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(1)Motive Force of the Cell Mass of the Cellular. Slime Mould Dictyostelium Discoideum: (I) Migrating pseudoplasmodium under dark condition (II) Migrating pseudoplasmodium under illuminated condition (III) Culminating fruiting body*i' By. Masanobu KITAMI*. .. Summary Here some preliminary results about the motive force of the cell mass of. D. discoideum are presented, which seem to be very significant in order to establish a model on following singular collective motions played by cells. The cells of D. discoideum show unificative collective motions, which are characterized as aggregation, migration, and culmination. These collective motions are likely to be very important and essential for their morphogenetic movements; however, very little is known about the mechanism of such collective motions. Many investigations and detailed explanations on particular life cycle of D. dis-. coideum and some experimental observations on the subjects above mentioned have been described in some books (Bonner, 1967; Loomis 1975; Cappuccinelli & Ashworth, 1977; Maeda & Maeda, 1978; Yamada, 1980). In this paper, attention will be paied mainly to migration and culmination.. The present measurements were performed by an original method utilizing the centrifugal force. The maximum value of the motive force of a migrating pseudoplasmodium in the dark was estimated as O.6-1.6dyn. Besides, we were successful in estimating the motive force of that in phototactic migration, whose i. estimation had not yet been reported. It was estimated as O.8-2.4dyn, which was about 1.5 times Iarger than that in the dark. Also, illumination with light from the front increased the motive force, whereas illumination from the back caused quite opposite results. That is to say, light illumination could give direct. influence upon their motive force. These results seem to suggest the necessity of taking into consideration the direct effect of light illumination upon the motility of the cells in the migrating pseudoplasmodium. In the present study, " Department of Physics, Faculty of Education, Yokohama National University, Tokiwadai 156, Hodogaya-ku, Yokohama 240, Japan. "i) A preliminary report of some of these results has been presented by Kitami in IX Congress of the International Society of Development Biologists (Basle/Switzerland) (Kitami, 1981)..

(2) 26 M. KITAMI. for the similar purpose, the effects of the "Artificial gravitation" upon culmina-. tion were also investigated by utilizing the centrifugal force. When the "Artificial gravitation" opposing the direction of culmination was increased, the efficiency of culmination decreased distinctly until hardly any cell mass could culminate at about 40-50 G. This measured value could be converted to O.3-1.0 dyn, which indicated the motive force of the cell rnass in the culminating fruit-. ing body. This value was almost comparable to that of a migrating pseudoplasmodium. Some interesting behaviors of the cell mass concerning culmination under various gravitational conditions were also observed. From these observa-. tions, the following suggestions were made. Assuming that the culmination mechanism has two steps, i.e., an upward lifting motion of the cell mass and its elongation, the "Artificial gravitation" seems to affect the upward lifting motion of the cell mass in the culminating fruiting body. Moreover, the motive. force of both migrating and culminating might be brought about by similar mechanisms and/or similar types of cells. On the basis of these results, a study on the model to elucidate their collective motions is in progress, and it will be reported shortly.. (I) Migrating pseudoplasmodium under dark condition Introductioit.. A pseudoplasmodium of D. discaidaum has a simple structure, i.e., it contains up to 105 cells which is surrounded by a slime sheath. By collective motion. of the cells, however, it can migrate several centimeters on the substratum for a few days. It has also been known that the pseudoplasmodium migrates while secreting from its whole surface the slime sheath, which is left behind as the. pseudoplasmodium moves on. That is to say, the pseudoplasmodium migrates by moving through a tunnel of the slime sheath that it makes for itself.. Some experimental observations and hypothetical models have been established in order to explain the mechanism of its migration (Bonner et al., 1950; Francis, 1964; Loomis, 1972; Poff & Loomis, 1973; Poff et al., 1973, 1974; Yamamoto, 1977; Maeda, 1977). Nevertheless, since no conclusive evidence is available on the mechanism of its migration, it is not yet elucidated whether it is the slime sheath or the interior cells that control such collective motion as migration.. On the other hand, because of the interest in morphogenetic movement of D. discoideum cell, observations of movement of the interior cells of the psedo-. plasmodiumand estimations of the motive force of the migrating pseudoplasmodium have been reported. Individual cell movement in later structures of D. discoideum (late aggregate, migrating pseudoplasmodium, regulating pseudoplasmodium pieces, and culminating pseudoplasmodium) have been observed, and they have suggested a tentative conclusion that the aggregation control competences regulate cell behavior throughout the D. discoideum development (Durston. & Vork, 1979). The rate of the migrating pseudoplasmodium is dependent on. 't.

(3) Motive Force of the Cell Mass of D. discoideum .27 its size, being greater in a large pseudoplamodium than in a small one (Bonner et al., 1953). It has been suggested by Francis that the motive force of the. migrating pseudoplasmodium is determined by the total number of cells and that the frictional drag is dependent on the surface area (refer to the publication edited by Loomis, 1975). However, these reports heretofore have been highly qualitative because there have been very little quantitative data on the estima-. tion of the motive force of the migrating pseudoplasmodium. It is essential to measure the motive force of the migrating pseudoplasmodium quantitatively as one of the means to study the mechanism of its migration as well as for elucidation of the morphogenetic movement of the cells in the pseudoplasmodium. Recently, the motive force of the pseudoplasmodium of D. discoideum has been measured as 5.85×106 dyn/cm3 (Inouye & Takeuchi, 1980). or 8-27cmHg (Yamamoto & Kamiya, 1971) by using the doublechamber method which is used for the me' asurement of the motive force of the protoplasmic streaming of Physarum polycephalum (Kamiya, 1953). It should be noted that their works have taken the lead of the quantitative measurement on the motive force of it and have informed many interest evidences; howeveit, we have an anxiety that the values above mentioned are too large. Actually, they are ne'arly one or two orders-of-magnitude larger than those of other organisms (Inouye & Takeuchi, 1980). One reason for this difference may be based on the serious difuculty in exerting exact pressure upon only the pseudoplasmodium in the capillary tube, since the capillary tube is too thin. Though the double-chamber method is the more sophisticated for the estimation of the motive force of the protoplasmic streaming, it has tendency of giving larger values for that of the pseudoplasmodium of D. discoideum because of the capillary effect of the thin. agar tube. Full particulars will be discussed later. ' '･. ,. Therefore, we consider that many examinations to estimate the motive force of the migrating pseudoplasmodium of D. discoideum, which are made by different methods, are needed. In the present work, the dependence of the velocity of the migrating pseudoplasmodium on the external force, which could give infiuence upon the motive force of the cell mass in the pseudoplasmodium, was measured by an original method utilizing the centrifugal force to investigate quantitatively the motility of the migrating pseudoplasmodium. On the basis of these data, we were successful in estimating the motive force of the migrating pseudoplasmodium under dark condition.. Materials and Methods. D. discoideum strain NC4 was incubated at 200C with Klebsiella aerogenes on the nutrient agar (Bonner, 1947), When D. discoideum amoebae constructed the lumps preceding "finger-like structure", about ten of them were collected and transferred onto the surface of the nonnutrient agar (2% wt/vol) in the measurement vessel constantly kept at 250C. They were then dissociated softly by a looped hair''and evenly..spread in a line O.1cm in width and 15cm in.

