Repair of functional and ultrastructural alterations after thermal injury of Physarum polycephalum

Repair of thermal injury of Physarum polycephalum Schw. plasmodia has been studied by light and electron microscopy. As a result of heating the plasmodia for 10 min at 42°C both the unordered and shuttle protoplasmic streaming were arrested; the outer plasmodial membrane showed alterations at sites of contact with water; the onset of the next mitosis was considerably delayed. The plasmodial ultrastructure was markedly disturbed, including disappearance of the granular component of the nucleoili and a compact, almost fibrillar structure of the latter. The mitochondria became distorted and their intracristal spaces enlarged while the outer and inner membranes appeared in some places to be separated. Glycogen particles disappeared from the cytoplasm. Recovery of both types of protoplasmic streaming of the motility of the plasmodium, of the resistance of its membrane to contact with water, and of the ability of the organism to go through the cell cycle went all hand in hand with the normalization of the structure of nucleoli, mitochondria and cytoplasm. All of the functional and structural characteristics are normalized within ca. 9 h following heating.     

Effects of supraoptimal temperatures on the myxomycete Phyrasum polycephalum

Summary The effects of heat shock on the protoplasmic streaming, respiration and leakage of plasmodial constituents absorbing at 260 nm (products of nucleic acid metabolism), 280 nm (products of protein metabolism), and 415 nm (the yellow pigments of the plasmodia) were studied in plasmodia of the myxomycete Physarum polycephalum.Plasmodia grown on a semidefined medium displayed a lower primary thermoresistance of the protoplasmic streaming, and had a lower Q ₁₀ coefficient of the heat injury of this function compared to those grown on rolled oats. They are able to repair thermal injuries during heating. The primary thermoresistance of the protoplasmic streaming is not changed during the mitotic cycle.A 10 min heating at 32°C lowers the rate of protoplasmic streaming and results in a leakage of plasmodial pigments. After a 10 min exposure at 37–38°C the protoplasmic streaming is stopped, the respiration reduced, and products of nucleic acid metabolism are detectable in the heating fluid. Leakage of protein metabolits was observed after 10 min heatshocks at 41°C. A heating of the plasmodia to 47–50°C caused the highest level of leaked substances and the complete cessation of respiration.In contrast to higher plants, the respiration and leakage of the pigments are thermolabile indicators of the condition of Physarum polycephalum plasmodia.     

Protoplasmic streaming during the cell cycle of Physarum polycephalum

It has been reported that protoplasmic streaming stops during the synchronous mitosis exhibited by growing plasmodia of P. polycephalum. Our data reveal that at no time during the mitotic cycle did streaming stop. However, during a 3–5 min period at anaphase the percent of each oscillation period accounted for by an outward flow was precisely equal in duration to the corresponding inward flow. At all other periods the duration of outward flow exceeded that of inward flow. Plasmodial migration or locomotion was briefly arrested at telophase, although shuttle streaming persisted.     

Locomotive mechanism of Physarum plasmodia based on spatiotemporal analysis of protoplasmic streaming

We investigate how an amoeba mechanically moves its own center of gravity using the model organism Physarum plasmodium. Time-dependent velocity fields of protoplasmic streaming over the whole plasmodia were measured with a particle image velocimetry program developed for this work. Combining these data with measurements of the simultaneous movements of the plasmodia revealed a simple physical mechanism of locomotion. The shuttle streaming of the protoplasm was not truly symmetric due to the peristalsis-like movements of the plasmodium. This asymmetry meant that the transport capacity of the stream was not equal in both directions, and a net forward displacement of the center of gravity resulted. The generality of this as a mechanism for amoeboid locomotion is discussed.     

