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Based on the knowledge about subcellular morphogenetic processes in the acellular slime mold Physarum polycephalum, we hypothesized that during differentiation of undifferentiated endoplasm to the highly differentiated complex structure of the contractile apparatus of this organism, the regularity of oscillating contractions must improve. We measured the endogenous contraction automaticity starting from the de novo generation within minutes after sampling small portions of undifferentiated endoplasm. The standard deviation of the normalized period duration of these samples was compared to the respective values of radial contractions of differentiated protoplasmic plasmodial strands. The mean normalized standard deviation in endoplasmic drops was 28.3% ± 12.2%. Respective values in protoplasmic strands were 10.0% ± 3.7%. The difference between the experimental groups was highly significant (p<<0.0001). We interpret the verification of our hypothesis as an indication that the very regular oscillating contractions in fully differentiated stages of Physarum require the complex structure of the sophisticated contractile apparatus, represented by the circular plasmalemma invagination system of protoplasmic strands, while the regularity is lower in stages, where the differentiation is still in progress. We believe that this is due to deficits in coordination capabilities, which need a directional and spatially oriented protoplasmic streaming as a precondition. Copyright 2006 Elsevier. Volltext f. Abonnenten.
1. Introduction
Physarum polycephalum has widely been used as a model organism to study non-muscle actomyosin. Oscillating contractions of the plasmodial ectoplasm generate hydrostatic pressure gradients which drive a vigorous shuttle streaming of the plasmodial endoplasm. This shuttle streaming serves locomotion, coordination of contraction rhythmicity and transport of organelles and other substances.
The structural basis of endogenous oscillating contractions is a contractile apparatus represented by a system of plasmalemma invaginations associated with extensive actomyosin sheets and fibrils (Achenbach and Wohlfarth-Bottermann 1981a). The interaction of these two components is a precondition for the de novo generation and continuation of oscillating contractions. At first, small portions of endoplasm are free of plasmalemma invaginations, and the actomyosin system is present as a low-viscosity solution of actin and myosin. Within seconds after isolation of a small portion of endoplasm, a new plasmamembrane is formed (Wohlfarth-Bottermann and Stockem 1970). Immediately, the formation of plasmalemma invaginations starts in combination with actomyosin complexes to associate with the newly formed invaginations. As a result, a 4-5 fold surface enlargement of the plasmamembrane occurs (Achenbach et al. 1979) within about 15 minutes. This subcellular morphogenesis represents the initial differentiation of the contractile apparatus, which starts to contract in an oscillatory manner at this time point (Götz v. Olenhusen et al. 1979).
Fig. 1. Phase contrast aspect of an endoplasmic drop at the age of about 15 min, i.e. oscillating contractions have just begun. Note irregularly distributed plasmalemma invaginations (some marked with “i”) and endoplasm-like areas (*).
Figure 1 represents a cross section of the sphere-like portion of endoplasm about 10 min after its generation. Regions of endoplasm delineated by invaginations of the plasmamembrane (i) are marked by asterisks, to show the irregular distribution and size.
This should be compared with figure 2, which represents a cross section of a plasmodial strand, i.e. a linear structure of several centimeters in length with a throughgoing shuttle streaming within one endoplasmic channel (marked by an asterisk and delineated by layers of plasmalemma invaginations (arrowheads)) spatially oriented in the longitudinal direction of the strand. We believe that this difference between the stages, i.e. the early endoplasmic drop with irregularly sized and distributed endoplasmic regions on the one hand, and the differentiated protoplasmic strand with one throughgoing endoplasmic channel oriented in a polarized manner, on the other hand, is the structural basis and reason for the difference in regularity of oscillations.
Fig. 2. Phase contrast aspect of a cross sectioned thin protoplasmic strand with the endoplasmic channel (*) separated from the peripheral ectoplasm (ec) by the circular plasmalemma invagination system (arrowheads).
Endoplasmic streaming is one of the most important factors for coordination and synchronization of oscillating contractions in this organism, i. e. it acts as a mechanical coupling for the plasmodial monorhythmicity (Achenbach and Wohlfarth-Bottermann 1981b). Hence, we wanted to test the hypothesis, whether regularity of the contraction rhythm depends on the progress of morphogenesis of the contractile apparatus and the existence of a regular shuttle streaming. As a parameter to estimate the regularity of contractions, we used the standard deviations of period duration normalized to the mean period duration of an individual specimen.
2. Methods
2.1. Cell Culture
Plasmodia of Physarum polycephalum (M3CVII, ATCC 204388) were obtained from microsclerotia (microspheres, generously provided by Prof. Dr. E. Holler, Institut für Biophysik und Physikalische Biochemie, University Regensburg). Samples of microspheres were placed on 1% agar containing 10% semi-defined culture medium (which is used for the axenic culture of plasmodia as a permanent shaken culture), and allowed to reactivate for about 3 days. When small migrating plasmodia had formed on the agar surface, the plasmodia were cut out with a portion of the agar substrate and placed one each in a 30 x 45 cm plastic culture vessel covered with moist filter paper under non-sterile conditions. A portion of rolled oats was sprinkled on top of the plasmodia and on the surrounding filter paper substrate. In the course of several days, the plasmodia grew to the size of several cm2 while feeding on the rolled oats. Pieces of about 5 cm2 were then cut from the filter paper overgrown with a compact mass of plasmodium, and placed on agar plates (1% agar in Aqua dest.). The starving plasmodia were then allowed to migrate on the agar surface over night, and used as experimental material the next day.