(4) 28. M. KITAMI length at about center of the agar field. When the transferred and dissociated lumps of cells had form pseudoplasmodia, the centrifugal force was applied. The measurement vessel was square in shape (6.5×3.5 cm2) and had a double wall, the height of the inner wall was 1.0cm, and the outer wall was slightly higher. ,The inside region surrounded by the inner wall was fi11ed with 2% nonnutrient agar to about O.5cm in depth, in which an iron mesh was set to anchor the agar. In order to take a good photograph of the loci of the migrating pseudoplasmodia, the bottom of the vessel was covered with black aluminium foil. The interspace of the double wall was fi11ed with water and the vessel was covered with a lid to keep the relative humidity in the vessel at 100%. The vessel was shut off completely from light by covering it with black colored aluminium foil. Thus prepared, four vessels were set on the modified rotor of a centrifuge, the agar surface being in the rotation plane.. The mean velocity of the migrating pseudoplasmodium was obtained by the method similar to that used in the double exposure photographs: At intervals of four hours over a period of twenty-four hours, the rotation was stopped and the loci of all migrating pseudoplasmodia on the plate in four vessels were photographed. A total of seven frames (scenes) of the loci extending with the lapes of time were obtained. Mean velocity for each migrating pseudoplasmodia was obtained by the following method, i. e., seven frames (scenes) were projected on the screen in order and the extention of each locus photographed every four hours was traced, and then the mean velocities of each migrating pseudoplasmodium were calculated by measuring the length of such extention. In order to have the maximum value of the motive force as exact as possible, the loci of two or three most rapidly migrating pseudoplasmodia, which migrated rectilinearly in the direction Parallel or antiparallel to the centrifugal force in each vessel, were adopted, The allowance of the definition of parallel or antiparallel was within ±15 deg. from the direction of the centrifugal force. By such procedures, four or five runs for each measurement were carried out.. Results and Discussion. Fig. 1 shows the behavior of the migrating pseudoplasmodia under following. conditions: The external force acted upon them to the right (Fe.). i) The ･ velocities of the pseudoplasmodia migrating parallel to the direction of the external force were obtained from such loci as (a), ii) those migrating antiParallel. to it were obtained from such loci as (b). The maximum motive force of the migrating pseudoplasmodium was estimated from the data ii). Two examples are shown in this figure, i. e., external forces are 20 G and 50 G. h. The "Mass" of a pseudoplasmodium used in this experiment was weighed directly as about 2.0×10-5 gram, the size of it was about 700 ptm in length and. about200ptm in diameter. Moreover, in order to make sure of the above mentioned value, the "Mass" of it was estimated as 0.7×10-5gram by the calculation shown in Appendix 1). The order-of-magnitude agreement between measured. e.

(5) Motive Force of the Cell Mass of D. discoideum. 29. .. Fex: 20 G. Cb):-EE{:-. ::-v). .:; :(a). t-----. t'. .. Fex: 50 G. ,. (b)`;;'::':'g.dsasi:s:--.;:: SLS. ..lf-}}F(a). Fig. 1. Behavior of the migrating pseudoplasmodia under following conditions: The external force acts upon them to -----)the right (Fex). i) The velocities of the pseudoplasmodia migrating Parallel to the direction of the external force are obtained from such loci as (a), ii) those migrating antiParaglel to it are obtained from such loci as (b). The Maxi-. mum motive force of the migrating pseudoplasmodium is estimated from the data ii). Two examples are shown in this figures, i.e., external forces are 20 G and 50 G.. and estimated values provided the propriety of the weighed value mentioned above. So, the "Mass" of a pseudoplasmodium in the present study was concluded as O.7×10'5-2.0×10-5 gram.. The focus of this work lies on the hypothesis that the velocity of the. '. migrating pseudoplasmodium is proportional to the vector sum of the internal motive force and the applied external froce. It is not clear whether the internal ,. motive force is constant or not, but we can adopt the simplest idea; that is, balancing the external force exerted upon the migrating pseudoplasmodium with its motive force, its velocity would be reduced to zero. To be more precise, the movement of a pseudoplasmodium can be expressed in a simple equation:. -> ---> m･dv/dt= --" FM +f(") + Lx where m; "Mass"ofapseudoplasmdoium, di; velocity of the migrating pseudoplasmodium, - motive force of the migrating pseudoplasmodium, Llf; - external force applied to the pseudoplasmodium, Eex; f(zi); intrinsic resistant force inside the pseudoplasmodium expressed as a function of di..

