at the lamella(Chew et al., 2002). When MLCK is inhibited with ML7, turnover of focalcomplexes is decreased (Webb et al., 2004) and the lamellipodial extension isblocked (Brahmbhatt and Klemke, 2003). In addition to an effect on focal adhesions,tension generated by NM II in stress fibers also causes a periodic contraction of thelamellipodia in migrating cells dependent on matrix rigidity, fibronectin binding,and MLCK activity (Giannone et al., 2004; Giannone et al., 2007). Phosphory-lation and activation of NM II through Rho kinase, which occurs at the cell center,is required for maturation of stress fibers and focal adhesions in this area of thecell (Katoh et al., 2001; Totsukawa et al., 2004). In addition to providing contrac-tility in stress fibers and tension on focal adhesions (Chrzanowska-Wodnicka andBurridge, 1996), NM II also acts as a sensor of the stiffness of the extracellularmatrix. Experiments with mesenchymal stem cells show that stiffness of the extra-cellular matrix contributes to neurogenic, myogenic, and osteogenic differentiation

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and that blebbistatin or MLCK inhibition can block the ability of the stem cells tosense the extracellular matrix (Engler et al., 2006).

Current investigations are directed towards understanding the different oroverlapping roles of the three NM II isoforms. Studies of spreading of the breastcancer cell line, MDA-MB-231, show increased phosphorylation and recruitmentof NM II to the lamellipodia in cells plated on fibronectin and support a key rolefor NM II-B in cell spreading (Betapudi et al., 2006). Fibroblasts isolated fromNM II-B null mice exhibit multiple, unstable protrusions and migrate with a lackof persistence, indicating that NM II-B is involved in directing cell movementby coordinating or suppressing side protrusions and stabilizing cell polarity (Loet al., 2004). Movement of NM II-A to the front of bovine aortic endothelial cellscan be blocked by blebbistatin treatment, but accumulation of II-B at the cell rearis unaffected (Kolega, 2006). Kolega proposes a division of functions between NMII-A associated dynamic actin networks such as those at the front of migratingcells and stable force sensing structures associated with NM II-B at the cell rear inthese cells. Genetic deletion or siRNA-mediated reduction of NM II-A enhances thespeed of non-directional migration of several cell types, stabilizes microtubules nearthe leading edge, and reduces cell contractility and the number of focal adhesions(Even-Ram et al., 2007). Some of these NM II-A deficient cells show markeddefects in retraction at the cell rear. Effects on microtubules indicate a role for NMII-A in crosstalk between dynamic microtubules and the actomyosin network, whichis only partially compensated by NM II-B after microtubule depolymerization.Inhibition or downregulation of the kinesin motor, Eg5, rescues the increased cellmigration due to absence of NM II-A suggesting the interplay of microtubule- andactomyosin-based systems.

Neuronal growth cones act as pathfinders to guide the growing neuron. WhenNM II in the neuronal growth cone is inhibited with blebbistatin, retrograde actinflow at the leading edge decreases by 51% and actin-bundle severing also decreases(Medeiros et al., 2006). During growth-cone collapse or retraction, however, centralactin-bundle contraction mediated by NM II is RhoA/Rho kinase-dependent (Zhanget al., 2003). In growth cones isolated from NM II-B null mice, retrograde flowincreases over the retrograde flow of growth cones from wild-type littermates(Brown and Bridgman, 2003) and filopodial-mediated traction force is reduced(Bridgman et al., 2001). NM II-A appears to partially compensate for the lackof II-B in these growth cones. Studies in Neuro-2A cells using antisense oligosto NM II-A or II-B are consistent with a role for NM II-A in neurite retractionand a contribution of II-B to neurite extension (Wylie et al., 1998; Wylie andChantler, 2003).

In the final step of cell migration, the back of the cell retracts and detaches fromthe substrate. In response to RhoA signals mediated by Rho kinase at the back ofthe cell (Worthylake et al., 2001; Alblas et al., 2001), stress fibers are contractedby NM II. Inability to retract the trailing edge has also been observed when MLCKor Rho kinase is inhibited showing the need for an activated form of myosin at theback of the cell (Eddy et al., 2000; Somlyo et al., 2000). The isoform dependence


of tail retraction, as with other NM II functions, cannot be clearly assigned andmay be dependent on cell type. NM II-B accumulation and tail retraction at theback of bovine aortic endothelial cells are blocked when Rho kinase is inhibitedby either a dominant negative construct or Y-27632. The phenotype is reversed byexpression of a constitutively active MLCK (Kolega, 2003). NM II-A localizationat the front of these migrating cells is unaffected. On the other hand, a strikingfeature of NM II-A null embryonic stem cells, which contain II-B, is the longtrailing tails (Even-Ram et al., 2007).

There is a coordination required between the structures that mediate cell-celladhesion and those of migration and cell-matrix adhesion and the study of thecontrol mechanisms involved in these processes is of particular interest. During theepithelial to mesenchymal transition, a normal as well as pathological process, cellsdisengage from each other and begin to migrate over each other, between othercells or on the extracellular matrix. Src family kinases have been proposed as apart of the crosstalk between the two systems, which converges at NM II (reviewedin Avizienyte and Frame, 2005) and which will likely involve the Rho family ofGTPases and actomyosin cytoskeleton remodeling.

The process of cell migration has been examined in cell culture, and now increas-ingly in three-dimensional matrices (reviewed in Even-Ram and Yamada, 2005;Meshel et al., 2005) and in intact animals (reviewed in Yamaguchi et al., 2005). Newtechniques for visualizing and recording this fundamental process are clarifying ourunderstanding and posing new questions for future studies.

