Evidence for actin transformation during the contraction-relaxation cycle of cytoplasmic actomyosin: Cycle blockade by Phalloidin injection

1) The injection of a mushroom drug, Phalloidin (750 microgram -1 mg/ml), into the endoplasmic channel of Physarum veins induces an irreversible blockade of the intrinsic contraction-relaxation automaticity of the ectoplasmic tube wall, as measured by tensiometrical methods. 2) The morphological responses to Phalloidin injection include an increase and condensation of cytoplasmic actomyosin sheets bordering the plasmalemma invaginations within the ectoplasmic tube and a more pronounced dense layer of "groundplasm" in the cell cortex. This is in accordance with experiments with other cells as well as with Physarum. 3) The addition of marker particles to the injection solution revealed that the injected substances can be brought into direct contact with the contractile substrate, before newly formed membranes separate off the injection fluid. 4) Since Phalloidin irreversibly transforms oligomeric actin into a filamentous "Phalloidin-actin complex" and because this transformation does not hinder the actin in activating myosin ATPase, it is concluded that the contraction-relaxation cycle of cytoplasmic actomyosin in Physarum involves actin transformations. If these transformations are hindered, e.g. by Phalloidin, one stage and thereby the whole cycle is sustained which results in a blockade of the intrinsic contraction automaticity. 5) The functional importance of actin transformations in the congraction physiology of cytoplasmic actomyosins and cell motility phenomena is discussed.     

Interaction between cell shape and contraction pattern in the Physarum plasmodium

The relationship between cell shape and rhythmic contractile activity in the large amoeboid organism Physarum polycephalum was studied. The organism develops intricate networks of veins in which protoplasmic sol moved to and fro very regularly. When migrating on plain agar, the plasmodium extends like a sheet and develops dendritic veins toward the rear. After a particular stimulation, the vein organization changes into veinless or vein-network structures. In both structures, the mixing rate of the protoplasm, which is related to communication among contraction oscillators, decreased compared with that of the dendritic one. Accompanying these changes in vein structure, the spatio-temporal pattern of the rhythmic contraction changed into a small-structured pattern from a synchronized one. In the above process, cell shape affects the contraction pattern, but, conversely, the contraction pattern effects the cell shape. To demonstrate this, a phase difference in the rhythmic contraction was induced artificially by entraining the intrinsic rhythm to external temperature oscillations. New veins then formed along the direction parallel to the phase difference of the rhythm. Consequently, the vein organization of the cell interacts with the contractile activity to form a feedback loop in a mechanism of contraction pattern formation.     

Reaction-diffusion-advection model for pattern formation of rhythmic contraction in a giant amoeboid cell of the physarum plasmodium

The plasmodium of Physarum polycephalum is a large amoeboid organism showing rhythmic contraction everywhere within an organism, and moves by forming spatio-temporal patterns of the rhythmic contraction. We propose a reaction-diffusion-advection model for the pattern formation. This model is constructed under physiological suggestions that the chemical oscillator acts as a clock regulating the rhythmic contraction and interacts spatially not only by diffusion but also by advection of protoplasm. Behavior of the model is studied by numerical calculation, especially the effects of the advection term on a simple reaction-diffusion system. The advection effect reproduces experimentally observed phenomena of fluctuating propagation of the contraction wave. Concept of the reaction-diffusion-advection system is promising for modeling the mechanism of amoeboid behaviour in the Physarum plasmodium. Copyright 1999 Academic Press.     

Chemically induced changes in the morphology, dynamic activity and cytoskeletal organization of Physarum cell fragments

Spherical cell fragments derived from Physarum polycephalum by caffeine-treatment were used as an experimental system to investigate the influence of 15 externally applied substances on the general morphology, motile behavior and cytoskeletal organization of the acellular slime mold. In comparison to controls, the most obvious changes observed after chemical stimulation proved to be cytokinetic activities, ameboid-like movement phenomena, intense cell surface dynamics and formation of cytoplasmic actin fibrils. The results demonstrate the high adaptability of the microfilament system in Physarum even when subjected to extreme conditions in the external environment.     

