Actomyosin System Research Articles

Emerging drugs for ocular hypertension

Introduction: Glaucoma is a prevalent ocular disease with characteristic optic disc and visual field changes. Globally, it is the second most common cause of visual disability, and the most common cause of irreversible and preventable blindness. Ocular hypertension (OH) occurs where intraocular pressure elevation occurs in the absence of glaucomatous disc and visual field changes. OH is a strong risk factor for glaucoma. Ocular hypotensive medications are the mainstay of glaucoma and OH treatment, and their use modifies the course of the disease by preventing onset and progression of damage.Areas covered: Prostaglandin analogs, β-blockers, α-agonists, carbonic anhydrase inhibitors and parasympathomimetics are available in our glaucoma armamentarium and are reviewed. Novel agents have evolved as our understanding of the complex mechanisms involved in aqueous humor production and obstacles to aqueous outflow increases. Potential future candidates appear to act on enhancing trabecular meshwork outflow: the Rho-kinase inhibitors, ion-channel modulators and chelating agents. Further work is needed on other promising agents: serotonergics, melatonins, cannabinoids, adenosine agonists, components of the actomyosin system, nucleotide analogs and gene silencing. Methods to improve side effect profiles or efficacy of currently available therapies are also being developed. As glaucoma treatment adherence is poor, novel drug delivery methods might address this challenge.Expert opinion: Although there are good intraocular pressure-lowering medications available, novel mechanisms and drug delivery modes may provide more effective glaucoma control in future.

Involvement of Elongation Factor-1α in Cytokinesis without Actomyosin Contractile Ring in the Primitive Red Alga <i>Cyanidioschyzon merolae</i>

Cytokinesis is a pivotal event in cell division in all living organisms. In mammals, cytokinesis progresses via constriction of the contractile ring, a bundle of actin filaments and myosin that is located on the equatorial plane. The eukaryotic elongation factor 1α (eEF-1α) is associated with the contractile ring in sea urchin eggs. Although eEF-1α is a ubiquitous protein translation factor and is highly conserved in eukaryotes, archaea, and prokaryotes, the archaebacterial EF-1α and the prokaryotic EF-1α ortholog EF-Tu do not function in cytokinesis, suggesting that the association between the contractile ring and EF-1α appeared at about the same time as the establishment of eukaryotic cells. However, the role of EF-1α in cytokinesis in primitive cells is unclear. In this study, we show that the primitive alga Cyanidioschyzon merolae elongation factor-lα (CmEF-1α) is localized in the contractile division plane, but not in the actomyosin contractile ring. The genome of C. merolae contains 2 unexpressed actin genes and lacks a myosin gene. Immunoblotting analyses revealed that the protein level of CmEF-1α remained constant during the G1 to M phase, and then peaked during cytokinesis. Immunofluorescent microscopy revealed 2 patterns of localization of CmEF-1α during the cell cycle: it was dispersed throughout the cytoplasm from G1 to early M phase, and then localized to the contractile region during cytokinesis. From the results of current study on cytokinesis in the primitive red algae C. merolae, our findings led us to hypothesize that when first established in the lower eukaryotes, cytokinesis fundamentally relied on eEF-1α, and the actomyosin system was acquired later during evolution.

Effect of H-7 on Secondary Cataract After Phacoemulsification in the Live Rabbit Eye

This study is aimed to determine if the serine-threonine kinase inhibitor H-7 inhibits secondary cataract after phacoemulsification in the live rabbit eye. Eighteen rabbits underwent extracapsular lens extraction by phacoemulsification in 1 eye. The eye was treated with intravitreal H-7 (300 or 1,200 μM; n = 6 or 5) or balanced salt solution (BSS) (n = 7) immediately after the surgery and twice weekly for 10 weeks. Each eye received slit lamp biomicroscopy once a week, during which posterior capsule opacification (PCO) was evaluated. The eye was then enucleated and the lens capsule was prepared, fixed, and imaged. PCO was evaluated again on the isolated lens capsule under a phase microscope. Soemmering's ring area (SRA) and the entire lens capsule area were measured from capsule images on a computer and the percentage of SRA (PSRA) in the entire capsule area was calculated. Wet weight of the capsule (WW) was determined on a balance. No significant difference in PCO was observed in any comparison. No significant differences in SRA, PSRA, and WW were observed between the 300 μM H-7-treated eye and the BSS-treated eye. However, SRA, PSRA, and WW in the 1,200 μM H-7-treated eye were significantly smaller than those in the BSS-treated eye [28.3 ± 16.2 vs. 61.4 ± 8.86 mm(2) (P = 0.001), 33% ± 20% vs. 65% ± 15% (P = 0.01), and 65.6 ± 27.9 vs. 127.0 ±37.3 mg (P = 0.01)]. Intravitreal H-7 (1,200 μM) significantly inhibits Soemmering's ring formation in the live rabbit eye, suggesting that agents that inhibit the actomyosin system in cells may prevent secondary cataract after phacoemulsification.