Change of contractile behavior in plasmodia ofPhysarum polycephalum during mitosis

Summary Oscillations of ectoplasmic contraction in plasmodia of the myxomycetePhysarum polycephalum growing on agar containing semidefined medium were studied to determine if the contractile force is altered during the synchronous mitosis. In interphase the regular oscillations of contraction in the plasmodial sheet had an average period of 0.93 minutes in plasmodia growing at 24 °C. During mitosis the amplitude of these oscillations gradually decreased, ceasing for an average time of 2.7 minutes in 74% of the 23 plasmodia studied. Cessation of oscillating contractions in mitosis was accompanied by a decrease in the width of the channels embedded in the plasmodial sheet, and a decrease in the velocity of endoplasmic shuttle streaming usually to a complete standstill. Of 13 plasmodia in which the mitotic stage was very accurately determined, the stop in oscillating contractions occurred during metaphase in 10 plasmodia, and in prometaphase, anaphase, telophase in the 3 others. The cessation of contractile oscillations or of streaming did not occur absolutely simultaneously during mitosis in widely separated locations within one plasmodium, indicating mitotic asynchrony over a period of a few minutes within each plasmodium. We suggest that the halt of plasmodial migration during mitosis reported by others is caused by a decrease or cessation at slightly different times in the amplitude of ectoplasmic contractile oscillations in different areas of a plasmodium in mitosis resulting in an overall lack of coordination of endoplasmic flow throughout the plasmodium, thus temporarily halting migration. Possible physiological mechanisms linking a decrease in actomyosin contraction with the metaphase stage of mitosis are discussed.     

Explaining the “Pulse of Protoplasm”: The search for molecular mechanisms of protoplasmic streaming

Explanations for protoplasmic streaming began with appeals to contraction in the eighteenth century and ended with appeals to contraction in the twentieth. During the intervening years, biologists prop...     

Oscillations in cell shape and size during locomotion and in contractile activities of Physarum polycephalum, Dictyostelium discoideum, Amoeba proteus and macrophages

Changes in cell shape and size were measured during locomotion, together with the motive force of the protoplasmic streaming, in various amoeboid cells in different stages of their life cycle, and under various environmental conditions. The variations in these measurements with time were examined by Fourier spectral analysis. Notwithstanding a change in cell type in the life cycle of P. polycephalum, myxamoebae and tiny plasmodia showed a similar time pattern of locomotion, exhibiting oscillations having a mixture of several periods. A regular oscillation with protoplasmic streaming appeared in the plasmodium only above a critical cell size. D. discoideum amoebae oscillated with two periods of a few minutes in preaggregation stage, but with a period of 10 min in aggregation stage, the latter being induced by cAMP. Macrophages and A. proteus also oscillated with periods of a few minutes. Periods of all these oscillations were prolonged severalfold by respiratory inhibition with NaCN, but were unaffected by glycolytic inhibition with 2-deoxyglucose. Cell fragments of A. proteus containing fewer granules oscillated more slowly and with a larger amplitude than those containing more granules. Among the granules, the nucleus was excluded as a possible modifier of the oscillation. The oscillation in Physarum plasmodium was reversibly suppressed by combining respiratory and ATPase inhibitions in mitochondria with NaCN and oligomycin, intracellular ATP concentration being kept at an appropriate level. The present results show that amoeboid motility, as well as cell shape, is oscillatory and that mitochondria are involved in time keeping.     

Steady and transient behaviors of protoplasmic streaming in Nitella internodal cell

Steady and transient behaviors of protoplasmic streaming in Nitella internodal cell have been investigated for various temperatures from 30°C to near 0°C. It has been found that steady velocity of the streaming linearly decreases with increasing inverse temperature but its proportionality coefficient changes at ~ 10°C. Velocity distribution, which reflects temporal fluctuations of the protoplasmic streaming, is nonGaussian and its half width becomes larger at higher temperatures. On the other hand, recovery of the protoplasmic streaming, which is observed after stopping the streaming with a current stimulus to the internodal cell, has been found to show more clear sigmoidal time courses at higher temperatures.     

