Plasmalemma invaginations as characteristic constituents of plasmodia of Physarum Polycephalum

The plasmodia of the acellular slime mould Physarum polycephalum cultivated on a moist substratum such as filter paper or agar gel exhibit a characteristic growing pattern. Migrating and feeding plasmodia consist of a more or less coherent, apparently solid protoplasmic mass at the front, while in the middle and in the retracting end the protoplasm is organized in the form of a network of communicating strands. These strands resemble veins. The wall of the vein, termed ectoplasm, surrounds an endoplasmic core. Within the predominantly stationary wall of ectoplasm, endoplasmic shuttle streaming is responsible for mass transport and thereby for the locomotion of the entire plasmodium.

Because the protoplasmic veins or strands are used as models for the study of the basic mechanism of protoplasmic streaming and obviously are of importance for the physiology of the multinucleate plasmodium, the organization of protoplasmic strands was studied in plasmodia growing on different substrates and fed with different food. The special aim of this work was to analyse the fine structure of the plasmodium in situ, i.e. to reveal the organizational pattern of the vegetative stage of Physarum in close contact with the substrate, without detaching the plasmodium from the original supporting layer. For comparison, the studies were extended to motile plasmodia growing on wet filter paper according to the culture method of Camp (1936).

The occurrence of plasmalemma invaginations has been described earlier by Wohlfarth-Bottermann (1963), Rhea (1966), Usui (1971) and Daniel & Järlfors (1972) in Physarum polycephalum and by Bauer (1967) and Stiemerling (1970) in Physarum confertum.

Though many physiological and biochemical investigations have been carried out on the vegetative stages of Physarum polycephalum, especially within the last decade, the morphology is not yet fully investigated even at the light-microscopical level.

Two strains of Physarum polycephalum were employed in this investigation: (1) Normal strain of Physarum polycephalum used in this laboratory. Sterile axenic cultures were grown in shaken flasks containing a semi-defined medium. Plasmodial cultures destined for investigation were started by transferring microplasmodia from the axenic cultures to filter paper or agar plates. (2) Physarum polycephalum, homothallic plasmodium, strain C5-1/501 (Dr J. Dee, Genetics Department, University of Leicester, England). This strain is especially favourable for observations of living material because pigment synthesis seems to be very slow. If the plasmodia are subcultured twice weekly, their translucence favours light-microscopical investigations.

Plasmodia of different sizes and shapes including mass cultures were grown on filter paper or on agar gel. Three different methods were applied: (1) Cultures on wet filter paper with rolled oats or oatmeal as food according to Camp (1936). (2) Cultures on 1 % agar plates containing yeast extract, tryptone and glucose (semi-defined medium (Daniel & Baldwin, 1964)) as used for the axenic cultures. (3) Cultures on 1 % agar plates with rolled oats or oatmeal as food.

Living plasmodia growing on their substratum, usually agar, were studied by differential interference-contrast and phase-contrast (Zeiss).

Plasmodia were fixed in situ by exposure to one of the following fixatives: (1) 1 01 2 % OsO₄ + 1 % K₂Cr₂O₇, pH 6·9–7·1 (Wohlfarth-Bottermann, 1957);(2)2% OsO₄+o·5% HgCl_a, pH 6·7 (Parducz, 1952); (3) 6 or 3% glutaraldehyde, pH 7·0 (0·1 M cacodylate buffer); (4) 4% paraformaldehyde, pH 6-8 (o-i M cacodylate buffer). They were then dehydrated in a graded ethanol series and embedded in styrene-methacrylate.

Serial 1–2 μm ‘semithin’ sections for light-microscopic examination and thin sections for fine-structural investigation were cut using an LKB ultramicrotome. Normally no staining techniques were applied. Staining of the semithin sections with 0·25 % toluidine blue, 1 % gentian violet or 1 % thionin enhances the contrast of the plasmalemma invaginations.

A Philips EM 200 electron microscope was used.

Fig. 3 shows a differential interference-contrast picture of a living, branched plasmodial strand growing on agar with a partially defined growth medium. Streaming of the endoplasmic core is indicated by the streaks of the moving endoplasm. Surface indentations within the ectoplasmic wall are visible only in strands of small and intermediate sizes which provide appropriate optical conditions.

Two serial cross-sections through a very small strand (Fig. 6) show a more or less rounded central core, enclosed by an outer region with infoldings of the outer strand plasmalemma.

