Accumulation Of Myosin Research Articles

The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

UNC-45 is a UCS (UNC-45/CRO1/She4P) class chaperone necessary for myosin folding and/or accumulation, but its requirement for maintaining cardiac contractility has not been explored. Given the prevalence of myosin mutations in eliciting cardiomyopathy, chaperones like UNC-45 are likely to be equally critical in provoking or modulating myosin-associated cardiomyopathy. Here, we used the Drosophila heart model to examine its role in cardiac physiology, in conjunction with RNAi-mediated gene silencing specifically in the heart in vivo. Analysis of cardiac physiology was carried out using high-speed video recording in conjunction with movement analysis algorithms. unc-45 knockdown resulted in severely compromised cardiac function in adults as evidenced by prolonged diastolic and systolic intervals, and increased incidence of arrhythmias and extreme dilation; the latter was accompanied by a significant reduction in muscle contractility. Structural analysis showed reduced myofibrils, myofibrillar disarray, and greatly decreased cardiac myosin accumulation. Cardiac unc-45 silencing also dramatically reduced life-span. In contrast, third instar larval and young pupal hearts showed mild cardiac abnormalities, as severe cardiac defects only developed during metamorphosis. Furthermore, cardiac unc-45 silencing in the adult heart (after metamorphosis) led to less severe phenotypes. This suggests that UNC-45 is mostly required for myosin accumulation/folding during remodeling of the forming adult heart. The cardiac defects, myosin deficit and decreased life-span in flies upon heart-specific unc-45 knockdown were significantly rescued by UNC-45 over-expression. Our results are the first to demonstrate a cardiac-specific requirement of a chaperone in Drosophila, suggestive of a critical role of UNC-45 in cardiomyopathies, including those associated with unfolded proteins in the failing human heart. The dilated cardiomyopathy phenotype associated with UNC-45 deficiency is mimicked by myosin knockdown suggesting that UNC-45 plays a crucial role in stabilizing myosin and possibly preventing human cardiomyopathies associated with functional deficiencies of myosin.

Functional Characterization of Mutations in the Myosin Vb Gene Associated With Microvillus Inclusion Disease

Microvillus inclusion disease (MVID) is a rare autosomal recessive enteropathy characterized by intractable diarrhea and malabsorption. Recently, various MYO5B gene mutations have been identified in patients with MVID. Interestingly, several patients with MVID showed only a MYO5B mutation in 1 allele (heterozygous) or no mutations in the MYO5B gene, illustrating the need to further functionally characterize the cell biological effects of the MYO5B mutations. The genomic DNA of 9 patients diagnosed as having MVID was screened for MYO5B mutations, and quantitative polymerase chain reaction and immunohistochemistry on the material of 2 patients was performed to investigate resultant cellular consequences. We demonstrate for the first time that MYO5B mutations can be correlated with altered myosin Vb messenger RNA expression and with an aberrant subcellular distribution of the myosin Vb protein. Moreover, we demonstrate that the typical and myosin Vb-controlled accumulation of Rab11a- and FIP5-positive recycling endosomes in the apical cytoplasm of the cells is abolished in MVID enterocytes, which is indicative of altered myosin Vb function. Moreover, we report 8 novel MYO5B mutations in 9 patients of various ethnic backgrounds with MVID, including compound heterozygous mutations. Our functional analysis indicates that MYO5B mutations can be correlated with an aberrant subcellular distribution of the myosin Vb protein, and apical recycling endosomes, which, together with the additional compound heterozygous mutations, significantly strengthen the link between MYO5B and MVID.