Thermotaxis and protoplasmic oscillations inPhysarum plasmodia analysed in a novel device generating stable linear temperature gradients

Summary The application of sublethal temperature gradients offers a simple, non-invasive means for in vivo studies of thermotaxis and other temperature-dependent processes in various organisms. Development, for instance, can be dramatically desynchronized, and the resulting development gradients allow to analyze physiological inter-dependencies between locally separated subsystems. For this purpose a simple device has been developed, by which a stable linear gradient of 8 °C/cm is established on an inert metal sheet with the aid of Peltier elements. The effects of linear temperature gradients on fusion, growth, and migration of plasmodia of the slime moldPhysarum polycephalum was filmed by 16 mm film time-lapse technique, and their local contraction—relaxation cycles analysed by multistrip kymography, which represents a graphic documentation of the spatio-temporal pattern of protoplasmic movements that occur along well-defined regions within the giant cell.Physarum plasmodia preferentially fuse, and grow, in the range of 24–26 °C. Different parts of a single macroplasmodium can simultaneously show positive and negative thermotaxis. The contraction—relaxation cycles generating the protoplasmic shuttle streaming within the network of veins essentially depend on local temperatures and are instantaneously desynchronized by the temperature gradient. Thus they cannot be controlled by a central pacemaker or an overall electric signal. However, there is a strong tendency to locally synchronize the various oscillation frequencies present within the giant cell if temperature differences do not exceed 2 °C.     

Observations on an ATP-sensitive protein system from the plasmodia of a myxomycete

1. Extracts of the plasmodia of the myxomycete, Physarum polycephalum, exhibit reversible decreases in viscosity in response to the addition of ATP under appropriate conditions. The protoplasm material prepared by extraction with KCl solution can apparently exist in either a high or a low viscosity state. As prepared, it is in the low viscosity condition. Rapid and extensive increases in viscosity of the extract are brought about by addition of AMP, inorganic phosphate, or, under certain conditions, of ATP. Only after the high viscosity state has been attained does addition of appropriate quantities of ATP cause a reversible decrease in viscosity. 2. The active principle of crude plasmodial extracts may be concentrated by fractional precipitation with ammonium sulfate and is found in the fraction precipitated between 30 and 40 per cent saturation. This material possesses a higher viscosity than does the original crude extract and is apparently in the high viscosity state since the addition of ATP causes an immediate reversible decrease in viscosity. 3. The ATP-sensitive fraction of myxomycete plasmodia possesses a viscosity which is dependent upon its previous thermal treatment. Extracts incubated at 0° for a period of a few hours increase greatly in viscosity when they are returned to 24.5°. This increased viscosity is structural in nature, is destroyed by mechanical agitation of the solution, and may be reversibly destroyed by addition of ATP. 4. It is suggested that the ATP-responsive protein of myxomycete plasmodia may be related to sol-gel transformations which have been observed in intact plasmodia and may participate in the protoplasmic streaming of the intact organism. This suggestion is based upon the following facts: (a) the protoplasmic streaming of myxomycete plasmodia is increased by microinjection of ATP; (b) the gel portion of the cytoplasm at the site of the microinjection of ATP is extensively converted to the sol state. The changes in structure of the intact cytoplasm are thus similar in nature to the changes exhibited in response to ATP by the purified ATP-sensitive protein. 5. The ATP-sensitive protein of myxomycete plasmodia appears to undergo reversible aggregation to form a high viscosity state. The function of ATP is to break down the aggregates thus formed. Since a specific ATPase activity is associated with the purified material, added ATP is gradually destroyed and recovery of viscosity attends the spontaneous reconstitution of aggregates.     

Tracking the unfolding and refolding pathways of outer membrane protein porin from Paracoccus denitrificans

We have investigated outer membrane protein porin from Paracoccus denitrificans for its stability against heat and pH. Pathways of unfolding and refolding have been analyzed. Porin incubated at pH 12.5 and above undergoes a slow unfolding into an unordered structure. The unfolded protein could be refolded into a nativelike structure that is functionally active but with distinct deviation from the native protein. This nativelike structure exhibited an entirely different thermal stability. Although aggregation is normally considered a structural "dead-end", the possibility of opening an aggregated porin and forming a functionally active structure was analyzed here. Porin aggregates on heating above 86.2 degrees C. Incubating the heat-aggregated protein at high pH (> or = 12.5) leads to a slow opening of the protein into an unordered structure. It was possible to refold this unordered protein into a trimeric nativelike structure which was capable of forming active pores. However, the thermal stability of the refolded porin was unlike that of the native porin. To understand the basic mechanism behind the unfolding processes, the protein was subjected to heating at various pH values. It was observed that at pH > or = 12.5 the protein does not aggregate upon heating; instead, it opens into an unordered structure. We conclude that at high pH values, the electrostatic interactions of various amino acid residues are perturbed which leads to unfolding into an unordered structure. This study shows for the first time an entirely new unfolding and refolding pathway for porin.     