(6) 30 M. KrpAMI. In the above equation, the inertial term (m･dv/dt) can be ignored, since the velocity of pseudoplasmodium migration is extremely slow, The definit nature of the resistant force (f(D)) is not known, but it can be considered as a function. of velocity, because it is determined by the interior cell movement of the pseudoplasmodium. Whether the motive force is constant or is variable depending on the external force, when the velocity-decreasing rectilinearly with in-. crease in the external force-drops to zero, the applied external force (Fex) should be equal to the maximum motive force of the migrating pseudoplasmodium. .. (FM). The changes in velocity shown in Fig. 2 are consistent with the hypothesis. ,. ' described above. ' Fig. 2 shows the changes in the velocities of the migrating pseudoplasmodia under two conditions when varying strengths of the external force were exerted upon them. i) Pseudoplasmodia migrate parallel to the direction of the external force (solid line A-B). ii) They migrate antiParallel to it (solid line A-C). The left and right halves in this figure show the antiparallel and parallel directions of the external force, respectively. In practice, the abscissa indicates. the acceleration in terms of G, aiming to emphasize the external force, but it. can be converted to the unit of force by multiplying by the "Mass" of the pseudoplasmodium. The ordinate shows the velocity of the migrating pseudoVe1octty (rm/h) antlparallel. 2e o. parallel. " tt. ,. o. e e. e. A. A. L. o. o,. o. e. : '. B. e. e. o c. 100 50 O 50 100 Centrtfugal acceleration (G) G: 98o cmlsec2. Fig. 2. Changes of the velocities of the migrating pseudoplasmodia under two conditions: i) Pseudoplasmodia migrate Parallel to the direction of the external force (solid line A-B). ii) They migrate antiParallel to it (solid line A-C). The left and righthalvesin this figure show the antiparallel and parallel directions of external force, respectively. The abscissa indicates the centrifugal acceleration in unit of G, aiming to emphasize the external force, and the ordinate shows the velocity of the migrating pseudoplasmodia. Individual point represents the mean velocity which is calculated by collecting data from two or three most rapidly migrating pseudoplasmodia in each plate of four or five runs.. ,.

(7) Motive Force of the Cell Mass of D. discoideum 31 plasmodia. Individual point represents the mean velocity which is calculated by collecting data from two or three most rapidly migrating pseudoplasmodia in each plate of four or five runs. The acceleration of the centrifugal motion was varied from 10G to 70 G, G being 980cm/sec2. i) At point A in Fig. 2, the extrapolated value of the velocity to the external force zero is about 1.4 mm/hour and is consistent with other works under similar condition (e.g., Yamamoto, 1977). When the external force was increased to a greater extent, it set the migrating pseudoplasmodium free from the bondage of its slime sheath and the interior cells rushed out eventua!ly (see broken line. .. starting from B). The velocities of the pseudoplasmodia migrat.iin. g Parallel to the direction of '. the external force seemed to be independent of the external force (see solid line A-B). This may be explained by the assumption that the rate of the slime sheath production is constant, except in cases where condition around the pseudoplasmodia (e.g., light, temperature, humidity, and chemical substances) are. changed. In other words, the velocity of the migrating pseudoplasmodium is restricted by its own rate of sheath production regardless of the external force,. and the observed value which is nearly constant is interpreted as the upper. limit of its velocity in the dark. ･ ii) The velocities of the pseudoplasmodia migrating antiParallel to the direction of the external force were clearly dependent upon the external force, as had been expected (see solid line A-C). On the basis of both this measurement and the aforementioned hypothesis, i.e., the extrapolated value of the applied external force (the acceleration of the centrifugal motion) to the velocity zero was about 80 G (see point C in Fig. 2), the maximum value of the motive force. of the migrating pseudoplasmodium in the dark was obtained. It was about O.6-1.6dyn when the "Mass" of it was O.7×10r5-2.0×10-5gram.. Formula Fe.=nG･Mp=FM wasdefined. where Fex; external fo rce applied to the pseudoplasmodium, FM; motive force of the migrating pseudoplasmodium, i/. Mp; "Mass" of a pseudoplasmodium, nG; "Artificial acceleration" measured actually in this measurement. It was measured as about 80 G, G; acceleration of gravity, 980cm/sec2.. Hence, the maximum motive force of the migrating pseudoplasmodium in the dark could be estimated as O.6-1.6 dyn.. It was converted to O.4×10`-1.0×10` dyn/cm2, which was almost comparable to the motive force of the protoplasmic streaming of Physarum polycephalum (see Appendix 2)). Moreover, in order to compare the value thus estimated with that obtained previously by Inouye & Takeuchi, it was converted to the motive. froce per unit volume of a pseudoplasmodium, which turned out to be O.5×1051.5×105dyn/cm3 (see Appendix 2)). As had been expected it was nearly one or.

(8) 32 M. KITAMI. two orders-of-magnitude smaller than that they had obtained.. The exact reason for such a difference remains unknown, but one reason for this may have been based on the serious difficulty in exerting exact pressure. upon only the pseudoplasmodium in the agar capillary tube, because the capillary. tube was too thin, and because water lay between the surface of the agar tube. and the pseudoplasmodium. In the case of the protoplasmic streaming of Myxomycete, pressures between the twQ chambers were exerted efficiently upon the plasmodia, because the plasmodia were stretched and exposed to the pressures in both chambers.. The monitored pressure inside the chamber was the pressure applied to the plasmodium in the chamber, and that pressure was exactly equal to that exerted to stop the protoplasmic streaming in the capillary.. That is to say, the protoplasmic streaming,in the capillary tube not only was not affected by the capillary effect of the thin tube but also was controlled only. by the pressures applied to the plasmodia in both chambers.. On the contrary, in the case of the pseudoplasmodium of D. discoideum, it was anticipated to be more diflicult to apply the pressure on the pseudoplasmo-dium, because both the anterior tip and the posterior end of it were not in the chambers but in the thin capillary tube. Therefore, a water layer would form between the pseudoplasmodium and the inner-surface of the agar tube, and the pressure ,would be applied to ･this water-surrounded pseudoplasmodium in the capillary tube. It was shwon in a photograph in their report (Inouye & Takeuchi, 1980). Addition of this capillary effect (surface tention, adhesive power). by the surrounding water in the narrow tube may make the motive force to apper greater than its real value. Moreover, the existence of the layers of agar, water, slime sheath, and the interior cells together in the narrow tube. may complicate the movement of the pseudoplasmodium in the tube, inducing the pseudoplasmodium itself to exhibit the capillary effect.. Though the double chamber method is the more sophisticated for the motive force of the protoplasmic streaming, it has such tendency of giving larger value for the motive force of the pseudoplasmodium of D. discoideum, In this centrifugal method, whereas, the direction of migration and also the troublesome question of the pressure in the capillary tube were not restricted. as in the doublechamber method, so the condition of migration of the pseudoplasmodium became closer to the natural condition. We believe that the value thus estimated by utilizing the cenurifugal method approaches pretty well to the real motive force of the migrating pseudoplasmodium of D, discoideum. '. We have now shown that the motive force of the migrating pseudoplasmodium is comparable to that of the protoplasmic streaming of Physarum polyceyhalum plasmodium and is about one order-of-magnitude larger than that of. an ameba (Chaos chaos: the motive force of it was about 2.0×103dyn/cm2; Kamiya, 1964). It seems that production of the motive force of the migrating pseudoplasmodium is brought about by the interior cells of it, since the contractile proteins. ,.