DAAM1 Is a Formin Required for Centrosome Re-Orientation during Cell Migration

BackgroundDisheveled-associated activator of morphogenesis 1 (DAAM1) is a formin acting downstream of Wnt signaling that is important for planar cell polarity. It has been shown to promote proper cell polarization during embryonic development in both Xenopus and Drosophila. Importantly, DAAM1 binds to Disheveled (Dvl) and thus functions downstream of the Frizzled receptors. Little is known of how DAAM1 is localized and functions in mammalian cells. We investigate here how DAAM1 affects migration and polarization of cultured cells and conclude that it plays a key role in centrosome polarity.Methodology/Principal FindingsUsing a specific antibody to DAAM1, we find that the protein localizes to the acto-myosin system and co-localizes with ventral myosin IIB-containing actin stress fibers. These fibers are particularly evident in the sub-nuclear region. An N-terminal region of DAAM1 is responsible for this targeting and the DAAM1(1-440) protein can interact with myosin IIB fibers independently of either F-actin or RhoA binding. We also demonstrate that DAAM1 depletion inhibits Golgi reorientation in wound healing assays. Wound-edge cells exhibit multiple protrusions characteristic of unpolarized cell migration. Finally, in U2OS cells lines stably expressing DAAM1, we observe an enhanced myosin IIB stress fiber network which opposes cell migration.Conclusions/SignificanceThis work highlights the importance of DAAM1 in processes underlying cell polarity and suggests that it acts in part by affecting the function of acto-myosin IIB system. It also emphasizes the importance of the N-terminal half of DAAM1. DAAM1 depletion strongly blocks centrosomal re-polarization, supporting the concept that DAAM1 signaling cooperates with the established Cdc42 associated polarity complex. These findings are also consistent with the observation that ablation of myosin IIB but not myosin IIA results in polarity defects downstream of Wnt signaling. The structure-function analysis of DAAM1 in cultured cells parallels more complex morphological events in the developing embryo.

Second Harmonic Generation Microscopy Probes Different States of Motor Protein Interaction in Myofibrils

The second harmonic generation (SHG) signal intensity sourced from skeletal muscle myosin II strongly depends on the polarization of the incident laser beam relative to the muscle fiber axis. This dependence is related to the second-order susceptibility χ(2), which can be described by a single component ratio γ under generally assumed symmetries. We precisely extracted γ from SHG polarization dependence curves with an extended focal field model. In murine myofibrillar preparations, we have found two distinct polarization dependencies: With the actomyosin system in the rigor state, γrig has a mean value of γrig = 0.52 (SD = 0.04, n = 55); in a relaxed state where myosin is not bound to actin, γrel has a mean value of γrel = 0.24 (SD = 0.07, n = 70). We observed a similar value in an activated state where the myosin power stroke was pharmacologically inhibited using N-benzyl-p-toluene sulfonamide. In summary, different actomyosin states can be visualized noninvasively with SHG microscopy. Specifically, SHG even allows us to distinguish different actin-bound states of myosin II using γ as a parameter.