7.3.3. Cytokinesis


Cytokinesis is the final event of the cell-division cycle and the process wherebythe cytoplasm of a single cell is divided to spawn two daughter cells. Cytoki-nesis usually begins with furrowing of the equatorial plasma membrane (cleavagefurrow) between the separated sister chromatids shortly after anaphase in mitosis.Accumulation of NM II as well as actin filaments in the cleavage furrow of dividingcells, often called the contractile ring, and the involvement of NM II activityin cytokinesis were first demonstrated in the 1970’s using the eggs of a marineorganism and mammalian cultured cells (Mabuchi and Okuno, 1977; Fujiwara andPollard, 1976). Since then, the role of NM II in cytokinesis has been studied inmany eukaryotic organisms including lower eukaryotes, invertebrates and verte-brates. Recent genome-wide screening in C. elegans and Drosophila also confirmsthe essential role of NM II in cytokinesis (Eggert et al., 2004; Echard et al., 2004;Skop et al., 2004; Sonnichsen et al., 2005). However, regulation of NM II activ-ities, including the actin-activated MgATPase activity, actin-filament gliding andfilament assembly differs considerably among the various species. Whereas NM IIsfrom mammals, flies and worms share a similar regulatory mechanism involvingMLC20 phosphorylation (see Section 7.2.4), yeast and slime mold use differentmechanisms to regulate NM II activity. This section focuses on mammalian NM II.

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7.3.3.2. NM II localization and dynamics during cytokinesis

Temporal and spatial movements of NM II have been studied by a number of labora-tories using different techniques including time-lapse fluorescence microscopy ofliving cells and antibody staining of fixed cells. Although there are three NMHCgenes in mammalian genomes, many earlier studies do not distinguish among theseisoforms.

Taylor’s laboratory has reported a comprehensive analysis of NM II filamentdynamics during cytokinesis by probing Swiss 3T3 cells with microinjectedfluorescent-labeled smooth-muscle myosin II, which can be incorporated intoendogenous NM II filaments (DeBiasio et al., 1996). Following chromosomeseparation during anaphase and telophase, short myosin filaments moved direc-tionally toward the equatorial region of the cell. The concentration of myosinfilaments increases in the equatorial region. At the same time, myosin filamentsin the polar regions move with a random orientation. In the equatorial region,myosin filaments are oriented parallel, perpendicular and at various angles to theequator. Myosin-containing fibers exist not only in the cortical region, but arealso distributed throughout the lumen of the cleavage furrow. Actin filaments alsoundergo dynamic remodeling and reorientation during cytokinesis. The arrays ofactin filaments in the equatorial region include a cortical meshwork of longitudinaland equatorial filaments and cortically associated filaments that project into thelumen (Fishkind and Wang, 1993). Interestingly, the extent of filament alignmentappears to correlate with the degree of cell-cell and cell-substratum interactions.

All of the differently oriented myosin-containing fibers in the cleavage furrowcontract during cytokinesis. During progression of cytokinesis, myosin-containingfibers are shortened and transported out of the cleavage furrow into the nearbycentral cytoplasm facing the two nascent daughter cells. In the case of adhesivecells, the concentration of myosin filaments increases in the polar regions over timeduring mid to late cytokinesis. The daughter cells then spread on the substratumand migrate in opposite directions.

The traction forces generated during cytokinesis have also been measured usinga modification of the silicone-rubber substratum method (Burton and Taylor, 1997).The formation of the cleavage furrow results from increased force generation atthe equatorial region rather than relaxation at the pole regions. The traction forcesubsequently mediates cytofission of the intracellular bridge in adhesive cells. Thisis similar to tail retraction in freely migrating cells.

As noted above (Section 7.2.4), the force-generating actomyosin MgATPaseactivity is regulated through phosphorylation of MLC20 at Ser19 with or withoutphosphorylation on Thr18. This phosphorylation also promotes myosin filamentformation. Therefore, phosphorylation of MLC20 at Ser19 (and Thr18� serves as anindicator of the active state of NM II. Studies using a fluorescent biosensor forMLC20 phosphorylation at Ser19 in living cells demonstrate that global phosphory-lation of MLC20 on Ser19 is initiated at anaphase when cortical myosin transportstarts toward the equatorial region (DeBiasio et al., 1996). The phosphorylation ofMLC20 remains high in the cleavage furrow through telophase and into cytokinesis,


whereas this phosphorylation decreases at the poles. The timing and pattern ofphosphorylation is the same as the shortening of the myosin-containing fibers in thecleavage furrow. After the cells are spread in late cytokinesis, MLC20 phosphory-lation remains near the intercellular bridge and it is also detected in the transverseregion between the leading edge and the nucleus of the two nascent daughter cells,similar to migrating cells. Studies with phospho-Ser19-specific antibodies agree withthe above observations (Matsumura et al., 1998). The staining of the equatorialregion with these antibodies can be seen only after chromosome separation at the latestage of anaphase. However, this staining precedes the ingression of the cleavagefurrow. In telophase, phospho-Ser19 antibody staining is seen at the cleavage furrow,where it persists until the end of cytokinesis. In addition, di-phosphorylated MLC20

at Ser19 and Thr18 also accumulates at the cleavage furrow (Yamashiro et al., 2003).In contrast, MLC20 is partially phosphorylated at Ser1 and/or Ser2 instead of Ser19

in metaphase cells (Yamakita et al., 1994). Phosphorylation of Ser1 and Ser2,unlike Ser19 phosphorylation, does not activate NM II. Phosphorylation at Ser19 inthe equatorial region at late anaphase is detected at the same time that NM II isdetected in this region. Moreover, actin-filament assembly in the equatorial regionalso occurs simultaneously. These observations suggest that NM II is already or isimmediately activated when it is recruited to the equatorial region. However, studieswith an inhibitor of myosin ATPase activity, blebbistatin, suggest that recruitmentof NM II to the equatorial region does not require myosin ATPase activity (Straightet al., 2003). Consistent with this, phosphorylation of MLC20 at Ser19 is observedprior to furrow ingression. There is a report, however, that the filament-assemblydomain of the NM II rod is required for NM II recruitment to the cleavage furrow(Ikebe et al., 2001). These studies suggest that MLC20 phosphorylation at Ser19

during an early stage of equatorial recruitment functions to accelerate NM II filamentassembly. For furrow ingression, NM II ATPase activity is required since, in thepresence of blebbistatin, cells fail to divide (Straight et al., 2003).