(9) Motive Force of the Cell Mass of D. discoideum 33 have been observed in the motile D. discoideum cells (Clarke et al., 1975; Eckert et al., 1977) and the velocity (motive force) of it is dependent on its size (volume)･. (Bonner et al., 1953; Inouye & Takeuchi 1980). '. (II) Migrating pseudoplasmodium under illuminated condition Introduction.. It has been well known that the cells of D. discoideum show collective motion characterized as migration at the pseudoplasmodial stage. A pseudo-. '. '. plasmodium of D. discoideum has a simple structure, i.e., it contains up to 105 cells which is surrounded by a slime sheath. By collective motion of the cells, however, it can migrate several centimeters on the substratum for a few days. Also, it is widely accepted that the pseudoplasmodium migrates while secreting from its whole surface the slime sheath, which is left behind as the pseudo-. plasmodium moves on. That is to say, the pseudoplasmodium migrates by moving through a tunnel of the slime sheath that it makes for itself.. In addition, the migrating pseudoplasmodium exhibits an extremely sensitive phototactic response. Behavior of the migrating pseudoplasmodium is usually a random walk migration, but it becomes oriented and migrates toward the light source when the pseudoplasmodium is exposed to unilateral light (Bonner et al., 1950). It exhibits the highest response to light in the ranges of 400-450nm and. 550-600nm by migrating toward the light source (Poff & Loomis, 1973). A photoresponsive pigment, which has a absorption spectrum consistent with the action spectrum of phototactic migration of the pseudoplasmodium, has been purified from D. discoideum cell (Poff et al., 1973, 1974). In phototactic migra-. t. tion, the basis of the oriented migration is a turning movement of the pseudoplasmodium, initiated by the turning of the anterior tip of it toward the light source (Poff & Loomis, 1973). This mechanism of turning is similar in concept to the following phenomenon: "A tracked vehicle makes a turn by the difference in speed between the outside and inside tracks" (Loomis, 1975). Some experimental observations and hypothetical models have been established in order to explain the mechanism of pseudoplasmodium migration (Bonner et al., 1950; Francis, 1964; Loomis, 1972; Poff & Loomis, 1973; Poff et al., 1973, 1974; Maeda, 1977;Hader & Poff, 1979a, 1979b; Durston & Vork, 1979). Nevertheless. since no conclusive evidence is available on pseudoplasmodium migration nor its oriented migration, it is not elucidated whether it is the slime sheath or the interior cells that control such collective motion as oriented migration (photo-taxis).. Therefore, it is essential to measure the motive force of the migrating pseudoplasmodium as one of the means to study pseudoplasmodium migration and the mechanism of its oriented migration. The dependence of the velocity of the migrating pseudoplasmodium upon the external force, which could give influence on the motive force of the cell mass.

(10) 34 M. KITAMI. in the pseudoplasmodium, was estimated by an original method utilizing the centrifugal force to investigate quantitatively the motility of the migrating pseudoplasmodium (Kitami, 1981). .As the results of our previous study, the maximum value of the motive force of the migrating pseudoplasmodium in the dark condition was estimated as about O.6-1.6dyn (O.5×105-1.5×105dyn/cm3). It was also suggested that the speed of the slime sheath production was constant, except in cases where condition around the pseudoplasmodia (i.e., light, temperature, humidity, and chemical substances) were changed. In other words, the velocity of the migrating pseudoplasmodium was restricted by its own rate of sheath production regardless of the external force, and the observed value which was nearly constant was interpreted as the upper limit of the velocity in the dark (Kitami, 1981). In order to measure the motive force of the pseudo,. plasmodium in phototactic migration, in the present work, this centrifugal method was utilized.. We were successful in estimating the motive force of it in phototactic migra-. tion, whose estimation had not yet been reported. Furthermore, some interesting results were obtained by measuring changes in the velocity of the pseudoplasmodia placed under two opposite conditions; i. e., combination of illuminated, and parallel or antiparallel to the direction of the external force.. Materials and Methods. The method used in the present study was similar to the previous one (see Part I): D. discoideum strain NC4 was incubated at 200C with Klebsiella aerogenes on the nutrient agar (Bonner, 1947). When D. discoideum amoebae constructed the lumps preceding "finger-like structure", about ten of them were collected and transferred onto the surface of the nonnutrient agar (2% wt/vol) in the measurement vessel constantly kept at 250C. They were then dissociated softly by a looped hair and evenly spread in a line O.1cm in width and 1.5cm in length at about center of the agar field. When the transferred and dissociated lumps of cells had form pseudoplasmodia, the centrifugal force was applied.. The measurement vessel was square in shape (6.5×3.5 cm2) and had a double wall, the height of the inner wall was 1.0cm, and the outer wall was slightly. higher. The inside region surrounded by the inner wall was fi11ed with 2% nonnutrient agar to about O.5cm in depth, in which an iron mesh was set to anchor the agar. In order to take a good photograph of the loci of the migrating pseudoplasmodia, the bottom of the vessel was covered with black aluminium foil. The interspace of the double wall waS fi11ed with water and the yessel was covered with a lid to keep the relative humidity in the vessel at 100%. Thus prepared, four vessels were set on the modified rotor of a centrifuge, the agar surface being in the rotation plane. The white light source was fixed in the center of the rotation plane and its intensity of illumination was about 400lx at the center, of each vessel which was 6cm apart from the light source.. rt.

