Cytoplasmic Myosin Research Articles

Enhanced expression of the myogenic factor Myocyte enhancer factor-2 in imaginal disc myoblasts activates a partial, but incomplete, muscle development program

The Myocyte enhancer factor-2 (MEF2) transcription factor plays a vital role in orchestrating muscle differentiation. While MEF2 cannot effectively induce myogenesis in naïve cells, it can potently accelerate myogenesis in mesodermal cells. This includes in Drosophila melanogaster imaginal disc myoblasts, where triggering premature muscle gene expression in these adult muscle progenitors has become a paradigm for understanding the regulation of the myogenic program. Here, we investigated the global consequences of MEF2 overexpression in the imaginal wing disc myoblasts, by combining RNA-sequencing with RT-qPCR and immunofluorescence. We observed the formation of sarcomere-like structures that contained both muscle and cytoplasmic myosin, and significant upregulation of muscle gene expression, especially genes essential for myofibril formation and function. These transcripts were functional since numerous myofibrillar proteins were detected in discs using immunofluorescence. Interestingly, muscle genes whose expression is restricted to the adult stages were not activated in these adult myoblasts. These studies confirm a broad activation of the myogenic program in response to MEF2 expression and suggest that additional regulatory factors are required for promoting the adult muscle-specific program. Our findings contribute to understanding the regulatory mechanisms governing muscle development and highlight the multifaceted role of MEF2 in orchestrating this intricate process.

The Protein Interactomes of CBFB-MYH11 and RUNX1-RUNX1T1 Suggest Different Mechanisms of Action for Initiating Acute Myeloid Leukemia

Core Binding Factor (CBF) leukemias are a subset of Acute Myeloid Leukemia (AML) initiated by translocation of genes encoding the heterodimeric core-binding factor (RUNX1 and CBFB) via generation of the oncogenic fusion genes, CBFB-MYH11 and RUNX1-RUNX1T1. While both are classified as favorable risk, relapse occurs in 40-50% of patients with conventional chemotherapy. To better understand the mechanisms by which these fusions initiate AML, we used an MSCV-based, GFP-tagged retroviral vector to introduce a highly active biotin ligase ("TurboID", or "TID") fused to either CBFB-MYH11 ("CM") or RUNX1-RUNX1T1 (AML1-ETO/"AE") at both the N-and C-termini of the proteins into primary murine hematopoietic progenitor cells. With this system, proteins within 10 angstroms of TurboID-fusion proteins are efficiently labeled with biotin, enriched with a streptavidin bead pulldown, and identified using mass spectrometry. We first defined top protein interactors with CBFB-MYH11-TID compared to TurboID protein only ("TID"), and plotted the strength of interactions in the heat map shown in Figure 1A, where each row represents an independent biological replicate (n=11 for TID, n=6 for CM-TID, and n=6 for AE-TID), and each column represents an interacting protein. N- and C-terminal TurboID fusions behaved similarly and are shown together in the figure. AML1-ETO-TID interactions with the same interacting proteins are "passively" plotted, revealing that 11 proteins interacted with both fusions (including RUNX1, RUNX2, and CBFB itself - as expected). AML1-ETO had a unique set of interacting proteins (data not shown) that were primarily nuclear localized, as defined by the Human Protein Atlas (e.g. BCOR, NCOR1, SIN3A, etc.). In contrast, most CBFB-MYH11-interacting proteins were unique for that fusion (including multiple myosin proteins; see Figure 1A), and most were annotated as non-nuclear in the Protein Atlas (denoted with an asterisk in the figure). The predicted cytoplasmic localization of CBFB-MYH11-TID was confirmed by immunofluorescence using an antibody directed against the TID protein. We independently confirmed these results using a CBFB-MYH11-GFP fusion protein, which likewise localized in cytoplasmic aggregates, while a CBFB-GFP fusion was found exclusively in the nucleus. GFP-MYH11 was localized to the cytoplasm, indicating that the MYH11 portion of CBFB-MYH11 directs cytoplasmic localization in a dominant fashion, perhaps by interacting with other cytoplasmic myosin heavy chains. Immunofluorescent staining for endogenous RUNX1 in cells expressing CBFB-MYH11-GFP showed that RUNX1 co-localized with the cytoplasmic CBFB-MYH11, strongly suggesting that the fusion protein acts as a "trap" to sequester RUNX1 in the cytoplasm. To test this hypothesis, we generated a CBFB-MYH11 variant with an N104A mutation in CBFB that is known to disrupt RUNX1 binding. We confirmed complete loss of the CBFB-MYH11 interaction with RUNX1 and RUNX2 using the TurboID system (Figure 1B), and documented restoration of nuclear localization of RUNX1 by immunofluorescence. Using bulk RNA-seq on days 4 and 7 after transduction, we found that WT CBFB-MYH11 overexpression dysregulated many genes (562 up, 352 down, >2-fold change, FDR<0.05), including Runx1 and Runx2, both of which were upregulated. Importantly, the expression phenotype was completely eliminated in cells transduced with the N104A mutated CBFB-MYH11, suggesting that it was dependent on the cytoplasmic sequestration of RUNX1 and/or RUNX2. Finally, analysis of the existing human AML TCGA and Bloodspot transcriptional datasets confirmed upregulation of RUNX1 (p<0.0002 in both datasets) and RUNX2 (p<0.001 in TCGA) in CBFB-MYH11 AMLs relative to non-CBFB-MYH11 AMLs, suggesting that these genes may be activated by a feedback loop that is regulated by functional RUNX1/2 protein levels in the nucleus. In sum, these data suggest that CBFB-MYH11 alters the function of RUNX1 and RUNX2 by sequestering them in the cytoplasm, rather than by directly altering their activity as transcription factors. Thus, novel approaches designed to release mislocalized RUNX1/2 from CBFB-MYH11 cytoplasmic complexes should be explored as a novel therapeutic approach for CBFB-MYH11 AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MYH9 is a novel cancer stem cell marker and prognostic indicator in esophageal cancer that promotes oncogenesis through the PI3K/AKT/mTOR axis.