Drosophila UNC-45 accumulates in embryonic blastoderm and in muscles, and is essential for muscle myosin stability

UNC-45 is a chaperone that facilitates folding of myosin motor domains. We have used Drosophila melanogaster to investigate the role of UNC-45 in muscle development and function. Drosophila UNC-45 (dUNC-45) is expressed at all developmental stages. It colocalizes with non-muscle myosin in embryonic blastoderm of 2-hour-old embryos. At 14 hours, it accumulates most strongly in embryonic striated muscles, similarly to muscle myosin. dUNC-45 localizes to the Z-discs of sarcomeres in third instar larval body-wall muscles. We produced a dunc-45 mutant in which zygotic expression is disrupted. This results in nearly undetectable dUNC-45 levels in maturing embryos as well as late embryonic lethality. Muscle myosin accumulation is robust in dunc-45 mutant embryos at 14 hours. However, myosin is dramatically decreased in the body-wall muscles of 22-hour-old mutant embryos. Furthermore, electron microscopy showed only a few thick filaments and irregular thick–thin filament lattice spacing. The lethality, defective protein accumulation, and ultrastructural abnormalities are rescued with a wild-type dunc-45 transgene, indicating that the mutant phenotypes arise from the dUNC-45 deficiency. Overall, our data indicate that dUNC-45 is important for myosin accumulation and muscle function. Furthermore, our results suggest that dUNC-45 acts post-translationally for proper myosin folding and maturation.

Engineering the Mechanobiological Oscillations of Single Cells

Recently, we found that cytoskeletal proteins, such as myosin-II and cortexillin-I, cooperatively accumulate to highly deformed regions in Dictyostelium cells and the accumulation extent increases with increasing forces. Accompanying the protein accumulation, the cellular deformation decreases even as the external forces are kept constant. We argue that the accumulation of cytoskeletal proteins sense the mechanical stimuli and accumulate locally to enhance the local mechanical resistance of the cell, i.e. the cortical stiffness, which leads to diminishing cellular deformation. We suggest that the underlying mechanism of the cooperative accumulation could be that the binding of myosin to actin enhances cortexillin binding to actin filaments. Especially, myosin, undoubtedly a force sensor, binds to actin filaments in a force-dependent manner. On the other hand, cortexillin maintains the mechanical integrity of the whole actin network and anchors it to the cell membrane. Additionally, we found that cells are able to repeat the “increased deformation-protein accumulation-decreased deformation” cycle while the external force does not change. We propose two mechanisms for the restarting of the cycles. The first is that the myosin accumulation itself leads to less force experienced by each myosin, which results in the falloff of the accumulated myosins and the subsequent cortical softening, leading to larger deformation again. The second is that blebs, which behave as increased deformation, are sometimes formed when either the cortex-membrane linkage is too weak or the local myosin associated contractility is too strong. To test these hypotheses, we use chemicals, genetic tools and their combinations to modulate the expression level, the localization and function of cytoskeletal proteins and therefore tune the cortical stiffness, the cortex-membrane linkage and the period of the cellular oscillations in a quantitative way.

DrosophilaUNC-45 accumulates in embryonic blastoderm and in muscles, and is essential for muscle myosin stability

UNC-45 is a chaperone that facilitates folding of myosin motor domains. We have used Drosophila melanogaster to investigate the role of UNC-45 in muscle development and function. Drosophila UNC-45 (dUNC-45) is expressed at all developmental stages. It colocalizes with non-muscle myosin in embryonic blastoderm of 2-hour-old embryos. At 14 hours, it accumulates most strongly in embryonic striated muscles, similarly to muscle myosin. dUNC-45 localizes to the Z-discs of sarcomeres in third instar larval body-wall muscles. We produced a dunc-45 mutant in which zygotic expression is disrupted. This results in nearly undetectable dUNC-45 levels in maturing embryos as well as late embryonic lethality. Muscle myosin accumulation is robust in dunc-45 mutant embryos at 14 hours. However, myosin is dramatically decreased in the body-wall muscles of 22-hour-old mutant embryos. Furthermore, electron microscopy showed only a few thick filaments and irregular thick-thin filament lattice spacing. The lethality, defective protein accumulation, and ultrastructural abnormalities are rescued with a wild-type dunc-45 transgene, indicating that the mutant phenotypes arise from the dUNC-45 deficiency. Overall, our data indicate that dUNC-45 is important for myosin accumulation and muscle function. Furthermore, our results suggest that dUNC-45 acts post-translationally for proper myosin folding and maturation.