(11) Motive Force of the Cell Mass of D. discoideum 35 Two of these vessels had a window to allow light in, others were shut off completely from light by covering them with black colored aluminium foil for references.. The mean velocity of the migrating pseudoplasmodium was obtained by the method similar to that used in the double exposure photographs: At intervals of four hours over a period of twenty-four hougs, the rotation was stopped and the loci of all migrating pseudoplasmodia on the agar plate in the vessels were photographed. A total of seven frames (scenes) of the loci extending with the lapes of time were obtained. Mean velocity for each migrating pseudoplasmodia was obtained by the following method, i. e., seven frames (scenes) were projected on the screen in order and the extention of each locus photographed every four '. hours was traced, and then the mean velocities of each migrating pseudoplasmodium were calculated by measuring the length of such extention. In order to have the maximum value of the motive force as exact as possible, the loci of two or three most rapidly migrating pseudoplasmodia, which migrated rectilinearly in the direction Parallel or antiParallel to the centrifugal force in each vessel,. were adopted. The allowance of the difinition of parallel or antiparallel was within ±15 deg. from the direction of the centrifugal force. By such procedures,. four or five runs were carried out for each measurement.. Results and Discussion. Fig. 3 shows the behavior of the migrating pseudoplasmodia under following. - and light conditions: The external force acted upon them to the right (F,.) was illuminated from the left (L). in this figure. i) The velocities of the pseudoplasmodia migrating parallel to the direction of the external force with exposure to light from the bacle were obtained from such loci as (a), ii) and those migrat-. ing antiparallel to it with exposure to light from the front were obtained from. such loci as (b). Four or five runs for each measurement were carried out. The maximum motive force of the pseudoplasmodium in phototactic migration was estimated from the data ii), in which the loci were noticed to be longer. '. than those in the right halves because of their phototactic migration. Two examples are shown in this figure, i. e., external forces are 20 G and 50 G and in. illuminated condition. '. The "Mass" of a pseudoplasmodium used in this experiment was concluded. as O.7×10-5-2.0×10-5gram (see part I). Also, the focus of this work lies on the same hypothesis to the previous one (see part I). That is to say, whether .the motive force is constant or is variable depending on the external force, when the velOcity-decresing rectilinearly with increase in the external force-drops. to zero, the applied external force (F,.) should be equal to the maximum motive force of the pseudoplasmodium in phototactic migration (F' M' ). Fig. 4 shows the changes of the velocities of the migrating pseudoplasmodia. under two conditions when varying strengths of the external force were exerted upon them: i) Pseudoplasmodia migrate Parallel to the direction of the.

(12) M. KITAMI. 36. .---･-. L. ). -s..:::... ..:･:(a). 'x, ,== ;:. ¥t..xA. '. --. t tt --e-e. (b). -b. Fex: 20 G. ') - yt)>. 1.. L (b)':':':.:-V1llf{llltr.ti:':-;':-;'i(a). > Fex: 50 G Fig. 3. Behavior of the migrating pseudoplasmodia under following conditions: The external force acts upon them to the right (Fex) and light is illuminated from the left (L) in this figure. i) The velocities of the pseudoplasmodia. - -->. migrating Paraglel to the direction of the external force with exposure to light from the bacle are obtained from such loci as (a), ii) and those migrating antiParallel to it with expo-. sure to light from the front are obtained from such loci as (b). Four or five runs for each measurement are carried out.. The maximum motive force of the pseudoplasmodium in phototactic migration is estimated from the data ii), in which. the loci are noticed to be longer than those in the right halves because of their phototactic migration. Two examples are shown in this figure, i.e., external forces are 20G and 50 G and in illuminated condition.. external force with exposure to light illumination from the back (solid line A-B).. ii) They migrate antiparallel to it with exposure to light illumination from the. front (solid line C-D). The data in the dark condition, which were performed in the previous study, are also shown in order to compare with the present data in illuminated condition, i. e., they migrate parallel to it in the darfe (solid. line A'-B') and antiparallel to it in the dark (solid line C'-D'). The left and right halves in this figure show the antiparallel and parallel directions of the external force, respectively. (as) show the case of the migrations in the dark and (O) are the data for migrations with exposure to light [illumination. The abscissa indicates the centrifugal acceleration in unit of G, aiming to emphasize the external force, but it can be converted to the unit of force by multiplying by the "Mass" of a pseudoplasmodium. The ordinate shows the velocities of the. `.

(13) Motive Force of the Cell Mass of D. discoideum. 37. Veloctty (mmlh) antlparallel. 2,. D. o. e. o. e. para11e1. o. A. D' le. c C' ,. J". e. B:t. --oe .. B. e. o. o. '. o e. '. o. e A. o,. o. 100 50 O 50 IOO. Centrifugal acceleratlon (G) G:98o cinlsec2. ,. Fig. 4. Changes of the velocities of the migrating pseudoplasmodia under two conditions when varying strengths of the external force are exerted upon them: i) Pseudoplasmodia migrate Parallel to the direction of the external force with exposure to light illumination from the bacle (solid line A-B). ii) They migrate antiParallel to it with exposure to light illumination from thefront (solid line C-D). The data in the dark condition, which were performed in the previous study (part I),are also shown in order to compare with the present data in illuminated condition, ie., they migrate Parallel to it in the dark (solid line A'-B') and antiParallel to it in the dark (solid line C'-D'). The left and right halves in this figure show the antiparallel and parallei directions of the external force, respectively. (e) show the case of the migrations in the dark and (O) are the data.for migrations with exposure to light illumination. The abscissa indicates the centrifugal acceleration in unit of G, aiming to emphasize the external force, but it can be converted to the unit of force by multiplying by the "Mass" of a pseudoplasmodium. The ordinate shows the velocities of the migrating pseudoplasmodia. Individual point represents the mean velocity which is calculated by collecting data from two or three most rapidly migrating pseudoplasmodia in each plate of four or five runs. The acceleration of the centrifugal motion is varied from 10 G to 70 G, G being 980cm/sec2.. migrating pseudoplasmodia. Individual point represents the mean velocity which is calculated by collecting data from two or three most rapidly migrating pseUdoplasmodia in each plate of four or five runs. The aCceleration of the centrifugal motion was varied from 10G to 70 G, G being 980cm/sec2. i) Illuminating the pseudoplasmodia from the back usually results in aturning response of the migration toward the ligh't s'ource. But, from this data, it. was adopted that the pseudoplasmodia continued the migratioA in the same direction as before without turning over (see Fig. 3). The velocities of the pseudoplasmodia migrating in parallel direction to the･external force with. eX;･.

