No. 1] Proc. Japan Acad., 43 (1967) 45 12. Movement o f the Myxomycete Plasmodium. III Artificial Polarization in Endoplasm Distribution in a Plasmodium and its Bearing on Protoplasmic Streaming By Takako TAKATA, Reiko NAGAI, and Noburo KAMIYA Department of Biology,Faculty of Science,Osaka University (Comm. by Tetsu SAKAMURA,M,J,A., Jan. 12, 1967) The endoplasmic streaming in the myxomycete plasmodium can be readily accelerated, retarded or reversed on application of local pressure. Artificial control of the rate and direction of the streaming may be best demonstrated taking advantage of the double-chamber method. If a certain amount of sufficiently large air pressure, whether positive or negative, is kept applied to one half of a dumbbell-shaped plasmodium while the other half remains in the atmospheric pressure, the endoplasm at the neck between the two halves rushes to whichever half of the plasmodium placed under a lower pressure. With this treatment, one half of the plasmodium becomes rich in endoplasm while the other half becomes poor in it. It is the purpose of the present paper thus to polarize the distribution of endoplasm within a plasmodium and to find its possible effect on the generation of the motive force as well as on the ultrastructure of the plasmodium. Plasmodia of Physarum polycephalum served as material. Experiments have been conducted using an ordinary double- chamber described previously (Kamiya 1942, 1953, 1959). After having measured the motive force under normal condition, a constant pressure amounting to 30-40 cm of water column, which is larger than the maximal value of the motive force developed in a normal slime mold, is kept applied for 15-60 minutes. Under this condition, where the motive force is no longer measurable, endoplasm is forced to move rapidly at first to one half of the plasmodium facing the lower pressure. This passive flow gradually slackens. Later a weak, short-lasting streaming against the external pressure gradient begins to take place showing that the motive force in that direction has augmented in the meantime to overcome the pressure gradient applied externally. With this procedure, at any rate, a considerable part of the endoplasm in one half of the plasmodium exposed to a higher pressure is transported to the other half leaving behind the ectoplasmic gel 46 T. TAKATA,R. NAGAI,and N. KAMIYA [Vol. 43, Thus we have a dumbbell-shaped plasmodium with endoplasm-rich and endoplasm-poor halves connected with a single strand. Fig. 1 shows this state, where the right half of the plasmodium in compartment B has withered away while the left half in compartment A has swollen with the inflowing endoplasm. Since ectoplasmic gel remains in situ in this case, the spreading area of each half of the plasmodium does not change appreciably. Hence the endoplasm-rich half becomes thick and opaque with the inflowing endoplasm while the endoplasm-poor half becomes thin and transparent. Under such a situation, the endoplasm always comes back rapidly as soon as the pressure gradient is removed between A and B. Instead of making the endoplasm free to flow by eliminating the one-sided pressure, however, we now keep applying the balance-pressure just sufficient to stop the endoplasm in the connecting strand. This balance-pressure, which changes according to a rhythmic pattern, is a measure of the motive force developed in the plasmodium whose endoplasmic distribution is polarized by the external pressure gradient. Fig. 2, where the motive force is represented by the balance- Fig. 2. The motive force production in a plasmodium whose endoplasm is dislocated by the externally applied pressure gradient. The left curve: control; the shaded part: period during which air pressure amounting to 30 cm of water column was applied to B for 17.5 min. (time scale is discontinued); the right curve: the motive force after the distribution of endoplasm has been polarized. pressure applied to compartment B, shows one of the results of the experiments of this kind. The curve during the first 12.5 minutes represents spontaneous changes of the motive force under normal condition. After this a constant air pressure amounting to 3.0 cm of water was kept applied to compartment B for 17.5 minutes, during No. 1] Polarization in Endoplasm Distribution in Plasmodium 47 which period the measurement of the motive force was interrupted. The motive force measurement was then followed. From Fig. 2 it is noted that the motive force was extremely high right after the removal of the constant one-sided pressure and that it did not cross the base-line. It took 5.5 minutes before the undulating curve came down and crossed the base-line at first, where the direction of the motive force reversed. We must remind here that this curve was obtained under such a condition that the endoplasm was not allowed to