Evidence for regulation of actin synthesis in cytochalasin D-treated HEp-2 cells

In HEp-2 cells treated with 0.2 or 2.0 μM cytochalasin D (CD), the relative rate of actin synthesis increased for about 12 h and then reached a plateau; this increase was suppressed by actinomycin D (AD). When CD was washed from cells which had been treated for 20 h, the elevated rate of actin synthesis declined to the control value within ca 4 h, as the actin-containing cytoskeletal components rearranged by CD recovered their normal morphology. Subsequently, actin synthesis was depressed below control values for a prolonged period; during recovery from 2 h treatment with CD, this depression was of much shorter duration. Re-addition of CD to cells after a 3 h recovery period again induced the cytoskeletal alterations characteristic of CD treatment but did not reverse the prior decline in the rate of actin synthesis. In HEp-2 cells treated with cycloheximide during exposure to CD for 20 h, the relative rate of actin synthesis measured after removal of cycloheximide was twofold higher than with CD alone and such cells exhibited a twofold slower decline in the rate of actin synthesis during recovery from CD in the continued presence of cycloheximide. These effects of cycloheximide, which resemble observations on “super-induction”, suggest that actin synthesis in CD-treated and recovering HEp-2 cells may be regulated by a repressor protein. The possibility that the proposed repressor protein is actin and that actin may thus be a feedback inhibitor of its own synthesis is discussed.     

Coordinated cortical ER remodeling facilitates actomyosin ring assembly

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The actomyosin ATPase: a two-state system.

Studies of the interaction between actin and myosin subfragment 1 (S1) in solution have shown that the association reaction takes place in at least two steps. Initially the association is relatively weak to form a complex called the A state which can then isomerize to the R state. The rate and equilibrium constants for the isomerization have been measured and are shown to depend upon the nucleotide bound to the S1 ATPase site; with ATP bound the A state is preferred but as ATP is hydrolysed and the products are sequentially released then the complex gradually shifts to the A state. An extensive series of experiments have characterized the A-to-R isomerization both in solution and in contracting muscle fibres and have shown it to be closely associated with the key events in the ATP-driven contraction cycle: the conformational change from the A to the R state can be monitored by fluorescent probes on either actin or the nucleotide; the isomerization can be perturbed by increases in hydrostatic pressure; the actin-induced acceleration of the rate of product release from myosin is coupled to the A-to-R isomerization; tropomyosin may control actin and myosin interaction by controlling the isomerization step and finally pressure perturbations of contracting muscle fibres shows there to be a close coupling between the isomerization of acto.S1 and the force generating event of muscle contraction.     

The cortical microfilament system of lymphoblasts displays a periodic oscillatory activity in the absence of microtubules: implications for cell polarity

For an understanding of the role of microtubules in the definition of cell polarity, we have studied the cell surface motility of human lymphoblasts (KE37 cell line) using video microscopy, time-lapse photography, and immunofluorescent localization of F-actin and myosin. Polarized cell surface motility occurs in association with a constriction ring which forms on the centrosome side of the cell: the cytoplasm flows from the ring zone towards membrane veils which keep protruding in the same general direction. This association is ensured by microtubules: in their absence the ring is conspicuous and moves periodically back and forth across the cell, while a protrusion of membrane occurs alternately at each end of the cell when the ring is at the other. This oscillatory activity is correlated with a striking redistribution of myosin towards a cortical localization and appears to be due to the alternate flow of cortical myosin associated with the ring and to the periodic assembly of actin coupled with membrane protrusion. The ring cycle involves the progressive recruitment of myosin from a polar accumulation, or cap, its transportation across the cell and its accumulation in a new cap at the other end of the cell, suggesting an assembly-disassembly process. Inhibition of actin assembly induces, on the other hand, a dramatic microtubule-dependent cell elongation with definite polarity, likely to involve the interaction of microtubules with the cell cortex. We conclude that the polarized cell surface motility in KE37 cells is based on the periodic oscillatory activity of the actin system: a myosin-powered equatorial contraction and an actin-based membrane protrusion are concerted at the cell level and occur at opposite ends of the cell in absence of microtubules. This defines a polarity which reverses periodically as the ring moves across the cell. Microtubules impose a stable cell polarity by suppressing the ring movement. A permanent association of the myosin-powered contraction and the membrane protrusion is established which results in the unidirectional activity of the actin system. Microtubules exert their effect by controlling the recruitment of cytoplasmic myosin into the cortex, probably through their direct interaction with the cortical microfilament system.     