Effects of caffeine and D2O on persistence and de novo generation of intrinsic oscillatory contraction automaticity in Physarum

The present investigation was performed in an attempt to contribute to answering the question whether the plasmalemma of the plasmodial stage of Physarum represents the site of a trigger mechanism for the oscillating contraction activity of cytoplasmic actomyosin. The effects of the following substances on persistence of tensiometrically measured longitudinal and radial activities of Physarum veins and on de novo generation of activities in experimentally generated drops were studied: caffeine, theophylline, acetylcholinium chloride, procaine, physostigminium salicylate, iso-ompa, nifedipin, sodium nitroprusside, potassium thiocyanate, D2O; as well as the effects of ions such as La+++ and high outer concentrations of Na+ and K+. Some of the substances were applied simultaneously for comparison externally (by bathing solutions) and internally (by injection). The experimental data speak against the existence of electrogenic rhythmical Ca++, Na+ or K+ pumps across the plasmalemma which could have a triggering function for the oscillation. The contraction activities of the cytoplasmic actomyosin seem to represent a spontaneous endogeneous oscillation which can be modulated via the plasmalemma during chemotaxis.     

Oscillating contractions in protoplasmic strands of Physarum: infared reflexion as a non-invasive registration technique

An inexpensive electronic device employing a miniaturised infrared reflection detector is described which enables the automatic registration of intrinsic radial contraction activities of protoplasmic strands of Physarum growing on their original substrate. Advantages and shortcoming of the new registration technique are discussed in relation to recent tensiometric techniques applied to protoplasmic strands for analysing the contraction physiology of cytoplasmic actomyosins, the force generating substrate of the contraction automaticity in the Physarum system.     

Dynamics of the profiles of plasmodial veins ofPhysarum polycephalum during the pulsation cycle

Summary The cross-sectional profiles of isolated veins ofPhysarum polycephalum plasmodia, winded round agar rods, were investigated by analysis of time-lapse films. The whole profile follows the same principal contraction-relaxation rhythm, but slight desynchronization and amplitude differences are found around the contour. As a result, the profile periodically changes its shape in the course of pulsation cycles, its lateral slopes becoming more convex in the expanded state than during contraction. Factors responsible for such geometrical deformations and their bearing for morphometrical studies of the motory activity of plasmodium are discussed.Study supported by the Research Project II.1 of the Polish Academy of Science.     

Membrane biophysics of chemoreception and taxis in the plasmodium of Physarum polycephalum

The threshold phenomena observed in chemoreception and taxis of the plasmodium of Physarum polycephalum were analyzed on the basis of physical chemistry. Various physicochemical concepts and rules, e.g. the Schulze-Hardy rule, the lyotropic number and the hydrophobic interactions, were shown to be applicable reasonably well to the physiological functions in Physarum. It was stressed that the structural change of the surface membrane induced by reception of chemical stimuli plays a decisive role in recognition and sensitivity to the external stimuli as well as the appearance of tactic movement in the amoeboid motility of Physarum.     

Reactivation of cell-free models of Physarum plasmodia after myosin reconstitution

Thin-spread glycerol-extracted Physarum plasmodia were treated with N-ethylmaleimide (NEM) to block myosin-ATPase and contractility. After supplementing the models with purified plasmodial myosin, they could be reactivated and contracted upon addition of ATP. Fluorescently labeled actomyosin fibers ruptured during contraction, resulting in beaded or rod-like contraction centers. Glycerol-extracted plasmodia lose their negative Ca++-dependence during extraction. Reconstitution of NEM-treated models with plasmodial myosin partly restored this Ca++-sensitivity. Thus, either myosin or a factor associated with it seems to be involved in the Ca++-dependent regulation of cytoplasmic actomyosin contraction in Physarum. NEM-blocked models reconstituted with skeletal muscle myosin were not reactivated by ATP. The same plasmodia subsequently incubated with plasmodial myosin were able to contract.     