(14) 38 ', M.KITAMI posure to light illumz'nation from the back, were undoubtedly dependent upon the external force (see solid line A-B).. ii) The velocities of the pseudoplasmodia migrating toward the light source in antiParaglel direction to the external force were undoubtedly dependent upon the external force, as had been expected (see solid line C-D). The changes in velocity were consistent with the hypothesis described above. On the basis of both this measurement and the aforementioned hypothesis; i, e., the extrapolated value of the applied external force (the acceleration of the centrifugal motion) to the velocity zero was about 120 G (see point C in Fig. 4), the maximum value of the motive force of the pseudoplasmodium in phototactic migration was ob-. tained. It was about O.8-2.4dyn, which was nearly 1.5times larger than the value in the dark estimated in our previous study. This rate of increase was comparable to the preceding result that light illumination stimulated the velocity. of its migration up to 75% (Poff & Loomis, 1973). So as to estimate the motive force above mentioned,. formula Fex==nG･Mp=FM wasdefined, where Fex; external force applied to the pseudoplasmodium, I712lf; motive force of the pseudoplasmodium in phototactic migration,. M,; "Mass" of a pseudoplasmodium, which was O.7×10-5-2.0×10-5 gram as described before (See partI), nG;' "Artificial acceleration of the centrifugal motion" measured actually in this study. It was m.easured as about 120 G, G; acceleration of gravity, 980cm/sec2.. '. Hence, the motive force of its phototactic migration could be estimated as O.82.4 dyn.. As was described before, the epitome of mechanism of its turning response toward the light source (phototaxis) has generally been taken to be the followings: The light illumination on the unilateral side of the pseudoplasmodium is focused onto the distal side by the "Lens etifect" (Shropshire, 1962), inducing difiierence in light intensity between two sides of it (Francis, 1964; Poiif & Loomis, 1973). This probably brings about the velocity difference in two sides (Francis, 1964). The velocity in outside of the turning pseudoplasmodium is possibly faster than that in inside, because the velocity of the migrating pseudoplasmodium is generally increased by light illumination from the front (e.g., Poff & Loomis, 1973). At present, there are, at least, two possibilities to give rise to the velocity difference; (a) it is probably induced by the difference in speed of the slime sheath production in both sides (Poff & Loomis, 1973), since migration rate of the individual cell dissociated from the pseudoplasmodium has been reported to be unresponsive to light illumination (Samuel, 1961; Francis, 1964) or (b) recent studies have been reported that movement of each D. disocideum cell is responsive to light illumination (Hader & Poff, 1979a, 1979b), suggesting a possibility of direct effect of light illumination on the difference in. ,.

(15) Motive Force of the Cell Mass of D. discoideum 39 motility of cells in both sides of it. Which factor, the difference in speed of slime sheath production or in motility of the interior cells, can contribute to its. turning response (oriented migration)? On the basis of our results, it is no doubt. that light illumination from both the front and the back can directly influence upon the motility of the migrating pseudoplasmodia.. When the pseudoplasmodia exposed to light illumination were compared with those in the dark, light illumination from the front increased their velocities and their increments from those in the dark showed nearly constant values (see solid lines C-D and C'-D' for comparison). Possible reasons, at least, for this increment of its velocity seem to be both (A) because of speed of the slime sheath production and (B) because of motility of the interior cells, those may be mainly stimulated at the illuminated anterior part of the migrating pseudoplasmodium. Therefore, in regretable, although the motive force of the pseudoplasmodium in phototactic migration can be estimated (see the result ii)), con-. clusive information to make us determine eather of the two reasons above mentioned has not been taken out from these data. On the contrary, light illumination from the back caused quite opposite results; i. e., their velocities were decreased and their decrements from those in. the dark dependent strongly upon the external force (see solid lines A-B and A'-B' for comparison). As the condition of the applied external force (parallel to the direction of its migration) in this case (solid line A-B) was the same to. that in the dark (solid line A'-B'), their decrements from those in the dark were obviously caused by only light illumination from the back. Moreover, this effect of light illumination from the back seemed to affect directly to its motive. force and/or mechanism of its migration, since their decrements were large when the external force was small (see points A and A' for comparison) and they got smaller with an increase in the external force. These data seem to be great interest so as to research the true character of its turning response (phototaxis).. It is thought to be chosen, from these results, whether it is the slime sheath or the interior cells that control this difference in velocity in both sides of it ,. during its turning response. It will be discussed in the near future, in the following report, a possibility. that changes in the motility of the interior cells in both sides of the pseudoplasmodium, inducing by light illumination, is directly participating in the turn--. ing response of it during phototactic migration.. (III) Culminating fruiting body Introduction. Another expression of the collective motions is culmination of the fruiting. body. At the end of the pseudoplasmodial stage, the pseudoplasmodium stops its migration and its anterior tip moves vertically in preparation for the final.