A cross-section through a strand with a greater diameter (Fig. 4) shows an essentially similar structure. The rounded central core is limited by an extensive extracellular labyrinth system having open connexions with the outer plasmalemma invaginations. In other words, the plasmalemma invaginations form an irregularly branched system of clefts and pockets throughout the ectoplasmic wall of the strand. The central core, which appears circular in cross-section, is limited by rounded-up pockets of this plasmalemma system which form a circular, clearcut peripheral layer or border. Occasional interruptions of this border provide protoplasmic bridges between the endoplasmic core and the ectoplasmic wall. Nuclei and vacuoles are found in the branched ectoplasmic leaflets of the outer part, as well as in the central endoplasmic core.

Two serial semithin sections through a branching point of 2 deviating strands are depicted in Figs. 7 and 8, showing a larger strand in cross-section (centre) and the side branch in longitudinal section (left side). The central core and its limitation by the branched extracellular indentation system are recognizable in both strands, irrespective of the direction of sectioning.

In order to test whether this extensive system of plasmalemma invaginations is dependent on the kind of food or substrate, plasmodia cultured on agar and on filter paper and fed with solid food were fixed in situ and investigated for comparison with the same technique.

Plasmodia grown on agar and fed with rolled oats exhibit the same structural features of the invagination system as those grown on agar containing a semi-defined nutrition medium. In plasmodia grown on filter paper some differences were observed. Fig. 9 shows a cross-section of a plasmodial strand 0-4 mm in diameter fixed in situ and cut with the supporting filter paper. Cellulose fibres are seen in the lower part of Fig. 9. Despite the low magnification, the plasmalemma invaginations are clearly detectable, but restricted to a relatively small outer zone of the vein. Often the invagination system is strongly diminished: it is lacking in some places at the region of contact between vein and filter paper. Small pseudopodia-like processes are anchored within the meshes of the cellulose fibres (Fig. 9 and Fig. 2, p. 28).

The phase-contrast microscope reveals fibrils (Wohlfarth-Bottermann, 1962) in cross-sections of strands of intermediate and larger diameter in the periphery of the endoplasmic core (Figs. 10, 11). Many fibrils run adjacent to the invaginations and have close contact with the rounded plasmalemma pockets facing the central core (Figs. 10 and 11, arrows). These fibrils have a circular or spiral course transverse to the longitudinal direction of the strand and are consistently found at the junction between ectoplasm and endoplasm. This means that they are located between the innermost pocket of the plasmalemma invagination system and the streaming endoplasma. In addition, many longitudinally, spirally or otherwise oriented fibrils are present within the outer strand cortex. These fibrils too have insertion points on plasmalemma invaginations. Many invaginations are sandwiched on both sides by fibrillar material arranged into lamellae. From a functional point of view, there is no doubt that the fibrils, which are assumed to be contractile (see Discussion), exert an intensive tension on the plasmalemma infoldings. In protoplasmic strands of medium and maximum diameters, the number of circular and longitudinal fibrils which are recognizable in the phase-contrast microscope is extremely high.

All strands, irrespective of the culture method used, are surrounded by a slime layer which is somewhat difficult to visualize in OsO₄-fixed, unstained preparations in the phase-contrast microscope (Fig. 4, arrow). The contrast of the slime layer can be enhanced by staining 1μtm sections with toluidine blue, gentian violet or thionin.

The invaginations of the strands are poorly preserved after aldehyde fixation: both 3 % glutaraldehyde and 4 % paraformaldehyde fail to demonstrate any clear distinction between an endoplasmic core and an outer layer with plasmalemma invaginations. Although 6 % glutaraldehyde yields better results, this fixative is still inferior to osmium tetroxide or mixtures containing osmium. The addition of 2 mM ATP to 4 % paraformaldehyde brings about a structural arrangement suggesting that the cytoplasmic leaflets together with the invagination system have been retracted before fixation was completed by the slowly acting fixative. It is only after this fixation procedure that plasmalemma invaginations are found to extend into the endoplasmic core. In addition, a gap appears between the slime layer and the circumference of the strand, indicating that the cytoplasmic leaflets have been retracted to a certain degree after exposure to the fixative.

In order to answer the question of whether or not the occurrence of the system of plasmalemma invaginations is dependent upon the diameter of the strands, measurements were made on living plasmodia, embedded plasmodia and on semithin sections.

In plasmodia grown on the agar substrate (semi-defined medium or oats) the diameters of the strands vary between 40 and 250 μm. The strand diameters of plasmodia grown on filter paper were found to vary between 200 μm and 1·5 mm. In all strands of these very different calibres, the plasmalemma invagination system was present. Its thicknesses varied between 10 and 60 μm, irrespective of the culture method used. Furthermore, there seems to be no correlation between the diameters of the strands, and the depth of their plasmalemma invagination system.