Tissue elongation requires oscillating contractions of a basal actomyosin network

Understanding how molecular dynamics lead to cellular behaviors that ultimately sculpt organs and tissues is a major challenge not only in basic developmental biology but also in tissue engineering and regenerative medicine. Here we use live imaging to show that the basal surfaces of Drosophila follicle cells undergo a series of directional, oscillating contractions driven by periodic myosin accumulation on a polarized actin network. Inhibition of the actomyosin contractions or their coupling to extracellular matrix (ECM) blocked elongation of the whole tissue, whereas enhancement of the contractions exaggerated it. Myosin accumulated in a periodic manner prior to each contraction and was regulated by the small GTPase Rho, its downstream kinase ROCK and cytosolic calcium. Disrupting the link between the actin cytoskeleton and the ECM decreased, while enhancing cell-ECM adhesion increased, the amplitude and period of the contractions. In contrast, disrupting cell-cell adhesions resulted in loss of the actin network. Our findings reveal a novel mechanism controlling organ shape and a new model for the study of the effects of oscillatory actomyosin activity within a coherent cell sheet.

TheC. eleganshomolog ofDrosophilaLethal giant larvae functions redundantly with PAR-2 to maintain polarity in the early embryo

Polarity is essential for generating cell diversity. The one-cell C. elegans embryo serves as a model for studying the establishment and maintenance of polarity. In the early embryo, a myosin II-dependent contraction of the cortical meshwork asymmetrically distributes the highly conserved PDZ proteins PAR-3 and PAR-6, as well as an atypical protein kinase C (PKC-3), to the anterior. The RING-finger protein PAR-2 becomes enriched on the posterior cortex and prevents these three proteins from returning to the posterior. In addition to the PAR proteins, other proteins are required for polarity in many metazoans. One example is the conserved Drosophila tumor-suppressor protein Lethal giant larvae (Lgl). In Drosophila and mammals, Lgl contributes to the maintenance of cell polarity and plays a role in asymmetric cell division. We have found that the C. elegans homolog of Lgl, LGL-1, has a role in polarity but is not essential. It localizes asymmetrically to the posterior of the early embryo in a PKC-3-dependent manner, and functions redundantly with PAR-2 to maintain polarity. Furthermore, overexpression of LGL-1 is sufficient to rescue loss of PAR-2 function. LGL-1 negatively regulates the accumulation of myosin (NMY-2) on the posterior cortex, representing a possible mechanism by which LGL-1 might contribute to polarity maintenance.

Determinants of Myosin II Cortical Localization during Cytokinesis

Myosin II is an essential component of the contractile ring that divides the cell during cytokinesis. Previous work showed that regulatory light chain (RLC) phosphorylation is required for localization of myosin at the cellular equator. However, the molecular mechanisms that concentrate myosin at the site of furrow formation remain unclear. By analyzing the spatiotemporal dynamics of mutant myosin subunits in Drosophila S2 cells, we show that myosin accumulates at the equator through stabilization of interactions between the cortex and myosin filaments and that the motor domain is dispensable for localization. Filament stabilization is tightly controlled by RLC phosphorylation. However, we show that regulatory mechanisms other than RLC phosphorylation contribute to myosin accumulation at three different stages: (1) turnover of thick filaments throughout the cell cycle, (2) myosin heavy chain-based control of myosin assembly at the metaphase-anaphase transition, and (3) redistribution and/or activation of myosin binding sites at the equator during anaphase. Surprisingly, the third event can occur to a degree in a Rho-independent fashion, gathering preassembled filaments to the equatorial zone via cortical flow. We conclude that multiple regulatory pathways cooperate to control myosin localization during mitosis and cytokinesis to ensure that this essential biological process is as robust as possible.

