- Email: [email protected]
The role of cell surface components in the morphogenesis of the cellular slime molds
TIBS - June 1976
130 would inhibit the respiratory chain. On adding the purine nucleotide. the conductance decreased to levels similar to those of liver or heart mitochondria. It therefore appears that there is a proton-conducting ‘channel’ crossing the inner membrane of these mitochondria, which can be switched off by adding a low concentration of a purine nucleotide to the incubation medium. It has been found that the nucleotides bind very specifically to a site on the outer face of the inner membrane of brown fat mitochondria (see [4]), and in so doing block the conductance of the pathway. Although of significance, physiological doubtful chloride also appears to be conducted by the channel (see [4]). To explain this dual specificity it is probable that instead of protons passing into the matrix on the channel, the experimentally indistinguishable efflux of hydroxyl ions from the matrix occurs. While it is unlikely that the physiological modulator of the proton conductance is simply a variation in the cytoplasmic level of certain purine nucleotides, it is very likely that this channel represents the final molecular site of thermogenesis in the tissue, short-circuiting the proton circuit, and allowing rapid respiration to occur in the absence of ATP synthesis. It is signilicant that the channel may possibly’be induced at birth and on cold-adaptation of non-hibernators, while hibernators appear to retain the channel regardless of the environment (see [3,4]).
7 Hohorst,
References Joel, C. D. (1965) in Handbook qf’Ph.vsiology, AdiA. E. and Cahill. G. F., eds) pose Ti.ssw(Reynold, Section 5, pp. 59-85, American Physiological Society, Washington D.C. Lindberg, O., ed. (1970) Brown Adipose Tissue, Elsevier, Amsterdam Flatmark, T. and Pederson, J.I. (1975) Biochim. Biophys.
Acru 4 16, 53-103
Nicholls, D.G. (1976) FE&S Lrtt. 61, 103-110 Mitchell, P. (1966) Chrmiosmoric Coupling in Oxidutiw crud Photosynrhefic Phosphoryiulion, Glynn Research, Bodmin, Cornwall Coupling und Mitchell, P. (1968) Chemiosmofic Energy Transduction, Glynn Research, Bodmin, Cornwall
Sqier’s
H-J.
and
Z. Ph?siol.
Rafael,
J. (1968)
Hoppe-
Chem. 349, 268-270
Rafael, J., Ludolph, H-J. and Hohorst, H-J. (1969) Hoppe-Seylrr’s Z. PIz~siol. Chunz. 350, 1121-1131 9 Mitchell, P. and Moyle. J. (1967) Biochem. J. 105, I14771 162
10 Nicholls,
D.G.
(1974) Eur. J. Biochem.
50, 305-
315
H. ( 1975) Bioenrrgetics 7, 61~ 74 I1 Rottenberg, l2 Bernson, V. and Nicholls, D.G. (1974) Eur. J. Biochem.
47. 5 17-525
The role of cell surface components in the morphogenesis of the cellular slime molds William A. Frazier The interaction of surface-bound lectins with complementary carbohydrate-containing receptors seems to be the basis for species-specific cell-cell adhesion in cellular slime molds.
The cellular slime molds, and in particular the species Dictyostehium discoideum, have been for several years a popular and extensively studied model system for eukaryotic development [ 11.The ease with which large numbers of homogeneous cells can be culOxidation at 4” C tured and synchronously stimulated to There is one final hurdle to be overcome begin their developmental program has made these organisms prime subjects for by brown fat mi.tochondria from hibernators to fulfil their physiological function, the study of several aspects of developand that is the ability to function at very ment, including the control of enzyme expression and the regulation of RNA synlow temperatures to protect and aid arouthesis [2]. Many mutants in various devesal of the hibernating animal. Here again lopmental functions have been described brown fat mitochondria are perfectly adapted for their role. At low temperaand a large body of morphological data tures, the citric acid cycle of brown fat has provided extensive correlations of mitochondria can scarcely function [ 121, phenotypic expression with such genetic perhaps due to the many diffusion-limited lesions [2,3]. The life cycle of D. discoideum indicates steps occurring in the matrix. In the absence of an alternative pathway thermothe many developmental functions which genesis would therefore be impossible, as may be studied in this organism (Fig. 1). acetyl-CoA from &oxidation could not be Single amoebae (haploid, 7 chromosomes) further metabolized. Fortunately for the feed on bacteria and grow and multiply hibernator, an alternative pathway exists as vegetative or non-social cells. Upon exhaustion of the food supply, the amoebae whereby acetyl-CoA is simply hydrolysed begin differentiation [4]. Cells aggregate to acetate. Fatty acid oxidation to acetate can still occur at 4’ C [ 121, and it may be in response to pulses of cyclic AMP, forming clusters of lo4 to lo5 cells in 610 h. that one of the reasons for the periodic arousal of hibernators is to give the peri- During this period, the cells also become cohesive and form stable intercellular attapheral tissues a chance to oxidize acetate accumulating in the animal, in much chments so that a multicellular organism the same way as the liver removes lactate Depurtments of’ Biological Chemistry and Neurobioaccumulated in the body after violent logy, Division of Biology and Biomedical Sciences, Washington University, St Louis, Miss. 63110, U.S.A. exercise.