MYH9 encodes the heavy chain of nonmuscle myosin IIA, a ubiquitously expressed cytoplasmic myosin that regulates the actin cytoskeleton, cell migration, cell polarization, and signal transduction in cancer cells. Here, we investigated the role of MYH9 in cancer stem cells (CSCs) associated with esophageal cancer (EC).The subcellular localization of MYH9 was investigated in SKGT-4 cells through immunofluorescent analysis. MYH9+ and MYH9- spheroid cells were derived from SKGT-4 cells by flow cytometry and compared for self-renewal capacity, tumorigenicity, CD133 positivity, cisplatin resistance, and phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR) activity. MYH9 messenger RNA expression was assessed in 30 EC patients by quantitativereverse transcription-polymerase chain reaction. Kaplan-Meier curves were plotted to explore the influence of MYH9 on EC survival.MYH9 localized to the plasma membrane, cytoplasm, and nucleus of SKGT-4 cells. Spheroid cells displayed higher MYH9 expression and positivity compared to parental SKGT-4 cells. MYH9+ cells showed strong CSC characteristics, including in vivo tumorigenicity, migration, invasion, cisplatin resistance, and CD133+ positivity. MYH9 activated the PI3K/AKT/mTOR axis in CSCs and was upregulated in EC patients with poor survival. Collectively, these data show that MYH9 significantly promotes tumorigenesis by regulating PI3K/AKT/mTOR signaling in EC. MYH9 expression remarkably correlates with poor prognosis and represents a novel biomarker and drug target for the diagnosis and treatment of EC.

Mechanism of Ultra-Fast Actin-Myosin Sliding Producing Cytoplasmic Streaming in Giant Algal Cell, Studied Using the Centrifuge Microscope