SAX-7/L1CAM and HMR-1/cadherin function redundantly in blastomere compaction and non-muscle myosin accumulation during Caenorhabditis elegans gastrulation

Gastrulation is the first major morphogenetic movement in development and requires dynamic regulation of cell adhesion and the cytoskeleton. Caenorhabditis elegans gastrulation begins with the migration of the two endodermal precursors, Ea and Ep, from the surface of the embryo into the interior. Ea/Ep migration provides a relatively simple system to examine the intersection of cell adhesion, cell signaling, and cell movement. Ea/Ep ingression depends on correct cell fate specification and polarization, apical myosin accumulation, and Wnt activated actomyosin contraction that drives apical constriction and ingression (Lee et al., 2006; Nance et al., 2005). Here, we show that Ea/Ep ingression also requires the function of either HMR-1/cadherin or SAX-7/L1CAM. Both cadherin complex components and L1CAM are localized at all sites of cell–cell contact during gastrulation. Either system is sufficient for Ea/Ep ingression, but loss of both together leads to a failure of apical constriction and ingression. Similar results are seen with isolated blastomeres. Ea/Ep are properly specified and appear to display correct apical–basal polarity in sax-7(eq1);hmr-1(RNAi) embryos. Significantly, in sax-7(eq1);hmr-1(RNAi) embryos, Ea and Ep fail to accumulate myosin (NMY-2∷GFP) at their apical surfaces, but in either sax-7(eq1) or hmr-1(RNAi) embryos, apical myosin accumulation is comparable to wild type. Thus, the cadherin and L1CAM adhesion systems are redundantly required for localized myosin accumulation and hence for actomyosin contractility during gastrulation. We also show that sax-7 and hmr-1 function are redundantly required for Wnt-dependent spindle polarization during division of the ABar blastomere, indicating that these cell surface proteins redundantly regulate multiple developmental events in early embryos.

Heat Stress in Rat Adriamycin Cardiomyopathy: Heat Shock Protein 25 and Myosin Accumulation

In order to evaluate the effects of hyperthermia on adriamycin cardiomyopathy and its relationship with heat shock protein induction and myosin accumulation, female Sprague-Dawley rats (21–24 days) were randomized into four groups: the control, adriamycin, temperature and temperature-adriamycin groups. Adriamycin was injected i.v. at a dose of 27 mg/Kg (0.1 ml). The rats were exposed to a temperature of 45ºC for 35 min, followed by a recovery (1 h) at room temperature prior to adriamycin treatment. Body weight was recorded weekly. The thickness of the ventricular wall and percentage of cellular damage were biometrically and ultrastructurally evaluated, respectively. Heat shock protein 25 and myosin accumulation were determined through Western blot analysis. The determinations were carried out monthly until the third month after treatment. At eight and twelve weeks after treatment, the thickness of the ventricular wall seemed to decrease in the adriamycin-treated rats in relation to the other groups. An electron microscopic analysis of the adriamycin group’s left ventricular wall samples, showed more sarcomeric changes and loss of myofibrils than the control, temperature and temperature-adriamycin groups. At 24 hours after treatment with adriamycin, higher levels of heat shock protein 25 and myosin were observed (week 0) in the temperature-adriamycin group than in the control and adriamycin groups (4, 8 and 12 weeks). Hyperthermia was confirmed by a multivariate approach to induce heat shock protein 25 and myosin, which would strengthen cardiac-sarcomeric myosin arrangement.

UNC-45 Knock-Down in Drosophila Heart Targets Myosin Accumulation and Yields Severe Myofibrillar Disarray and Cardiac Dysfunction

