Nonmuscle Myosin II Isoform and Domain Specificity During

Nonmuscle myosin II isoform and domain speciﬁcity during early mouse development

Aibing Wanga, Xuefei Maa, Mary Anne Contia, Chengyu Liub, Sachiyo Kawamotoa, and Robert S. Adelsteina,1

aLaboratory of Molecular Cardiology, and bTransgenic Mouse Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1583

Edited* by Thomas D. Pollard, Yale University, New Haven, CT, and approved July 7, 2010 (received for review March 26, 2010) Nonmuscle myosins (NMs) II-A and II-B are essential for embryonic quire the cross-linking properties of myosin could be replaced by mouse development, but their specific roles are not completely another isoform, but those functions dependent on myosin’s defined. Here we examine the isoforms and their domain specifi- motor activity were not substitutable because of a difference in cally in vivo and in vitro by studying mice and cells in which kinetic properties. This hypothesis remains to be further tested, nonmuscle myosin heavy chain (NMHC) II-A is genetically replaced especially in regard to substitution of NM II-A by II-B. Fur- by NMHC II-B or chimeric NMHC IIs that exchange the rod and head thermore, studies using chimeric NM IIs, which contain func- domains of NM II-A and II-B. In contrast with the failure of visceral tional domains from two different isoforms, can provide more endoderm formation resulting in embryonic day (E)6.5 lethality of direct evidence to substantiate this idea and also help to un- A−/A− mice, replacement with NM II-B or chimeric NM IIs restores derstand their domain specificities. a normal visceral endoderm. This finding is consistent with NM II’s To test this hypothesis in vivo and in vitro, we used a genetic- role in cell adhesion and also confirms an essential, isoform- replacement strategy (15, 16) to study NM II in mouse embryos independent requirement for NM II in visceral endoderm function. and in cells isolated from these embryos. We generated the The knock-in mice die between E9.5 and 12.5 because of defects in following four mouse lines (Fig. S1 A–C) in which the Myh9 first placenta formation associated with abnormal angiogenesis and coding exon is disrupted by: (i) cDNA encoding GFP-tagged cell migration, revealing a unique function for NM II-A in placenta human NMHC II-B (GFP-hNMHC II-B, Ab*/Ab* mice); (ii) development. In vitro results further support a requirement for cDNA encoding chimeric GFP-hNMHC II-AB (the N-terminal NM II-A in directed cell migration and focal adhesion formation. domain of NMHC II-A fused to the C-terminal II-B domain, These findings demonstrate an isoform-specific role for NM II-A Aab/Aab mice); (iii) GFP-hNMHC II-BA (the N-terminal do- during these processes, making replacement by another isoform, main of NMHC II-B fused to the C-terminal II-A domain, Aba/ or chimeric NM II isoforms, less successful. The failure of these Aba mice); and (iv) as a control, cDNA encoding mCherry- substitutions is not only related to the different kinetic properties hNMHC II-A (AmCh/AmCh mice) was likewise inserted into the of NM II-A and II-B, but also to their subcellular localization de- same site of the Myh9 locus. Each of these expression cassettes termined by the C-terminal domain. These results highlight the was placed under control of the NMHC II-A promoter. There- functions of the N-terminal motor and C-terminal rod domains of fore, mutant mice or cells lack endogenous NM II-A but express NM II and their different roles in cell-cell and cell-matrix adhesion. knock-in proteins (Fig. S1D). Our results support a critical role for NM II in visceral endoderm development. They reveal cell migration | genetic substitution | placenta development | visceral a unique function for NM II-A in placenta development and endoderm formation | chimeric myosin II support a requirement for NM II-A in directed cell migration and focal adhesion formation in vitro and in vivo. onmuscle myosin II (NM II) is a major cytoskeletal protein Results and Discussion that interacts with actin to contribute to cellular processes, N An Essential but Isoform-Independent Role for NM II in Visceral such as cell migration (1–4), cell adhesion (5–8), and cytokinesis CELL BIOLOGY (9). In mammals there are three NM II isoforms, each composed Endoderm Formation. Thegenerationoffourmutantknock-in SI Materials and Methods of two identical heavy chains and two pairs of light chains. Three mouse lines is described in and shown in separate genes (Myh9, Myh10, Myh14) encode the nonmuscle Fig. S1. All heterozygotes from the different lines are in- distinguishable from their wild-type littermates. They were myosin heavy chains (NMHCs; NMHC II-A, II-B, and II-C), mCh mCh which together with the light chains are referred to as NM II-A, crossed to produce homozygous embryos. Control A /A II-B, and II-C. The three NM II isoforms not only show con- mice are born at the expected Mendelian frequency and are normal, demonstrating that the phenotypes observed in the mu- siderable homology in primary structure, but also have a similar tant mice are not a result of genetic manipulations of the Myh9 molecular structure in that each NM II contains two structurally locus (Table S1). The ratio of the GFP-NM II-B to the endoge- defined regions: a globular region at the N-terminal end har- α nous II-B in MEF cells is 2.8:1, indicative that GFP-NM II-B is boring MgATPase and actin binding activities, and an -helical D fi expressed under control of the NMHC II-A promoter (Fig. S1 , coiled-coil C-terminal tail region that mediates lament assem- right-most lane). Fig. S1E indicates that expression of the chi- bly (10). The in vivo functions of two of the isoforms have been meric NM IIs are similar to GFP-NM II-B in Ab*/Ab* mice. studied following germline ablation, revealing markedly different We first determined whether knock-in NM II-B or chimeric phenotypes: death by embryonic day (E)6.5 because of a failure NM IIs could functionally replace NM II-A, and rescue the cell- in cell-cell adhesion and visceral endoderm formation in the case of NM II-A and lethality by E14.5, resulting from cardiac and