In addition to cleavage furrow contraction, recent studies suggest that NM IIMgATPase activity (or MLC20 phosphorylation) may be involved in turnover ofactin in the contractile ring of the cleavage furrow (Murthy and Wadsworth, 2005;Guha et al., 2005). Fluorescence recovery after photo-bleaching (FRAP) studieshave demonstrated that contractile ring proteins, such as actin, are highly dynamicduring cytokinesis with a half-life in the order of 10 seconds. Treatment of cellswith blebbistatin or inhibitors of MLC20 phosphorylation, which interfere withcytokinesis, results in delay of actin turnover at the cleavage furrow. These studiessuggest that the contractile ring proteins are turning over dynamically and that thecontinuous disassembly and reassembly of the actin filaments may play a role infurrow ingression.

7.3.3.3. Signaling molecules regulating NM II activities during cytokinesis

A major modification of NM II regulating its activities during cytokinesis isphosphorylation of MLC20 at Ser19 and Thr18. Phosphorylation of the NMHC canmodulate NM II activity and assembly (see Section 7.2.4), but there is no report

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studying NMHC phosphorylation during cytokinesis in mammalian cells. A fewproteins that are capable of interacting with NM II during cytokinesis have beenreported. These molecules will be discussed in the following section (Section7.3.3.4). This section focuses on molecules that regulate MLC20 phosphorylation.

To date, three protein kinases, Rho kinase, citron kinase and smoothmuscle/nonmuscle myosin light chain kinase (MLCK), and one phosphatase,myosin phosphatase, are known to change the extent of MLC20 phosphorylationat Ser19 and Thr18 during cytokinesis. Activation of MLCK requires its bindingto the Ca2+/calmodulin complex. Therefore, MLC20 phosphorylation by MLCK ismediated by a Ca2+-signaling pathway. On the other hand, the other three enzymesare targets of active RhoA. Modulation of each of these three enzymes mediatedby a RhoA-signaling pathway leads to an increase of MLC20 phosphorylation.

Thesmoothmuscle/nonmuscleMLCKgenegenerates twokinases, ahighmolecularweight isoform (long MLCK) and a low molecular weight isoform (short MLCK)by using two alternative promoters. The long MLCK, which is transcribed froman upstream promoter, contains identical sequences to the short MLCK with theaddition of a unique amino-terminal extension of about 900 amino-acid residues(Birukov et al., 1998). Long MLCK is expressed predominantly in nonmuscle cellsand proliferating muscle cells, whereas short MLCK is expressed predominantly insmooth-muscle cells. The relative abundance of the long and short MLCKs variesamong different cell types. It has been reported that the long MLCK is enrichedthroughout the cell cortex during metaphase and that the concentration of the longMLCK increases in the equatorial cortex and cleavage furrow during late anaphaseand telophase (Poperechnaya et al., 2000). The unique amino-terminal extension,along with a region common to the amino-terminal of short MLCK, is requiredto target the long MLCK to the cleavage furrow. However, the molecules respon-sible for targeting long MLCK to the cleavage furrow are presently unknown. Theshort MLCK does not accumulate in the cleavage furrow and is distributed diffuselyduring the cell cycle. On the other hand, using fluorescent resonant energy transfer(FRET), the activation state of short MLCK, which is bound to Ca2+/calmodulin,has been measured during anaphase and cytokinesis (Chew et al., 2002). Signif-icant MLCK enrichment occurs during anaphase at the equator of the spindle whereMLCK shows moderate levels of calmodulin binding. Immediately before constrictionof the cleavage furrow, MLCK appears to be maximally activated. This increasein activity at the cleavage furrow is immediately followed by recruitment of activeMLCK to the pole regions of the daughter cells. The amounts of MLCK decreaseat the spindle midzone following the onset of cleavage furrow contraction, butMLCK remains maximally activated until cleavage is complete. MLCK at the polesshows high levels of calmodulin binding, especially within the lamella and ruffles.Although this study used the short MLCK as a biosensor, it measures the relativespecific activity per MLCK as well as protein amounts (Chew et al., 2002). Therefore,the timing and location of MLCK activation are applicable to the long MLCK tosome extent, since Ca2+/calmodulin is a common activator to both the short andlong MLCKs. It would be of interest to repeat these experiments using long MLCK.


Changes in the intracellular Ca2+ levels, which are of critical importance duringthe mammalian cell cycle, remain to be elucidated. In the case of large fertilizedeggs, such as sea urchin or Xenopus, a local increase of calcium, called a calciumtransient, has been observed at various time points correlating with cell cycle eventsincluding metaphase-anaphase transition (anaphase onset) and cytokinesis (Ciapaet al., 1994; Chang and Meng, 1995; Webb et al., 1997; Lucero et al., 2006). Thereis evidence that a localized elevation of Ca2+ is spatially and temporally associatedwith the formation of the cleavage furrow. However, similar evidence is lacking formammalian somatic cells. Studies with these cells usually measure global changes,not local changes, in intracellular Ca2+ levels. A gradual increase of Ca2+ hasbeen observed starting in anaphase and continuing until a late stage in cytokinesisin mammalian cells (Ratan et al., 1988; Tombes and Borisy, 1989). Therefore,global activation of MLCK can occur starting at anaphase. On the other hand, theCa2+/calmodulin-activated MLCK activity can be inhibited by phosphorylation ofMLCK catalyzed by other protein kinases, including p21-activated kinase (PAK;Sanders et al., 1999). Rac1, which can activate PAK, is itself activated at the polarcortex at late anaphase and telophase (Yoshizaki et al., 2003). This Rac1 activationat the polar regions may result in an inhibition of MLCK activity at the polarregions, thereby leaving MLCK activity high at the equatorial region.