In giant intermodal cells of green algae Chara collaria, cytoplasmic streaming is produced by ATP-dependent sliding between myosin heads extending from amorphous cytoplasmic organelles and actin filament arrays (actin cables) fixed on chloroplast rows. The velocity of cytoplasmic streaming is many times faster than the maximum myofilament sliding in skeletal muscle. In this article, we compared steady-state force-velocity (P-V) relations between cytoplasmic myosin and skeletal and cardiac muscle myosins using the centrifuge microscope, in which myosincoated latex beads were made to slide along the actin cables under various centrifugal forces. In contrast with the hyperbolic P-V relation of actin-myosin sliding in skeletal and cardiac myosins, the P-V relation of cytoplasmic myosin versus actin cable sliding was a straight line, indicating a very large duty ratio and a very small rate of chemomechanical energy conversion. Possible mechanisms of the ultra-fast actin-myosin sliding are discussed.Highlights• The velocity of cytoplasmic streaming, caused by ATP-dependent sliding between cytoplasmic myosin and actin cables in giant algal cells is many times faster than ATP-dependent actin-myosin sliding in skeletal and cardiac muscles.• The mechanism of ultra-fast actin-myosin sliding was studied using the centrifuge microscope, in which beads coated with cytoplasmic myosin were made to slide along actin cables under various centrifugal forces serving as loads against cytoplasmic myosin versus actin cable sliding.• Unlike the hyperbolic force-velocity (P-V) relation of skeletal and cardiac muscle actin-myosin sliding, the P-V relation of cytoplasmic actin myosin sliding was a straight line irrespective of the force generated by cytoplasmic myosin.• These results indicate a very large duty ratio and a very small efficiency of chemo-mechanical energy conversion in cytoplasmic actin-myosin sliding.

Myosin substitution rate is affected by the amount of cytosolic myosin in cultured muscle cells.

In striated muscles, approximately 300 myosin molecules form a single thick filament in myofibrils. Each myosin is continuously displaced by another myosin to maintain the thick filament structure. Our previous study using a fluorescence recovery after photobleaching (FRAP) technique showed that the myosin replacement rate is decreased by inhibition of protein synthesis, but myosin is still exchangeable. This result prompted us to examine whether myosin in the cytoplasm is involved in myosin replacement in myofibrils. To address this, FRAP was measured in green fluorescent protein (GFP)-tagged myosin heavy chain 3 (Myh3) expressing myotubes that were treated with streptolysin-O (SLO), which forms pores specifically in the plasma membrane to induce leakage of cytoplasmic proteins. Our biochemical data demonstrated that the cytoplasmic myosin content was reduced in SLO-permeabilized semi-intact myotubes. Furthermore, FRAP experiments showed a sluggish substitution rate of GFP-Myh3 in SLO-permeabilized myotubes. Taken together, these results demonstrate that the myosin substitution rate is significantly reduced by a decreased amount of myosin in the cytoplasm and that cytoplasmic myosin contributes to myosin replacement in myofibrils.

Tropomyosin‐Mediated Regulation of Cytoplasmic Myosins

The ability of the actin-based cytoskeleton to rapidly reorganize is critical for maintaining cell organization and viability. The plethora of activities in which actin polymers participate require different biophysical properties, which can vary significantly between the different events that often occur simultaneously at separate cellular locations. In order to modify the biophysical properties of an actin polymer for a particular function, the cell contains diverse actin-binding proteins that modulate the growth, regulation and molecular interactions of actin-based structures according to functional requirements. In metazoan and yeast cells, tropomyosin is a key regulator of actin-based structures. Cells have the capacity to produce multiple tropomyosin isoforms, each capable of specifically associating as copolymers with actin at distinct cellular locations to fine-tune the functional properties of discrete actin structures. Here, we present a unifying theory in which tropomyosin isoforms critically define the surface landscape of copolymers with cytoplasmic β- or γ-actin. Decoration of filamentous actin with different tropomyosin isoforms determines the identity and modulates the activity of the interacting myosin motor proteins. Conversely, changes in the nucleotide state of actin and posttranslational modifications affect the composition, morphology, subcellular localization and allosteric coupling of the associated actin-based superstructures.

Cytoplasmic myosin-exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability.

The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease.

