Dynamics of the cytoskeleton in Amoeba proteus. III. Influence of microinjected antibodies on the organization and function of the microfilament system

Affinity-purified antibodies against actin, myosin, alpha-actinin and vinculin cross-reacted with corresponding proteins from Amoeba proteus in immunoblotting experiments. Antibody staining of cells fixed during locomotion revealed different distribution patterns with a local concentration of anti-actin in the intermediate and of anti-myosin in the uroid region. Anti-alpha-actinin labeled a thin layer at the internal face of the plasma membrane, whereas anti-vinculin was distinctly concentrated at the base of advancing pseudopodia. Microinjection of different control solutions or antibodies against actin, myosin and alpha-actinin neither influenced the normal morphology and motile activity of amoebae nor changed the cellular distribution pattern of complementary antigens. However, antibodies against vinculin disorganized controlled locomotion and altered the spatial morphology of the microfilament system as well as the localization of the vinculin antigen thus pointing to a function of this protein in adhesion and locomotion of A. proteus. The results of the present paper show similarities to observations on mammalian tissue culture cells.     

Pinocytosis and locomotion of amoebae. XIX. Immunocytochemical demonstration of actin and myosin in Amoeba proteus

The spatial distribution of cytoplasmic actin and myosin in 1. normal locomoting, 2. immobilized, and 3. pinocytosing Amoeba proteus was demonstrated by indirect immunofluorescence microscopy. In orthotactic and polytactic cells fixed during normal locomotion actin is mainly located in a cortical layer delineating the granuloplasm from the peripheral hyaloplasm. In cell areas lacking a hyaloplasmic sheet the actin layer immediately borders the plasma membrane. The amount of actin within the continuous layer seems to increase from the advancing front to the middle cell region and to decrease again toward the uroid. The distribution of myosin is largely congruent to the display of actin, with the exception that the myosin-based fluorescence of the cortical layer gradually increases from the front to the uroid. A considerable amount of actin and myosin is also distributed around the nucleus and the contractile vacuole. In immobilized cells contracted by the external application of 10(-4)M procaine hydrochloride the cortical layer distinctly increases in thickness. In contrast to normal locomoting cells actin and myosin show a uniform distribution within the cell cortex along the entire surface. In pinocytosing cells, up to three cortical layers conspicuously rich in actin are produced during the process of channel formation. One of these layers is located in close proximity to the plasma membrane of the pinocytotic channels and the vacuoles. The immunocytochemical results are discussed with respect to earlier observations on the distribution of actin and myosin in Amoeba proteus as obtained by other methods.     

Analysis of the Actin–Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation

While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin–myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin–myosin network in the lamellipodial/cell body transition zone.     

Myosin and actin in melanophore-like variants of goldfish erythrophoroma cells: their intracellular distribution and possible association with pigment translocation

Summary The occurrence and intracellular distribution of myosin and actin in melanophore-like cells derived from a goldfish erythrophoroma cell line have been studied by means of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblot and immunofluorescence using antisera against chick gizzard myosin heavy chain and carp skeletal muscle actin. SDS-PAGE of the cell extracts separates out one band at 200 kDalton; this is conjugated with the anti-myosin antiserum. Immunofluorescence using the anti-myosin antiserum discloses that myosin in these cells occurs in two forms: discrete, minute clusters and thin filaments bearing a resemblance to stress fibers. The former is distributed evenly over the entire cytoplasm in the cells with dispersed pigments and, upon pigment aggregation, accumulates densely around collapsed melanosomes. The latter runs as thin bundles either radially along the cell center-to-periphery axis or connecting the corners of cell margins; it gives a similar profile in all states of the motile response. Immunofluorescence using the antiactin antiserum or rhodamine-conjugated phalloidin discloses that actin is similarly distributed to myosin, suggesting its possible existence as actomyosin. Simultaneous translocation of the amorphous forms of myosin and actin with melanosomes indicates that they may be involved in pigment migration.     