flow in the connecting strand between A and B (cf. Fig. 1). From this figure we understand that there was a strong tendency of the endoplasm to come back at first but it gradually faded away even when endoplasm was prevented from going back. After 35 minutes on the time scale the motive force curve represents an almost normal pattern. This shows that the temporary disturbance of the motive force generation caused by the forced dislocation of the endoplasm has been readjusted in the meantime. The response of the plasmodium to the local pressure is always alike in its basic feature. Forced polarization of the endoplasm distribution induces the production of the motive force of the back streaming which gradually disappears if the endoplasmic flow is clamped at the connecting strand by the balance-pressure. We are however still ignorant about the cause of this phenomenon. Nor do we know which half, endoplasm-rich or endoplasm-poor half, is mainly responible for producing the high motive force for back streaming. Elastic rebound of the cortex caused by its expansion brought about by inflowing endoplasm is not likely to be the cause in the light of the experiments to be reported subsequently. With the idea that this phenomenon may have to do with a more deep-seated nature in the cytoplasm, we investigated the fine structure of the plasmodium in the endoplasm-rich and endoplasm- poor halves of a polarized plasmodium. The material was fixed within a minute or two after the external pressure gradient had been removed. Techniques of fixation and further procedures of preparation of the specimen were the same as stated before (Nagai and Kamiya 1966). In the ectoplasmic layer of the endoplasm-rich half of the plasmodium we find fibrillar bundles well developed in the ground cytoplasm. They are extremely similar to those in the normal plasmodium. We are also often encountered with a case in which the fibrillar bundles are attached to the vacuolar membrane and plasmalemma at their terminals, a fact which has been correctly pointed out by Wohlfarth-Bottermann (1964, 1965). Fig. 3 shows 4$ T. TAKATA, R. NAGAI, and N. KAMIYA [Vol. 43, Fig. 3. Fibrillar differentiation of the cytoplasm near the periphery of the endoplasm-rich half of the plasmodium. Fig. 4. Aggregate of globular-looking cytoplasmic components around the vacuole in the endoplasm-poor half of the plasmodium. a part of the endoplasm-rich half of the plasmodium close to its advancing front in a section parallel to the agar substratum on No. 1] Polarization in Endoplasm Distribution in Plasmodium 49 which the organism was creeping. Here fibrillar bundles branch off from those running along the membrane. In the endoplasm-poor half, on the other hand, the situation is quite different. There are scarcely any fibrillar differentiations in the ground cytoplasm. Instead, we observe aggregates consisting of more or less globular substances similar in staining to those of the fibril bundles. Further, it is to be noticed that they are found in regions just where fibrillar bundles are likely to have been present, especially frequently around the vacuole (Fig. 4). From these facts we are inclined to believe that the filaments composing the fibrillar bundles have been converted into another form which appears to be globular, when the endoplasm has been squeezed out to disturb the normal endoplasm-ectoplasm ratio. As already pointed out in the foregoing paper of this series (Nagai and Kamiya 1966), fibrillar differentiation in the plasmodium probably represents bundles of filaments of actin which was suc- cessfully isolated from Physarum polycephalum and characterized by Hatano and Oosawa (1966, 1967). In the present context, it is especially worthy of note that Hatano, Totsuka, and Oosawa (1967) further showed that there are, besides F-actin, another state of actin polymer, or "Mg-polymer," which is formed in the presence of Mg. Unlike plasmodium F-actin, Mg-polymer of the actin is low in viscosity suggesting that it is globular rather than fibrillar. Though anything definite is still to be investigated, the dense region around the vacuole in Fig. 4 may represent an aggregate of Mg-polymer found by Hatano et al. We have already stated that under polarized state of the plasmodium, where endoplasm-ectoplasm ratio is made widely different between the two halves of one and the same plasmodium, strong motive force is generated to bring the endoplasm back from the endoplasm-rich half to the endoplasm-poor half. If fibrous differen- tiation is a sine qua non for the generation of the motive force, the above fact may show that the cause of the strong tendency of the endoplasm to come back in to be sought in the endoplasm-rich half and not the other half. In other words, the motive force is likely to act as a "push" from behind rather than as a "pull" from the front.