MOLECULAR MOTORS AS NOVEL TARGETS TO BLOCK GLIOMA DISPERSION AND PROLIFERATION

BACKGROUND: Two of the defining features of glioblastoma are their ability to invade brain and to proliferate. While both processes are stimulated by the multiple signaling cascades that are activated in GBM, the redundancy present in these pathways has limited the clinical efficacy of kinase inhibitors. An alternative would be to directly target the cellular machinery that drives dispersion and proliferation. This machinery includes “molecular motors”—a superfamily of enzymes that generate the forces needed for a wide variety of cellular physiologies. In this study, we have examined two members from this family that can be targeted with clinically available drugs—cytoplasmic myosin II, which drives cellular motility, and Eg5, which drives the separation of the chromosomes during mitosis. METHODS: Rodent proneural glioblastomas were generated by intracranial injection of PDGF-expressing bi-cistronic retroviruses, and tumor cell invasion in vitro and ex vivo was examined as described (Ivkovic et al., Mol. Biol. Cell, 23, 533-42; Isermann et al., Curr. Protoc. Cell Biol., 2012). Patient-derived human glioma stem cells (GSCs) were isolated and maintained as described (Cheng et al., Cell, 153, 139-52). RESULTS: Myosin II: Myosin II isoforms are upregulated in all four subtypes of GBM, and expression correlates inversely with survival. We find that PDGF abolishes the anti-invasive effect of the EGFR inhibitior erlotinib on EGF-stimulated tumor dispersion, and, likewise, EGF abolishes the corresponding effect of the PDGFRα inhibitor imatinib on PDGF-stimulated tumor migration. However, blebbistatin, an allosteric inhibitor of myosin II, effectively blocks glioma dispersion even with simultaneous activation of both pathways. Furthermore, the rho kinase inhibitor fasudil, which inhibits myosin II and is used clinically for the treatment of pulmonary hypertension, also blocks PDGF-stimulated tumor invasion. Eg5: We find that Eg5 is consistently overexpressed in GSCs, compared to non-GSCs and that GSCs exhibit a greater sensitivity to ispinesib, a clinically available Eg5 inhibitor, compared to non-GSCs. Eg5 inhibition compromises the tumor initiation capability of GSCs as well as tumor progression in an orthotopic xenograft model. CONCLUSIONS: Prior efforts to block glioma invasion and proliferation have been hampered both by therapy-resistant GSCs and the redundancy present in pro-migratory signaling cascades. Our results indicate that molecular motors represent a set of targets whose inhibition blocks glioma dispersion and proliferation regardless of the activity of upstream growth and dispersion-promoting pathways and they imply that further pre-clinical and clinical development of molecular motor inhibitors in malignant glioma is warranted. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level

Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ~14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (~20-25%) motor. The ADP release step (~0.35 s(-1)) of NMIIB is only ~3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s(-1)). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of single- and double-headed myosin fragments and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished using total internal reflection fluorescence (TIRF) with fluorescence imaging with 1-nm accuracy (FIONA) and dual-beam optical trapping. At a physiological ATP concentration (1 mm), the rate of detachment of the single-headed and double-headed molecules was similar (~0.4 s(-1)). Using optical tweezers we found that the power stroke sizes of single- and double-headed heavy meromyosin (HMM) were each ~6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast, robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady-state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments.

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level

Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ∼14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (∼20-25%) motor. The ADP release step (∼0.35 s-1), of NMIIB is only ∼3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s-1), and as a result acto-NMIIB has the highest ADP-affinity reported so far for the myosin superfamily (<0.15 μM). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of a single- and double-headed myosin fragments, and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished by the use of TIRF using FIONA and dual-beam optical trapping. At a physiological ATP concentration (1 mM), the rate of detachment of the single-headed and double-headed molecules was similar (∼0.4 s-1). using optical tweezers we found that the power-stroke sizes of single- and double-headed HMM were ∼6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments.

A Cytoplasmic Complex Mediates Specific mRNA Recognition and Localization in Yeast

Author SummaryIn eukaryotes, the majority of cells are asymmetric and a way to establish such polarity is directional transport of macromolecules along cytoskeletal filaments. Among the cargoes transported, mRNAs play an essential role, as their localized translation contributes significantly to the generation of asymmetry. To date, hundreds of asymmetrically localized mRNAs in various organisms have been identified. These mRNAs are recognized by RNA-binding proteins and incorporated into large motor-containing messenger ribonucleoprotein particles (mRNPs) whose molecular assembly is poorly understood. In this study, we used the well-characterized process of ASH1-mRNA transport in Saccharomyces cerevisiae to address the question of how localizing mRNAs are recognized and specifically incorporated into mRNPs. Surprisingly, we found that the previously implicated mRNA-binding proteins She2p and Puf6p do not bind to cargo mRNAs with high specificity. Instead, the cytoplasmic motor-adapter protein She3p is responsible for synergistic cargo binding with She2p and for the stable incorporation of specific localizing mRNA into the transport complex. We propose that the specific recognition of localizing mRNAs happens at the very last step of cytoplasmic mRNP maturation. Other organisms might employ similar mechanisms to establish cellular polarity.