Rho kinase, citron kinase and the myosin-binding subunit (MBS) of myosinphosphatase can bind directly to the active form of RhoA, GTP-bound RhoA(Leung et al., 1995; Matsui et al., 1996; Kimura et al., 1996; Madaule et al., 1998).Therefore, following activation of RhoA in the equatorial cell membrane duringlate anaphase, these three enzymes are recruited to the equatorial cortex, albeit atslightly different times. Rho kinase is recruited at late anaphase, presumably earlierthan citron kinase, and stays at the cleavage furrow during cytokinesis (Kosakoet al., 1999). Citron kinase is localized at the cleavage furrow at telophase, staysthere during cytokinesis and is concentrated at the midbody until the completionof cytokinesis (Eda et al., 2001). Rho kinase is activated by active RhoA (Leunget al., 1995; Matsui et al., 1996) and based on the structural similarity between thetwo enzymes, citron kinase is also predicted to be activated by RhoA. However,there is no clear biochemical data for this. Citron kinase can show kinase activity invitro to some extent in the absence of the active RhoA (Yamashiro et al., 2003). BothRho kinase and citron kinase are capable of phosphorylating MLC20 at Ser19 andThr18 (Amano et al., 1996; Yamashiro et al., 2003). Citron kinase has an increasedpropensity to phosphorylate Thr18 following phosphorylation of Ser19 compared toRho kinase and MLCK. In addition to mono-phosphorylated MLC20 (on Ser19�,di-phosphorylated MLC20 (on Ser19 and Thr18� is detected at the cleavage furrowin late cytokinesis.

Myosin phosphatase, which is composed of MBS, a type 1 phosphatase catalyticsubunit and a regulatory subunit, can also serve as a substrate for Rho kinase, butnot citron kinase. Without phosphorylation of MBS, myosin phosphatase is consti-tutively active. MBS itself is also a target of the active RhoA. The binding of MBSwith active RhoA does not affect myosin phosphatase activity, but it translocates

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MBS to the equatorial membrane, where Rho kinase is also recruited by the activeRhoA. Rho kinase phosphorylates MBS mainly on Thr695/Thr697 (chicken/rat) andThr850/Thr855 (chicken/rat; Kawano et al., 1999; Feng et al., 1999). Phosphory-lation of the former site results in inhibition of the catalytic activity, whereasphosphorylation at the latter site results in dissociation of MBS from myosin (Fenget al., 1999; Velasco et al., 2002). Therefore, phosphorylation of either site leadsto inhibition of MLC20 dephosphorylation. An additional site, Ser849/Ser854/Ser852

(chicken/rat/human), of MBS can be phosphorylated uniquely by Rho kinase.MBS phosphorylated at this site has been demonstrated to accumulate in thecleavage furrow (Kawano et al., 1999). Interestingly, MBS can be phosphorylatedat Ser430/Ser435 (chicken/rat) by other mitotic kinase(s) at prometaphase (Totsukawaet al., 1999). This phosphorylation leads to enhancement of the phosphatase activity,an effect opposite to that of Rho kinase. Collectively, the RhoA-activation pathwayleads to an increase in MLC20 phosphorylation, thereby to activation of NM II.Among the three RhoA-dependent events: MBS phosphorylation by Rho kinase,direct phosphorylation of MLC20 by Rho kinase and MLC20 phosphorylation bycitron kinase, the event which is dominant during cytokinesis has not been deter-mined. Chemical inhibition of Rho kinase, exogenous expression of a dominantnegative form of Rho kinase and depletion of Rho kinase by siRNAs all inhibit theaccumulation of MLC20 phosphorylation in the cleavage furrow and inhibit cytoki-nesis to some extent (Kosako et al., 2000; Yokoyama et al., 2005). On the otherhand, exogenous expression of a truncated citron kinase interferes with the laterstages of cytokinesis and results in a high frequency of multinucleation (Madauleet al., 1998). Whether MLC20 phosphorylation is affected by inhibiting citron kinaseor not has not been determined.

Accumulating evidence supports RhoA activation as a key event for furrowformation and ingression during cytokinesis in a number of organisms includingmammals (reviewed in Piekny et al., 2005). Recent studies successfully demon-strate RhoA accumulation at the equatorial cortex in late anaphase (Yoshizakiet al., 2003; Yuce et al., 2005). Other lines of evidence suggest that the locationof the cleavage furrow is determined by the position of the mitotic spindle,especially the central spindle, a set of anti-parallel microtubules that become bundledbetween the separating chromosomes during anaphase and serve to concentrate keyregulators of cytokinesis (reviewed in Glotzer, 2005 and D’Avino et al., 2005).The crucial molecules mediating the central spindle signals, which activate RhoAat the equatorial cortex, are the centralspindlin complex and a RhoA guaninenucleotide exchange factor (GEF). The centralspindlin complex is composed ofa kinesin-6 family protein, MKLP1 and a Rho family GTPase activating protein(GAP), CYK-4. During anaphase, centralspindlin becomes highly concentratedin the central spindle where it is required for central spindle assembly. Similarto all small Rho family GTPases, RhoA activation requires a GEF. Among alarge family of GEFs, ECT2 is a critical RhoGEF for cytokinesis in mammaliancells (Tatsumoto et al., 1999). ECT2 concentrates on the central spindle and isrequired for accumulation of RhoA at the equatorial cortex. Depletion of ECT2


by siRNA blocks furrow formation and actomyosin contractile ring formation asdoes depletion of RhoA. Recent studies have demonstrated a cell cycle-dependentassociation of ECT2 with CYK-4, providing a physical link at the central spindleamong the centralspindlin complex (MKLP1 and CYK-4), ECT2, and RhoA (Yuceet al., 2005; Kamijo et al., 2006). This link appears to explain the timing andlocation of RhoA activation. Another RhoGEF, MyoGEF, has also been reportedto be localized to the cleavage furrow during cytokinesis (Wu et al., 2006). Inter-estingly, MyoGEF binds directly to NM II. As described above, the active RhoAactivates Rho kinase and citron kinase. In addition, RhoA activates formin, anactin-nucleating factor, to accelerate unbranched actin filament formation at thecleavage furrow.

