Actomyosin motility on nanostructured surfaces

We have here, for the first time, used nanofabrication techniques to reproduce aspects of the ordered actomyosin arrangement in a muscle cell. The adsorption of functional heavy meromyosin (HMM) to five different resist polymers was first assessed. One group of resists (MRL-6000.1XP and ZEP-520) consistently exhibited high quality motility of actin filaments after incubation with HMM. A second group (PMMA-200, PMMA-950, and MRI-9030) generally gave low quality of motility with only few smoothly moving filaments. Based on these findings electron beam lithography was applied to a bi-layer resist system with PMMA-950 on top of MRL-6000.1XP. Grooves (100-200nm wide) in the PMMA layer were created to expose the MRL-6000.1XP surface for adsorption of HMM and guidance of actin filament motility. This guidance was quite efficient allowing no U-turns of the filaments and approximately 20 times higher density of moving filaments in the grooves than on the surrounding PMMA.     

Actomyosin systems of biological motility

Evolution of notions on the molecular mechanism of muscle contraction and other events based on the actin—myosin interaction, from the middle of XX century to the present time, is briefly reviewed, including recent views on the functioning of the myosin head as a molecular motor. The results of structural and functional studies on the myosin head performed by the author and his colleagues using differential scanning calorimetry are also reviewed.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1447–1462.Original Russian Text Copyright © 2004 by Levitsky.In memory of my teacher Boris F. Poglazov     

Actomyosin kinetics and in vitro motility of wild-type Drosophila actin and the effects of two mutations in the Act88F gene.

Two missense mutations of the flight muscle-specific actin gene of Drosophila melanogaster, Act88F, assemble into normally structured myofibrils but affect the flight ability of flies and the mechanical kinetics of isolated muscle fibers. We describe the isolation of actin from different homozygous Act88F strains, including wild-type, an Act88F null mutant (KM88), and two Act88F single point mutations (E316K and G368E), their biochemical interactions with rabbit myosin subfragment 1 (S1), and behavior with rabbit myosin and heavy meromyosin in in vitro motility assays. The rabbit and wild-type Drosophila actins have different association rate constants with S1 (2.64 and 1.77 microM-1 s-1, respectively) and in vitro motilities (2.51, 1.60 microns s-1) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increasing from 0.11 to 0.17 microM) and a reduced velocity in vitro (reduced by 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with respect to the recently published atomic models for the actomyosin structure and our findings that G368E fibers show a reduced rate constant for delayed tension development and increased fiber stiffness. We interpret these results as possibly caused either by effects on A1 myosin light chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed with respect to its likely position within the tropomyosin binding site of actin.     

Fourier's law of heat transfer and its implication to cell motility

Fourier's law of heat transfer addressing temperature differences is intrinsically selective in favoring the mitigation of the differences proceeding as fast as possible. We present an experimental demonstration of such selective behavior of material origin. When an actin filament equipped with nano-scale heat acceptors was placed under heat pulsation, it demonstrated a unidirectional movement without the presence of myosin or ATP. The prime factor for the unidirectional movement was the temperature differences between the locally heated portions on the actin filament and the cooler material bodies in the surroundings. The unidirectional movement could be enhanced in the process of mitigating the temperature differences as fast as possible.     