Toxoplasma gondii transmembrane microneme proteins and their modular design

Host cell invasion by the Apicomplexa critically relies on regulated secretion of transmembrane micronemal proteins (TM-MICs). Toxoplasma gondii possesses functionally non-redundant MIC complexes that participate in gliding motility, host cell attachment, moving junction formation, rhoptry secretion and invasion. The TM-MICs are released onto the parasite's surface as complexes capable of interacting with host cell receptors. Additionally, TgMIC2 simultaneously connects to the actomyosin system via binding to aldolase. During invasion these adhesive complexes are shed from the surface notably via intramembrane cleavage of the TM-MICs by a rhomboid protease. Some TM-MICs act as escorters and assure trafficking of the complexes to the micronemes. We have investigated the properties of TgMIC6, TgMIC8, TgMIC8.2, TgAMA1 and the new micronemal protein TgMIC16 with respect to interaction with aldolase, susceptibility to rhomboid cleavage and presence of trafficking signals. We conclude that several TM-MICs lack targeting information within their C-terminal domains, indicating that trafficking depends on yet unidentified proteins interacting with their ectodomains. Most TM-MICs serve as substrates for a rhomboid protease and some of them are able to bind to aldolase. We also show that the residues responsible for binding to aldolase are essential for TgAMA1 but dispensable for TgMIC6 function during invasion.

Cinemechanometry (CMM): A Method to Determine the Forces that Drive Morphogenetic Movements from Time-Lapse Images

Although cell-level mechanical forces are crucial to tissue self-organization in contexts ranging from embryo development to cancer metastases to regenerative engineering, the absence of methods to map them over time has been a major obstacle to new understanding. Here, we present a technique for constructing detailed, dynamic maps of the forces driving morphogenetic events from time-lapse images. Forces in the cell are considered to be separable into unknown active driving forces and known passive forces, where actomyosin systems and microtubules contribute primarily to the first group and intermediate filaments and cytoplasm to the latter. A finite-element procedure is used to estimate the field of forces that must be applied to the passive components to produce their observed incremental deformations. This field is assumed to be generated by active forces resolved along user-defined line segments whose location, often along cell edges, is informed by the underlying biology. The magnitudes and signs of these forces are determined by a mathematical inverse method. The efficacy of the approach is demonstrated using noisy synthetic data from a cross section of a generic invagination and from a planar aggregate that involves two cell types, edge forces that vary with time and a neighbor change.

Rab6 and myosin II at the cutting edge of membrane fission

Rab GTPases regulate the dynamics of transport carriers by participating in their translocation across the cytoplasm, and in their docking and fusion with acceptor compartments. An interaction between Golgi-associated Rab6 and myosin II has now been shown to regulate the fission of Rab6-positive carriers, illuminating a previously unappreciated role for Rab6 and the actomyosin system in carrier biogenesis.

Class XI Myosins Are Required for Development, Cell Expansion, and F-Actin Organization in Arabidopsis

The actomyosin system is conserved throughout eukaryotes. Although F-actin is essential for cell growth and plant development, roles of the associated myosins are poorly understood. Using multiple gene knockouts in Arabidopsis thaliana, we investigated functional profiles of five class XI myosins, XI-K, XI-1, XI-2, XI-B, and XI-I. Plants lacking three myosins XI showed stunted growth and delayed flowering, whereas elimination of four myosins further exacerbated these defects. Loss of myosins led to decreased leaf cell expansion, with the most severe defects observed in the larger leaf cells. Root hair length in myosin-deficient plants was reduced approximately 10-fold, with quadruple knockouts showing morphological abnormalities. It was also found that trafficking of Golgi and peroxisomes was entirely myosin dependent. Surprisingly, myosins were required for proper organization of F-actin and the associated endoplasmic reticulum networks, revealing a novel, architectural function of the class XI myosins. These results establish critical roles of myosin-driven transport and F-actin organization during polarized and diffuse cell growth and indicate that myosins are key factors in plant growth and development.