7.3.3.4. NM II interacting proteins relevant to cytokinesis

How is NM II targeted to the equatorial region and how does it remain in thecontractile ring during cytokinesis? MLC20 phosphorylation at Ser19 enhancesmyosin filament self-assembly. Is this high degree of phosphorylation sufficient tolocalize NM II filaments at the equatorial cortex where Rho kinase is activated andmyosin phosphatase is inactivated? A recent study using cultured Drosophila cellsshows that NM II containing an unphosphorylatable MLC20 is unable to localizeat the cleavage furrow, whereas NM II containing a phospho-mimic MLC20 (i.e.,irreversibly phosphorylated) can be targeted to the cleavage furrow (Dean andSpudich, 2006). These findings suggest that, although phosphorylation of MLC20

is required, the phosphorylation reaction does not necessarily occur at the cleavagefurrow. NM II with phosphorylated MLC20 is not localized at the equatorial regionuntil late anaphase. Therefore, self-assembly of NM II due to MLC20 phosphory-lation is not the only mechanism responsible for targeting of NM II to the cleavagefurrow. NM II could be recruited by some other molecules via direct binding. Anumber of molecules, which are also localized in the cleavage furrow and have theability to bind directly to NM II, have been reported.

Anillin is an actin-filament bundling protein and has a pleckstrin homologydomain, which targets the protein to the cell membrane. Anillin accumulates at theequatorial cortex at an early time during cytokinesis and becomes a component ofthe contractile ring of dividing cells. Of note, anillin can interact directly with NMII in a MLC20 phosphorylation-dependent manner (Straight et al., 2005). It can bindto NM II that has been phosphorylated on MLC20, but not to unphosphorylatedNM II. In anillin-depleted cells, NM II is initially localized to the equatorial regionand the furrow starts to ingress. Halfway through furrow ingression, however, theincipient daughter cells begin to dramatically change diameter, growing on one sideand shrinking on the other, as the cytoplasm is extruded back and forth throughthe constricted furrow. At this time, the constricted furrow does not stay betweentwo daughter nuclei. The location of NM II is no longer confined to the originalcortex between the two separated chromosomes. NM II moves to be concentrated atsites in the cortex where abnormal contractions are occurring. When the abnormaloscillating contractions subside, the cleavage furrow regresses, resulting in failure of

246 CONTI ET AL.

cytokinesis. Therefore, anillin constrains NM II contractility to the original cleavagefurrow during the late stage of cytokinesis, presumably by direct-interaction ofanillin with the MLC20 phosphorylated NM II.

A novel NM II-binding protein, MyoGEF, has also been reported (Wuet al., 2006). Among Rho-family small GTPases, MyoGEF activates RhoA, butnot Rac1. MyoGEF localizes at the equatorial cortex and the central spindle inanaphase cells. This localization of MyoGEF is in contrast with the localization ofECT2, which is localized only at the central spindle, but not at the equatorial cortex.MyoGEF binds directly to the rod region of NM II-A and co-localizes with NM IIin anaphase cells. Depletion of MyoGEF results in multinucleation. These resultssuggest an attractive model in which MyoGEF-RhoA and NM II act as a functionalunit at the cleavage furrow to advance furrow ingression during cytokinesis.

7.3.3.5. NM II isoforms and other factors affecting cytokinesis

The majority of studies on the function of NM II during cytokinesis have notdistinguished among the isoforms of NM II. Since there are multiple genes encodingeach NMHC, MLC20 and MLC17, the potential combinations of subunits result in alarge number of putative NM II isoforms. Because of a lack of studies dealing withisoforms of MLC20 and MLC17, only isoforms of the NMHC will be consideredhere. As noted above, there are three genes encoding the NMHC protein, NMHCII-A, B and C in mammals. Both NMHC II-A and B accumulate in the cleavagefurrow in HeLa cells (Maupin et al., 1994). A cytokinesis defect in Cos-7 cells inwhich NMHC II-B is depleted by siRNA can be rescued by exogenous expressionof each of the NMHC II-A, B and C isoforms (Bao et al., 2005). These observationssuggest that NM II isoforms play a redundant role in cytokinesis in HeLa andCos-7 cells. However, this may not be true in all types of cells in culture or inintact animals. A recent study using the A549 lung carcinoma cell line demonstratesthat a particular isoform of NMHC II-C, NMHC II-C1, which is generated byalternative RNA splicing and contains an insert of eight amino acids in loop 1(see Figure 7.1C), is required for proper cytokinesis (Jana et al., 2006). A549cells depleted of this isoform can form a cleavage furrow, but the completion ofcytokinesis is prolonged even when the total NM II content is compensated forby other NMHC isoforms. The NM II-C1 isoform is localized to the intercellularbridge in the late stage of cytokinesis, whereas other NM II isoforms localize to thepolar regions. This differential distribution of NM II isoforms seems important forlung tumor A549 cells, but how the different NM II isoforms segregate in generalinto different regions of cells during cytokinesis is not known. Together with thefact that expression of NMHC II-C1 is elevated in a number of epithelial cancercell lines compared to the normal cultured cells and in primary tumors comparedto the normal surrounding tissues, these data indicate that the NM II-C1 may beinvolved in abnormal cell proliferation (Jana et al., 2006).

Mice that have been ablated for each of the NMHC isoforms have been generated.Among them, the embryonic cardiac myocytes in NMHC II-B null mice show adefect in cytokinesis with a high frequency of multinucleation (Takeda et al., 2003).


After E8.5, cardiac myocytes lose detectable amounts of NMHC II-A (Ma andAdelstein, unpublished observation), but still express NMHC II-C. Moreover, whenNMHC II-B is replaced by II-A in mice, the cytokinesis defect remains in thecardiac myocytes suggesting that NMHC II-B plays a specific role in these cells(Bao et al., 2007). The above examples show that isoforms of NM II can haveunique cytokinesis functions in different cell contexts.

Cell type-dependent differences in Rho-family GTPase signaling during cytoki-nesis have also been described. The importance of RhoA activation at the cleavagefurrow has been emphasized using a number of cell lines including HeLa cells. InRat 1A cells, however, suppression of Rac1 by CYK-4, a Rac GAP, appears to bemore important in the central spindle than activation of RhoA by the RhoA GEF,ECT2 (Yoshizaki et al., 2004). Rac1 is known to activate PAK, which phospho-rylates MLCK and inhibits MLCK activity (Sanders et al., 1999). Therefore, Rac1suppression in the central spindle may result in an increase in MLCK activityin the equatorial cortex. The balance between Rac1 and RhoA activities at thecleavage furrow is critical for the progression of cytokinesis (reviewed in D’Avinoet al., 2005). Progression of cytokinesis requires high RhoA activity or low Rac1activity in the cleavage furrow. The dominant pathway seems dependent on cell type.