is constructed (12 hours) called a pseudoplasmodium, slug or grex. The cells do not fuse, in contrast to the Myxogastria (Myxomycetes), or acellular (true) slime molds [ 1,5]. Under certain conditions, aggregating cells may form macrocysts rather than slugs, thus initiating a sexual phase during which meiosis occurs and which gives rise to normal haploid amoebae [6]. More commonly, the slug is formed, migrates for a time, and then further differentiatiates so that the front one-third of the cells form a plant-like stalk, bearing aloft the remaining two-thirds of the cells which become spores [4]. These are very resistant to adverse conditions, and when germination occurs, each gives rise to a single haploid amoeba, thus completing the life cycle. At this stage, cell fusion may occur with a very low frequency (about 10d4) yielding diploid cells in a para-sexual cycle 171.
In recent years, several groups have focused their investigations on events which occur during the initial aggregation phase and which involve functions mediated at the cell surface. These are the morphogenetic functions of chemotaxis and selective cell adhesion, processes which, in some form, play a role in the morphogenesis of all higher organisms. Since the discovery of a chemoattractant or acrasin by Bonner [8] and its subsequent identification as cyclic AMP [9], much
131
TIBS - June 1976
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elegant work has been done in defining the chemotactic process and in identifying the macromolecular components which generate [lo], detect [ 11,121 and relay [ 131 the chemotactic signal. The net result of this information relay system is that cells move toward aggregation centers in pulsatile fashion in response to radially propagated waves of cyclic AMP [12,13]. The primary purpose of this review is to summarize the current view of the mechanism by which this aggregation results in a stable, multicellular organism, that is, the nature of intercellular adhesive bonds. Characteristics of cell adhesion Concomitant with the appearance of the chemotactic apparatus, differentiating slime mold cells acquire the ability to
adhere to one another. This cohesiveness can be assayed quantitatively by monitoring changes in the light scattering properties of a cell suspension [ 141or by swirling cells in an EDTA-containing buffer and determining the number and size of aggregates formed with a Coulter particle counter [15]. The presence of EDTA eliminates the slight adhesion observed with non-differentiated, vegetative cells. If slime mold cells’are separated from their bacterial food supply and gyrated in dilute phosphate buffer dr plated on moist millipore filters, significant EDTA-resistant cohesiveness appears after about 6 h. At about the same time, the chemotactic system becomes functional and aggregation is complete by about 12 h. Coincident with the completion of aggregation, the binding capacity of the
cells for cyclic AMP decreases [ 11,161,suggesting a ‘down regulation’ of the chemotactic receptors. This may represent a mechanism for turning off chemotaxis after aggregation is complete. Further evidence that cell-cell contact may have a role in regulating differentiation is found in the effects of membranes from cohesive cells on the expression of six enzymes appearing during development. The addition of these membranes to cells at the beginning of differentiation blocked the acquisition of aggregation competence [2] perhaps indicating blockage of the chemotactic apparatus. Four of the six enzymes were not made at all, one appeared at the normal time and rate and another, alkaline phosphatase, appeared early and reached twice its normal peak activity [2]. These consequences of membrane interaction may indicate that the sequence of events is important for the proper expression of surface mediated differentiative changes. The aspect of cell aggregation in the slime molds which is most relevant to their use as a model system for cell adhesion in higher organisms is species specificity. If labeled vegetative amoebae of different species are mixed together and differentiation allowed to proceed, the slugs which result contain only one species of cell. Thus, segregation or sorting out of different species has occurred. In some cases this process is aided by the fact that the mixed species employ different chemotactic agents (acrasins) [ 161 so that the geographical location of the aggregation centers is distinct. However, in other com-
TIBS - June 1976
132 binations of two closely related species such as D. discoideum and D. mucoroides which both appear to use cyclic AMP as their acrasin [ 161, mixed aggregates form which then give rise to two separate slugs of homo-specific cell type [17,18]. This clearly indicates that true sorting out has occurred and that a cell recognition apparatus per se does indeed exist.