Septum Development in Neurospora crassa: The Septal Actomyosin Tangle

Septum formation in Neurospora crassa was studied by fluorescent tagging of actin, myosin, tropomyosin, formin, fimbrin, BUD-4, and CHS-1. In chronological order, we recognized three septum development stages: 1) septal actomyosin tangle (SAT) assembly, 2) contractile actomyosin ring (CAR) formation, 3) CAR constriction together with plasma membrane ingrowth and cell wall construction. Septation began with the assembly of a conspicuous tangle of cortical actin cables (SAT) in the septation site >5 min before plasma membrane ingrowth. Tropomyosin and myosin were detected as components of the SAT from the outset. The SAT gradually condensed to form a proto-CAR that preceded CAR formation. During septum development, the contractile actomyosin ring remained associated with the advancing edge of the septum. Formin and BUD-4 were recruited during the transition from SAT to CAR and CHS-1 appeared two min before CAR constriction. Actin patches containing fimbrin were observed surrounding the ingrowing septum, an indication of endocytic activity. Although the trigger of SAT assembly remains unclear, the regularity of septation both in space and time gives us reason to believe that the initiation of the septation process is integrated with the mechanisms that control both the cell cycle and the overall growth of hyphae, despite the asynchronous nature of mitosis in N. crassa.     

Actomyosin contractililty: Force power drives tumor growth

Comment on: Samuel MS et al. Cancer Cell 2011; 19:776-91.     

Patterns in Space: Coordinating Adhesion and Actomyosin Contractility at E-cadherin Junctions

Abstract

Cadherin adhesion receptors are fundamental determinants of tissue organization in health and disease. Increasingly, we have come to appreciate that classical cadherins exert their biological actions through active cooperation with the contractile actin cytoskeleton. Rather than being passive resistors of detachment forces, cadherins can regulate the assembly and mechanics of the contractile apparatus itself. Moreover, coordinate spatial patterning of adhesion and contractility is emerging as a determinant of morphogenesis. Here we review recent developments in cadherins and actin cytoskeleton cooperativity, by focusing on E-cadherin adhesive patterning in the epithelia. Next, we discuss the underlying principles of cellular rearrangement during Drosophila germband extension and epithelial cell extrusion, as models of how planar and apical–lateral patterns of contractility organize tissue architecture.     

Actomyosin organization during cytokinesis: reversible translocation and differential redistribution in Dictyostelium

Synchronized cultures of Dictyostelium discoideum were used to study organizational changes of the cytoskeleton during mitotic cell division. The agar-overlay technique (Yumura et al.: J. Cell Biol. 99:894-899, 1984) was employed for immunofluorescence localization and video microscopic observation of living mitotic cells. The mitotic phase was defined by changes in chromosome configuration by using a double stain with the fluorescent dye DAPI. This study showed that the actin- and myosin-containing cytoskeleton was reversibly redistributed between the cortical ectoplasm and the endoplasm during prophase and telophase. Both actin and myosin filaments were dissociated from the cell cortex in prophase. Most of the actin and myosin was filamentous and remained in the endoplasm until telophase. Saltatory movements of organelles stopped suddenly, coincident with the breakdown of the cytoplasmic microtubule network. This change in the microtubule system was temporally coupled with the disappearance of actomyosin from the cortex. At the same time, the local vibrating movement of particles almost stopped, suggesting that the viscoelastic nature of the endoplasm was altered. In the late anaphase, actin and myosin relocalized to the cortical ectoplasm. Early in this phase, myosin filaments were localized specifically at the anticipated cleavage furrow region of the cleavage furrow, whereas actin filaments were redistributed more uniformly in the cell cortex, with an extremely large accumulation in the polar pseudopods. Subsequently the actin formed an orderly parallel array of cables along with myosin filaments in the contractile ring. The spatial segregation of actin and myosin in late anaphase was clearly demonstrated by multipolar cell division of artificially induced giant cells. Actin was relocalized in both the polar and the proximal constricting regions whereas myosin was only localized in the center of each pair of daughter microtubule networks where the cleavage furrow was formed. This study demonstrates that actin and myosin are reorganized by a temporally coordinated but spatially different mechanism during cytokinesis of Dictyostelium.     

Actomyosin rings: the riddle of the sphincter.

Contractile rings consisting of actin filaments and myosin-2 have long been known to power cytokinesis, but recent work has shown that single-cell and multicellular actomyosin rings participate in processes ranging from wound healing to extrusion of apoptotic cells from epithelia.     