In addition to cell type, environmental factors, such as the extracellular matrixand cell-cell contact, also affect cytokinesis. It has been reported that the extent ofalignment of actin filaments at the contractile ring appears to be correlated withthe degree of cell-cell and cell-substratum interaction. For example, alignmentsof actin filaments at the cleavage furrow are different between dorsal and ventralcortexes (Fishkind and Wang, 1993). Moreover, in the case of adherent cells culturedrelatively sparsely, cells become spread on the substratum and lamellae form atthe two opposite poles as cleavage furrow ingression advances. The two daughtercells then begin to migrate in opposite directions and start pulling each other. Inorder to complete cytokinesis, constriction of the contractile ring at the cleavagefurrow is accompanied by this traction force, which is generated by the daughtercells on the substratum (Jana et al., 2006). Generation of traction force couldbe affected by extracellular matrix proteins and contact with neighboring cells,as well as cellular adhesion and migration properties. In many mammalian cells,both constriction of the contractile ring and traction force are mediated by NM IIactivities. However, there is a report that highly adhesive cells, such as NRK cellsand HT1080 fibrosarcoma cells, plated on collagen or fibronectin-coated plates,can divide using only NM II-independent traction force (Kanada et al., 2005). Inthe context of tissues or intact animals, cells are surrounded three-dimensionallyby neighboring cells and extracellular matrix. Future studies are needed to examinethe effects of the various environmental factors in order to understand the role ofNM II in cytokinesis in the intact animal.

As described above, MLCK, Rho kinase, citron kinase and myosin phosphataseare thought to play a role in the regulation of NM II during cytokinesis. Therelevance of these enzymes to cytokinesis in the animal context can be evaluatedin null mice. The mouse genome contains a single citron kinase gene, a single

248 CONTI ET AL.

smooth muscle/nonmuscle MLCK gene, two Rho kinase genes and a single smoothmuscle/nonmuscle MBS gene for myosin phosphatase. Citron kinase-null miceshow a cytokinesis defect in particular types of neural cells in the brain (Di Cuntoet al., 2000). In the case of Rho kinases, null mice for each of two genes do notshow an obvious cytokinesis defect (Thumkeo et al., 2003; Shimizu et al., 2005). Adetailed analysis of the expression of two Rho kinase genes in different tissues andcells is required to interpret the results of these null mice and it may be necessaryto produce null mice for both Rho kinases or conditional null mice in order tounderstand the in vivo function of the Rho kinases in cytokinesis. MLCK-nullmouse embryos seem to develop to normal size, suggesting that MLCK may not beessential in cell proliferation in most tissues (Somlyo et al., 2004). However, detailedhistological studies have not yet been reported. MBS-null mice have also beenproduced. A preliminary study shows MBS ablation results in embryonic lethalitybefore E7.5 and no live embryos have been found yet (Okamoto et al., 2005; seeSection 7.4.1).

Finally, there are a few reports suggesting that NM II may play a role inother phases of cell division in addition to cytokinesis. NM II-dependent corticalmovement is required for centrosome separation and positioning, thereby allowingNM II to play a role in mitotic spindle assembly (Rosenblatt et al., 2004). A numberof studies have described detection of NMHC II and phosphorylated MLC20 at thespindle poles using specific antibodies (Kelley et al., 1996; Matsumura et al., 1998).Overexpression of an unphosphorylatable MLC20 or treatment with an inhibitor ofNM II MgATPase activity, blebbistatin, causes abnormal karyokinesis (Komatsuet al., 2000; Kanada et al., 2005). These observations suggest a role for NM II inkaryokinesis.

7.4. EXPERIMENTS IN LIVE ANIMALS

7.4.1. During Development and in the Adult

The presence of three different isoforms of NMHC IIs in mammalian cells posesunique problems and presents major opportunities to decipher the function of NMII. At present, it is not clear at which point in evolution animals with three isoformsemerged. Drosophila, similar to Dictyostelium, has only a single gene product,which appears to resemble both human MYH9 (II-A) and MYH10 (II-B) geneproducts by amino-acid comparison. Xenopus contains at least two isoforms, II-Aand II-B (Kelley et al., 1996) and, at present, it does not appear to have II-C.This section will deal with the current knowledge of the function of the threedifferent NM II isoforms present in mammalian cells. The emphasis will be onexperiments carried out in mice as well as information obtained from humans whohave mutations in their MYH9 and 14 genes. To date, no humans with defects in theMYH10 gene have been described, although mice with a point mutation (Arg709Cys)have been generated (Ma et al., 2004; Ma et al., 2007).

A major advance was made in understanding the role of myosin in Dictyosteliumwhen NM II was ablated by homologous recombination (De Lozanne and


Spudich, 1987). Similarly, mutation of the single isoform of NM II in Drosophilaenhanced our understanding of the role of NM II in the early development of thisinvertebrate (Young et al., 1993; Peralta et al., 2007; Franke et al., 2005b). Allthree isoforms appear to be ubiquitous in their distribution in mammals duringembryonic development, although NM II-C is not present in the early embryo.However, II-C can be detected using antibodies by E11.5 in mice (see Figure 7.2;Golomb et al., 2004).

An interesting question that is presently under investigation relates not only tothe distribution of the various isoforms in the developing and adult mammal, butalso to the quantity of each isoform in any given cell. This is of importance sincethe relative amount of each isoform can have a direct effect on the cell phenotypeand the results of lowering a specific isoform using the readily available techniqueof siRNA can differ depending on the relative amount of the isoform. For example,analysis of Cos-7 cells shows that over 90% of NM II is II-B and the remainderis II-C (there is no II-A). Lowering II-B using siRNA has a marked effect oncytokinesis in that 68% of the cells become multinucleated. Lowering II-C has noeffect, but this most likely reflects the small amount of II-C (less than 10% ofII-B) in these cells. It does not necessarily reflect whether II-C can or cannot playa role in cytokinesis in cells in which this isoform is more abundant (e.g., in thelung tumor cell line A549, see Section 7.3.3.5). It is also important to appreciatethat immunoblots, unless accompanied by ancillary quantitative techniques (Baoet al., 2005), do not readily provide quantitative data by themselves.