The cortical motor system

The cortical motor system of primates is formed by a mosaic of anatomically and functionally distinct areas. These areas are not only involved in motor functions, but also play a role in functions formerly attributed to higher order associative cortical areas. In the present review, we discuss three types of higher functions carried out by the motor cortical areas: sensory-motor transformations, action understanding, and decision processing regarding action execution. We submit that generating internal representations of actions is central to cortical motor function. External contingencies and motivational factors determine then whether these action representations are transformed into actual actions.     

Dynamic modes of the cortical actomyosin gel during cell locomotion and division

Tight regulation of the contractility of the actomyosin cortex is essential for proper cell locomotion and division. Enhanced contractility leads, for example, to aberrations in the positioning of the mitotic spindle or to anomalous migration modes that allow tumor cells to escape anti-dissemination treatments. Spherical membrane protrusions called blebs occasionally appear during cell migration, cell division or apoptosis. We have shown that the cortex ruptures at sites where actomyosin cortical contractility is increased, leading to the formation of blebs. Here, we propose that bleb formation, which releases cortical tension, can be used as a reporter of cortical contractility. We go on to analyze the implications of spontaneous cortical contractile behaviors on cell locomotion and division and we particularly emphasize that variations in actomyosin contractility can account for a variety of migration modes.     

ATP and calmodulin dependent actomyosin aggregates induced by cytochalasin D in goldfish retinal ganglion cell axons in vitro

Growing retinal ganglion cell (RGC) axons of the goldfish have mobile varicosities, which play a role in rapid bulk redistribution of axoplasm (Koenig, Kinsman, Repasky, and Sultz, 1985; Edmonds and Koenig, 1987). Varicosities contain a tubulo-vesicular SER embedded in an actin-containing cytomatrix (Koenig et al., 1985). Cytochalasin D (CD) induces the formation of focal cytoskeletal aggregates throughout preterminal axons and especially in varicosities. The aggregates are visible when labelled with fluoroscein isothiocyanate (FITC)-conjugated phalloidin. Double-labelling experiments show that Texas red-myosin or rhodamine isothiocyanate (RITC)-calmodulin immunofluorescence co-localizes with FITC-phalloidin-labelled aggregates. Formation of aggregates is blocked by calmidazolium, a calmodulin antagonist. Axon models permeabilized with digitonin retain the capacity to form focal aggregates in response to CD, when ATP or adenosine-5'-O(3-thiotriphosphate) (ATP-gamma S) is present in the permeabilization buffer, but not when 5'-adenylylimidodiphosphate (AMP-PNP) is present. The latter result indicates that formation of focal aggregates depends on ATP. The findings suggest that the formation of focal aggregates in immature axons is a manifestation of actomyosin interactions after free actin-filament ends are generated by CD treatment.     

Increased translatability in a cell-free system of RNA extracted from actinomycin D-treated cultures

RNA extracted from myogenic cultures treated with actinomycin D was found to be more active in stimulating protein synthesis in the wheat germ cell-free system than RNA from untreated cultures. The rate of incorporation of amino acids was up to 30% higher and the synthesis of actin and of myosin light chains increased by up to 50% when RNA from actinomycin-treated cultures was used. A cell-free system product which affects the rate of translation does not seem to be involved in this phenomenon.     

Aurora B‐dependent polarization of the cortical actomyosin network during mitotic exit

The isotropic metaphase actin cortex progressively polarizes as the anaphase spindle elongates during mitotic exit. This involves the loss of actomyosin cortex from opposing cell poles and the accumulation of an actomyosin belt at the cell centre. Although these spatially distinct cortical remodelling events are coordinated in time, here we show that they are independent of each other. Thus, actomyosin is lost from opposing poles in anaphase cells that lack an actomyosin ring owing to centralspindlin depletion. In examining potential regulators of this process, we identify a role for Aurora B kinase in actin clearance at cell poles. Upon combining Aurora B inhibition with centralspindlin depletion, cells exiting mitosis fail to change shape and remain completely spherical. Additionally, we demonstrate a requirement for Aurora B in the clearance of cortical actin close to anaphase chromatin in cells exiting mitosis with a bipolar spindle and in monopolar cells forced to divide while flat. Altogether, these data suggest a novel role for Aurora B activity in facilitating DNA‐mediated polar relaxation at anaphase, polarization of the actomyosin cortex, and cell division.     