Oriented thick and thin filaments in amoeba proteus

Actin and myosin filaments as a foundation of contractile systems are well established from ameba to man (3). Wolpert et al. (19) isolated by differential centrifugation from Amoeba proteus a motile fraction composed of filaments which moved upon the addition of ATP. Actin filaments are found in amebas (1, 12, 13) which react with vertebrate heavy meromyosin (HMM), forming arrowhead complexes as vertebrate actin (3, 9), and are prominent within the ectoplasmic tube where some of them are attached to the plasmalemma (1, 12). Thick and thin filaments possessing the morphological characteristics of myosin and actin have been obtained from isolated ameba cytoplasm (18, 19). In addition, there are filaments exhibiting ATPase activity in amebas which react with actin (12, 16, 17). However, giant ameba (Chaos-proteus) shapes are difficult to preserve, and the excellent contributions referred to above are limited by visible distortions occurring in the amebas (rounding up, pseudopods disappearing, and cellular organelles swelling) upon fixation. Achievement of normal ameboid shape in recent glycerination work (15) led us to attempt other electron microscope fixation techniques, resulting in a surprising preservation of A. proteus with a unique orientation of thick and thin filaments in the ectoplasmic region.     

Comparative maps of motion and assembly of filamentous actin and myosin II in migrating cells

To understand the mechanism of cell migration, one needs to know how the parts of the motile machinery of the cell are assembled and how they move with respect to each other. Actin and myosin II are thought to be the major structural and force-generating components of this machinery (Mitchison and Cramer, 1996; Parent, 2004). The movement of myosin II along actin filaments is thought to generate contractile force contributing to cell translocation, but the relative motion of the two proteins has not been investigated. We use fluorescence speckle and conventional fluorescence microscopy, image analysis, and computer tracking techniques to generate comparative velocity and assembly maps of actin and myosin II over the entire cell in a simple model system of persistently migrating fish epidermal keratocytes. The results demonstrate contrasting polarized assembly patterns of the two components, indicate force generation at the lamellipodium-cell body transition zone, and suggest a mechanism of anisotropic network contraction via sliding of myosin II assemblies along divergent actin filaments.     

Immunolocalization of a putative unconventional myosin on the surface of motile mitochondria in locust photoreceptors

Light stimulation of locust (Schistocerca gregaria) photoreceptors results in an actin-dependent translocation of mitochondria towards the photoreceptive microvilli and an antagonistic movement of endoplasmic reticulum towards the cell body. Using immunocytochemical techniques, we have tried to identify myosin-like motors that may drive the light-induced organelle motility. A monoclonal antibody against the motor domain of Acanthamoeba myosin identifies a prominent 110-kDa protein on Western blots of locust retina. Cross-reactivity with two polyclonal anti-myosin antibodies and a monoclonal anti-myosin-I-antibody, together with ATP-dependent binding to actin filaments, provides evidence that the 110-kDa protein is an unconventional myosin. By indirect immunofluorescence, the 110-kDa protein has been localized to both photoreceptors and pigment cells within the retina. In the photoreceptor cells, the 110-kDa protein is bound to the surface of mitochondria. This putative unconventional myosin may thus be a motor protein involved in the light-induced translocation of mitochondria in photoreceptors.     

Visualization of actin polymerization and depolymerization cycles during polyamine-induced cytokinesis in living Amoeba proteus

Summary Microinjection of spermine induces cytokinesis of Amoeba proteus. Within 30–60 s after spermine injection cells form one, or less commonly, two cleavage furrows and within the following 4–10 min the constrictions are completed. The resulting nucleated cell parts show normal streaming and locomotion, whereas the non-nucleated cell parts remain stationary and later degenerate.The intracellular distribution of fully polymerization-competent fluorescently labelled muscle actin was followed by image intensification. Double injection experiments initially using labelled actin and 30 min later spermine revealed a ring-like structure of enhanced fluorescence corresponding to the constricting cleavage furrow. Immediately after cleavage was completed, the ring disappeared. Electron microscopy of cells fixed during spermine-induced cytokinesis showed numerous well aligned actin and myosin filaments in the developing cleavage furrow. These filaments are a specialized manifestation of the cell cortex.The results demonstrate that cycles of actin and myosin polymerization and depolymerization and the parallel alignment of preexisting filaments (crosslinking) represent a basic mechanism in the generation of the motive force during cytokinesis.     

Recruitment of a myosin heavy chain kinase to actin-rich protrusions in Dictyostelium

Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives the disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin-dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading-edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion.     