Nature of the cell adhesion apparatus Gerisch and his colleagues have taken an immunological approach to define some properties of the surface membrane of differentiated cells which are relevant to adhesion. Antibody was prepared to cohesive D. discoideum cells, adsorbed with vegetative amoebae and fragmented to yield FAB. These FAB fragments were found to block EDTA-resistant cell cohesion by preventing the end-to-end contact found in streams of cells entering aggregates [19]. The cell surface antigen is termed contact site A. Another surface antigen, contact site B, is present in both vegetative and cohesive cells [20]. However, univalent antibodies to this antigen block cell adhesion by preventing side-toside contact. Thus, two apparent classes of contact ‘sites’ with distinct spatial distributions on the cell surface are involved in cell adhesion. Recently, progress has been made in purifying these contact sites from detergent extracts of particulate fractions of D. discoideum [21]. A different, and perhaps more direct approach, to the study of the macromolecular species responsible for cell cohesion was initiated by the observation that, as six species of slime mold develop aggregation competence, there appear in crude extracts of the cohesive cells proteins which are potent agglutinins of erythrocytes [22]. These agglutinins are multivalent carbohydrate binding proteins which have distinct sugar specificity in each species [22,23] even though they are similar in subunit molecular weights, amino acid compositions and, in some cases, their tryptic peptide fingerprints [23]. The relevance of these lectins to the cell adhesion process has been investigated in detail in the two species D. discoideum and Polysphondelium pallidurn. The lectin activity of D. discoideum cells accumulates during differentiation with a time course very similar to the development of cohesiveness [ 151 and the appearance of contact sites A [19]. The activity has been purified using Sepharose 4-B, a galactose-containing polymer, as an affinity adsorbent [24,25]. The purified protein was found to contain two lectins, both with a native molecular weight near 100,000. The major lectin, designated discoidin I, has a subunit molecular weight
of 26,000 and the minor lectin, called discoidin II, has a subunit weight of 25,000 [25]. The two lectins are also distinguishable on the basis of their isoelectric points, amino acid compositions, tryptic peptide maps, the types of red cells which they agglutinate, the simple saccharides which inhibit their agglutination activity, and their time course of appearance during differentiation [25]. Lactose is the best inhibitor of discoidin II and N-acetyl-D-galactosamine is the best inhibitor of discoidin I. Glucose and mannose do not inhibit either lectin [25]. The developmentally regulated lectin activity of P. pallidurn (called pallidin) has been purified by affinity adsorbtion on formalinized (fixed) human type 0 and rabbit erythrocytes [23,26,27]. The native lectin aggregates extensively forming decamers of about 250,000 mol. wt and higher aggregates. The proteins purified with either type of red cells have a subunit molecular weight of 25,000 [23,27]. However, the lectin purified with rabbit erythrocytes appears to consist primarily of subunits with a slightly basic isoelectric point while that purified with type 0 erythrocytes consists of species with acidic isoelectric points. Furthermore, the two products have slightly different sugar specificities, although both are very sensitive to inhibition by lactose and melibiose. While the structural differences of these two forms of pallidin are as yet unclear, it does appear that at least two distinct types of subunits (with identical molecular weights) are made. There also appears to be a carbohydrate binding protein, a dimer of 18,000dalton subunits, which binds N-acetyl-Dglucosamine present in P. pallidurn cells. It is a very poor red cell agglutinin, but does appear to function in some aspect of cell adhesion. The presence of lectins on the surface of D. discoideum has been inferred in two ways. First, cohesive cells form mixed aggregates or rosettes with