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.     

A study of protoplasmic streaming in Nitella by laser Doppler spectroscopy.

Laser light scattered from particles in the streaming protoplasm of a living cell is shifted in frequency by the Doppler effect. The spectrum of the scattered light can be measured and interpreted to infer details of the velocity distribution in the protoplasm. We have developed this approach to study the protoplasmic streaming in the fresh-water alga Nitella. Our results indicate a characteristic flow pattern to which diffusion makes a negligible contribution. No difference in the velocity of particles of different size is indicated. The streaming velocity linearly with temperature with a supraoptimal temperature of 34 degrees C, and the velocity distribution becomes narrower at high temperatures. The protoplasmic streaming can be inhibited by laser light, and this effect has been used to study the photoresponse of the algae. Using beam diameters of about 50 mum, we have shown that the inhibition is very local, becoming minimal at a displacement of about 200 mum in the upstream direction and 400 mum in the downstream direction. Prolonged exposure produces a bleached area free of chloroplasts, which is three orders of magnitude less sensitive to photoinhibition.     

Ca2+ effect on protoplasmic streaming in Nitella internodal cell

Ca²⁺ ion effect on protoplasmic streaming in an internodal cell of Nitella has been investigated for various temperatures. We have found that the protoplasmic streaming at low temperature is remarkably affected by the Ca²⁺ ions in the internodal cell but larger concentrations of the Ca²⁺ ions are needed to suppress the streaming velocity at higher temperatures. These streaming behaviors of the protoplasm, furthermore, have been elucidated on the basis of the reaction equations which take into account ATP hydrolysis due to actin-myosin molecules and inactivity of the molecules due to the Ca²⁺ ions.     

Morphological anomalies induced by antimicrotubule agents inBryopsis plumosa

Summary Antimicrotubule agents, colchicine, vinblastine, and griseofulvin, induced conspicuous morphological anomalies inBryopsis plumosa. First, following cessation of protoplasmic streaming within 15 minutes, elongation stopped in a few hours. Second, innumerable protrusions or new growth points generated over the cell flank in a few days. Similar phenomena were observed in the cells which were subjected to high pressure or low temperature both of which are known to disrupt microtubule.These phenomena were investigated with light and electron microscopy. It is suggested that inhibition of microtubule dependent protoplasmic streaming which may function as an intracellular transport system causes such morphological anomalies.     

STIMULATION BY COLD IN NITELLA

Sudden local chilling causes action currents to be set up in Nitella and in Chara, an effect which does not follow gradual local chilling. This may be due to a partial solidification of the non-aqueous protoplasmic surfaces which makes them susceptible to rupture by the protoplasmic streaming. This movement continues usually for several minutes after the chilling, whereas if stimulation occurs at all it occurs immediately on chilling. It is found that a chilled spot is much more sensitive to mechanical stimulation than is a spot at room temperature. Chilling is accompanied by a rise of resistance, a lowered rate of recovery following stimulation, and usually by a falling off in the magnitude of the action curve.     