细弱绒泡菌的生活史

利用燕麦-琼脂培养、基物培养及扫描电镜技术研究了细弱绒泡菌的个体发育过程,在燕麦琼脂培养基上完成了从孢子到孢子的生活史。结果表明,细弱绒泡菌生活史包括单核的黏变形体或游动胞、多核的营养体原质团以及孢子形成阶段。孢子球形,表面具细小疣点。孢子萌发为裂式,释放1黏变形体。黏变形体行变形运动,在有水的条件下,可转变为游动胞。成熟原质团橘黄色。原质团类型为显型,具有扇形网络状菌脉。成熟原质团可形成多个孢囊。琼脂培养基上获得的细弱绒泡菌孢子与野生型相似,并具有可育性。     

Towards Physarum Robots： Computing and Manipulating on Water Surface

Plasmodium of Physarum polycephalum is an ideal biological substrate for implementing concurrent and parallel com-putation, including combinatorial geometry and optimization on graphs. The scoping experiments on Physarum computing in conditions of minimal friction, on the water surface were performed. The laboratory and computer experimental results show that plasmodium of Physarum is capable of computing a basic spanning tree and manipulating of light-weight objects. We speculate that our results pave the pathways towards the design and implementation of amorphous biological robots.     

Leukocyte adhesion to walls of the rat pial venules in normoxia and during brain ischemia

With the help of contact optic system direct observations were carried out on leukocyte movements and on the acts of their adhesion to the inner surface of the walls of pial venules of the rats in normoxia and ischemia resulting from ligating two carotid arteries. After ligating the carotid artery and subsequent gradual development of ischemia, an abrupt increase in the number of the acts of adhesion to the walls of pial venules was shown to occur, the leukocytes adhere to each other inside the venules. A complete occlusion of venules and small veins results, which can be one of the reasons for the animal death in brain ischemia.     

Acceleration-sensitivity threshold of Physarum

Free-living cells show distinct gravisensitivities and often use the gravity ('g') vector for their spatial orientation. The rhythmic contractions of the ameboid Myxomycete (acellular slime mold) Physarum polycephalum are a sensitive parameter which can be modified by external stimuli. Space experiments and ground-based 0 x g simulation studies established that the contraction period transiently decreases after a transition from 1 x g to 0 x g with a back-regulating process starting after 30 min. For determination of the threshold of acceleration sensitivity, a slow-rotating centrifuge microscope (NIZEMI--Niedergeschwindigkeits-Zentrifugenmikroskop) was used, providing in space accelerations from 0 x g to 1.5 x g. A stepwise acceleration increase revealed that the lowest acceleration level capable of inducing a response was 0.1 x g. The response to the acceleration increase was an increase in contraction period, in contrast to a stimulus deprivation, which led to a period decrease. The time schedule of the acceleration responses and back-regulating process seems to be fixed, suggesting that every acceleration being above the threshold can induce a complete response-regulation process. The low acceleration-sensitivity threshold favors rather large and dense cell organelles as candidates for the gravity receptor in Physarum.     

Reactivation of cell-free models of endoplasmic drops from Physarum polycephalum after glycerol extraction at low ionic strength

The effect of calcium ions on the reactivation of cytoplasmic actomyosin contraction in cell-free models of endoplasmic drops from Physarum polycephalum after glycerol extraction at low ionic strength depends on the duration of the extraction procedure: Ca++ prevents contraction in 20-h extracted specimens, whereas after several days of extraction this Ca++-sensitivity is lost. These results indicate an inhibitory effect of Ca++ on cytoplasmic actomyosin contraction.     

