Conditional deletion of nonmuscle myosin II-A in mouse tongue epithelium results in squamous cell carcinoma.

To investigate the contribution of nonmuscle myosin II-A (NM II-A) to early cardiac development we crossed Myh9 floxed mice and Nkx2.5 cre-recombinase mice. Nkx2.5 is expressed in the early heart (E7.5) and later in the tongue epithelium. Mice homozygous for deletion of NM II-A (A(Nkx)/A(Nkx)) are born at the expected ratio with normal hearts, but consistently develop an invasive squamous cell carcinoma (SCC) of the tongue (32/32 A(Nkx)/A(Nkx)) as early as E17.5. To assess reproducibility a second, independent line of Myh9 floxed mice derived from a different embryonic stem cell clone was tested. This second line also develops SCC indistinguishable from the first (15/15). In A(Nkx)/A(Nkx) mouse tongue epithelium, genetic deletion of NM II-A does not affect stabilization of TP53, unlike a previous report for SCC. We attribute the consistent, early formation of SCC with high penetrance to the role of NM II in maintaining mitotic stability during karyokinesis.

PKC412 normalizes mutation-related keratin filament disruption and hepatic injury in mice by promoting keratin-myosin binding.

UNLABELLED: Keratins, among other cytoskeletal intermediate filament proteins, are mutated at a highly conserved arginine with consequent severe disease phenotypes due to disruption of keratin filament organization. We screened a kinase inhibitor library, using A549 cells that are transduced with a lentivirus keratin 18 (K18) construct, to identify compounds that normalize filament disruption due to K18 Arg90Cys mutation at the conserved arginine. High-throughput screening showed that PKC412, a multikinase inhibitor, ameliorated K18 Arg90Cys-mediated keratin filament disruption in cells and in the livers of previously described transgenic mice that overexpress K18 Arg90Cys. Furthermore, PKC412 protected cultured A549 cells that express mutant or wild-type K18 and mouse livers of the K18 Arg90Cys-overexpressing transgenic mice from Fas-induced apoptosis. Proteomic analysis of proteins that associated with keratins after exposure of K18-expressing A549 cells to PKC412 showed that nonmuscle myosin heavy chain-IIA (NMHC-IIA) partitions with the keratin fraction. The nonmuscle myosin-IIA (NM-IIA) association with keratins was confirmed by immune staining and by coimmunoprecipitation. The keratin-myosin association is myosin dephosphorylation-dependent; occurs with K8, the obligate K18 partner; is enhanced by PKC412 in cells and mouse liver; and is blocked by hyperphosphorylation conditions in cultured cells and mouse liver. Furthermore, NMHC-IIA knockdown inhibits PKC412-mediated normalization of K18 R90C filaments. CONCLUSION: The inhibitor PKC412 normalizes K18 Arg90Cys mutation-induced filament disruption and disorganization by enhancing keratin association with NM-IIA in a myosin dephosphorylation-regulated manner. Targeting of intermediate filament disorganization by compounds that alter keratin interaction with their associated proteins offers a potential novel therapeutic approach for keratin and possibly other intermediate filament protein-associated diseases.

Nonmuscle myosin II is required for internalization of the epidermal growth factor receptor and modulation of downstream signaling.

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) is an important process for regulating signal transduction, cellular dynamics, and cell-cell communication. Here, we demonstrate that nonmuscle myosin II (NM II) is required for the internalization of the EGFR and to trigger the EGFR-dependent activation of ERK and AKT. The EGFR was identified as a protein that interacts with NM II by co-immunoprecipitation and mass spectrometry analysis. This interaction requires both the regulatory light chain 20 (RLC20) of NM II and the kinase domain of the EGFR. Two paralogs of NM II, NM II-A, and NM II-B can act to internalize the EGFR, depending on the cell type and paralog content of the cell line. Loss (siRNA) or inhibition (25 µm blebbistatin) of NM II attenuates the internalization of the EGFR and impairs EGFR-dependent activation of ERK and AKT. Both internalization of the EGFR and downstream signaling to ERK and AKT can be partially restored in siRNA-treated cells by introduction of wild type (WT) GFP-NM II, but cannot be restored by motor mutant NM II. Taken together, these results suggest that NM II plays a role in the internalization of the EGFR and EGFR-mediated signaling pathways.

Ablation of nonmuscle myosin II-B and II-C reveals a role for nonmuscle myosin II in cardiac myocyte karyokinesis.

Ablation of nonmuscle myosin (NM) II-A or NM II-B results in mouse embryonic lethality. Here, we report the results of ablating NM II-C as well as NM II-C/II-B together in mice. NM II-C ablated mice survive to adulthood and show no obvious defects compared with wild-type littermates. However, ablation of NM II-C in mice expressing only 12% of wild-type amounts of NM II-B results in a marked increase in cardiac myocyte hypertrophy compared with the NM II-B hypomorphic mice alone. In addition, these hearts develop interstitial fibrosis associated with diffuse N-cadherin and ß-catenin localization at the intercalated discs, where both NM II-B and II-C are normally concentrated. When both NM II-C and II-B are ablated the B-C-/B-C- cardiac myocytes show major defects in karyokinesis. More than 90% of B-C-/B-C- myocytes demonstrate defects in chromatid segregation and mitotic spindle formation accompanied by increased stability of microtubules and abnormal formation of multiple centrosomes. This requirement for NM II in karyokinesis is further demonstrated in the HL-1 cell line derived from mouse atrial myocytes, by using small interfering RNA knockdown of NM II or treatment with the myosin inhibitor blebbistatin. Our study shows that NM II is involved in regulating cardiac myocyte karyokinesis by affecting microtubule dynamics.