Actin dynamics in Amoeba proteus motility

Summary. We studied the distribution of the endogenous Arp2/3 complex in Amoeba proteus and visualised the ratio of filamentous (F-actin) to total actin in living cells. The presented results show that in the highly motile Amoeba proteus, Arp2/3 complex-dependent actin polymerisation is involved in the formation of the branching network of the contractile layer, adhesive structures, and perinuclear cytoskeleton. The aggregation of the Arp2/3 complex in the cortical network, with the exception of the uroid and advancing fronts, and the spatial orientation of microfilaments at the leading edge suggest that actin polymerisation in this area is not sufficient to provide the driving force for membrane displacement. The examined proteins were enriched in the pinocytotic pseudopodia and the perinuclear cytoskeleton in pinocytotic amoebae. In migrating amoebae, the course of changes in F-actin concentration corresponded with the distribution of tension in the cell cortex. The maximum level of F-actin in migrating amoebae was observed in the middle-posterior region and in the front of retracting pseudopodia. Arp2/3 complex-dependent actin polymerisation did not seem to influence F-actin concentration. The strongly condensed state of the microfilament system could be attributed to strong isometric contraction of the cortical layer accompanied by its retraction from distal cell regions. Isotonic contraction was limited to the uroid. Correspondence and reprints: Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, ulica Pasteura 3, 02-093 Warszawa, Poland.     

Dynamin2 Organizes Lamellipodial Actin Networks to Orchestrate Lamellar Actomyosin

Actin networks in migrating cells exist as several interdependent structures: sheet-like networks of branched actin filaments in lamellipodia; arrays of bundled actin filaments co-assembled with myosin II in lamellae; and actin filaments that engage focal adhesions. How these dynamic networks are integrated and coordinated to maintain a coherent actin cytoskeleton in migrating cells is not known. We show that the large GTPase dynamin2 is enriched in the distal lamellipod where it regulates lamellipodial actin networks as they form and flow in U2-OS cells. Within lamellipodia, dynamin2 regulated the spatiotemporal distributions of α-actinin and cortactin, two actin-binding proteins that specify actin network architecture. Dynamin2''s action on lamellipodial F-actin influenced the formation and retrograde flow of lamellar actomyosin via direct and indirect interactions with actin filaments and a finely tuned GTP hydrolysis activity. Expression in dynamin2-depleted cells of a mutant dynamin2 protein that restores endocytic activity, but not activities that remodel actin filaments, demonstrated that actin filament remodeling by dynamin2 did not depend of its functions in endocytosis. Thus, dynamin2 acts within lamellipodia to organize actin filaments and regulate assembly and flow of lamellar actomyosin. We hypothesize that through its actions on lamellipodial F-actin, dynamin2 generates F-actin structures that give rise to lamellar actomyosin and for efficient coupling of F-actin at focal adhesions. In this way, dynamin2 orchestrates the global actin cytoskeleton.     

Coupled myosin VI motors facilitate unidirectional movement on an F-actin network

Unconventional myosins interact with the dense cortical actin network during processes such as membrane trafficking, cell migration, and mechanotransduction. Our understanding of unconventional myosin function is derived largely from assays that examine the interaction of a single myosin with a single actin filament. In this study, we have developed a model system to study the interaction between multiple tethered unconventional myosins and a model F-actin cortex, namely the lamellipodium of a migrating fish epidermal keratocyte. Using myosin VI, which moves toward the pointed end of actin filaments, we directly determine the polarity of the extracted keratocyte lamellipodium from the cell periphery to the cell nucleus. We use a combination of experimentation and simulation to demonstrate that multiple myosin VI molecules can coordinate to efficiently transport vesicle-size cargo over 10 µm of the dense interlaced actin network. Furthermore, several molecules of monomeric myosin VI, which are nonprocessive in single molecule assays, can coordinate to transport cargo with similar speeds as dimers.     

ACTIN AND MYOSIN REGULATE PSEUDOPODIA OF PORPHYRA PULCHELLA (RHODOPHYTA) ARCHEOSPORES1

Archeospores of Porphyra pulchella Ackland, J. A. West et Zuccarello (Rhodophyta) display amoeboid and gliding motility. Time‐lapse videomicroscopy revealed that amoeboid cells extend and retract pseudopodia as they translocate through the media. We investigated the involvement of actin and myosin in generating the force for amoeboid motility using immunofluorescence, time‐lapse videomicroscopy, and cytoskeletal inhibitors. Actin filaments were seen as short and long rodlike bundles around the periphery of spores. The actin inhibitors cytochalasin D (CD) and latrunculin B (Lat B), and the myosin inhibitor butanedione monoxime (BDM) disrupted the actin filament network and reversibly inhibited pseudopodial activity, resulting in the rounding and immobilization of spores. It was uncertain whether forward translocation of archeospores resumed following drug removal. These results demonstrate that actin and myosin have a role in generating force for pseudopodial activity. This is the first report of cytoskeletal involvement in red algal cell movement. The involvement of actin and myosin in forward translocation of amoeboid archeospores can only be speculated upon.     