UNC-45 belongs to the UCS (UNC-45, CRO1, and She4p) domain protein family, whose members interact with various myosins and are required for myosin function. The in vivo RNAi approach using the Gal4/UAS system permits us to knock down Drosophila unc-45 (dunc-45) transcripts in a tissue-specific manner. DUNC-45 knock-down with muscle-specific driver 24bGal4 leads to embryonic and larval lethality. However, DUNC-45 knock-down in the heart allows survival to adulthood, but lifespan is dramatically reduced, with ∼90% dead within three weeks. Optical heartbeat analysis of semi-intact hearts from various age adults was carried out using high-speed video and movement analysis algorithms. Analysis of several cardiac parameters (e.g., diastolic and systolic diameters, heart period and arrythmicity index) showed severely compromised cardiac function. For example, ∼80% of 1, 2 and 3 week old knock-down hearts do not show contraction or relaxation of normally highly-contractile regions (primarily in abdominal segment three). The remainder of the heart shows a greatly reduced and irregular beating pattern. Another striking response to DUNC-45 knock-down was dilation of the third segment of the heart. For example, the average diastolic diameter for one week old control fly heart is 76 μm. However, in the one-week-old knock-down heart the average diastolic diameter is 128 μm. Cardiac arrhythmias were observed in one-week-old flies, whereas arrhythmias were only observed after three weeks of age in wild type flies. Immunofluorescent images of relaxed hearts using myosin antibody showed irregular heart muscle patterning and missing myofibrils in DUNC-45 knock-down adults. Heart defects of the DUNC-45 knock-down heart were rescued to some extent by transgenic over-expression of DUNC-45. As UNC-45 is essential for accumulation of myosin, its knock-down in the heart results in myofibrillar disarray and reduced contraction.

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

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

AMPK inhibits myoblast differentiation through a PGC-1α-dependent mechanism

Elevated phosphorylation of AMP-activated protein kinase (AMPK) has been shown to inhibit skeletal muscle growth in both culture and animal models, but its role in differentiation of muscle cells is less clear. p21 is known to have an important role in differentiation, but AMPK's role regulating p21 in differentiation in muscle cultures is unknown. Therefore, the purpose of this study was to determine the role of p21 in differentiation of skeletal muscle cells under conditions of elevated AMPK phosphorylation. Treating C(2)C(12) myoblast cultures with 1 mM 5-aminoimidazole-4-carboxamide 1-beta-D-ribonucleoside (AICAR) for up to 24 h induced AMPK phosphorylation. Activation of AMPK reduced p21 protein and mRNA expression, which was associated with reduced G(1)/S cell cycle transition and p21 promoter activity. AICAR-treated myoblasts undergoing differentiation also had reduced p21 protein expression, reduced myotube formation, and myosin accumulation. When myotube cultures were treated with AICAR for 24 h, p21, myosin protein expression, and MyoD were significantly reduced. Myotube atrophy was also apparent compared with control conditions. Addition of compound C, an AMPK inhibitor, attenuated AICAR's negative effects on the myotube cultures. The nuclear expression of p21 protein appeared to be more affected by AICAR-treated myotubes than the cytosolic portion of p21 protein, which was attenuated with compound C treatment. Further analysis revealed that AICAR treatment increased PGC-1alpha and decreased FOXO3A protein expression, which was reversed with compound C cotreatment. Knockdown of PGC-1alpha with shRNA corroborated the compound C data, preserving nuclear FOXO3A and p21 protein expression. These data demonstrate that AICAR-induced AMPK phosphorylation inhibits cell cycle transition, reducing differentiation of myoblasts into myotubes, through PGC-1alpha-FOXO3A-p21.

The actin-binding and bundling protein, EPLIN, is required for cytokinesis

Cytokinesis involves two phases: 1) membrane ingression followed by 2) membrane abscission. The ingression phase generates a cleavage furrow and this requires co-operative function of the actin-myosin II contractile ring and septin filaments. We demonstrate that the actin-binding protein, EPLIN, locates to the cleavage furrow during cytokinesis and this is possibly via association with the contractile ring components, myosin II, and the septin, Sept2. Depletion of EPLIN results in formation of multinucleated cells and this is associated with inefficient accumulation of active myosin II (MRLCS19) and Sept2 and their regulatory small GTPases, RhoA and Cdc42, respectively, to the cleavage furrow during the final stages of cytokinesis. We suggest that EPLIN may function during cytokinesis to maintain local accumulation of key cytokinesis proteins at the furrow.