Rab and actomyosin-dependent fission of transport vesicles at the Golgi complex

Trafficking between membrane compartments is a characteristic of eukaryotic cells and relies on transport carriers that bud and fission from a donor membrane, before being transported and fusing with the correct acceptor compartment. Rab GTPases ensure specificity and directionality of trafficking steps by regulating the movement of transport carriers along cytoskeletal tracks, and the recruitment of tethering factors required for the docking and fusion processes. Here we show that Rab6, a Golgi-associated Rab, forms a complex with myosin II, contributes to its localization at the Golgi complex and, unexpectedly, controls the fission of Rab6 vesicles. Inhibition of either Rab6 or myosin II function impairs both the fission of Rab6 transport carriers from Golgi membranes and the trafficking of anterograde and retrograde cargo from the Golgi. These effects are consistent with myosin II being an effector of Rab6 in these processes. Our results provide evidence that the actomyosin system is required in vesicle biogenesis at the Golgi, and uncover a function for Rab GTPases in vesicle fission.

Motoring around the plant cell: insights from plant myosins

Organelle movement in plants cells is extremely dynamic. Movement is driven by the acto-myosin system. Higher plant myosins fall into two classes: classes XI and VIII. Localization studies have highlighted that myosins are present throughout the cytosol, label motile puncta and decorate the nuclear envelope and plasma membrane. Functional studies through expression of dominant-negative myosin variants, RNAi (RNA interference) and T-DNA insertional analysis have shown that class XI myosins are required for organelle movement. Intriguingly, organelle movement is also linked to Arabidopsis growth and development. The present review tackles current findings relating to plant organelle movement and the role of myosins.

Does an Actomyosin Supplement the Axoneme n Mammalian Sperm

Boar sperm fixed in paraformaldehyde, pemeabilized in cold acetone and incubated in the presence of DAPI and Rhodamine-conjugated phalloidin or with fluorescein-conjugated cytochalasin D show brilliant blue staining of the nucleus and brilliant red or green fluorescence respectively in the middle piece and tail regions of the sperm. The results suggest the presence of F-actin in the boar sperm middle piece. Immunocytochemical studies also show the presence of tropomyosin in the middle piece region of boar sperm. Western Blot studies, using lysates of isolated boar sperm middle pieces and tails, indicate prominent sperm-actin bands with similar electrophoretic patterns of rat cardiac and striated muscle. The results strongly suggest that a 41 kDa actin, similar to cardiac or striated muscle actin, is present in sperm middle pieces and corroborates the phalloidin and cytochalasin D localization by fluorescence microscopy. We suggest that the simple sliding filament mechanism of the axoneme may not, by itself, impose the propulsive forces needed to propel large spermatozoa through the viscous prefertilization milieu. We further propose that an actomyosin system associated with the microtubule/dynein complex might play a role in sustained motility of mammalian spermatozoa.

Spontaneous Oscillations of a Minimal Acto-Myosin System Under Elastic Loading

Spontaneous mechanical oscillations occur in various types of biological systems where groups of motor molecules are elastically coupled to their environment. By using an optical trap to oppose the gliding motion of a single bead-tailed actin filament over a substrate densely coated with heavy meromyosin molecules, we mimicked this condition in vitro. We show that this minimal acto-myosin system can oscillate spontaneously. Our finding accords quantitatively with a general theoretical framework where oscillatory instabilities emerge generically from the collective dynamics of molecular motors under load.

Both actin and myosin inhibitors affect spindle architecture in PtK1 cells: does an actomyosin system contribute to mitotic spindle forces by regulating attachment and movements of chromosomes in mammalian cells?

Immunocytochemical techniques are used to analyze the effects of both an actin and myosin inhibitor on spindle architecture in PtK(1) cells to understand why both these inhibitors slow or block chromosome motion and detach chromosomes. Cytochalasin J, an actin inhibitor and a myosin inhibitor, 2, 3 butanedione 2-monoxime, have similar effects on changes in spindle organization. Using primary antibodies and stains, changes are studied in microtubule (MT), actin, myosin, and chromatin localization. Treatment of mitotic cells with both inhibitors results in detachment or misalignment of chromosomes from the spindle and a prominent buckling of MTs within the spindle, particularly evident in kinetochore fibers. Evidence is presented to suggest that an actomyosin system may help to regulate the initial and continued attachment of chromosomes to the mammalian spindle and could also influence spindle checkpoint(s).