Characterization of Single- and Double-Headed Non-Muscle Myosin IIB Heavy Meromyosins using Single Molecule Techniques

Non-muscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays important role in cell motility by maintaining cortical tension. It forms bipolar filaments with ∼14 myosin molecules on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (∼20-25%) motor. The ADP release step (∼0.35 s-1), of NMIIB is only ∼3 times faster than the rate-limiting phosphate release (0.13 s-1), and as a result acto-NMIIB has the highest ADP-affinity reported so far for the myosin superfamily (<0.15 μM). To examine the kinetics of NMIIB at single-molecule level we used a dual-beam optical tweezers. A surface-immobilized bead was coated with single- (NMIIB-SH-HMM) and double-headed (NMIIB-HMM) heavy meromyosin-like molecules. We measured the lifetimes of unitary actomyosin interactions and determined the actin-detachment kinetics at two ATP concentrations. Results showed that at physiological ATP concentration (1 mM), the rate of detachment of acto-NMIIB-SH-HMM interactions was ∼0.51 s-1, similar to the ADP release rate and steady-state ATPase rate reported from solution kinetic studies. Decreasing the ATP concentration to 1 μM did not alter this rate of detachment (∼0.43 s-1). In case of NMIIB-HMM the detachment rates were ∼0.43 s-1 (1 mM ATP) and ∼0.28 s-1 (1 μM ATP). Also, we found that the power-stroke of NMIIB-SH-HMM and NMIIB-HMM were about 8 nm. No signs of processive stepping were observed in case of NMIIB-HMM. The detachment rates calculated from landing assays using NMIIB-SH-HMM-GFP and NMIIB-HMM-GFP were 0.58 and 0.57 s-1 at 1 mM ATP. The double-headed molecules were not motile, but we observed robust motility of minifilaments of full-length NMIIB-GFP. We will discuss our single-molecule results from the perspective of the essential cellular functions of NMIIB in cell locomotion, tension generation and maintenance.

Coherent anti-Stokes Raman scattering microscopy of human smooth muscle cells in bioengineered tissue scaffolds

The integration of living, human smooth muscle cells in biosynthesized cellulose scaffolds was monitored by nonlinear microscopy toward contractile artificial blood vessels. Combined coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy was applied for studies of the cell interaction with the biopolymer network. CARS microscopy probing CH(2)-groups at 2845 cm(-1) permitted three-dimensional imaging of the cells with high contrast for lipid-rich intracellular structures. SHG microscopy visualized the fibers of the cellulose scaffold, together with a small signal obtained from the cytoplasmic myosin of the muscle cells. From the overlay images we conclude a close interaction between cells and cellulose fibers. We followed the cell migration into the three-dimensional structure, illustrating that while the cells submerge into the scaffold they extrude filopodia on top of the surface. A comparison between compact and porous scaffolds reveals a migration depth of <10 μm for the former, whereas the porous type shows cells further submerged into the cellulose. Thus, the scaffold architecture determines the degree of cell integration. We conclude that the unique ability of nonlinear microscopy to visualize the three-dimensional composition of living, soft matter makes it an ideal instrument within tissue engineering.

Some CLL Cells Bind Myosin-Exposed Apoptotic Cells. Exposure of Cytoplasmic Myosin Results From Transfer of Caspase-3 Dependent Cleavage Products to the Outer Cell Membrane