brain defects following II-B ablation (7, 11, 12). These results Author contributions: A.W., X.M., and R.S.A. designed research; A.W., X.M., M.A.C., and C.L. suggest that both isoforms are essential for mouse development. performed research; A.W., X.M., M.A.C., S.K., and R.S.A. analyzed data; and A.W., X.M., Because most cells contain more than one isoform, their specific M.A.C., S.K., and R.S.A. wrote the paper. in vivo roles during embryogenesis are unclear. Previous work The authors declare no conflict of interest. has shown that some defects associated with the loss of NM II-B *This Direct Submission article had a prearranged editor. could be rescued in vivo by a motor-impaired II-B or when NM 1To whom correspondence should be addressed. E-mail: [email protected]. fi II-A is expressed from the II-B locus (13, 14). These ndings This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. resulted in the hypothesis that the functions of NM IIs that re- 1073/pnas.1004023107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1004023107 PNAS | August 17, 2010 | vol. 107 | no. 33 | 14645–14650 Downloaded by guest on September 27, 2021 cell adhesion defects of the visceral endoderm associated with II- a similar phenotype based on their appearance, as exemplified by Adeficiency at E6.5. Ab*/Ab*, Aab/Aab,Aba/Aba (collectively Ab*/Ab* embryos. These embryos display pale yolk sacs with referred to as “mutant”) and A+/A+ embryo sections were fewer visible vessels compared with the A+/A+ yolk sacs (Fig. stained with antibodies to NMHC II-A or II-B together with E- 2A). Whole-mount PECAM-1 staining also reveals that Ab*/Ab* − − cadherin (Fig. 1 and Fig. S2). In contrast to A /A embryos, embryos have a less intricate vascular network in the head and which have unidentifiable cell layers and a disorganized visceral trunk regions compared with the well-developed and hierarchi- endoderm marked by GATA4 staining of the nuclei (compare cally organized vascular architecture of A+/A+ counterparts Fig. 1 J with I), all mutant embryos (Fig. 1 E and G; Fig. S2 E, G, (Fig. 2B). These findings suggest that although vasculogenesis I, K) appear normal, with a polarized columnar visceral endo- occurs in the Ab*/Ab* yolk sacs, there is a defect in subsequent derm similar to A+/A+ embryos (Fig. 1 A and C and Fig. S2 A angiogenesis, which results in the impairment of the blood supply and C). Moreover, the cell-cell borders of the visceral endoderm to the embryo. In addition, Ab*/Ab* embryos show growth re- of A+/A+ and mutant embryos contain E-cadherin (arrows in tardation, which becomes more pronounced with age (Fig. S3). − − Fig. 1I and Fig. S2), which is not present in the A /A GATA4- However, this defect in growth is not a result of abnormalities in positive cells (arrows, Fig. 1J). As noted previously (7), wild-type cell proliferation or apoptosis, as shown in Fig. S4A, which shows visceral endoderm expresses NM II-A at the cell-cell boundaries no difference in the BrdU and TUNEL staining between Ab*/Ab* but has no detectable II-B (Fig. 1 B and D, arrows), whereas in and wild-type embryos at E9.5. mutant visceral endoderm, NM II-B or chimeric NM IIs are expressed in place of NM II-A (Fig. 1 F and H and Fig. S2 F, H, Specific Requirement for NM II-A in Placenta Development. The J, L, arrows). The presence of NM II-B or the chimeric NM IIs defects found in the yolk sac suggested that similar abnormalities restores normal cell-cell adhesion to the visceral endoderm. in vascular development might be observed in the placenta, − − Furthermore, unlike A /A mice, all of the mutant mice undergo which could be the cause of lethality. Mouse placental de- − − gastrulation, confirming that the early lethality of A /A mice is velopment involves at least three steps: formation of the allantois the result of a failure in formation of a functional visceral en- and the chorion, chorioallantoic fusion, and vascularization (17). doderm. This finding is consistent with a role for NM IIs in cell- We therefore examined the placenta of mutant mice between cell adhesion, a process that requires the cross-linking properties E8.5 and E10.5. Similar to A+/A+ mice, all of the mutant em- of NM II, therefore allowing substitution between the different bryos form proper allantoic structures (Fig. S3A, arrows); the NM II isoforms because of their similar structural properties. fusion of the allantois to the chorionic plate around E8.5 appears − − These results also confirm that the E6.5 lethality of A /A em- to be normal too. However, subsequent vascularization of these bryos is caused by the absence of both NM II-B and II-C from the mutant placentas was abnormal. At E10.5, A+/A+ placentas visceral endoderm, rendering it particularly vulnerable to NM II- acquire the typical trilaminar structure composed of a distal Aablation. circumferential giant cell layer, a middle spongiotrophoblast layer, and a proximal labyrinthine layer with a network of fetal Substitution for NM II-A by II-B or Chimeric NM IIs is Lethal. No capillaries and maternal blood sinuses (Fig. 3A, enlarged in C). mutant homozygous mice were found at weaning, indicating that In contrast, embryonic blood vessels of Ab*/Ab*orAba/Aba mice the development of the mutant mice is arrested at earlier stages fail to invade the labyrinthine layer and remain restricted to the (Table S1). Ab*/Ab* and Aba/Aba embryos die between E9.5 and chorioallantoic region. Consistent with this, Ab*/Ab*andAba/Aba E10.5, whereas Aab/Aab embryos die between E11.5 and E12.5. placentas are thinner and more compact, composed pre- Despite the differences in life span, the mutant embryos exhibit dominantly of clusters of cuboidal trophoblasts, making the la-