(16) 40 M. KITAMI. differentiation which results in the formation of the fruiting body. When the culmination starts, a funnel made of a cellulose-containing ring which forms the stalk sheath appears near the tip. The peripheral cells near the tip are lifted up along the funnel, and being obstructed by the reticulately packed cells around. the tip, they fall into the funnel, and then become vacuolized and lay down the･. cellulose walls. As a result of the repetition of this. .. process, the vacuolized cells are piled up in order and "-. .ri thestalkrisesup.Thisprocessislike"afountain running backward" (Loomis, 1975). In this process, the cells seem to be lifted up along the rising stalk by accepting or producing some type.of force. The shape of the stalk of D. discoideum usually. Fcu. resembles a cone-shaped tower constructed with bilding blocks. The number of the cel!s composing each steps of the stalk decrease as they are piled up to construct. A'. the fruiting body. About this problem, in the present. study, an interest phenomenon was observed; that is, the shape of the stalk formed on the substratum without culmination into the air was cylindrical.. In order to obtain the fundamental information Fag se. .e). v- :t. tv Fig. 5.. The carica-. for the collective motion of the cells in the culminating fruiting body, the effect of the "Artificial gravita-. tion" upon the culminating fruiting body was investigated by utilizing the centrifugal force; consequently, the motive force of the cell mass at the culmination stage was estimated, whose any informa-. ture shows that balancing the motive force to. tions have not yet been available, by balancing the. plied external force. . until the cell mass force ("Artificial gravitation" F.,). - to apculminate (Fcu). (Artificial gravitation;. Fag). in the culminating. fruiting body.. . to applied external motive force to culminate (Fb.) would be able to culminate no further (Fig.5). The motive force thus obtained was compared with that of the migrating pseudoplasmodium.. Materials and Methods. the incubation of D. discoideum strain NC4 was the same to The method of the previous one. When D. discoideum amoebae constructed the hemispherical. Iumps preceding"Mexican hat", about ten of them were transferred separately by a looped hair ontothe surface of the nonnutrient agar (2% wt/vol) in the measurement vesselconstantly kept at 250C. After an hour, the centrifugal force ("Artificial gravitation") was applied. The measurement vessel was square. in shape (6.5×3.5cm2) and the nonnutrient agar was poured into about O.3cm prepared, four vessels were set on the modified rotor of a cenin depth. Thus trifuge, the agarsurface being perpendicular to the 'rotation plane. They were. '.

(17) MotiveForceoftheCellMassofD.discoideum ･41 .t. rotated for 12hours at 25eC. All of those vessels were shut off completely from light. By using such arrengement as the centrifugal force (external force) could be exerted perpendicularly upon the surface of the agar, the external force could be affected in opposite direction to the motive force of the cell mass in the culminating fruiting body.. After 12hours of rotation, the states of the culmination of the 'fruiting bodies on the surface of the agar were obtained by the photomicroscope, then the relative culmination efficiency to the control under 1G was obtained. The definition of the relative culmination efficiency (%) is. culmination ethciency under "Artificial gravitation" × 100 culmination efficiency under "1 G" .. The culmination efficiency (%) is defined as. number of the normal fruiting bodies number of the lumps transferred × 100 Results and Discussion.. '. Fig. 6 shows the "Artificial gravitation"' dependence of the efiiciency of the. culmination of the fruiting body. The abscissa indicates the magnitude of the "Artificial gravitation" in unit of G, G being 980cm/sec2. The ordinate indicates. '. G. 9.. 5. ･- 100. v. ',". e. ￠o. -H'. `. efficiency. 1. g O. ' rela. culmi.. E- 50. as･. o. .S ee. IO 15 20 30 40 50. 100. 85 70 37 40 l4 2. l. ".. e 1 10 20 -30 40 50 Artificial gravitation; G=980 crn/s 2. Fig. 6. The "Artificial gravitation" dependence of the culmination efficiency of the fruiting body. The abscissa-indicates the magnitude of the "Artificial gravitation" in unit of G, G being 980 cm/sec2. The ordinate indicates the relative.culmination efficiency (% of a control at 1G)..

(18) 42 M. KITAMI. the relative culmination efficiency (% of a control at 1G). Usually, the cell mass of D. discoideum are able to construct the fruiting. body within about ten hours in the culmination stage. In the present study, however, when the "Artificial gravitation" against the direction of culmination was increased, the ecaciency of culmination decreased distinctly and hardly any cell masses could culminate at about 40-50 G. Meanwhile, among the cell masses which could not culminate, some had been resting at the period of the preculmintion stage (they were mound in shape) and others had'been migrating as the pseudoplasmodia, forming stalks on the agar (usually, the pseudoplasmoda of D. disccideum do not migrate while forming the stalks). Moreover, although the shape of the stalks culminated into the air were cone-shaped tower, that remain-. ing on the agar were cylindrical. It must be noted that, when every disculminated cell mass under such various gravitational conditions were held at 1G again, almost all of them had constructed perfect fruiting bodies and their culmination efficiencies had regained the values at 1G. Using this method, it. may be possible to control the morphogenesis and/or the time range of the culmination stage to some extent.. We supposed that the culminating fruiting bady should have two stePs in its culmination mechanism: The first step is represented by the cell movement along the rising stalk; that is to say, the cell mass are lifted up along the funnel by accepting or producing some type of force before falling into the funnel at the top area of the sorus of the fruiting body. The second step, elongation of such fallen cell as the prestalk cell of the cell mass vacuolized in. the funnel, is analogous to elongation of the knotcell of a bamboo. Therefore, the first step might be sensitive to the gravitational force, since it is proposed that the elongating force per se would be hardly affected by the gravitational force.. On the other hand, the motive force of the cell mass in the culminating fruiting body was estimated as O.3-1.0dyn, whose any estimations had not yet been reported, provided the "Mass" of the cell mass lifting up along the stalk. was about O.7×10-5-2.0×10-5gram because the "Mass" of the cell mass in the fruiting body at the starting time of culmination can be supposed to be almost equal to that of a pseudoplasmodium. In order to estimate the maximum motive force of the cell mass in the culminating fruiting body,. Formula F=nG･M, wasdefined, where F; motive force of the cell mass in the culminating fruiting body, Ms; "Mass" of the cell mass obtained the above mentioned suppositon, nG; "Artificial gravitation" exerted actually upon the culMinating fruit-. ing body. It was measured as about 50 G against the appoximate maximum value of the motive force of culmination, G; accelaration of gravity, 980cm/sec2..