Myosin VI,an actin motor for membrane traffic and cell migration

The actin cytoskeleton and associated myosin motor proteins are essential for the transport and steady-state localization of vesicles and organelles and for the dynamic remodeling of the plasma membrane as well as for the maintenance of differentiated cell-surface structures. Myosin VI may be expected to have unique cellular functions, because it moves, unlike almost all other myosins, towards the minus end of actin filaments. Localization and functional studies indicate that myosin VI plays a role in a variety of different intracellular processes, such as endocytosis and secretion as well as cell migration. These diverse functions of myosin VI are mediated by interaction with a range of different binding partners .     

Myosin VI, a new force in clathrin mediated endocytosis.

The integrity of the actin cytoskeleton and associated motor proteins are essential for the efficient functioning of clathrin mediated endocytosis at least in polarised cells. Myosin VI, the only motor protein so far identified that moves towards the minus end of actin filaments, is the first motor protein to be shown to associate with clathrin coated pits/vesicles at the plasma membrane and to modulate clathrin mediated endocytosis. Recent kinetic studies suggest that myosin VI may move processively along actin filaments providing clues about its functions in the cell. The possible role(s) of myosin VI in the sequential steps involved in receptor mediated endocytosis are discussed.     

Phosphorylation of Amoeba G-actin and its effect on actin polymerization

Mass culture of Amoeba proteus enabled us to do biochemical studies on this organism. Actin and profilin were purified from Amoeba to examine actin phosphorylation and polymerization. The apparent molecular weight of Amoeba actin was 44,000, and its isoelectric point was 5.8. The apparent molecular weight of Amoeba profilin was 12,000, and its isoelectric point was 4.9. It reduced the rate of actin polymerization as reported in the cases of profilins from other organisms. A protein of Mr = 44,000 (44 K protein) was phosphorylated in a Ca2+-dependent manner in cell homogenate of Amoeba without being inhibited by calmodulin antagonists. Using the homogenate as a kinase, purified Amoeba G-actin could be phosphorylated in proportion to the amount of actin. However, neither Amoeba F-actin nor rabbit skeletal muscle G-actin was phosphorylated. The phosphorylation of Amoeba actin with a kinase partially purified from A. proteus increased with dilution of the actin concentration. When Amoeba profilin was added, more than 80% of the actin was phosphorylated. By viscometry, electron microscopy, and ultracentrifugation analysis it was demonstrated that Amoeba G-actin phosphorylated in the presence of profilin and kinase did not polymerize in this solution. High-performance liquid chromatography analysis showed that phosphorylated Amoeba actin remained in a monomeric state even under conditions favorable for actin polymerization.     

The contractile basis of ameboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts

The role of calcium and magnesium-ATP on the structure and contractility in motile extracts of Amoeba proteus and plasmalemma-ectoplasm "ghosts" of Chaos carolinensis has been investigated by correlating light and electron microscope observations with turbidity and birefringence measurements. The extract is nonmotile and contains very few F-actin filaments and myosin aggregates when prepared in the presence of both low calcium ion and ATP concentrations at an ionic strength of I = 0.05, pH 6.8. The addition of 1.0 mM magnesium chloride, 1.0 mM ATP, in the presence of a low calcium ion concentration (relaxation solution) induced the formation of some fibrous bundles of actin without contracting, whereas the addition of a micromolar concentration of calcium in addition to 1.0 mM magnesium-ATP (contraction solution) (Taylor, D. L., J. S. Condeelis, P. L. Moore, and R. D. Allen. 1973. J. Cell Biol. 59:378-394) initiated the formation of large arrays of F-actin filaments followed by contractions. Furthermore, plasmalemma-ectoplasm ghosts prepared in the relaxation solution exhibited very few straight F-actin filaments and myosin aggregates. In contrast, plasmalemmaectoplasm ghosts treated with the contraction solution contained many straight F-actin filaments and myosin aggregates. The increase in the structure of ameba cytoplasm at the endoplasm-ectoplasm interface can be explained by a combination of the transformation of actin from a less filamentous to a more structured filamentous state possibly involving the cross-linking of actin to form fibrillar arrays (see above-mentioned reference) followed by contractions of the actin and myosin along an undetermined distance of the endoplasm and/or ectoplasm.     