erythrocytes and these are specifically dissociated by the sugars that inhibit the agglutination activity of the purified lectins. This aggregation is not due to lectin leaking from ruptured slime mold cells [22]. Secondly, if rabbit antibody prepared against purilied discoidin I and II is reacted with cells followed by fluorescein of ferritin labeled anti-rabbit IgG, labeling of the surface of cohesive, but not vegetative, cells is found. If the cells are in suspension at room temperature or 37” C, this surface label forms patches, and later, caps [28]. Evidence for the involvement of this surface lectin in cell adhesion is found in the observation that sugars which inhibit the red cell agglutination of purified lectin also inhibit the endogenous cohesiveness of
living or heat-killed slime mold cells [22]. Recently it has been shown that the aggregation of predifferentiated P. pallidurn cells is blocked by the addition of the purilied pallidins. Furthermore, glycoproteins, and glycopeptides derived from them, which contain terminal or penultimate galactose residues inhibit this aggregation at very low concentrations (S. Rosen, unpublished observations). These glycoproteins and glycopeptides also inhibit the red cell agglutination by purified pallidins and inhibit the binding of 1251-labeled pallidin (purified on type 0 red cells) to glutaraldehyde fixed cohesive P. pallidurn cells (W. A. Frazier, unpublished). Reitherman et al. [29] demonstrated that glutaraldehyde fixation of cohesive D. discoideum and P. pallidurn cells destroys the endogenous cohesiveness of these cells. However, there remains on the surface of the cells a receptor for the lectins. In the case of D. discoideum cells the binding of discoidin I or II (each with a KU of about 1 x log M-i) to this receptor (5 x lo5 per cell) results in agglutination of the cells. Even high concentrations of discoidin I or II would not agglutinate vegetative cells. However, with increasing times of differentiation up to 9 h, the cells become progressively more readily agglutinable by both lectins. Pallidin also agglutinated 9-h D. discoideum cells better differentiated than vegetative cells, but the affinity of its binding was lower than the discoidins [29]. Interestingly, Ricinus communis agglutinin I which, like the discoidins has a specificity for galactose residues, agglutinated fixed differentiated but not vegetative D. discoideum cells. Concanavalin A agglutinated cohesive and vegetative cells equally well, and wheat germ agglutinin was a much better agglutinin of vegetative than cohesive cells. These results indicate an alteration in the distribution or composition of surface carbohydrate residues during differentiation. In contrast to the discoidins, pallidin (purified on type 0 cells) does not agglutinate fixed cohesive P. pallidurn cells. It does, however, bind to these cells (3.2 x lo5 sites per cell) with an apparent KU of 4 x log M r, and to fixed cohesive D. discoideum cells with a K, of 5 x 10s M -l, nearly an order of magnitude weaker. Both discoidin I and II bind to fixed cohesive P. pallidurn cells with a K, of 2 x lO8, ten times weaker than they bind to D. discoideum cells. The sum of these experiments indicates that there are high affinity lectin receptors which appear on the surface of slime mold cells as they develop aggregation competence [29]. Furthermore, in the species tested the interaction of the homo-specific combination of lectin and receptor is of greater affinity than any
133
TIBS - June 1976
hetero-specific combination. This result suggests that the observed species specilicity of cell aggregation may be due to the similarly specific high affinity combination of cell surface lectin with a complementary cell surface receptor. Perspectives