A second example of the importance of the amount of NM II can be seen in thegeneration of hypomorphic mice. These mice, which express decreased amounts ofgene product due to the placement of the cassette encoding Neomycin resistancein the affected gene, are particularly informative in studying the function of NMII-B. The generation of mice expressing 12% of the normal amount of II-B inthe heart and mice expressing 6% of the normal amount of II-B reveals a genedosage effect correlating with the decrease in expression. Thus, whereas II-B-ablated mice die before or at birth, the severe (6%) hypomorphs survive for 30days, exhibiting similar but somewhat milder cardiac and brain defects and theless severe hypomorphs (12%) survive to adulthood allowing the further study ofdefects in the heart and brain (Uren et al., 2000).

Ablation of NM II-A results in early lethality (E6.5) in mice with defects inthe visceral endoderm and in cell-cell adhesion. The failure in the developmentof the visceral endoderm in the II-A null mice could reflect the absence of bothNM II-A and II-B from this cell layer, thus rendering it particularly vulnerableto loss of a number of functions, and may also explain why it never differen-tiates into a polarized secretory epithelium. Absence of a functioning visceralendoderm may explain why NM II-A-ablated embryos fail to undergo gastrulation.A second major defect noted in the developing embryo as well as in II-A nullembryonic stem cells is a failure in cell-cell adhesion (Conti et al., 2004 andsee Section 7.3.1.2). This was manifested by cells spontaneously detaching fromembryoid bodies and by a decrease in cadherin and catenin at cell-cell boundaries

250 CONTI ET AL.

as revealed by immunofluorescence microscopy. Immunoblots show no loss ofthese proteins in these cells so the loss observed by microscopy is attributed totheir diffuse redistribution throughout the cell rather than degradation. The loss ofcadherin and catenin from the cell-cell boundaries of both embryos and embryoidbodies seen with II-A null cells was duplicated using siRNA to lower NMHC II-AmRNA in wild-type embryonic stem cells. These findings suggest that tension byNM II-A on actin filaments is required to stabilize the adhesion complex and areconsistent with the work of others using cultured cell lines (Shewan et al., 2005).

Ablation of NMHC II-B results in major defects in the brain and heart andresults in death between E14.5 and birth (Tullio et al., 1997; Tullio et al., 2001).The phenotype observed suggests that NM II-B cannot be solely responsible forall neuronal migration in vivo, since the brain for the most part develops normally.This may be attributed to the presence of NM II-C and possibly NM II-A in thesecells. Of note is that generation of point mutant (Arg709Cys) hypomorphic miceresults in defects in the migration of three specific groups of neurons: cerebellargranular cells, pontine neurons and facial neurons (Ma et al., 2004).

The major brain defect seen in II-B-ablated mice is hydrocephalus, the accumu-lation of excess cerebral spinal fluid (CSF) in the brain ventricles, which resultsin the destruction of brain tissue and architecture due to the marked increase inintracerebral pressure. Hydrocephalus, although treatable after birth (3/1000 livebirths), remains a serious cause of prenatal morbidity in humans. Recent workdemonstrates that the onset of hydrocephalus in NM II-B null mice is due to aloss of cell-cell adhesion in a mesh-like structure at the apical border of the cellslining the spinal canal beginning at El1.5 (Ma et al., 2007). The defect in the apicaladhesion complex in the cells lining the canal allows the underlying neuroepithelialcells to invade the canal and obstruct the circulation of the CSF. In addition toablation of NM II-B as noted above, a decrease in the amount of NM II-B by 70–80% can also result in hydrocephalus, although the onset is later and the severity oftissue destruction is decreased (Uren et al., 2000). Interestingly, hydrocephalus inNM II-B null mice can be rescued by expressing two different isoforms of NM II.Homologous recombination was used to ablate NMHC II-B with cDNA encodingNMHC II-A, thereby placing II-A under control of the endogenous II-B promoter.This results in NM II-A expression throughout the brain and spinal cord and inthe localization and expression of II-A in place of II-B in the mesh-like structurebordering the spinal canal. Despite significant differences in the kinetic propertiesbetween NM II-A and II-B (see Table 7.1), NM II-A is able to restore normal celladhesion to the cells lining the canal and thereby to prevent hydrocephalus (Baoet al., 2007).

Hydrocephalus is also rescued by expressing wild-type quantities (but notdecreased amounts) of a mutant NMHC II-B (Arg709Cys) in the cells lining thespinal canal. This mutant form of myosin has a decreased actin-activated MgATPaseactivity and cannot propel actin in the in vitro motility assay, although it bindstightly to actin (Kim et al., 2005; see Table 7.1). The ability of this mutant myosinto rescue hydrocephalus demonstrates the importance of the scaffolding properties


of myosin rather than its enzymatic properties in restoring cell-cell adhesion to thecells bordering the spinal canal. Although both NM II-A and the mutant Arg709CysII-B are capable of rescuing the cell adhesion defect in the brain that leads to hydro-cephalus, neither of these isoforms can completely rescue the defects in neuronalmigration found for the facial and pontine neurons in II-B-deficient mice (Maet al., 2007; Bao et al., 2007). These results reflect the two different roles NM II-Bplays in neuronal cell function, both as a motor and as a scaffold.

The cardiac defects found in the NM II-B ablated mice include a membranousventricular septal defect, translocation of the aorta which together with thepulmonary artery originates in the right ventricle, and a defect in cytokinesisresulting in a 70% reduction in the number of cardiac myocytes but not non-myocytes in the heart. The decrease in myocytes is accompanied by the enlargementof the cardiac myocytes (Takeda et al., 2003). These mice die from a failure incardiac function (Tullio et al., 1997).