Fig. 1. Ab*/Ab* embryos exhibit normal visceral endoderm at E6.5. Sections from E6.5 A+/A+ (A–D) and Ab*/Ab*(E–H) embryos are stained with antibodies detecting E-cadherin (red) and NM II-A (green) or NM II-B (green). B, D, F, and H are enlarged from A, C, E, and G. There is no difference in the morphology, including embryo size and cell-layer organization, between A+/A+ and Ab*/Ab* embryos. Of note, the wild-type visceral endoderm expresses NM II-A (B, arrows) but lacks II-B (D, arrows), whereas Ab*/Ab* embryos lack NM II-A (F, arrows) and instead express II-B at the cell boundaries of the visceral endoderm (H, arrows). E6.5 A+/A+ (I) and A−/A− (J) embryo sections are stained with antibodies to E-cadherin (red) and GATA4 (green), a speciﬁc marker of visceral endoderm. DAPI (blue) stains the nuclei. (Scale bars, 100 μminG and J;20μminH.)

14646 | www.pnas.org/cgi/doi/10.1073/pnas.1004023107 Wang et al. Downloaded by guest on September 27, 2021 Fig. 2. Ab*/Ab* mutant mice have a defect in angiogenesis. (A) E9.5 and E10.5 A+/A+ and Ab*/Ab* yolk sacs. (Inset) A view of the entire embryo and yolk sac. Ab*/Ab* yolk sacs exhibit a pale appearance with fewer blood-filled vessels Fig. 3. Placenta defects in E10.5 Ab*/Ab* mice. (A and B) H&E-stained sec- compared with A+/A+ controls, indicating angiogenesis defects. (B) Whole- tions of A+/A+ and Ab*/Ab* placentas. At E10.5, Ab*/Ab* placentas are mount PECAM-1 staining (green) of embryonic vasculature of E10.5 A+/A+ and markedly thinner compared with the A+/A+ and lack fetal vessels in the Ab*/Ab* embryos. A less intricate vessel network is seen in the head and trunk labyrinthine layer. (C and D) Magnified from the red boxes in A and B.InC, of Ab*/Ab* embryos compared with A+/A+ controls. (Scale bar, 300 μm.) fetal blood vessels with nucleated erythrocytes (green arrow) invade and mix with maternal blood vessels (yellow arrow). In D, embryonic blood vessels (green arrow) do not invade but remain on the edge of the labyrinthine byrinthine and spongiotrophoblast layers difficult to discern (Fig. layer. (E and F) Endothelial cell distribution in A+/A+ and Ab*/Ab* placentas is 3 B and D and Fig. S5 C and F). Interestingly, invasion of em- visualized with a CD34 antibody (green). Almost no endothelial cells are seen bryonic blood vessels was observed in Aab/Aab placentas, but this in the labyrinthine layer of the Ab*/Ab* placenta when compared with the + + process was compromised, as shown by the reduced thickness of A /A placenta. Orientation in all panels is maternal side toward the top and the placenta and decreased internal space of fetal and maternal fetal side toward the bottom. Abbreviations: cp, chorionic plate; gc, giant vessels (Fig. S5 B and E). This finding is consistent with the in- cells; la, labyrinthine layer; m, maternal tissue; sp, spongiotrophoblast layer. DAPI stains nuclei in E and F. (Scale bars, 200 μminB and F;50μminD.) creased survival of Aab/Aab mice compared with Ab*/Ab*orAba/ Aba mice, indicating that the presence of the N-terminal domain of NM II-A supports increased placental maturity. Staining