The viability of samples was tested by placing the specimen on a plain agar surface at the end of experiments to test if a transformation into a migrating plasmodium occurred. This was the case in each of the experimental samples without exception.
Physarum is not subject to any concerns about contamination.
Macroplasmodia spreading on filter paper were allowed to transform to sclerotia by slowly dehydrating for several days. These sclerotia serve as a source for living plasmodia and are available from the authors on request.
2.2. Sample preparation
Endoplasmic drops of 0.5-1 mm in diameter were generated by puncturing protoplasmic strands with an insect needle. The time point of extrusion of the endoplasmic drop was marked on the registering chart recorder. After waiting for 15 seconds, the drop was lifted off the strand, placed on a concave microscopic slide, covered with A. dest. and a cover slip.
Radial contractions of protoplasmic strands (diameter 0.5-1 mm) were registered on samples in situ, i.e. sections of plasmodial strands on portions of agar of 1x2 cm in size placed on a microscopic slide. After cutting out, the plasmodial strands were allowed to recover in a moist chamber for 30 minutes.
2.3. Registration of contractile activity
The registration of contractile activity was performed by a home-made photocell equipment on the basis of a silicon blue cell mounted to the intermediate image plane of a conventional light microscope at low magnification (Fig. 3, Achenbach et al. 1981). The border of the endoplasmic drop and a smooth edge of the strand, respectively, were centered in the sensor area to allow registration of changes in light intensity due to the movement of the specimen.
Fig. 3. Setup of photocell registration in a conventional light microscope. The photocell (d) is mounted in the intermediate image plane (a) of the microscope. The measuring field is limited by the image of the illumination diaphragm (c). The periphery of the specimen (b) to be measured is positioned into the center of the photocell. Thus changes of position of the rim of specimen (dotted line) result in changes of light intensity detected by the photo cell.
Illumination of the microscope was driven by a constant voltage power supply at low voltage and filtered with a dark red light filter. All experiments were carried out under safety light conditions (red light dark room illumination).
The measuring signal was recorded by a mechanical chart recorder (Philips 8110) calibrated to a chart speed of 5 mm per minute. Charts were evaluated by determination of the time distance between contraction maxima (period duration).
2.4. Statistical analysis
The mean period duration from endoplasmic drop experiments and from protoplasmic strands were calculated and compared by Student’s t-test.
Because of variations in the mean period duration in different plasmodial samples, we normalized the standard deviations to the mean period duration, i.e. we calculated: standard deviation divided by mean period duration expressed in percent for each plasmodial sample and pooled these values to calculate the respective mean value hereof.
The normalized values were pooled and statistically compared with the respective values obtained from measurements of radial contractions of plasmodial strands.
2.5. Morphology
Phase contrast images depicted in figures 1 and 2 are semithin sections derived by a conventional embedding technique according to Achenbach and Wohlfarth-Bottermann, 1981a.
3. Results
With a total of 32 specimen measured, we could verify our hypothesis that the regularity of oscillating contraction is impaired in plasmodial stages, where the highly organized structure of the circular plasmalemma invagination system is lacking. This is the case in early stages of isolated endoplasm, where the subcellular morphogenesis of the contractile apparatus is in progress, and a directional orientation of endoplasmic channels is still lacking (Fig. 1).
We decided to use the standard deviations of the mean period duration of oscillating contractions as a parameter to estimate the regularity of this rhythmic process, hypothesizing that this value should be higher in endoplasmic drops as compared to the differentiated stage, represented by a protoplasmic strand. Two characteristic registrations are shown in figure 4.
Fig. 4. Characteristic registrations of oscillating contractions in an endoplasmic drop (A) and a protoplasmic strand (B). Time point of extrusion of the endoplasmic drop is marked with 0, and the starting point of registration is represented by the vertical dotted line at about 3 min. Ordinate: contraction (c) and relaxation (r), respectively. Note that oscillating contractions are generated de novo at about 10 min of drop age (A).
Fig. 5 compares the normalized standard deviations of the data calculated from endoplasmic drops (A, 28.3%) and protoplasmic strands (B, 10.0%), respectively. The difference between the two mean values was highly significant with p<<0.0001.
Fig. 5: Comparison of normalized standard deviations of period durations in endoplasmic drops (A) and protoplasmic strands (B). The difference between the two groups is significant (n=32).
A comparison of the mean values of period duration in the two groups revealed no significant difference (Fig. 6).
Fig. 6: Comparison of mean period duration (ordinate, min) of endoplasmic drops (A) with the respective values of protoplasmic strands (B). The difference is not significant (n=32).