Identification of the first in cis mutations in MYH9 disorder

Here, we report the first in cis mutations in exon 1 of the MYH9 gene in a patient with MYH9 disorder. The patient was a 5-yr-old girl with macrothrombocytopenia and conspicuous cytoplasmic inclusion bodies in neutrophils. Immunofluorescence analysis of neutrophil non-muscle myosin heavy chain-II A (NMMHC-IIA) indicated several cytoplasmic spots of NMMHC-IIA aggregates that were circular to oval in shape (type II pattern). Mutational analysis showed two mutations, c.99G > T and c.103C > G, which would result in p.W33C and p.P35A, respectively, in exon 1 of the MYH9 gene. In addition, concurrent mutations were present on the same chromosome. Inclusion bodies are usually faint or mostly invisible in MYH9 disorders with a mutation in exon 1. In this case, double mutations might have caused the large myosin protein aggregation and accumulation. Although not observed in this patient, the development of Alport manifestations should be monitored by careful follow-up.

Multiple Mechanisms for Accumulation of Myosin II Filaments at the Equator During Cytokinesis

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

Actin-based propulsive forces and myosin-II-based contractile forces in migratingDictyosteliumcells

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

Apical accumulation of Rho in the neural plate is important for neural plate cell shape change and neural tube formation.

Although Rho-GTPases are well-known regulators of cytoskeletal reorganization, their in vivo distribution and physiological functions have remained elusive. In this study, we found marked apical accumulation of Rho in developing chick embryos undergoing folding of the neural plate during neural tube formation, with similar accumulation of activated myosin II. The timing of accumulation and biochemical activation of both Rho and myosin II was coincident with the dynamics of neural tube formation. Inhibition of Rho disrupted its apical accumulation and led to defects in neural tube formation, with abnormal morphology of the neural plate. Continuous activation of Rho also altered neural tube formation. These results indicate that correct spatiotemporal regulation of Rho is essential for neural tube morphogenesis. Furthermore, we found that a key morphogenetic signaling pathway, the Wnt/PCP pathway, was implicated in the apical accumulation of Rho and regulation of cell shape in the neural plate, suggesting that this signal may be the spatiotemporal regulator of Rho in neural tube formation.

Inhibition of “self” engulfment through deactivation of myosin-II at the phagocytic synapse between human cells

Phagocytosis of foreign cells or particles by macrophages is a rapid process that is inefficient when faced with “self” cells that display CD47—although signaling mechanisms in self-recognition have remained largely unknown. With human macrophages, we show the phagocytic synapse at cell contacts involves a basal level of actin-driven phagocytosis that, in the absence of species-specific CD47 signaling, is made more efficient by phospho-activated myosin. We use “foreign” sheep red blood cells (RBCs) together with CD47-blocked, antibody-opsonized human RBCs in order to visualize synaptic accumulation of phosphotyrosine, paxillin, F-actin, and the major motor isoform, nonmuscle myosin-IIA. When CD47 is functional, the macrophage counter-receptor and phosphatase-activator SIRPα localizes to the synapse, suppressing accumulation of phosphotyrosine and myosin without affecting F-actin. On both RBCs and microbeads, human CD47 potently inhibits phagocytosis as does direct inhibition of myosin. CD47–SIRPα interaction initiates a dephosphorylation cascade directed in part at phosphotyrosine in myosin. A point mutation turns off this motor's contribution to phagocytosis, suggesting that self-recognition inhibits contractile engulfment.

A wave of EGFR signaling determines cell alignment and intercalation in theDrosophilatracheal placode

Invagination of organ placodes converts flat epithelia into three-dimensional organs. Cell tracing in the Drosophila tracheal placode revealed that, in the 30-minute period before invagination, cells enter mitotic quiescence and form short rows that encircle the future invagination site. The cells in the rows align to form a smooth boundary (;boundary smoothing'), accompanied by a transient increase in myosin at the boundary and cell intercalation oriented in parallel with the cellular rows. Cells then undergo apical constriction and invaginate, followed by radially oriented mitosis in the placode. Prior to invagination, ERK MAP kinase is activated in an outward circular wave, with the wave front often correlating with the smoothing cell boundaries. EGFR signaling is required for myosin accumulation and cell boundary smoothing, suggesting its propagation polarizes the planar cell rearrangement in the tracheal placode, and coordinates the timing and position of intrinsic cell internalization activities.