with NM IIs to rescue this defect, requires the presence of both CD34, a marker for endothelial cells, shows the blood vessels in functional domains: a specific motor domain and a specific CELL BIOLOGY + + E thelabyrinthinelayeroftheA /A placenta (Fig. 3 ). However, C-terminal domain. very few CD34-positive cells are found in the labyrinthine layer of E10.5 mutant mice (Fig. 3F and Fig. S5 H and I, compare with G). Mutant Mouse Embryonic Fibroblasts Exhibit Cell Migration Defects. Although formation of the labyrinthine layer fails and the tro- The failure of the mutant blood vessels to invade the labyrinthine phoblast compartment appears thinner in mutant placentas, no layer implies that the impaired vascularization might result from abnormality in cell proliferation and apoptosis is found in the a defect in cell migration. We therefore examined the migration mutant extraembryonic tissues (Fig. S4B). of mouse embryonic fibroblast (MEF) cells derived from wild- To further support the idea that defects in the mutant pla- type and mutant embryos. In a wound-healing assay to evaluate centas are a result of the specific loss of NM II-A, which is not the speed of cell migration, Ab*/Ab* and Aab/Aab MEFs closed rescued by expression of II-B (or chimeric NM IIs) from the the wound more rapidly than A+/A+ MEFs (Ab*/Ab*, 33.9 ± 2.8 Myh9 locus, we analyzed the expression of NM IIs in the placenta μm/h; Aab/Aab, 36.7 ± 3.9 μm/h; A+/A+ 24.2 ± 2.6 μm/h) (Fig. by immunofluorescence staining, which reveals an enriched and S7A). Interestingly, Aba/Aba cells migrate at 20.5 ± 4.8 μm/h, uniform expression of NM II-A at the fetal side of the A+/A+ a speed similar to A+/A+ cells. Previous studies reported that E9.5 placenta (Fig. S6A), consistent with the importance of NM the absence of NM II-A increases the speed of migration (3, 18). II-A in placenta development. Despite tracking the NM II-A Our results reveal that the C-terminal rod domain but not the N- tissue expression pattern in the mutant placenta (Fig. S6 A and terminal motor domain of II-A has an important effect on cell D), NM II-B or chimeric NM IIs cannot adequately substitute for migratory speed, because its absence in Ab*/Ab* and Aab/Aab II-A functions. Importantly, the placentas of E11.5 NM II-B- cells correlates with the increased speed of wound closure where − − ablated (B /B ) embryos do not show any obvious defects (Fig. the cells migrate as a sheet. On the other hand, using a transwell S5 K and M), providing further evidence of the specific re- assay to examine directional migration of single cells toward quirement for NM II-A. Collectively, these results suggest that serum, the number of MEFs from all three mutant lines mi- NM II-A plays a unique role in placenta development, a role grating across the transwell pores after 16 h was significantly less which is different from its functions in cell-cell adhesion in the than that of the A+/A+ MEFs (Fig. S7B). There is no significant visceral endoderm and, as shown by the failure of the chimeric difference among the three mutant MEFs, suggesting that both