AbstractAbstract 3900Many monoclonal antibodies (mAbs) produced by B-cell chronic lymphocytic leukemia (CLL) cells bind a subset of apoptotic cells that expose intracellular myosin on the cell surface. CLL patients with mAbs that bind these myosin-exposed apoptotic cells (MEACs) have shorter overall survival. Thus, understanding the mechanism of formation of MEACs and how CLL cells interact with MEACs may help elucidate the pathogenesis of CLL.To test if formation of MEACs is part of general apoptotic mechanisms, apoptosis was induced in Jurkat T cells by either the intrinsic or extrinsic pathways. The intrinsic pathway was either achieved spontaneously by culturing at high cell density or induced by camptothecin (CPT) treatment. The extrinsic pathway was induced by Fas ligand (FasL) or anti-Fas mAb treatment. Apoptosis and myosin exposition were analyzed by flow cytometry. All four methods of apoptosis induction produced MEACs after prolonged incubation as detailed below. CPT, FasL or anti-Fas mAb incubation for 4 hrs induced significant apoptosis (43-58%) with a detectable fraction of MEACs (9-12%). After incubation for 16 hrs or longer, the majority of apoptotic cells were MEACs (61-89%). Similarly, spontaneous apoptosis produced more MEACs after longer incubation (20% on day 1 versus 59–69% on days 2–4). Both early apoptotic cells, which flip phosphatidylserine (PS) from the inner to outer membrane surface yet retain membrane integrity (AnnexinV+, 7-actinomycin D (7AAD)-), and late apoptotic cells, which become membrane permeable (AnnexinV+, 7AAD+), demonstrate a subpopulation of MEACs that increases with longer incubation times. In contrast, MEACs are not detectable in non-apoptotic cells (AnnexinV-, 7AAD-).Thus, both intrinsic and extrinsic apoptotic pathways lead to MEAC formation, suggesting that a common downstream mediator may be involved. Caspase-3 activation mediates apoptotic PS exposure and membrane permeability. Therefore, we tested a caspase-3 inhibitor, Z-DEVD-FMK, and found that it significantly reduced both apoptosis and MEAC formation. For example, Z-DEVD-FMK reduced FasL induced apoptosis and MEAC formation from 74 to 14% and from 57 to 10%, respectively. In contrast, caspase-1 inhibitor, Z-YVAD-FMK, had no effect. To test if intracellular myosin is transferred from the cytoplasm to the cell membrane surface during apoptosis, cytoplasmic and membrane protein extracts were prepared, isolated by ultracentrifugation, and blotted with anti-myosin antibody. Two protein bands of the size expected for caspase-3 cleaved myosin (149 and 94 kDa) appeared in membrane extracts of apoptotic cells, but not of live cells. A protein band of the size expected for full-length myosin (250 kDa) predominated in cytoplasmic extracts of live cells. Furthermore, Z-DEVD-FMK inhibited the formation of the 149 and 94 kDa myosin bands in membrane extracts as well as the formation of caspase-3 dependant PARP cleavage products; the same treatment did not alter CD3 membrane protein or GAPDH cytoplasmic protein levels.Taken together, these results suggest that both intrinsic and extrinsic apoptotic pathways produce MEACs at a later stage in apoptosis that involves the common downstream caspase-3 activation. In turn, myosin fragmentation occurs with subsequent exposure to the cell membrane, where the myosin fragments can serve as a potential neoantigen that may be recognized by some CLL mAbs. Because the mAbs we have used in these analyses were originally integral components of CLL surface membranes, we hypothesized that CLL cells could bind MEACs. Indeed, CLL cells binding to MEACs were visualized by confocal microscopy. To determine the functional consequences of such binding, analyses of the effects of MEAC binding on CLL cell survival in vitro are underway. Disclosures:No relevant conflicts of interest to declare.

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.

Natural antibodies recognize cytoplasmic myosin that is cleaved and transferred to the cell membrane during apoptosis (89.4)

Abstract Natural antibodies (Abs) and Abs from patients with chronic lymphocytic leukemia (CLL) bind a subset of apoptotic cells that expose intracellular myosin on their surface. To test the mechanisms that produce myosin exposed apoptotic cells (MEACs), Jurkat T cells were induced to undergo intrinsic (spontaneous or camptothecin-induced) or extrinsic (Fas ligand- or anti-Fas-induced) apoptosis. After prolonged incubation by all methods, a high percentage of apoptotic cells became MEACs. Thus, both intrinsic and extrinsic apoptotic pathways lead to MEAC formation, suggesting that a common downstream mediator is involved. Consistent with this, a caspase-3 inhibitor reduced both apoptosis and MEAC formation, whereas a caspase-1 inhibitor had no effect. To test if caspase-3 cleavage resulted in membrane exposure of myosin, cytoplasmic and membrane protein extracts were examined for caspase-3 cleaved myosin fragments. MEAC extracts predominantly exhibited the expected 149 and 94 kDa myosin fragments in the membrane fraction. In contrast, live cell extracts exhibited predominantly the full-length myosin protein in the cytoplasmic fraction. These results indicate that MEAC formation occurs during intrinsic and extrinsic apoptosis when caspase-3 is activated. Caspase-3 cleavage of myosin produces fragments that reside in the cell membrane, where they become available to react with CLL and natural Abs.