Other important and related proteins that have been ablated in mice includeboth myosin light chain kinase (MLCK) and the myosin-binding subunit (MBS) ofmyosin phosphatase. Surprisingly, targeted disruption of both the long and shortisoforms of MLCK results in mice that develop to normal size, although they die 1–5 hours after birth. These mice show abnormalities in their coronary vessels, whichhave variable diameters and irregular shapes. Despite the lack of MLCK, culturedsmooth-muscle cells are able to contract and undergo MLC20 phosphorylation,presumably due to another kinase(s) present in the cells (Somlyo et al., 2004). Incontrast, ablation of the MBS of myosin phosphatase results in early (prior to E7.5)embryonic lethality in mice, indicating the absence of compensation from otherenzymes or a second critical function for MBS (Okamoto et al., 2005). Using siRNAdirected at lowering MBS, Xia et al. (2005) provide evidence that MBS plays a rolein actin assembly that is independent of its role in MLC20 dephosphorylation. Theauthors show that the proper expression of MBS (or a second variant of MBS foundin HeLa cells) is required to maintain normal cellular functions by simultaneouslycontrolling NM II activation (through MLC20 dephosphorylation) and cytoskeletalarchitecture.

7.4.2. NM II Associated Diseases

Single amino-acid mutations in MYH9 in humans result in defects in a numberof different cells and tissues. These include enlarged, but fewer blood plateletsresulting in bleeding defects, inflammation of the kidneys (nephritis), deafness,leukocyte inclusions and cataracts. The identified point mutations span the NMHCII-A molecule starting at amino acid Asn93 and ending at Arg1933 (Heath et al., 2001;Seri et al., 2003). As expected, despite their different locations, the single amino-acid mutations are limited to conserved parts of the molecule. While the samemutation can cause a number of different defects, the same tissues and organs, notedabove, are always affected. A relatively frequent mutation is Arg702Cys, which isconsistently associated with defects in the kidneys, hearing and platelets. Table 7.1

252 CONTI ET AL.

includes the effects of some of these mutations on the activities of HMM derivativesof the various NM II isoforms. The Table shows that the Asn93Lys mutant has amuch greater effect by lowering both the actin-activated MgATPase activity and invitro motility than does the Arg702Cys mutation (Hu et al., 2002). Working in vitrowith four different mutations in the myosin rod (Arg1165Cys, Asp1424Asn, Glu1841Lysand Arg1933Stop), Franke et al. (2005a) show that these mutants act dominantly tointerfere with the proper assembly of wild-type rod fragments.

Recent work provides new insights into the role played by NM II-A in thegeneration of platelets from megakaryocytes. Chen et al. (2007) show that NM II-Anull mouse embryonic stem cells differentiate into megakaryocytes that are fullycapable of proplatelet formation. On the other hand, elevation of NM II-A activityin these cells, either by exogenous expression or by mimicking phosphorylation ofMLC20, markedly attenuates proplatelet formation. Similarly, Chang et al. (2007)found that inhibiting Rho kinase or MLCK results in increased proplatelet formation.These experiments are of interest since they show that NM II-A activity appearsto negatively regulate proplatelet formation. Future experiments in this area shouldhelp to clarify the mechanism involved.

To date, there have been no reports of humans with any mutations in MYH10encoding NMHC II-B. Recently, mice with a point mutation, Arg709Cys, homol-ogous in location to a known mutation in NMHC II-A (Arg702Cys) have beengenerated (Ma et al., 2004; 2007). These mice are born with large omphalocelesand 50% of them are born with their hearts outside of the thoracic wall (Ma andAdelstein, unpublished data). This models a human syndrome called Pentalogy ofCantrell (Cantrell et al., 1958), a rare congenital abnormality (5/106births). Thedefect in ventral wall closure observed in these mice is reminiscent of the defect indorsal wall closure following ablation of NM II in Drosophila (Young et al., 1993:Franke et al., 2005b). Interestingly, Shimizu et al. (2005) have generated mice thatare ablated for Rho kinase I and find that these mice are born with open eyelids andomphaloceles. The authors demonstrate that Rho kinase I regulates closure of theventral body wall by inducing the formation and contraction of actomyosin bundles.These results are consistent with idea that NM II is one of the main effectors of theRhoA/Rho kinase pathway in vivo.

With respect to NM II-C, a mutation of the NMHC at Arg726Ser, a site homol-ogous to that found to be mutated in NMHC II-A (Arg702Cys), results in deafnessin humans showing that NM II-A and II-C both play roles in hearing (Donaudyet al., 2004). As noted above and in Table 7.1, the same mutation in the insertedHMM II-C1 has no effect on the actin-activated MgATPase activity but doesdecrease the in vitro motility of actin filaments by 75% (Kim et al., 2005; seeTable 7.1).

7.5. SUMMARY

The purpose of this chapter is to review the basic properties of the NM II geneproducts, particularly in mammalian cells. An effort is made to highlight some


of the recent advances made in our understanding of the role of NM II in threeimportant basic functions: cell adhesion, cell migration and cytokinesis. In the finalsection, the function of the three NM IIs in vivo that are in the live, intact animal ishighlighted. Recently, not a month passes without some new report on the role ofa NM II in an additional cellular process not previously reported (Lee et al., 2007).The future for understanding the various roles of NM IIs in health and diseaseappears extensive, challenging and exciting.

ACKNOWLEDGEMENTS

We want to thank James Sellers, Xuefei Ma and Siddhartha Jana for critical readingof the manuscript, Mihaly Kovacs for his helpful discussions on kinetics andCatherine Magruder for expert editorial assistance.

ABBREVIATIONS

NM II, nonmuscle myosin IINMHC, nonmuscle myosin heavy chain IIMHC, myosin heavy chainMLC20, 20kDa myosin light chainMLC17, 17kDa myosin light chainHMM, heavy meromyosinS-1, subfragment-1IVM, in vitro motilityMLCK, myosin light chain kinaseMBS, myosin-binding subunit of myosin phosphatasePAK, p21-activated protein kinasePKC, protein kinase CAJC, apical junction complexTJ, tight junctionGEF, GTP exchange factorGAP, GTP activating proteinCSF, cerebral spinal fluidE, embryonic daysiRNA, small interfering RNA

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[Proteins and Cell Regulation] Myosins Volume 7 || Non-Muscle Myosin II

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