Wang et al. PNAS | August 17, 2010 | vol. 107 | no. 33 | 14647 Downloaded by guest on September 27, 2021 domains contribute to single-cell migration through the pores of are disorganized and often fail to align with the direction of the chamber. The directionality of individual cells was further migration (Fig. 4 D, G,andJ). Interestingly, the actin cytoskeletal evaluated by time-lapse microscopy in 2D cell-culture dishes and structure of Aba/Aba cells appears more like the wild-type cells. persistence expressed as the ratio of net to total migrated dis- The staining for paxillin, a component of the focal-adhesion tance (N/T). The migration speed calculated from individual contacts, which anchors actin stress fibers, reveals abundant and cells (total distance/time) agrees with that derived from the well-spread focal adhesions throughout the A+/A+ cells (Fig. 4B). wound-healing assay. Interestingly, whether speed of migration In contrast, fewer and smaller focal adhesions are detected in the increases in Ab*/Ab* and Aab/Aab cells or is unchanged, as in mutant cells (Fig. 4 E, H,andK), especially in the Ab*/Ab*and Aba/Aba cells, their migratory persistence decreases significantly Aab/Aab cells. This decrease in both number and size of focal (Fig. S7C), suggesting that these mutant cells have a defect in adhesions is quantified in Table S2. The merge images indicate directed cell migration. Thus, our results indicate that either the that the stress fibers are anchored at both ends by focal adhesions in absence of the motor domain of NM II-A in Aba/Aba cells or the A+/A+ cells (Fig. 4C), whereas in the mutant cells, these structures lack of the NM II-A rod domain in Aab/Aab cells causes abnor- are in disarray and one or both ends of actin filaments are missing malities in cell migration, demonstrating that the two functional large focal-adhesion contacts, particularly in the Ab*/Ab*and domains collectively determine the specific function of NM II. Aab/Aab cells (Fig. 4 F, I,andL). In addition to reduced paxillin staining in focal adhesions, Ab*/Ab* cells exhibit almost no visible Mutant MEFs Display Reduced Focal Adhesions and Abnormal Stress phospho-Tyr118-paxillin immunofluorescence staining compared Fibers. To investigate the mechanism underlying the abnormali- with A+/A+ cells (Fig. 5A). Immunoblots demonstrate that the total ties of cell migration in the mutant cells, we examined actin cy- amount of focal-adhesion proteins, including paxillin and vinculin, toskeletal structure and focal adhesion formation, which are are unchanged, whereas the level of the phospho-Tyr118-paxillin, fi essential for cell migration. The distribution of actin stress bers indicative of paxillin activation and focal adhesion formation is and focal-adhesion contacts was visualized by staining with undetectable in the Ab*/Ab* cells (Fig.5B). Of note, when a plasmid phalloidin and antibodies to focal-adhesion proteins. Typical, encoding wild-type mCh-NMHC II-A is transfected into Ab*/Ab* fi + + robust and parallel stress bers are observed in the A /A cells cells, the focal adhesions are restored, confirming that the reduction A fi (Fig. 4 ), although mutant cells show thin actin laments that in focal adhesions is associated with the loss of NM II-A (Fig. 5C).