Evaluation of myosin VI, E-cadherin and beta-catenin immunostaining in renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) is a cancer of increasing incidence and mortality. Currently, there are no immunohistochemical prognostic markers for RCCs in routine use. The aim of this study was to examine for the first time the immunostaining of myosin VI in RCCs as well as its association with E-cadherin and beta-catenin immunostaining and the prognostic significance of these markers in RCCs.MethodsOur study population consisted of 152 patients who underwent surgery for RCCs between 1990 and 1999. The tumours were examined with three immunohistochemical markers: myosin VI, E-cadherin and beta-catenin.ResultsThe immunostaining for cytoplasmic myosin VI was common (72%). One-third of the tumours were immunopositive for nuclear myosin VI. Cytoplasmic myosin VI immunopositivity and nuclear beta-catenin immunostaining were associated with lower Fuhrman grades (p = 0.04 and p = 0.005, respectively), but not stages. There was no significant association between myosin VI immunostaining and the histological subtype of RCC. Nuclear myosin VI was associated with the nuclear expression of beta-catenin. A direct association could also be proven between membranous E-cadherin and cytoplasmic beta-catenin. Cytoplasmic myosin VI immunostaining was a marker of poorer prognosis in multivariate Cox regression model adjusted with stage and Fuhrman grade with hazard ratio 2.4 (95% confidence interval 1.1 to 5.0 with p = 0.024).ConclusionsCytoplasmic myosin VI immunopositivity and nuclear beta-catenin immunostaining were associated with lower Fuhrman grades, and there was a strong positive relationship between E-cadherin immunostaining and beta-catenin immunostaining in RCCs. Cytoplasmic myosin VI immunostaining was associated with poorer prognosis in RCCs.

Single Molecule Kinetic Measurements Of Non-muscle Myosin IIB Using Optical Tweezers

Non-muscle myosin IIB (NMIIB) is a cytoplasmic myosin which is ubiquitously expressed in eukaryotic cells, particularly in the central nervous system and cardiac tissues. Our previous solution kinetic study of the single headed, subfragment-1 (S1) construct of NMIIB showed that this myosin spends a significant amount of its actomyosin ATPase cycle in the strongly bound state. In the presence of actin, the rate of ADP release is slow (∼0.35 s−1), comparable to the steady-state ATPase rate (0.13 ± 0.01 s−1).

Maternal UNC-45 is involved in cytokinesis and colocalizes with non-muscle myosin in the early Caenorhabditis elegans embryo.

The Caenorhabditis elegans UNC-45 protein contains tetratricopeptide repeats and a domain with similarity to fungal proteins, and it differentially colocalizes with myosin heavy chain B in the body wall muscles of adult worms. Although it is essential for normal myosin filament assembly in body wall muscle development, strong mutants show a previously unexplained maternal effect. We show here that the UNC-45 protein is maternally contributed and is present in all cells of the early embryo whereas zygotic UNC-45 expression is only detected in the developing muscle cells. Embryos produced from adults with reduced germline expression of UNC-45 exhibit cytokinesis defects suggesting that UNC-45 has a novel role in the early embryo in addition to muscle development. Yeast two-hybrid screens show that UNC-45 can directly interact with NMY-2, a non-muscle type II myosin, and UNC-45 and NMY-2 colocalize at cell boundaries in early embryos. Localization of UNC-45 at these boundaries is dependent upon the presence of NMY-2. Our results suggest that UNC-45 interacts with more than one type of myosin and functions in the embryo to regulate cytoplasmic myosin assembly and/or stability during cytokinesis.