Myosin II isoforms identify distinct functional modules that support integrity of the epithelial zonula adherens.

Classic cadherin receptors cooperate with regulators of the actin cytoskeleton to control tissue organization in health and disease. At the apical junctions of epithelial cells, the cadherin ring of the zonula adherens (ZA) couples with a contiguous ring of actin filaments to support morphogenetic processes such as tissue integration and cellular morphology. However, the molecular mechanisms that coordinate adhesion and cytoskeleton at these junctions are poorly understood. Previously we identified non-muscle myosin II as a target of Rho signalling that supports cadherin junctions in mammalian epithelial cells. Myosin II has various cellular functions, which are increasingly attributable to the specific biophysical properties and regulation of its different isoforms. Here we report that myosin II isoforms have distinct and necessary roles at cadherin junctions. Although two of the three mammalian myosin II isoforms are found at the ZA, their localization is regulated by different upstream signalling pathways. Junctional localization of myosin IIA required E-cadherin adhesion, Rho/ROCK and myosin light-chain kinase, whereas junctional myosin IIB depended on Rap1. Further, these myosin II isoforms support E-cadherin junction integrity by different mechanisms. Myosin IIA RNA-mediated interference (RNAi) selectively perturbed the accumulation of E-cadherin in the apical ZA, decreased cadherin homophilic adhesion and disrupted cadherin clustering. In contrast, myosin IIB RNAi decreased filament content, altered dynamics, and increased the lateral movement of the perijunctional actin ring. Myosin IIA and IIB therefore identify two distinct functional modules, with different upstream signals that control junctional localization, and distinct functional effects. We propose that these two isoform-based modules cooperate to coordinate adhesion receptor and F-actin organization to form apical cadherin junctions.

Non-muscle myosin IIA differentially regulates intestinal epithelial cell restitution and matrix invasion.

Epithelial cell motility is critical for self-rejuvenation of normal intestinal mucosa, wound repair, and cancer metastasis. This process is regulated by the reorganization of the F-actin cytoskeleton, which is driven by a myosin II motor. However, the role of myosin II in regulating epithelial cell migration remains poorly understood. This study addressed the role of non-muscle myosin (NM) IIA in two different modes of epithelial cell migration: two-dimensional (2-D) migration that occurs during wound closure and three-dimensional (3-D) migration through a Matrigel matrix that occurs during cancer metastasis. Pharmacological inhibition or siRNA-mediated knockdown of NM IIA in SK-CO15 human colonic epithelial cells resulted in decreased 2-D migration and increased 3-D invasion. The attenuated 2-D migration was associated with increased cell adhesiveness to collagen and laminin and enhanced expression of beta1-integrin and paxillin. On the 2-D surface, NM IIA-deficient SK-CO15 cells failed to assemble focal adhesions and F-actin stress fibers. In contrast, the enhanced invasion of NM IIA-depleted cells was dependent on Raf-ERK1/2 signaling pathway activation, enhanced calpain activity, and increased calpain-2 expression. Our findings suggest that NM IIA promotes 2-D epithelial cell migration but antagonizes 3-D invasion. These observations indicate multiple functions for NM IIA, which, along with the regulation of the F-actin cytoskeleton and cell-matrix adhesions, involve previously unrecognized control of intracellular signaling and protein expression.

Rap1 activation in collagen phagocytosis is dependent on nonmuscle myosin II-A.

Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or beta1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.

The May-Hegglin anomaly gene MYH9 is a negative regulator of platelet biogenesis modulated by the Rho-ROCK pathway.

The gene implicated in the May-Hegglin anomaly and related macrothrombocytopenias, MYH9, encodes myosin-IIA, a protein that enables morphogenesis in diverse cell types. Defective myosin-IIA complexes are presumed to perturb megakaryocyte (MK) differentiation or generation of proplatelets. We observed that Myh9(-/-) mouse embryonic stem (ES) cells differentiate into MKs that are fully capable of proplatelet formation (PPF). In contrast, elevation of myosin-IIA activity, by exogenous expression or by mimicking constitutive phosphorylation of its regulatory myosin light chain (MLC), significantly attenuates PPF. This effect occurs only in the presence of myosin-IIA and implies that myosin-IIA influences thrombopoiesis negatively. MLC phosphorylation in MKs is regulated by Rho-associated kinase (ROCK), and consistent with our model, ROCK inhibition enhances PPF. Conversely, expression of AV14, a constitutive form of the ROCK activator Rho, blocks PPF, and this effect is rescued by simultaneous expression of a dominant inhibitory MLC form. Hematopoietic transplantation studies in mice confirm that interference with the putative Rho-ROCK-myosin-IIA pathway selectively decreases the number of circulating platelets. Our studies unveil a key regulatory pathway for platelet biogenesis and hint at Sdf-1/CXCL12 as one possible extracellular mediator. The unexpected mechanism for Myh9-associated thrombocytopenia may lead to new molecular approaches to manipulate thrombopoiesis.

Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk.

Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.