4. Discussion
Rhythmic movement phenomena have attracted the interest of scientists since the early days of cell biology. In striated muscle and smooth muscle systems, the source and location of oscillators as well as the processes of triggering contraction and relaxation, respectively, are quite well understood. The physiology and functional regulation of most non-muscle actomyosin systems, however, is hardly understood so far. Especially, the function of oscillating contractions of the cytoplasmic actomyosin system in Physarum has long been subject to worldwide research on this model system for non-muscle actomyosin. One feature of this system is the “inverse” calcium sensitivity of contraction, i.e. contraction is inhibited by an increase in calcium concentration, just opposite to all muscle actomyosin systems (Achenbach and Wohlfarth-Bottermann, 1986; for review see Kohama, 1992).
A second unsolved question in this system is the nature and location of the oscillator. So far, no indications for a electrophysiological, membrane-bound process have been found. However, all experimental data available favor a mechanical coupling by endoplasmic streaming via stress activation by hydraulic pressure (Achenbach and Wohlfarth-Bottermann, 1981b).
Time-lapse cinematographic observation of the de novo generation of oscillating contractions in endoplasmic drops of Physarum suggested that the rhythmicity is irregular at first, but improves in regularity in later stages, where a polarized locomotion is observed (Achenbach et al. 1985). This phenomenon of changes in regularity during morphogenesis of endoplasm into a migration plasmodium has never been verified by exact measurements. Hence, we hypothesized that this change in regularity of oscillations is reflected by differences in the standard deviations of period duration, if early “endoplasmic” contractions are compared with those measured in fully developed protoplasmic strands.
Although there is no significant difference in the overall period durations of the two samples compared (Fig. 6), there is a dramatic difference in the regularity of oscillations, as judged by the standard deviations of period duration of contractions in these two plasmodial stages (Fig. 5). This verification of our initial hypothesis may be a further indication for the importance of a fully developed contractile apparatus (Fig. 2) for the highly regular oscillating contractions in Physarum plasmodia (Fig. 4B). In earlier, less developed stages (Fig. 1) contractions are irregular due to lacking or irregularly distributed endoplasmic channels (Fig. 4A). Upon manifestation of some kind of directional polarization of endoplasmic streaming, regularity of contractions improves, and is highest in fully differentiated protoplasmic strands.
The structural basis for the highly regular oscillations in protoplasmic strands is a functioning shuttle streaming that acts as a pacemaker according to extensive investigations of the Wohlfarth-Bottermann group. As a consequence, we suspected that plasmodial stages lacking a polarized shuttle streaming as it is present in a plasmodial strand, should display oscillating contractions significantly less regular as compared to the plasmodial strands. In Physarum we have the unique possibility to prepare a plasmodial stage void of a throughgoing shuttle streaming. This plasmodial stage is represented by endoplasmic portions generated by puncturing plasmodial strands. At first, these endoplasmic drops are pure endoplasm, i.e. low viscosity protoplasm similar to the endoplasm within the endoplasmic channel of protoplasmic strands. Within some minutes, these portions of endoplasm differentiate to protoplasm, penetrated by plasmalemma invaginations eventually encircling newly developed endoplasmic regions (cf asterisks in fig. 1).
These endoplasmic regions are irregularly distributed within the endoplasmic drop. This is brought about by contractions, de novo generated at different sites of the drop, causing sol-gel-transformation at respective sites. Contractions starting at different sites interfere with each other, spoiling the regularity of period duration. Later the endoplasmic regions will tend to be more and more spatially oriented with respect to a locomotor direction of the developing plasmodium. This leads to an improvement of coordination of rhythms. The morphologically defined transition from this irregular distribution of endoplasmic regions to the highly polarized situation of a protoplasmic strand is characterized by a transition from irregular oscillating contractions to the highly regular oscillations in the protoplasmic strand.
The temporal sequence of these events can be summarized by the following flow chart: Endoplasm > Formation of ectoplasm by sol-gel-transformation and plasmalemma invaginations > de novo generation of contractions at different sites of the drop > Formation of endoplasmic regions irregularly distributed > Incomplete coordinator function of endoplasmic streaming > Interference of contractions spoils regularity due to lack of coordination > Improvement of spatial orientation of endoplasmic streamlets in combination with further differentiation of the contractile apparatus improves coordination of contractions > Formation of protoplasmic channels or protoplasmic strands with one polarized endoplasmic channel (front – uroid region) enables highly regular oscillating contractions.
Recent publications have attempted to fit the behavior of Physarum oscillating contractions to mechanochemical/mathematical models (Kobayashi et al. 2006; Teplov et al. 2005), supporting the idea of a mechanically based coordinating system. Kobayashi et al. (2006) discuss the importance of “plasmodial stiffness” rather than coupled oscillators to play a major role in plasmodial behavior. This somewhat diffuse term “plasmodial stiffness” might be related to structural changes occurring during plasmodial differentiation discussed above.
Acknowledgments
The authors wish to thank M. Streck, University of Bonn, Dr. Ingrid Block, German Space Agency, Cologne, Prof. Dr. W. Marwan, University of Magdeburg for providing experimental materials and Prof. Dr. E. Holler, University of Regensburg, for the generous gift of spherules, and his coworker, Mrs. Sonja Fuchs, for the preparation of spherules.
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