Induction of a plasmodial stage of Physarum without plasmalemma invaginations

Experimentally generated protoplasmic drops of Physarum show time-dependent differentiation processes, i.e. regeneration of plasmalemma, actomyosin fibrillogenesis and regeneration of the plasmalemma invagination system. According to Hatano (1970), caffeine treatment of drops results in a pinching off process of small translucent droplets in which specific effects of Ca++ on protoplasmic streaming phenomena were demonstrated. The light and electron microscopic investigation of the original drop reveal that the time-dependent differentiation processes, e.g. actomyosin fibrillogenesis, are not inhibited by caffeine. However, caffeine hinders the regeneration of the plasmalemma invaginations in the original drop (up to a drop age of 30--40 min). The experimental advantage of this stage of Physarum with full vitality, but without plasmalemma invaginations is discussed.     

Immunocytochemistry of the acellular slime mold Physarum polycephalum

Abstract. The polygonal arrangement of actomyosin fibrils in different stages of the acellular slime mold Physarum polycephalum is correlated with morphogenetic processes at the cell surface. Light and electron microscopic investigations on both endoplasmic drops and thin-spread small plasmodia demonstrate that the differentiation of a polygonal pattern depends on a transient deficiency of plasma membrane invaginations.
Glycerol-extracted specimens show condensation and drastic spatial changes in the organization of the polygonal net after addition of ATP, thus indicating contractile properties of this system. Observations with the polarizing microscope reveal rhythmic changes in fibrillar birefringence intensity corresponding to the protoplasmic streaming activity, i.e., birefringence increases during contraction and decreases during relaxation. Cell fusion experiments, local irradiation with blue light (450 nm), and chemical treatment by impeding the mitochondria1 function with DNP (2,4-di-nitrophenol) demonstrate morphological as well as physiological interdependences of the actomyosin system, the motive force generation, and the expression of a locomotor polarity in plasmodia of Physarum polycephalum.     

Propagated waves induced by gradients of physiological factors within plasmodia ofPhysarum polycephalum

Plasmodia ofPhysarum polycephalum were analyzed with the aid of cinematography and the infrared reflection technique for characterization of the phase behavior of their oscillating contraction activity, with special emphasis placed on the effects of temperature gradients. In response to temperature gradients, phase gradients were documented cinematographically as well as by infrared registration. A quantitative evaluation of the cinematographically recorded phenomena was carried out with the aid of streak photography. The phase gradient is directed across the region of the temperature gradient with a delay in phase toward the colder side. The correspondingly generated waves are as short as 1 mm and are propagated toward the colder region. A comparison of these waves with the known flickering phenomena in cinematographic films reveals a common nature of both.Supported by Deutsche Forschungsgemeinschaft.     

Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.     

A multiscale sliding filament model of lymphatic muscle pumping

The lymphatics maintain fluid balance by returning interstitial fluid to veins via contraction/compression of vessel segments with check valves. Disruption of lymphatic pumping can result in a condition called lymphedema with interstitial fluid accumulation. Lymphedema treatments are often ineffective, which is partially attributable to insufficient understanding of specialized lymphatic muscle lining the vessels. This muscle exhibits cardiac-like phasic contractions and smooth muscle-like tonic contractions to generate and regulate flow. To understand the relationship between this sub-cellular contractile machinery and organ-level pumping, we have developed a multiscale computational model of phasic and tonic contractions in lymphatic muscle and coupled it to a lymphangion pumping model. Our model uses the sliding filament model (Huxley in Prog Biophys Biophys Chem 7:255–318, 1957) and its adaptation for smooth muscle (Mijailovich in Biophys J 79(5):2667–2681, 2000). Multiple structural arrangements of contractile components and viscoelastic elements were trialed but only one provided physiologic results. We then coupled this model with our previous lumped parameter model of the lymphangion to relate results to experiments. We show that the model produces similar pressure, diameter, and flow tracings to experiments on rat mesenteric lymphatics. This model provides the first estimates of lymphatic muscle contraction energetics and the ability to assess the potential effects of sub-cellular level phenomena such as calcium oscillations on lymphangion outflow. The maximum efficiency value predicted (40%) is at the upper end of estimates for other muscle types. Spontaneous calcium oscillations during diastole were found to increase outflow up to approximately 50% in the range of frequencies and amplitudes tested.