Self-polarization and directional motility of cytoplasm

Background: Directional cell motility implies the presence of a steering mechanism and a functional asymmetry between the front and rear of the cell. How this functional asymmetry arises and is maintained during cell locomotion is, however, unclear. Lamellar fragments of fish epidermal keratocytes, which lack nuclei, microtubules and most organelles, present a simplified, perhaps minimal, system for analyzing this problem because they consist of little other than the motile machinery enclosed by a membrane and yet can move with remarkable speed and persistence.Results: We have produced two types of cellular fragments: discoid stationary fragments and polarized fragments undergoing locomotion. The organization and dynamics of the actin–myosin II system were isotropic in stationary fragments and anisotropic in the moving fragments. To investigate whether the creation of asymmetry could result in locomotion, a transient mechanical stimulus was applied to stationary fragments. The stimulus induced localized contraction and the formation of an actin–myosin II bundle at one edge of the fragment. Remarkably, stimulated fragments started to undergo locomotion and the locomotion and associated anisotropic organization of the actin–myosin II system were sustained after withdrawal of the stimulus.Conclusions: We propose a model in which lamellar cytoplasm is considered a dynamically bistable system capable of existing in a non-polarized or polarized state and interconvertible by mechanical stimulus. The model explains how the anisotropic organization of the lamellum is maintained in the process of locomotion. Polarized locomotion is sustained through a positive-feedback loop intrinsic to the actin–myosin II machinery: anisotropic organization of the machinery drives translocation, which then reinforces the asymmetry of the machinery, favoring further translocation.     

Regulating actin dynamics at membranes: a focus on dynamin

Dynamin, the large guanosine triphosphatase, is generally considered to have a key role in deforming membranes to create tubules or vesicles. Dynamin, particularly dynamin2 isoforms, also are localized with actin filaments, often at locations where cellular membranes undergo remodeling. Perturbing dynamin function interferes with endocytic traffic and actin function. Thus, dynamin may regulate actin filaments coordinately with its activities that remodel membranes. This review will highlight recent observations that provide clues to mechanisms whereby dynamin might coordinate membrane remodeling and actin filament dynamics during endocytic traffic, cell morphogenesis and cell migration.     

Actin activation of myosin heavy chain kinase A in Dictyostelium: a biochemical mechanism for the spatial regulation of myosin II filament disassembly

Studies in Dictyostelium discoideum have established that the cycle of myosin II bipolar filament assembly and disassembly controls the temporal and spatial localization of myosin II during critical cellular processes, such as cytokinesis and cell locomotion. Myosin heavy chain kinase A (MHCK A) is a key enzyme regulating myosin II filament disassembly through myosin heavy chain phosphorylation in Dictyostelium. Under various cellular conditions, MHCK A is recruited to actin-rich cortical sites and is preferentially enriched at sites of pseudopod formation, and thus MHCK A is proposed to play a role in regulating localized disassembly of myosin II filaments in the cell. MHCK A possesses an aminoterminal coiled-coil domain that participates in the oligomerization, cellular localization, and actin binding activities of the kinase. In the current study, we show that the interaction between the coiled-coil domain of MHCK A and filamentous actin leads to an approximately 40-fold increase in the initial rate of kinase catalytic activity. Actin-mediated activation of MHCK A involves increased rates of kinase autophosphorylation and requires the presence of the coiled-coil domain. Structure-function analyses revealed that the coiled-coil domain alone binds to actin filaments (apparent K(D) = 0.9 microm) and thus mediates the direct interaction with F-actin required for MHCK A activation. Collectively, these results indicate that MHCK A recruitment to actin-rich sites could lead to localized activation of the kinase via direct interaction with actin filaments, and thus this mode of kinase regulation may represent an important mechanism by which the cell achieves localized disassembly of myosin II filaments required for specific changes in cell shape.