Spontaneous Oscillations of a Minimal Actomyosin System under Elastic Loading

Spontaneous mechanical oscillations occur in various types of biological systems where groups of motor molecules are elastically coupled to their environment. By using an optical trap to oppose the gliding motion of a single bead-tailed actin filament over a substrate densely coated with myosin motors, we mimicked this condition in vitro. We show that this minimal actomyosin system can oscillate spontaneously. Our finding accords quantitatively with a general theoretical framework where oscillatory instabilities emerge generically from the collective dynamics of molecular motors under load.

Actomyosin contractility spatiotemporally regulates actin network dynamics in migrating cells

Coupling interactions among mechanical and biochemical factors are important for the realization of various cellular processes that determine cell migration. Although F-actin network dynamics has been the focus of many studies, it is not yet clear how mechanical forces generated by actomyosin contractility spatiotemporally regulate this fundamental aspect of cell migration. In this study, using a combination of fluorescent speckle microscopy and particle imaging velocimetry techniques, we perturbed the actomyosin system and examined quantitatively the consequence of actomyosin contractility on F-actin network flow and deformation in the lamellipodia of actively migrating fish keratocytes. F-actin flow fields were characterized by retrograde flow at the front and anterograde flow at the back of the lamellipodia, and the two flows merged to form a convergence zone of reduced flow intensity. Interestingly, activating or inhibiting actomyosin contractility altered network flow intensity and convergence, suggesting that network dynamics is directly regulated by actomyosin contractility. Moreover, quantitative analysis of F-actin network deformation revealed that the deformation was significantly negative and predominant in the direction of cell migration. Furthermore, perturbation experiments revealed that the deformation was a function of actomyosin contractility. Based on these results, we suggest that the actin cytoskeletal structure is a mechanically self-regulating system, and we propose an elaborate pathway for the spatiotemporal self-regulation of the actin cytoskeletal structure during cell migration. In the proposed pathway, mechanical forces generated by actomyosin interactions are considered central to the realization of the various mechanochemical processes that determine cell motility.

Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6alpha localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6alpha inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to "pull" the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6alpha.

Thermodynamic origin of cooperativity in actomyosin interactions: The coupling of short-range interactions with actin bending stiffness in an Ising-like model

We present Monte Carlo simulations for a molecular motor system found in virtually all eukaryotic cells, the acto-myosin motor system, composed of a group of organic macromolecules. Cell motors were mapped to an Ising-like model, where the interaction field is transmitted through a tropomyosin polymer chain. The presence of Ca2+ induces tropomyosin to block or unblock binding sites of the myosin motor leading to its activation or deactivation. We used the Metropolis algorithm to find the transient and the equilibrium states of the acto-myosin system composed of solvent, actin, tropomyosin, troponin, Ca2+, and myosin-S1 at a given temperature, including the spatial configuration of tropomyosin on the actin filament surface. Our model describes the short- and long-range cooperativity during actin-myosin binding which emerges from the bending stiffness of the tropomyosin complex. We found all transition rates between the states only using the interaction energy of the constituents. The agreement between our model and experimental data also supports the recent theory of flexible tropomyosin.

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.

Dynamic conformational changes in the FERM domain of FAK are involved in focal-adhesion behavior during cell spreading and motility

Focal adhesion kinase (FAK) controls cellular adhesion and motility processes by its tight link to integrin- and extracellular-matrix-mediated signaling. To explore the dynamics of the regulation of FAK, we constructed a FRET-based probe that visualizes conformational rearrangements of the FERM domain of FAK in living cells. The sensor reports on an integrin-mediated conformational change in FAK following cellular adhesion. The perturbation is kinase-independent and involves the polybasic KAKTLR sequence in the FERM domain. It is manifested by an increased FRET signal and is expressed primarily in focal adhesions, and to a lesser extent in the cytoplasm. The conformational change in the FERM domain of FAK is observed in two consecutive phases during spreading - early and late - and is enriched in fully adhered motile cells at growing and sliding peripheral focal-adhesion sites, but not in stable or retracting focal adhesions. Inhibition of the actomyosin system indicates the involvement of tension signaling induced by Rho-associated kinase, rather than by myosin light-chain kinase, in the modulation of the FERM response. We conclude that the heterogeneous conformation of the FERM domain in focal adhesions of migrating cells reflects a complex regulatory mechanism for FAK that appears to be under the influence of cellular traction forces.