Polyphasic character of ATP hydrolysis in actomyosin system.

The interaction of 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) with the metal ions (M2+) Mg2+, Ca2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 0.1 or 1.0 M, KNO3, 25 degrees C). Stability constants of the M(Tris)2+ complexes were determined; those constants which were measured by both methods agreed well. Ternary complexes containing ATP4- as a second ligand were also investigated and it is shown that in the presence of Tris, mixed-ligand complexes of the type M(ATP)(Tris)2- are formed. The values for delta log KM, where delta log KM = log KM(ATP)M(ATP)Tris--log KMM(Tris), are all negative, thus indicating that the interaction of Tris with M(ATP)2- is somewhat less pronounced than with M2+. However, it should be noted that even in mixed-ligand systems complex formation with Tris may still be considerable, hence great reservations should be exercised in employing Tris as a buffer in systems which also contain metal ions. Distributions of the complex species in dependence on pH are shown for several systems, and the structures of the binary M(Tris)2- and the ternary M(ATP)(Tris)2- complexes are discussed. The participation of a Tris-hydroxo group in complex formation is, at least for the M(Tris)2- species, quite evident.     

Cooperativity of the calcium switch of regulated rabbit actomyosin system

Summary The concentration range required for calcium activation of skeletal myofibrillar ATPase activity has previously been attributed to simultaneous binding of two calcium ions to each troponin. We present data representative of the majority of myofibrillar preparations and data with acto subfragment-1 (S-1) whose calcium activation of ATPase activity occurs over a much too narrow range of calcium concentrations to be so explained. S-1 binding significantly broadened the range of Ca²⁺ concentrations over which activation occurred but not to the extent that is associated with simultaneous binding of 2 calcium ions.     

The second meiosis occurs in cytochalasin D-treated eggs of Corbicula leana even though it is not observed in control androgenetic eggs because the maternal chromosomes and centrosomes are extruded at first meiosis

The hermaphroditic freshwater clam Corbicula leana reproduces by androgenesis. In the control (androgenetic development), all maternal chromosomes and maternal centrosomes at the meiotic poles were extruded as the two first polar bodies, and subsequently, second meiosis did not occur. But, in C. leana eggs treated with cytochalasin D (CD) to inhibit polar body extrusion, the second meiosis occurred. At metaphase-I, the spindle showed the typical bipolar structure and two spheroid centrosomes were located at its poles. All the maternal chromosomes were divided at anaphase-I, but they were not extruded as polar bodies due to the effects of CD. After completion of first meiosis, the maternal centrosomes split into four. At the second meiosis, twin or tetrapolar spindles were formed and two groups of maternal chromosomes divided into four sets of chromosomes. After the second meiosis, the spindle disassociated and the four maternal centrosomes disappeared. Four groups of maternal chromosomes transformed into the four female pronuclei. Male and female pronuclei became metaphase chromosomes of the first mitosis. The present study clearly indicates that typical meiosis systems still proceed in androgenetic triploid C. leana. We conclude that the androgenetic form may have arisen from the meiotic form.     

The effect upon actomyosin of stoichiometric amounts of adenosinetriphosphate regenerated in a coupled enzyme system

Pyruvate kinase and phosphoenolpyruvate, added to actomyosin, cause a maintenance of the response of the actomyosin to stoichiometric amounts of ATP. This steady state maintenance depends on the presence of Mg ions.     

Ca2+-ionophore-induced microvilli and cortical contractions inXenopus eggs. Evidence for involvement of actomyosin

Summary Using scanning electron microscopy, we show that the calcium ionophore A23187 has a profound effect on the surface morphology ofXenopus laevis eggs. The response to ionophore can be interpreted with respect to animal/vegetal polarity and the presence of an asymmetrically organized actomyosin-based contractile system in the egg cortex. When incubated in ionophore, the egg cortex contracts, pigment granules move towards the animal pole, and microvilli increase dramatically in size. While at first overall microvilli density decreases, many additional microvilli appear later in the animal hemisphere but not in the vegetal hemisphere. Eggs incubated in high concentrations of A23187 undergo the same surface changes at a faster rate, and rupture due to a massive cortical contraction. Local application of ionophore to the egg surface results in increased microvilli size and density in that area, with the animal hemisphere showing the greatest response. Since the effects of ionophore are inhibited by the actomyosin probe, N-ethylmaleimide-modified heavy meromyosin, actomyosin is implicated in the ionophore-induced surface changes.