Fig. 5. Focal adhesions in Ab*/Ab* cells. (A)Ab*/Ab* cells shows markedly Fig. 4. Mutant MEF cells display abnormal stress fibers and fewer focal decreased phospho-paxillin staining (white spots) compared with A+/A+ cells. adhesions. A+/A+ (A–C), Ab*/Ab* (D–F), Aab/Aab (G–I), and Aba/Aba (J–L) MEF (B) Immunoblot indicates the approximate equivalence of several focal ad- cells are stained with phalloidin (green) and paxillin antibody (red). Com- hesion proteins in A+/A+ and Ab*/Ab* MEF cells, whereas phosphorylation of pared with thicker, organized stress fibers in A+/A+ cells (A), mutant cells show Tyr118-paxillin is low or undetectable in the latter. (C) Focal adhesions are thinner actin filaments that are disorganized and often fail to align with the restored when wild-type mCh-NMHC II-A is introduced into Ab*/Ab* MEF direction of migration (D, G, and J). In contrast to abundant focal adhesions cells (NM II-A, red; vinculin, green). Compare vinculin staining of focal (paxillin staining) throughout the A+/A+ cells (B), fewer and smaller focal adhesions in transfected (T) and untransfected (Un) Ab*/Ab* cells. Nuclear adhesions are detected in the mutant cells (E, H, and K). (Scale bar, 50 μm.) staining seen with the II-A antibody in C is nonspecific. (Scale bar, 50 μm.)

14648 | www.pnas.org/cgi/doi/10.1073/pnas.1004023107 Wang et al. Downloaded by guest on September 27, 2021 Fig. 6. Localization of endogenous NM IIs and knock-in GFP-NMHC IIs in MEF cells. Heterozygous A+/Ab*(A–F), A+/Aab (G–L), and A+/Aba (M–R) MEF cells are stained with the following antibodies to detect endogenous NM IIs: NMHC II-A C terminus (A–C and G–I); II-B C terminus (P–R); NMHC II-A N terminus (M–O); or II-B N terminus (D–F and J–L). Knock-in GFP-NMHC IIs are visualized by the fused GFP. Endogenous NM II-B and knock-in GFP-NM II-B (F) or GFP-NM II-AB (L) colocalize in the MEF cells; Endogenous NM II-A and knock-in II-BA (O) colocalize in the MEFs, indicating the importance of the C-terminal domain in localization. Lack of colocalization, as seen in C, I, and R reﬂects the different localization of endogenous and knock-in isoforms of NM II. End, endogenous; KI, knock-in. (Scale bar, 30 μm.)

The above finding is consistent with a specific role for NM II-A dynamic processes, in which the actomyosin complex interacts in cell migration, as reflected by the requirement for II-A for actin with cell matrix adhesion proteins and which require not only cytoskeletal organization and focal adhesion formation. During motor activity but also correct subcellular localization, sub- these processes, both the N-terminal and C-terminal domains of stitution in vivo is unsuccessful (Table S3). Differences in these NM II-A are required. Neither the N-terminal motor domain of dynamic properties may reflect differences in the kinetic prop- NM II-A in the Aab/Aab cells or the C-terminal rod domain of II-A fi ba ba erties between NM II-A and II-B. These include signi cant dif- in the A /A cells can rescue the defects caused by the loss of ferences in the rate of ATP hydrolysis when myosin is bound to intact II-A. actin, which is approximately 3-fold greater in the case of NM II-