In contrast to this, the number of fibrils recognizable in the phase-contrast microscope within the ectoplasmic cortex is much higher in the thick protoplasmic strands with diameters of 0·5–1·5 mm, grown on filter paper, than in the thin strands of agar-grown plasmodia, which mostly produce veins with diameters between 40 and 250 μm.

A quantitative estimation of the magnitude of plasmalemma area invaginated within strands with different diameters was obtained from morphometric measurements of the ratio between outer (Po) and inner (Pi) plasmalemma surface areas. The following values resulted from the analysis of Figs. 4, 6, 7 and 9 (Table 1). Plasmalemma invaginations can also be revealed without fixation by observing very thin strands of living plasmodia in profile and surface view with the differentia] interference-contrast microscope and examining unfixed frozen sections with the phase-contrast microscope.

The main results of this study are diagrammatically presented in Figs. 1 and 2. An outer, quantitatively important part of the protoplasmic veins of Physarum polycephalum is highly fissured by pleomorphic plasmalemma invaginations. The innermost parts of these pockets surround a more or less cylindrical endoplasmic core. After fixation with mixtures containing OsO₄, this inner core was never found to contain plasmalemma indentations. The morphological aspect summarized in Figs, 1 and 2 is observable in strands of all sizes which can be found when culturing Physarum polycephalum on semi-defined agar, on non-nutritious agar with added single oat flakes or on filter paper according to the method of Camp (1936).

Rhea (1966) described ‘deeply penetrating invaginations’ in vegetative stages of Physarum polycephalum. Figs. 6 and 16 of his paper obviously show the same system of plasmalemma invaginations, but much less pronounced than that described here. ‘Indentations in motile microplasmodia’, as demonstrated by Usui (1971, Fig. 8), and ‘microchannels’ (Daniel & Järlfors, 1972), probably represent the same system.

Comparative examinations of I-μm sections with the phase-contrast microscope and consecutive thin sections with the electron microscope have revealed that the extracellular space within the outer part of the strands is continuously lined by the plasmalemma. With regard to the invagination system, the electron microscope has not revealed structural details other than those found by phase-contrast microscopy.

A comparative analysis of the living plasmodium on agar and the semithin sections reveals that the inner core containing no invaginations represents the endoplasmic channel engaged in shuttle streaming. Consequently, under culture conditions as applied here, there is a very clear structural distinction between the stationary ectoplasmic wall and the streaming endoplasmic core.

The invagination system is difficult to reveal with slowly acting fixatives such as 3% glutaraldehyde and 4% paraformaldehyde. This is even more pronounced when paraformaldehyde containing 2 mM ATP (Alléra & Wohlfarth-Bottermann, 1972) is applied. The interpretation, that slowly acting fixatives give the ectoplasmic wall opportunity to retract partly prior to definite fixation is in accordance with previous observations, that glutaraldehyde is a slowly acting fixative in comparison to fixatives containing osmium tetroxide (Wohlfarth-Bottermann & Komnick, 1966). This judgement is based on the longer period of time which glutaraldehyde needs to stop movement in comparison to osmium, when applied to various objects. If definite fixation is defined as the time needed between application of the fixative and the visible cessation of shuttle streaming, then, for example after the application of 4 % paraformaldehyde it takes 30–50 s before the cessation of all observable movement within the immersed strand.

Contraction processes of the ectoplasmic wall are of interest in respect to the problem of how the motive force for the shuttle streaming is generated along the protoplasmic veins (Komnick, Stockem & Wohlfarth-Bottermann, 1973). Rhythmic variations in the diameter of protoplasmic strands in correlation with shuttle streaming were clearly observed by cinematography; these variations in strand diameter suggest contraction processes. A contraction of the wall, in spite of its extremely fissured structure, should be capable of producing a hydrostatic pressure on the endoplasmic core. Upon relaxation of the wall, the pressure exerted on the endoplasmic core should diminish. Simultaneously, the counter pressure within the endoplasmic core, generated and transmitted from other regions of the plasmodium presumably causes an extension of the relaxing ectoplasmic wall.

The protoplasmic phase of the endoplasmic core is continuous with the protoplasmic phase within the leaflets of the ectoplasmic wall via small protoplasmic bridges. Through these connecting channels protoplasm can be transported back and forth to the outer ectoplasmic region, thus increasing or decreasing the thickness of the ectoplasmic wall.

