Dynamic phenomena in microspheres from thermal proteinoid

Comp. Biochem. Physiol., 1964, Vol. 11, pp. 317 to 321. Pergamon Press Ltd. Printed in Great Britain

DYNAMIC PHENOMENA IN MICROSPHERES FROM THERMAL PROTEINOID* S I D N E Y W. F O X and S H U H E I

YUYAMA

Institute for Space Biosciences, The Florida State University, Tallahassee, Florida (Received 27 M a y 1963)

A b s t r a c t - - T i m e lapse photomicrographic e+idence is presented for the occurrence in self-organized supramacromolecular units of: 1. Optical disappearance of interior material to leave outer boundary or membrane ; 2. Brownian motion within the unit; 3. Appearance of septa which divide the units. These phenomena occur in a suspension of particles formed from thermal polyamino acid. The dynamic phenomena are triggered by an increase in pH with phosphate-citrate buffer. The formed units and the phenomena they display arise from the material which is formed also in an experimental continuum, and thus provide a model of how some precellular phenomena might have arisen spontaneously. INTRODUCTION THERMAL proteinoids containing the eighteen amino acids found also in protein (Fox & Harada, 1958, 1960) have been shown to form microspheres. Because of their origin, composition, stainability and m a n y other properties (Fox e t al., 1959a, b; Fox, 1960; Fox et al., 1962; Fox & Yuyama, 1963a, b) they are unique as cell models. T h e y are of interest also as precell models alternative to the coacervate droplet (de Jong, 1949) which has been interpreted in a precellular context by Oparin (1957). T h e purpose of this paper is to record several dynamic p h e n o m e n a which may be c o m p a r e d with processes in contemporary bacteria. T h e presentation is entirely f r o m time-lapse photographic sequences; the principal observations are representative of m a n y time-lapse sequences and also of similar behavior patterns observed in this laboratory for a n u m b e r of years. M E T H O D S AND MATERIALS T h e preparation and characteristics of the thermal proteinoid (2 : 2: 1) used have been described (Fox & Harada, 1960). Microspheres were prepared by the * Contribution No. 15 of the Institute for Space Biosciences. Work supported by Grant C-397I of the National Institutes of Health, U.S. Public Health Service and by Grant NsG-173-62 of the National Aeronautics and Space Administration. Some of the material appears in outline form in the First Annual Report of the Institute for Space Biosciences (1962). 21

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hot-to-cold method (Fox et al., 1959a) although it has been learned that contact of amorphous proteinoid with cold water will automatically yield microspheres. A typical polymerization involved first heating 10 g of z-glutamic acid at 175-180°C until molten (about 30 rain). To this was then added 10 g of Dz-aspartic acid and 5 g of an equimolar mixture of DL-alanine, L-arginine monohydrochloride, L-cystine, glycine, L-histidine hydrochloride monohydrate, DL-isoleucine, Dz-leucine, L-lysine monohydrochloride, DL-methionine, DL-phenylalanine, z-proline, DL-serine, DL-threonine, DL-tryptophan, Dz-tyrosine and DL-valine. The solution was maintained at 170 + 2°C under nitrogen for 4 hr. The ambercolored vitreous mass was rubbed with water, which converted it to a yellow-brown granular precipitate. This material could be used directly for experiments in production of microspheres, or it could be washed by dialysis as a solid. The washed material yielded microparticles which were less plastic than those obtained from crude polymer. Yields were typically in the range of 10-25 per cent depending upon time of heating, degree of purification from smaller polyamino acids, etc. For the sequence shown in Figs. 1-16, the suspension of microspheres at a pH of about 3"0 was treated under the coverglass with a drop of McIlwain's acetatephosphate buffer of pH 6"0. The standard interval between frames was 30 sec. Accordingly, No. 161 is 80 min later than No. 1. The equipment employed was a Bolex camera photographing through a Leitz Ortholux microscope and regulated by a Sanders timer. The magnification used with the microscope was 450 x although Figs. 1-16 are larger than that magnification.

RESULTS The phenomena of most interest are indicated by arrows and the letters A-C. Only one frame in each entire sequence is designated. Separate frames are used for each letter and arrow merely to prevent crowding in the designations. The large microsphere, A, is representative of many series in which the interior disappears progressively and entirely, leaving behind the outer boundary or membrane. As this phenomenon is watched through the microscope, Brownian motion of the residual center can be observed. The effects of such Brownian motion are especially evident in pictures 7-16 which show the residual center at different positions relative to the geometric center. A second phenomenon is observed in a number of individual units. This is a tendency to cleave, or for centers to separate. Two microspheres which best illustrate this tendency are those at B and C. The change in B is most marked from No. 6 to No. 9 and from No. 10 to No. 16. The most marked changes in C are also evident in Nos. 11 to 15. These results provide at least a partial answer to the phenomena involved in producing twinned microspheres as presented in Figs. 3 and 4 in another paper (Fox & Yuyama, 1963b). Transformations of this type have been recorded in numerous time-lapse sequences.

FIGS. 1-16. Time-lapse photographs showing movements in particle A and cleavage in particles R and C. (Enlargements of 2, 3, 14 and 15 are on the next page.)