The data summarized here strongly argue that the protein-carbohydrate interaction of lectin and its receptor mediate the specificity and provide the mechanical strength of intercellular adhesion in aggregating cellular slime molds. Is the slime mold lectin-receptor system only intercellular glue, or does this interaction have a role in the initiation of subsequent differentiative events? The fact that the induction of many developmentally regulated enzymes in D. discoideum [ 1,2] depends on actual cell contact, not just on the development of aggrega.tion competence, suggests that the interaction of membrane components may trigger a certain phase of the developmental program. The profound effects of cohesive membranes on the regulation of enzymes indicate that membrane contact may be able to turn off some enzymes, as occurs in contact inhibition, as well as to initiate the production of others [2]. A good candidate for the mediator of these contact-dependent responses is the lectin receptor interaction. Whether cell recognition and adhesion in higher organisms is mediated by protein-carbohydrate interactions remains to be seen. However, the diverse nature of recognitive processes which have been found to employ the binding of proteins to carbohydrates, is suggestive of a generalized function for this type of recognition. These processes include viral-host cell recognition [30] yeast mating factor, the interaction of a number of bacterial (diphtheria, cholera, botulinurn) and plant (abrin, ricin) toxins with mammalian cells, and the recognition and uptake of desialated serum glycoproteins, including hormones, by liver [3 11.This latter case is particularly interesting in that the binding protein in liver plasma membranes has a specificity for galactose and N-acetyl-ogalactosamine like the slime mold lectins discoidin and pallidin. When the binding protein is solubilized it appears to aggregate, and in this form it is a red cell agglutinin as are the slime mold lectins. This analogy suggests that the agglutination activity of these lectins may be due to a fortuitous aggregation of the soluble proteins and not a true reflection of the oligomerit state of the carbohydrate binding proteins as they exist at the surface of the cell membrane. This same consideration may apply to the recently identified ‘electrolectin' found in extracts of Torpedo elec-
tric organ, chick embryo pectoral muscle and L16 myoblasts [32,33] as well as a membrane glycoprotein of tibroblasts, which, when solubilized, exhibits lectin activity [34]. References I Loomis, W. F. (1975) Dictyostelium discoideum. 4 Developmental System, Academic Press, New York 2 Jacobson. A. and Lodish, H. F. (1975) Annu. Rev. Genet. 9, 145-185 3 Newell, P. C. (1971) Essays in Biochemistr_v (P. N. Campbell, F. Dickens, eds), Vol. 7, pp. 87-126, Academic Press, New York 4 Bonner, J.T. (1971) Annu. Rev. Microbial. 25. 71 97 I i-li
5 Olive, L. S. (1975) The Mycetozoans, Academic Press, New York A. W. and Raper, K. B. (1973) Am. 6 Nickerson, J. Bat. 60, 190-19.7 7 Gingold, E. B. and Ashworth, J. M. (1974) J. Gen. Microbial. 84, 70-78 8 Bonner, J.T. (1947) J. Exp. Zoo/. 106, 1-26 9 Konijn, T. M., Van de Miene, J. G.C., Bonnet’, J. T. and Barkley, D. S. (1967) Proc. Natl. Acad. Sci. U.S.A. 58, 1152-l 154 IO Rossomando, E. F. and Sussman, M. (1972) Biothem. Biophys. Res. Commun. 47, 604610 I1 Green, A. A. and Newell, P. C. (1975) Cell 6, 129136 12 Malchow, D. and Gerisch, G. (1974) Proc. Null. Acad. Sri. U.S.A. 71,2423-2427 13 Malchow, D. and Gerisch, G. (1973) Biochem. Biophys. Res. Commun. 55. 20@-204 14 Beug, H. and Gerisch, G. (1972) J. Immunol. Methods 2, 49-57 I5 Rosen, S.. Kafka, J. A., Simpson, D. L. and Barondes, S. H. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 25542557 16 Mato, J.M. and Konijn, T.M. (1975) Biochim. Biophys. Acta 385, 173-179 17 Raper. K.B. and Thorn, C. (1941) Am. J. Bat. 28, 69
18 Bonner,
J. T. and Adams, M. S. (1958) J. &~?hrj~ol. Erp. Morphol. 6, 346 l9 Beug. H., Gerisch, G., Kempff. S.. Riedel, V. and Cremer. G. (1970) Exp. Crll Re.\. 63. 147- I58 lo Beug, H., Katz, F. E. and Gerisch, G. (1973) J. Cell Biol. 56, 647-658 21 Huesgen, A. and Gerisch. G. (1975) FEBS Let/. 56, 46-49 R. W. and Barondes. 22 Rosen S. D., Reitherman, S. H. (1975) E.up. Cell Rex 95. IW 166 R. W. 23 Frazier. W.A., Rosen, S. D., Reitherman. and Barondes, S. H. (1976) in Cell Surfircr Receptors (Bradshaw, R.A., ed.), Plenum Press, New York (in the press) 24 Simpson, D. L., Rosen, S. D. and Barondes, S. H. (I 974) Biochemisrry 13, 3487.