The rhythmic variations in strand diameter imply variations in the strand surface area. The membrane surplus provided by the plasmalemma invagination system probably facilitates these changes in surface area. The extracellular clefts within the ectoplasmic region of the strands can be interpreted in two ways: as plasmalemma invaginations into a primarily solid ectoplasmic wall; or more probably, as an extracellular space between interconnected pseudopodial-like processes, originating from the central core and extending into the surrounding medium.

There is no doubt that the ectoplasmic region is a highly dynamic structure, capable of performing rapid transformations. In the ectoplasmic wall of very small strands on agar, movement and streaming phenomena can be observed which are obviously independent of the direction and velocity of the shuttle streaming within the endoplasmic channel.

Concerning the fibrillar system, there is the difficulty that small fibrils present in microplasmodia and small strands cannot be seen by phase-contrast microscopy. Only fibrils of greater dimension can be detected by this method. This must be taken into account when considering the fact that, in protoplasmic strands with diameters between 40 and 200 μm, fibrillar differentiations are rarely observed with the phasecontrast microscope. The number of actomyosin fibrils (Altéra, Beck & Wohlfarth-Bottermann, 1971) which can be observed on semithin sections by phase-contrast microscopy increases with the diameter of the strands. Fibrillar differentiations of light-microscopical dimensions obviously are only needed in strands of greater diameters. Large strands (200μm–1·5 mm) which can be cultured according to the method of Camp (oatmeal on filter paper) demonstrate an extensive fibrillar arrangement within the ectoplasmic wall. In large strands with diameters exceeding 0·5 mm and more the fibrils insert most often at the plasmalemma invaginations.

As described previously (Wohlfarth-Bottermann, 1963), in protoplasmic drops fibrils were observed to insert at invaginations and vacuoles and to cause deformations of the vacuoles. In the light of the present findings, these vacuoles must be interpreted as plasmalemma invaginations, reaching deeply into the protoplasmic drops. Newly formed drops within the age range from o to 5 min do not contain invaginations. Ten-minute and older drops possess an extremely fissured system of plasmalemma invaginations with inserting fibrils. As Rhea (1966) pointed out, the fibrils in the strand are attached tangentially to the invagination membranes. The intimate connexion of plasmalemma invaginations and actomyosin fibrils will be described in detail in a subsequent paper.

The invagination system can be interpreted functionally as an enlargement of protoplasmic surface area for physiological reasons. The extensive infoldings in plasmodia growing on a partially defined nutrition agar without food particles may be functionally involved in food resorption and excretion processes (Figs. 4, 7, 8). Those strands with a comparatively reduced invagination system (Fig. 9) may be more engaged in mass transport of endoplasm than in food absorption. However it must be pointed out that thick strands having a much more pronounced invagination system than the reduced form shown in Fig. 9 were-also found.

The morphometric measurements (Table 1) show that the real surface area of protoplasmic strands is 2-to 10-fold larger than the outer strand surface visible in whole mount preparations. No clear correlation was found between thicknesses of strands and the depth of the invagination system.

The occurrence of plasmalemma invaginations is not restricted to the protoplasmic strands. The invaginations also represent the characteristic structural feature of the apparently solid protoplasmic sheet at the advancing front of a growing and migrating plasmodium (Fig. 5). This part, which appears as a coherent sheet of protoplasm in whole-mount preparations, is highly fissured by plasmalemma invaginations in the same way and to the same extent as the ectoplasmic regions of strands. This is in agreement with Rhea’s (1966) description, that the ‘advancing ends’ of the plasmodium ‘are not solid masses but intricate networks formed by deeply penetrating plicae and invaginations’.

The invagination system is a characteristic constituent of the plasmodial organization in Physarum polycephalum, suggesting that it is of considerable physiological importance. It is present throughout the growing plasmodia, except within their endoplasmic channels involved in shuttle streaming.

In Physarum confertum, Stiemerling (1970) described ‘flattened vacuoles’ around the endoplasmic channel (figs, 1b and 3b-d). Obviously these structures are identical with plasmalemma invaginations, but the invagination system seems to be less pronounced in this species, which produces smaller plasmodia.

‘Rows of esterolytic invaginations’ (Bauer, 1967) arranged more or less periodically transverse to the long axis of strands seem to suggest a periodic arrangement of invaginations in Physarum confertum. In Physarum polycephalum, indications of a periodical arrangement of the invagination system are vague.

The author is indebted to Dr R. Stiemerling for drawing the diagrams in Figs. 1 and 2, and Mrs B. Koeppen for skilful technical assistance. The investigation was supported by the ‘Landesamt für Forschung des Landes Nordrhein-Westfalen’.