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DISCUSSION The time-lapse sequence of Figs. 1-16 illustrates a striking difference in behavior between the outer layer and the interior. A provocative question of the constitutional difference between the outer layers and the inner material is thus posed. Studies of composition and of differences in physical state are under way. The formation of cell-like units from thermal polyamino acids was postulated (Fox, 1957) a year in advance of the first experiment yielding such microspheres on the basis of probable heat denaturation, in hot water, of the surface of globules of thermal polyamino acid. The correctness or incorrectness of this postulate of "denaturation" as the basis for the phenomenon has, however, not been established. The phenomenon depicted in A is also part of the basis for regarding the boundary as a membrane, as the membrane is ordinarily defined (Kruhoffer, 1961 ; Kuyper, 1962). The selective diffusion from the interior without disappearance of the material of the boundary itself is supported by electron micrographs (Fox & Fukushima, 1964). Of several kinds of cleavage observed in experiments with microspheres under various conditions in this laboratory, the type of division simulated in B and C and in other units in this time-lapse series resembles that of septate separation, as observed in the cocci (Bisset, 1955). Cell models which can be made to undergo cleavage have long been known (Thompson, 1943). Such widely diverse materials as mercury, soap and gelatingum arabic coacervates tend to form droplets which in turn tend to form smaller droplets. This tendency is evidently common in microspherical units above a given range of size, regardless of the material constitution of the droplet or bubble, with undoubtedly a different critical range of size for droplets of different constitution. Oparin has for instance pointed out that coacervate droplets divide into smaller ones when they are shaken (Oparin, 1957, p. 357). A crucial difference in this respect between the coacervate droplets and the proteinoid microspheres may be simply, as in other comparisons, that the microspheres from proteinoid have a degree of stability that results in relatively slow changes. This degree of stability is indicated also by the secfionability which permits electron micrography (Fox & Fukushima, 1964). A full evaluation of each of the various phenomena depicted will require at least a study of which other phenomena, some not recorded here, can be coupled with cleavage of the sort depicted; such evaluation may require extended research. Of particular interest, however, is the fact that a new cell model made from material more closely resembling cellular protein than any other synthetic polymer has in greater measure than other models the numerous known properties (see bibliography) of the cells being modeled. Among the discernible characteristics which appear to distinguish this model from some contemporary cells are (1) a different and less aqueous consistency, (2) absence of energy production, (3) absence of biological materials such as RNA and DNA and (4) absence of sensitivity to low concentrations of salt. Other differences must exist. Distinctions between the model and a first primitive cell are, of

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course, not subject to analysis since the total attributes of a primordial cell or pre-cell are unknown. None of the four recognized distinctions is either absolute or final. The possibility of altering the proteinoid microspheres in each of the known or unknown characteristics can, of course, be visualized. The changing status of understanding of the origin of nuclear material and of nucleic acids is illustrative. On one hand, some evidence has been accumulated for the thermal polymerizability of mononucleotides to polynucleotides under conditions which are the same as (Fox et al., 1962 ; Fox & Yuyama, 1963b) or very similar to (Calvin, 1962; Schramm, 1962) those employed for the polymerization of amino acids. On the other hand, the need for the usual nuclear phenomena in mitosis has been questioned (Harvey, 1960). Basically, many of the properties of true cells can increasingly be understood as due to the general self-organizing properties of macromolecules (cf. Wald, 1954; Mandelkern, 1956; Schmitt, 1956; Picken, 1960; Waugh, 1961; Calvin, 1962; Keller, 1962). The findings of these morphological phenomena in various polymers constitute a striking new chapter of knowledge. Some macromolecules have more morphogenic properties than others. The course of future research with cell models may well be in part that of finding which alterations in macromolecules such as proteinoids and which associations with other molecules decrease the gap between the model and that which is being modeled. Inasmuch as the phenomena illustrated in Figs. 1-16 result from an experiment in which the pH was altered, change in charge distribution in the macromolecules and in the microscopic units is probably among the causes of some of these kinds of behavior in the experiments represented in this paper. These data and interpretations point to the possibility that the salient answers to the problems of the first cell may derive primarily from an empirically gained understanding of polymer morphology instead of relying solely upon concepts of direct control of morphology by genes (cf. Horowitz & Miller, 1962), especially when viewed in the rigorous context of cellular origins. The concept of indirect control, however, of morphology by genes is compatible with a theory of coded control of amino acid sequence by nucleic acid and concomitant self-arranging of the protein macromolecules. When one considers as a sequence of inexorable processes the formation of amino acids, polymers of amino acids, formed microspherical units and a tendency of these latter to cleave to smaller units, as illustrated here, a versatile experimental model is available for study in the laboratory in a way that can be imputed to natural experiments on the primitive earth (Fox, 1960; Morrison, 1962; Fox & Yuyama, 1963b).

Acknowledgements--Others who have observed various stages in spontaneous cleavage of microspheres include Ronald F. Fox, Jean Kendrick, Kent Stewart and Charles Ray Windsor. The authors are indebted particularly to Robert McCauley for much photographic work.

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