THE CHANGING PATTERN OF BIREFRINGENCE IN PLASMODIA OF THE SLIME MOLD, PHYSARUM POLYCEPHALUM

Plasmodia of the acellular slime mold, Physarum polycephalum, reveal a complex and changing pattern of birefringence when examined with a sensitive polarizing microscope. Positively birefringent fibrils are found throughout the ectoplasmic region of the plasmodium. In the larger strands they may be oriented parallel to the strand axis, or arranged circularly or spirally along the periphery of endoplasmic channels. Some fibrils exist for only a few minutes, others for a longer period. Some, particularly the circular fibrils, undergo changes in birefringence as they undergo cyclic deformations. In the ramifying strand region and the advancing margin there is a tendency for fibrils of various sizes to become organized into mutually orthogonal arrays. In some plasmodia the channel wall material immediately adjacent to the endoplasm has been found to be birefringent. The sign of endoplasmic birefringence is negative, and its magnitude is apparently constant over the streaming cycle. The pattern of plasmodial birefringence and its changes during the shuttle streaming cycle of Physarum are considered in the light of several models designed to explain either cytoplasmic streaming alone or the entire gamut of plasmodial motions. The results of this and other recent physical studies suggest that both streaming and the various other motions of the plasmodium may very likely be explained in terms of coordinated contractions taking place in the fibrils which are rendered visible in polarized light.     

Morphogenesis and Disassembly of the Circular Plasmalemma Invagination System in Physarum polycephalum

During the morphogenesis of small plasmodia developing from endoplasmic drops, an extended plasmalemma invagination system is formed. This system is a characteristic constituent of the ectoplasm. The invaginations have different cytophysiological functions.
The transition from the initial very irregular plasmalemma indentations in protoplasmic drops to the highly organized circular invagination ring of protoplasmic strands, i.e., the differentiation as well as the disassembly of this circular invagination system in retracting endings of strands was investigated with the aid of the semithin- and ultrathin-sectioning technique.
Live observation of protoplasmic drops revealed that simultaneously with the onset of initially irregular oscillating contractions, small endoplasmic streamlets are generated. Subsequently, an improvement of the coordination of contraction activities leads to an oriented mass transport of protoplasm and thereby to locomotion. The growing endoplasmic channels continuously develop into the well-known structure of protoplasmic strands. Differentiation and disassembly of circular plasmalemma invaginations are based on processes of membrane invagination in combination with intracytosis and exocytosis.
The importance and correlations of the following phenomena for morphogenesis and differentiation are discussed: 1) the formation and distribution of the contractile apparatus, i.e., the system of cytoplasmic actomyosin fibrils, 2) plasmalemma invaginations, 3) the generation of oscillating contractions, and 4) the endoplasmic streaming.     

Alkali light chains are involved in stabilization of myosin head

1. Fast skeletal myosin subfragment 1 (S1) was separated into two isozymes, S1(A1) and S1(A2), based on the associated alkali light chain, and their thermostabilities were compared. 2. Inactivation rate constants of Ca2(+)-ATPase (at 30 and 35 degrees C) were higher and heat-induced turbidity increase at 340 nm (at 40 degrees C) was faster with S1(A1) than with S1(A2), indicating a higher stability of S1(A2). 3. When S1 isozymes were incubated in the presence of excess alkali light chain, turbidity increase was markedly reduced, depending on the amount of light chain added. 4. Results obtained strongly suggest that alkali light chains are involved in the maintenance of myosin head structure.     

An infrared spectroscopy study of acid stability and thermal unfolding process of granulocyte-colony stimulating factor

Temperature-dependent (25-80 degrees C) infrared (IR) spectra were obtained for recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) in aqueous solutions over the pD range of 5.5-2.1 to investigate its thermal stability at various pDs. Second derivative, Fourier self-deconvolution, and curve-fitting analyses were performed to analyze the obtained spectra. These spectral analyses demonstrated that in the thermal unfolding process the alpha-helix structure of rmethuG-CSF partially changes to an unordered structure and then the unordered structure forms aggregates. The temperature-dependent IR spectra revealed that the structure of rmethuG-CSF is the most stable at pD 2.5 in the pD range of 5.5-2.1. It has been suggested that the unordered structure formed before the marked structural change in the whole molecule is a perturbed form of the native structure of rmethuG-CSF and plays a role as a precursor for the aggregation. This alteration to the perturbed form is likely to be the first secondary structure change that occurs along the aggregation pathway. Of particular note is that the stability at pD 2.1 is slightly lower than that at pD 2.5, but that aggregates are formed at higher temperature at pD 2.1 than at pD 2.5, probably because the repulsive interaction between the unordered structure is stronger at pD 2.1.