Actomyosin ATPase activity in Atlantic hagfish muscles

Summary Incubation for Ca⁺⁺-activated myosin ATPase reveals three types of muscle fibres in m. parietalis of the Atlantic hagfish (Myxine glutinosa), while m. craniovelaris and m. longitudinalis linguae both contain one type of muscle fibres.The fast twitch white fibres of m. longitudinalis linguae and m. parietalis show relatively high ATPase activity, while the intermediate fibres of m. parietalis show low activity. Despite of being slow non-twitch, the superficial red fibres of m. parietalis and the fibres of m. craniovelaris show an ATPase activity even higher than that of the fast twitch muscle fibres.     

Actomyosin cross-linking by caldesmon in non-muscle cells

The role of myosin-binding in cytoskeletal arrangement of non-muscle low molecular weight caldesmon (l-caldesmon) was studied. The N-terminal myosin-binding domain of caldesmon N152 colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-caldesmon displacement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-caldesmon, suggesting it may represent a regulatory mechanism for actin-caldesmon interaction in vivo.     

Force to Divide: Structural and Mechanical Requirements for Actomyosin Ring Contraction

One of the unresolved questions in the field of cell division is how the actomyosin cytoskeleton remains structurally organized while generating the contractile force to divide one cell into two. In analogy to the actomyosin-based force production mechanism in striated muscle, it was originally proposed that contractile stress in the actomyosin ring is generated via a sliding filament mechanism within an organized sarcomere-like array. However, over the last 30 years, ultrastructural and functional studies have noted important distinctions between cytokinetic structures in dividing cells and muscle sarcomeres. Myosin-II motor activity is not always required, and there is evidence that actin depolymerization contributes to contraction. In this Review, the architecture and contractile dynamics of the actomyosin ring at the cell division plane will be discussed. We will report the interdisciplinary advances in the field as well as their integration into a mechanistic understanding of contraction in cell division and in other biological processes that rely on an actomyosin-based force-generating system.     

Actomyosin sliding is attenuated in contractile biomimetic cortices

Myosin II motors embedded within the actin cortex generate contractile forces to modulate cell shape in essential behaviors, including polarization, migration, and division. In sarcomeres, myosin II–mediated sliding of antiparallel F-actin is tightly coupled to myofibril contraction. By contrast, cortical F-actin is highly disordered in polarity, orientation, and length. How the disordered nature of the actin cortex affects actin and myosin movements and resultant contraction is unknown. Here we reconstitute a model cortex in vitro to monitor the relative movements of actin and myosin under conditions that promote or abrogate network contraction. In weakly contractile networks, myosin can translocate large distances across stationary F-actin. By contrast, the extent of relative actomyosin sliding is attenuated during contraction. Thus actomyosin sliding efficiently drives contraction in actomyosin networks despite the high degree of disorder. These results are consistent with the nominal degree of relative actomyosin movement observed in actomyosin assemblies in nonmuscle cells.     

Actomyosin interactions with insulin-storage granules in vitro.

Interactions between actomyosin and insulin storage granules isolated from rat islets of Langerhans have been examined in a simple system in vitro, which allows comparison of the sedimentation of the granules in the presence of absence of actomyosin in various conditions. Actomyosin altered granule-sedimentation rates in a manner consistent with the binding of the granules of actomyosin filaments. This interaction was enhanced by addition of ATP (1.5 mM) but unaltered by addition of CaCl2, by calmodulin or by calmodulin in the presence of 10 microM-CaCl2. Addition of EGTA (0.1 mM), cyclic AMP (10 microM) of cytochalasin B (10 microgram/ml) were also without effects in these conditions. Pre-incubation of granules with phospholipase c did not affect granule-actomyosin interaction. Ultrastructural studies showed close contacts between the membranes of the granules and actomyosin filaments. The results indicate the possibility that actomyosin might provide the motile force for granule translocation during the insulin secretory process.