-3493 25 Frazier, W.A.. Rosen, S. D., Reitherman, R. W. and Barondes. S. H. (1975) J. Eiol. C/rem. 250. 7714-7721 R. W., Rosen, S. D. and Barondes, 26 Reitherman, S. H. (1974) Nuture 248, 594-600 27 Simpson, P. L., Rosen, S. D. and Barondes, S. H. (1975) Biochim. Biophys. Acfa 412, 109-l 14 R. W., Rosen, S.D. 28 Chang, C-M., Reitherman, and Barondes, S.H. (1975) Exp. Cell Res. 95, 136142 R. W., Rosen, S.D.. Frazier. W.A. 29 Reitherman, and Barondes, S. H. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3541-3545 3. Schulze, I.T. (1973) Adv. Virus Rex 18, l-55 Bradshaw, R.A., ed. (1976) Cell Surfbce Recep31 tors, Plenum Press, New York (in the press) 32 Teichberg, V.I., Silman, I., Beitsch, D. D. and Resheff, G. (1975) Proc. Natl. Acud. Sci. U.S.A. 72. 1383. 1387 33 Nowak, T.P., Haywood, P.L. and Barondes. S. H. (1976) Biochem. Biophys. Res. Commun. (in the press) K. M., Yamada, S.S. and Pastan, 1. 34 Yamada, (1975) Proc. Null. Acad. Sri. U.S.A. 72. 315% 3162
Kinetics of protein synthesis in higher organisms in vivo Audrey
E. V. Haschemeyer
New methods have been developed for kinetic analysis of protein synthesis in animal tissues in vivo. Quantitative rate data can be obtained and the effects qf regulatory agents on initiation and elongation stages of pol_vpeptide chain synthesis studied independently. Protein Synthesis represents the last stage in genetic information transfer within a cell and, in’a quantitative sense, plays a significant role in the overall metabolism of animal tissues. Although the protein synthetic system has been the object of intense investigation for many years, most of our information is still of a qualitative nature, and certain basic questions remain unanswered. For example, what is the normal protein synthetic rate in animal tisHunter College of the City University of New York, 695 Park Avenue, New York, N.Y. 10021, U.S.A.
sues, relative to growth or other normal developmental processes? What role do changes in protein synthetic rate play in higher the variety of adaptations organisms make to perturbations of their environments? external internal or Answers to these questions have been difficult to come by, primarily because of poor experimental methodology for study in vivo and confusion generated by the extrapolation of protein synthesis results in vitro to the living animal. Before reviewing the new techniques developed recently for the study of protein synthesis in vivo, I
130 would inhibit the respiratory chain. On adding the purine nucleotide. the conductance decreased to levels similar to those of liver or heart mitochondria. It therefore appears that there is a proton-conducting ‘channel’ crossing the inner membrane of these mitochondria, which can be switched off by adding a low concentration of a purine nucleotide to the incubation medium. It has been found that the nucleotides bind very specifically to a site on the outer face of the inner membrane of brown fat mitochondria (see [4]), and in so doing block the conductance of the pathway. Although of significance, physiological doubtful chloride also appears to be conducted by the channel (see [4]). To explain this dual specificity it is probable that instead of protons passing into the matrix on the channel, the experimentally indistinguishable efflux of hydroxyl ions from the matrix occurs. While it is unlikely that the physiological modulator of the proton conductance is simply a variation in the cytoplasmic level of certain purine nucleotides, it is very likely that this channel represents the final molecular site of thermogenesis in the tissue, short-circuiting the proton circuit, and allowing rapid respiration to occur in the absence of ATP synthesis. It is signilicant that the channel may possibly’be induced at birth and on cold-adaptation of non-hibernators, while hibernators appear to retain the channel regardless of the environment (see [3,4]).
7 Hohorst,
References Joel, C. D. (1965) in Handbook qf’Ph.vsiology, AdiA. E. and Cahill. G. F., eds) pose Ti.ssw(Reynold, Section 5, pp. 59-85, American Physiological Society, Washington D.C. Lindberg, O., ed. (1970) Brown Adipose Tissue, Elsevier, Amsterdam Flatmark, T. and Pederson, J.I. (1975) Biochim. Biophys.
