Initial stages of tumor cell metastasis involve an epithelialmesenchyme transition that involves activation of amoeboid migration and loss of cell-cell adhesion. The actomyosin cytoskeleton has fundamental but poorly understood roles in these events. Myosin II, an abundant force-producing protein, has roles in cell body translocation and retraction of the posterior of the cell during migration. Recent studies have suggested that this protein may also have roles in leading edge protrusive events. The metastasis-promoting protein metastasin-1, a regulator of myosin II assembly, colocalizes with myosin IIA at the leading edge of cancer cells, suggesting direct roles for myosin II in metastatic behavior. We have assessed the roles of specific myosin II isoforms during lamellar spreading of MDA-MB-231 breast cancer cells on extracellular matrix. We find that the two major myosin II isoforms IIA and IIB are both expressed in these cells, and both are recruited dramatically to the lamellar margin during active spreading on fibronectin. There is also a transient increase in regulatory light chain phosphorylation that correlates the recruitment of myosin IIA and myosin IIB into this spreading margin. Pharmacologic inhibition of myosin II or myosin light chain kinase dramatically reduced spreading. Depletion of myosin IIA via small interfering RNA impaired migration but enhanced lamellar spreading, whereas depletion of myosin IIB impaired not only migration but also impaired initial rates of lamellar spreading. These results indicate that both isoforms are critical for the mechanics of cell migration, with myosin IIB seeming to have a preferential role in the mechanics of lamellar protrusion. (Cancer Res 2006; 66(9): 4725-33)

The human genetic code encrypted in thousands of genes holds the secret for synthesis of proteins that drive all biological processes necessary for normal life and death. Though the genetic ciphering remains unchanged through generations, some genes get disrupted, deleted and or mutated, manifesting diseases, and or disorders. Current treatment options—chemotherapy, protein therapy, radiotherapy, and surgery available for no more than 500 diseases—neither cure nor prevent genetic errors but often cause many side effects. However, gene therapy, colloquially called “living drug,” provides a one-time treatment option by rewriting or fixing errors in the natural genetic ciphering. Since gene therapy is predominantly a viral vector-based medicine, it has met with a fair bit of skepticism from both the science fraternity and patients. Now, thanks to advancements in gene editing and recombinant viral vector development, the interest of clinicians and pharmaceutical industries has been rekindled. With the advent of more than 12 different gene therapy drugs for curing cancer, blindness, immune, and neuronal disorders, this emerging experimental medicine has yet again come in the limelight. The present review article delves into the popular viral vectors used in gene therapy, advances, challenges, and perspectives.

In mammalian nonmuscle cells, the mechanisms controlling the localized formation of myosin-II filaments are not well defined. To investigate the mechanisms mediating filament assembly and disassembly during generalized motility and chemotaxis, we examined the EGF-dependent phosphorylation of the myosin-IIA heavy chain in human breast cancer cells. EGF stimulation of MDA-MB-231 cells resulted in transient increases in both the assembly and phosphorylation of the myosin-IIA heavy chains. In EGF-stimulated cells, the myosin-IIA heavy chain is phosphorylated on the casein kinase 2 site (S1943). Cells expressing green fluorescent protein-myosin-IIA heavy-chain S1943E and S1943D mutants displayed increased migration into a wound and enhanced EGF-stimulated lamellipod extension compared with cells expressing wild-type myosin-IIA. In contrast, cells expressing the S1943A mutant exhibited reduced migration and lamellipod extension. These observations support a direct role for myosin-IIA heavy-chain phosphorylation in mediating motility and chemotaxis.

Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly ␤ 2 GPI, and activate endothelial cells (ECs) in a ␤ 2 GPI-dependent manner after binding of ␤ 2 GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-␤ 2 GPI Ab-mediated EC activation are uncertain, although a role for a TLR4/myeloid differentiation factor 88-dependent pathway leading to activation of NF-B has been proposed. In the present study, we confirm a critical role for TLR4 in anti-␤ 2 GPI Ab-mediated EC activation and demonstrate that signaling through TLR4 is mediated through the assembly of a multiprotein signaling complex on the EC surface that includes annexin A2, TLR4, calreticulin, and nucleolin. An essential role for each of these proteins in cell activation is suggested by the fact that inhibiting the expression of each using specific siRNAs blocked EC activation mediated by APLAs/anti-␤ 2 GPI Abs. These results provide new evidence for novel proteinprotein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-␤ 2 GPI-associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome. (Blood. 2012; 119(3):884-893) IntroductionAntiphospholipid syndrome (APS) is characterized by thrombosis and recurrent fetal loss in patients with circulating antiphospholipid Abs (APLAs) and is the most important cause of acquired thrombophilia. [1][2][3] Prospective studies have demonstrated that patients with APS experience significant morbidity and mortality despite recommendations for indefinite anticoagulation. 4 The term "antiphospholipid" is actually a misnomer, because the majority of APLAs are directed against phospholipid-binding proteins, of which ␤ 2 -glycoprotein I (␤ 2 GPI) is the most common. 5,6 The clinical importance of anti-␤ 2 GPI Abs has been demonstrated in several previous reports, 7 and recent studies have shown that affinity-purified human anti-␤ 2 GPI Abs induce thrombosis in mice. 8 Despite the clinical importance of APS, however, its pathogenesis has not been well defined. 1,3,9 One mechanism by which APLAs/anti-␤ 2 GPI Abs may promote thrombosis is through ␤ 2 GPI-dependent activation of endothelial cells (ECs). [10][11][12] ECs play a critical role in the maintenance of blood fluidity through expression of anticoagulant proteins on their luminal surface and the elaboration of antithrombotic substances. 13 However, EC activation leads to loss of these anticoagulant properties and transformation to a pro-adhesive, procoagulant phenotype. 13 APLAs/anti-␤ 2 GPI Abs induce EC activation in vitro and in vivo, as determined by their ability to increase the expression of adhesion molecules (E-selectin, ICAM-1, VCAM-1), and tissue factor (TF) and to enhance the expression, synthesis, and/or secretion of pro-inflammatory cytokines and chemokines. 3,[10][11][12] These effects may account for the ab...

Myosin II filament assembly in Dictyostelium discoideum is regulated via phosphorylation of residues located in the carboxyl-terminal portion of the myosin II heavy chain (MHC) tail. A series of novel protein kinases in this system are capable of phosphorylating these residues in vitro, driving filament disassembly. Previous studies have demonstrated that at least three of these kinases (MHCK A, MHCK B, and MHCK C) display differential localization patterns in living cells. We have created a collection of single, double, and triple gene knockout cell lines for this family of kinases. Analysis of these lines reveals that three MHC kinases appear to represent the majority of cellular activity capable of driving myosin II filament disassembly, and reveals that cytokinesis defects increase with the number of kinases disrupted. Using biochemical fractionation of cytoskeletons and in vivo measurements via fluorescence recovery after photobleaching (FRAP), we find that myosin II overassembly increases incrementally in the mutants, with the MHCK A ؊ /B ؊ /C ؊ triple mutant showing severe myosin II overassembly. These studies suggest that the full complement of MHC kinases that significantly contribute to growth phase and cytokinesis myosin II disassembly in this organism has now been identified. INTRODUCTIONMyosin II plays fundamental roles in a variety of cellular contractile processes, ranging from cytokinesis to cell migration and developmental morphogenesis (Ridley et al., 2003;Baumann, 2004;Van Haastert and Devreotes, 2004). A critical feature of nonmuscle myosin II isoforms is that cellular function requires the assembly of this motor protein into bipolar filament structures that can interact with cortical actin arrays. Filament assembly in nonmuscle cells is dynamic and subject to both spatial and temporal regulation. In mammalian nonmuscle cells, phosphorylation of the myosin II regulatory light chain (RLC) is a widely cited model for assembly regulation, with RLC phosphorylation favoring filament assembly (Scholey et al., 1980). However, there is also strong experimental support for other models of mammalian nonmuscle myosin II assembly control, including evidence for monomer sequestration by the S100 protein metastasin 1 (Li et al., 2003), and biochemical evidence that MHC phosphorylation may modulate filament assembly (Murakami et al., 1998;Murakami et al., 2000). Definitive in vivo studies to distinguish relative contributions of each mechanism have as yet not been performed.The simple amoeba Dictyostelium discoideum contains a single myosin II heavy chain gene, which serves cellular roles that are conserved with those of myosin II in other organisms. Biochemical and cellular studies in this system have established that myosin II filament assembly in vivo involves a dynamic equilibrium between a large pool of disassembled molecules and a pool of assembled filaments that associate with the cortical cytoskeleton. This equilibrium appears regulated by a variety of events ranging from chemoattractant receptor st...