(19) Motive Force of the Cell Mass of D. discoideum 43 hence, the maximum motive force of the cell mass in the culminating fruiting body could be estimated as O.3-1.0dyn. It has not been possible to compare the motive force of the cell mass at the pseudoplasmodial stage with that at the culmination stage until the motive force of the culminating fruiting body was estimated. The motive force of the culminating fruiting body thus obtained had the similar order-of-magnitude to that of the migrating pseudoplasmodium. On the basis of these consideretions, we concluded that the "Artificial gravitation" affected upon the first step of the culmination; i. e., affected directly upon .. cell motility in the culminating fruiting body, and moreover, the motive forces. of both migration and culmination might be brought by similar mechanism and/or similar types of cells.. It will also.be discussed before long, in the follewing report, that a model to interpret the cell motility both in the migrating pseudoplasmodium and in the culminating fruiting body.. Appendix 1) The formula to calculate the "Mass" of a pseudoplasmodium was given as. C･Ma･Vp/Va where Ma; Dry weight ("Mass") of an amoebae of D. discoideum. It was measured as 5.5×10-!i gram by application of an interference microscope method (Hieda & Ishizaka, personal communication*2),. VP; The volume of a pseudoplasmodium used in the present study. It was calculated as 1.1×10-5cm3, since its shape could be regarded. ' asapproximatelyhemicolumnar,itslengthandwidthweremeasured as the values described before,. Va; The volume of an amoebae. It was calculated as 1.6×10-iOcm3, which was comparable to other data (Bonner & Frascella, 1953), assuming that it was a pancake in shape with 10 ptm in diameter. .. and 2pm in thickness, Vp/Va; The total number of the amoebae constituting a pseudoplasmodium. It was supposed that nearly 105 amoebae were in a pseudoplasmodium used in the present study, c; A constant value. It was assumed that 809oi of the constituted of an amoebae was water. hence, the "Mass" of a pseudoplasmodium used in the present study could be estimated as nearly O.7×10-5 gram.. *2) K. Hieda, Biophysics Laboratory, Rikkyo University; S. Ishizaka, Institute of Biological Sciences, University of Tsukuba; Biomass of Animals of Terrestrial Ecosystems in Japan, Rikkyo Univ. Press, (1978)..

(20) ;. 44 M. KITAMI Appendix 2) The "Balance pressure" of the protoplasmic streaming of Physarum polycePhalum plasmodia was measured by the double chamber method as 20cmH20 and less (Kamiya 1953). This measured value was converted to 2.0×10` dyn/cm2. The motive force of a pseudoplasmodium in the present study could be also converted to O.4×10`-1.0×10`dyn/cm2, since an area of a transverse section of a pseudoplasmodium could be calculated as 1.6×10-`cm2. The motive force per unit volume of a pseudoplasmdoium in this study was. calculated as O.5×105-15×105dyn/cm3, since the volume of a pseudoplasmodium could be calculated as 1.1×10"5cm3.. Acknowledgements I thank Professor Y. Nagahara and Professor T. Yamada for their valuable discussions. This work was greatly assisted by the efforts of Mr T. Kato, Miss M. Fukuda, Mr T. Sakai and Mr M. Ohta.. References BoNNER, J.T. (1947): J. Exp. Zool. 106, 1-26.. BoNNER, J.T., W.W. CLARK, C.L. NEELy, JR. & M.K. SLiFKiN (1950): J. Cell. comP. Phisiol. 36, 149-158. BoNNER, J.T. & E.B. FRAscELLA (1953): Biol. Bztll. 104, 297-300.. BoNNER, J.T., P.G. KooNTz & D. PAToN (1953): Mycologia 45, 235-240. BoNNER, J.T, (1967): The Cellular Slime Molds, 2nd edn. New Jersey: Princeton University Press. CAppucciNELLi, P. & J.M AsHvsioRTH (1977): DeveloPments in Cell Biology, Vol. 1 DeveloPment and Differentiation in the Cellular Slime Moulds. Amsterdam: ElsevierlNorth-Holland. CLARKE, M., G. ScHATTEN, D. MAziA & J,A. SpuDicH (1975): Proc. Nat. Acad. Sci. USA 72, 1758-1762. DuRsToN, A.J. & F. VoRK (1979): 1. Cell Sci. 36, 261-279. EcKERT, B.S., R.H. WARREN & R.W. RuBiN (1977): J. Cell Biol. 72, 339-350. FRANcis, D.W. (1964): J. Cell comp. PhNsiol.'64, 131-138. HADER, D-P. & K. PoFF (1979a): Photochem. Photobiol. 29, 1157-1162. H.?ilRDER, D-P. & K. PoFF (1979b): ExPl. Mycol. 3, 121-l31. INouyE, K. & I. TAKEucHi (1980): J. Cell Sci. 41, 53-64. KAMiyA, N. (1953): A. ReP. Sci. VVorles, Fac. Sci., Osalea Univ. 1, 53-83.. KAMiyA, N. (1964): Primitive Motile System in Cell Biology. (ed. by ALLEN, R.D. &. .. N.KAMiyA)pp.257-277NewYork:Acad.Press. KiTAMi, M. (1981): IX Congress of the International SocietN of DeveloPment Biologists. (Basle/Switzerland). '. LooMis, M.F. (1972): Nature, new Biol. 240, 6-9. LooMis, W.F. (ed.) (1975): Dictyostelium discoideum: A DeveloPmental System. New. York: Academic Press. MAEDA, Y. (1977): DeveloP., Growth and Diffea, 19, 201-205.. MAEDA, M. & Y. MAEDA (ed.) (1978): The Biology of Slime Moulds UP Biology: Tokyo Univ. Press (in Japanese).. s.

(21) Motive Force of the Cell Mass of D. discoideum 45 PoFF, K. & W.F. LooMis (1973): ExPl Cell Res. 82, 236-240. PoFF, K., W. BuTLER & W.F. LooMis (1973): Proc. nat. Acad. Sci. USA.70, 813-816. PoFF, K., W.F. LooMis & W. BuTLER (1974): 1. biol. Chem. 249, 2164-2168. SAMuEL, E.W. (1961): Devl Biol. 3, 317-335. SHRopsHiRE, W. (1962): J. gen. PhJ,siol. 45, 949-958.. YAMADA, T. (arr.) (1980): The Biology of Slime Moulds (ed. J.M. AsHwoRTH & J. DEE), Asakura-Arnold Biology; 16 (in Japanese). YAMAMoTo, M. (1977): DeveloP., Growth and Differ. 19, 93-102. YAMAMoTo, M. & N. KAMiyA (1971): JaP. J. Devl Biol. 25, 84-85 (in Japanese).. '.

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