Acru 4 16, 53-103
Nicholls, D.G. (1976) FE&S Lrtt. 61, 103-110 Mitchell, P. (1966) Chrmiosmoric Coupling in Oxidutiw crud Photosynrhefic Phosphoryiulion, Glynn Research, Bodmin, Cornwall Coupling und Mitchell, P. (1968) Chemiosmofic Energy Transduction, Glynn Research, Bodmin, Cornwall
Sqier’s
H-J.
and
Z. Ph?siol.
Rafael,
J. (1968)
Hoppe-
Chem. 349, 268-270
Rafael, J., Ludolph, H-J. and Hohorst, H-J. (1969) Hoppe-Seylrr’s Z. PIz~siol. Chunz. 350, 1121-1131 9 Mitchell, P. and Moyle. J. (1967) Biochem. J. 105, I14771 162
10 Nicholls,
D.G.
(1974) Eur. J. Biochem.
50, 305-
315
H. ( 1975) Bioenrrgetics 7, 61~ 74 I1 Rottenberg, l2 Bernson, V. and Nicholls, D.G. (1974) Eur. J. Biochem.
47. 5 17-525
The role of cell surface components in the morphogenesis of the cellular slime molds William A. Frazier The interaction of surface-bound lectins with complementary carbohydrate-containing receptors seems to be the basis for species-specific cell-cell adhesion in cellular slime molds.
The cellular slime molds, and in particular the species Dictyostehium discoideum, have been for several years a popular and extensively studied model system for eukaryotic development [ 11.The ease with which large numbers of homogeneous cells can be culOxidation at 4” C tured and synchronously stimulated to There is one final hurdle to be overcome begin their developmental program has made these organisms prime subjects for by brown fat mi.tochondria from hibernators to fulfil their physiological function, the study of several aspects of developand that is the ability to function at very ment, including the control of enzyme expression and the regulation of RNA synlow temperatures to protect and aid arouthesis [2]. Many mutants in various devesal of the hibernating animal. Here again lopmental functions have been described brown fat mitochondria are perfectly adapted for their role. At low temperaand a large body of morphological data tures, the citric acid cycle of brown fat has provided extensive correlations of mitochondria can scarcely function [ 121, phenotypic expression with such genetic perhaps due to the many diffusion-limited lesions [2,3]. The life cycle of D. discoideum indicates steps occurring in the matrix. In the absence of an alternative pathway thermothe many developmental functions which genesis would therefore be impossible, as may be studied in this organism (Fig. 1). acetyl-CoA from &oxidation could not be Single amoebae (haploid, 7 chromosomes) further metabolized. Fortunately for the feed on bacteria and grow and multiply hibernator, an alternative pathway exists as vegetative or non-social cells. Upon exhaustion of the food supply, the amoebae whereby acetyl-CoA is simply hydrolysed begin differentiation [4]. Cells aggregate to acetate. Fatty acid oxidation to acetate can still occur at 4’ C [ 121, and it may be in response to pulses of cyclic AMP, forming clusters of lo4 to lo5 cells in 610 h. that one of the reasons for the periodic arousal of hibernators is to give the peri- During this period, the cells also become cohesive and form stable intercellular attapheral tissues a chance to oxidize acetate accumulating in the animal, in much chments so that a multicellular organism the same way as the liver removes lactate Depurtments of’ Biological Chemistry and Neurobioaccumulated in the body after violent logy, Division of Biology and Biomedical Sciences, Washington University, St Louis, Miss. 63110, U.S.A. exercise.
is constructed (12 hours) called a pseudoplasmodium, slug or grex. The cells do not fuse, in contrast to the Myxogastria (Myxomycetes), or acellular (true) slime molds [ 1,5]. Under certain conditions, aggregating cells may form macrocysts rather than slugs, thus initiating a sexual phase during which meiosis occurs and which gives rise to normal haploid amoebae [6]. More commonly, the slug is formed, migrates for a time, and then further differentiatiates so that the front one-third of the cells form a plant-like stalk, bearing aloft the remaining two-thirds of the cells which become spores [4]. These are very resistant to adverse conditions, and when germination occurs, each gives rise to a single haploid amoeba, thus completing the life cycle. At this stage, cell fusion may occur with a very low frequency (about 10d4) yielding diploid cells in a para-sexual cycle 171.