Non-muscle cells express multiple myosin-II motor proteins myosin IIA, myosin IIB and myosin IIC transcribed from different loci in the human genome. Due to a significant homology in their sequences, these ubiquitously expressed myosin II motor proteins are believed to have overlapping cellular functions, but the mechanistic details are not elucidated. The present study uncovered a mechanism that coordinates the distinctly localized myosin IIA and myosin IIB with unexpected opposite mechanical roles in maneuvering lamellipodia extension, a critical step in the initiation of cell invasion, spreading, and migration. Myosin IIB motor protein by localizing at the front drives lamellipodia extension during cell spreading. On the other hand, myosin IIA localizes next to myosin IIB and attenuates or retracts lamellipodia extension. Myosin IIA and IIB increase cell adhesion by regulating focal contacts formation in the spreading margins and central part of the spreading cell, respectively. Spreading cells expressing both myosin IIA and myosin IIB motor proteins display an organized actin network consisting of retrograde filaments, arcs and central filaments attached to focal contacts. This organized actin network especially arcs and focal contacts formation in the spreading margins were lost in myosin IIÂ cells. Surprisingly, myosin IIB̂ cells displayed long parallel actin filaments connected to focal contacts in the spreading margins. Thus, with different roles in the regulation of the actin network and focal contacts formation, both myosin IIA and IIB determine the fate of lamellipodia extension during cell spreading.

Summary Background Platelet transfusion applications face severe challenges due to the limited availability and portability, high risk of contamination and short shelf-life of platelets. Therefore there is significant interest in synthetic platelet substitutes that can render hemostasis while avoiding these issues. Platelets promote hemostasis by injury site-selective adhesion and aggregation, and propagation of coagulation reactions on their membrane. Based on these mechanisms, we have developed a synthetic platelet technology (SynthoPlateTM) that integrates platelet-mimetic site-selective ‘adhesion’ and ‘aggregation’ functionalities via heteromultivalent surface-decoration of lipid vesicles with Von Willebrand Factor-binding, collagen-binding and active platelet integrin GPIIb-IIIa-binding peptides. Objective SynthoPlateTM was evaluated for its effects on platelets and plasma in vitro, and for systemic safety and hemostatic efficacy in severely thrombocytopenic mice in vivo. Methods In vitro, SynthoPlateTM was evaluated using aggregometry, fluorescence microscopy, microfluidics, and thrombin and fibrin generation assays. In vivo, SynthoPlateTM was evaluated for systemic safety using prothrombin and fibrin assays on plasma and for hemostatic effect on tail-transection bleeding time in severely thrombocytopenic (TCP) mice. Results SynthoPlateTM did not aggregate resting platelets or spontaneously promote coagulation in plasma, but could amplify recruitment and aggregation of active platelets at the bleeding site and thereby site-selectively enhance fibrin generation. SynthoPlateTM dose-dependently reduced bleeding time in TCP mice, to levels comparable to normal mice. SynthoPlateTM has a reasonable circulation residence time and is cleared mostly by the liver and spleen. Conclusion The results demonstrate the promise of SynthoPlateTM as a synthetic platelet substitute in transfusion treatment of platelet-related bleeding complications.

Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC) belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients' life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life.