A. In contrast, NM II-B has a higher duty ratio and affinity for CELL BIOLOGY Subcellular Localization of Knock-in NM II in Mutant MEF Cells. To ADP, which makes it particularly well suited to exert tension on address the question of whether a decrease of focal adhesions in fi – the mutant MEF cells is related to the alterations in the cellular actin laments for longer periods of time (22 24). Importantly, distribution of NM IIs, we studied the in vitro subcellular our results reveal that the essential role of NM II in visceral localization of knock-in GFP-NM II-B or chimeric NM IIs in endoderm formation and function is isoform-independent, A+/Ab*, A+/Aab, and A+/Aba heterozygous cells in which both whereas placenta development depends on the specific proper- the endogenous NMHC II-A and knock-in NMHC IIs are under ties of NM II-A. control of the NMHC II-A promoter. GFP-NM II-BA like NM II-A, and GFP-NM II-AB like NM II-B, display distinct sub- Materials and Methods cellular localizations: GFP-NM II-BA colocalizes to regions of Targeting Constructs. All mouse procedures were carried out in accordance the MEFs with endogenous II-A (Fig. 6, compare O to R), with National Heart, Lung, and Blood Institute Animal Care and Use Com- whereas GFP-NM II-B or GFP-NM II-AB colocalize to regions mittee guidelines. To create the knock-in/knockout constructs for homolo- F L C gous recombination, DNA fragments flanking exon2, the first coding exon of of the MEFs with endogenous II-B (Fig. 6 compare and to fi and I). These results agree with previous reports (19–21) and the mouse Myh9 gene, were ampli ed from a 129/Sv genomic BAC clone confirm, using an endogenous promoter, the observation that harboring the complete Myh9 locus (25). The arms consisted of a 4-kb fragment 5′ of the initiating ATG and a 1.7-kb fragment 3′ of the ATG co- subcellular localization is determined by the C-terminal domain don. The targeting constructs are depicted in Fig. S1A and consist of the 5′ of NMHC II. These results also indicate the important effects of arm, a cDNA cassette encoding mCherry (mCh)-human NMHC II-A or GFP- the C-terminal domain of NM II on focal adhesion formation human NMHC II-B or chimeric GFP-human NMHC II-AB or GFP-human NMHC and help explain our observation of an increase in focal adhe- II-BA, followed by SV40 polyA, a Neor cassette flanked by two loxP sites, the ba ba sions in A /A cells compared with the other mutant cell types. 3′ arm, and the thymidine kinase cassette. Chimeric GFP- NMHC II-AB in- This study supports the idea that, when the cross-linking cluding amino acids 1 to 836 of II-A and 844 to 1,977 of II-B, and II-BA function of NM II is required, the isoforms are interchangeable containing amino acids 1 to 843 of II-B and 837 to 1,961 of II-A were gen- in vivo, such as during the process of cell-cell adhesion when the erated by PCR with the templates GFP-NMHC II-A and GFP-NMHC II-B, de- actomyosin complex interacts with cell-cell adhesion proteins. scribed previously (26). Nucleotide sequences of the cloned DNA fragments However, during cell migration and focal adhesion formation, were confirmed in all cases by sequencing.

Wang et al. PNAS | August 17, 2010 | vol. 107 | no. 33 | 14649 Downloaded by guest on September 27, 2021 Histology and Immunofluorescence Staining. Embryos and placentas were an N-terminal NMHC II-A or II-B antibody was used and is noted in the figure dissected, fixed in 4% PFA overnight at 4 °C, dehydrated by methanol series, legends. Fluorescently labeled secondary antibodies (Molecular Probes) were − and stored in 100% methanol at 20 °C. Embryos and placentas were em- used and the slides were mounted with coverslips using Prolong Gold anti- fi μ bedded in paraf n, sectioned at 5 m on silanized slides, and stained with fade reagent (Molecular Probes). H&E (Histoserv). Alternatively, for immunofluorescence staining, antigen retrieval was performed as described before (27). These sections were then ACKNOWLEDGMENTS. We thank Dr. Christian A. Combs and Daniela Malide blocked with 10% normal goat serum and probed overnight at 4 °C with (Light Microscopy Core Facility, National Heart, Lung, and Blood Institute) primary antibodies: NMHC II-A, II-B (28), E-cadherin (Sigma, 1:100 or BD- for professional skills and advice regarding microscopy-related experiments Biosciences, 1:250), GATA4 (Santa Cruz, 1:50), CD34 (BD-Biosciences, 1:200), performed in this study. This work was funded by the intramural program of BrdU (Sigma, 1:500) or p-Histone-H3 (Santa Cruz, 1:200). Where appropriate, National Heart, Lung, and Blood Institute, National Institutes of Health.

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