In recent years, several groups have focused their investigations on events which occur during the initial aggregation phase and which involve functions mediated at the cell surface. These are the morphogenetic functions of chemotaxis and selective cell adhesion, processes which, in some form, play a role in the morphogenesis of all higher organisms. Since the discovery of a chemoattractant or acrasin by Bonner [8] and its subsequent identification as cyclic AMP [9], much
131
TIBS - June 1976
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c@ t
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A
Haploid amoebae
\
Formation & Segregation of Diploid Cells
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/-
Appearance
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,
Macrocyst
[
Germin
&Q
haplolds
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Rupiwe pm,ary
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1
01 and
~~ ,_ ._I,^
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26hr nw-stalk -.-“\
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24hr
20-21 hr
elegant work has been done in defining the chemotactic process and in identifying the macromolecular components which generate [lo], detect [ 11,121 and relay [ 131 the chemotactic signal. The net result of this information relay system is that cells move toward aggregation centers in pulsatile fashion in response to radially propagated waves of cyclic AMP [12,13]. The primary purpose of this review is to summarize the current view of the mechanism by which this aggregation results in a stable, multicellular organism, that is, the nature of intercellular adhesive bonds. Characteristics of cell adhesion Concomitant with the appearance of the chemotactic apparatus, differentiating slime mold cells acquire the ability to
adhere to one another. This cohesiveness can be assayed quantitatively by monitoring changes in the light scattering properties of a cell suspension [ 141or by swirling cells in an EDTA-containing buffer and determining the number and size of aggregates formed with a Coulter particle counter [15]. The presence of EDTA eliminates the slight adhesion observed with non-differentiated, vegetative cells. If slime mold cells’are separated from their bacterial food supply and gyrated in dilute phosphate buffer dr plated on moist millipore filters, significant EDTA-resistant cohesiveness appears after about 6 h. At about the same time, the chemotactic system becomes functional and aggregation is complete by about 12 h. Coincident with the completion of aggregation, the binding capacity of the
cells for cyclic AMP decreases [ 11,161,suggesting a ‘down regulation’ of the chemotactic receptors. This may represent a mechanism for turning off chemotaxis after aggregation is complete. Further evidence that cell-cell contact may have a role in regulating differentiation is found in the effects of membranes from cohesive cells on the expression of six enzymes appearing during development. The addition of these membranes to cells at the beginning of differentiation blocked the acquisition of aggregation competence [2] perhaps indicating blockage of the chemotactic apparatus. Four of the six enzymes were not made at all, one appeared at the normal time and rate and another, alkaline phosphatase, appeared early and reached twice its normal peak activity [2]. These consequences of membrane interaction may indicate that the sequence of events is important for the proper expression of surface mediated differentiative changes. The aspect of cell aggregation in the slime molds which is most relevant to their use as a model system for cell adhesion in higher organisms is species specificity. If labeled vegetative amoebae of different species are mixed together and differentiation allowed to proceed, the slugs which result contain only one species of cell. Thus, segregation or sorting out of different species has occurred. In some cases this process is aided by the fact that the mixed species employ different chemotactic agents (acrasins) [ 161 so that the geographical location of the aggregation centers is distinct. However, in other com-
TIBS - June 1976
132 binations of two closely related species such as D. discoideum and D. mucoroides which both appear to use cyclic AMP as their acrasin [ 161, mixed aggregates form which then give rise to two separate slugs of homo-specific cell type [17,18]. This clearly indicates that true sorting out has occurred and that a cell recognition apparatus per se does indeed exist.
Nature of the cell adhesion apparatus Gerisch and his colleagues have taken an immunological approach to define some properties of the surface membrane of differentiated cells which are relevant to adhesion. Antibody was prepared to cohesive D. discoideum cells, adsorbed with vegetative amoebae and fragmented to yield FAB. These FAB fragments were found to block EDTA-resistant cell cohesion by preventing the end-to-end contact found in streams of cells entering aggregates [19]. The cell surface antigen is termed contact site A. Another surface antigen, contact site B, is present in both vegetative and cohesive cells [20]. However, univalent antibodies to this antigen block cell adhesion by preventing side-toside contact. Thus, two apparent classes of contact ‘sites’ with distinct spatial distributions on the cell surface are involved in cell adhesion. Recently, progress has been made in purifying these contact sites from detergent extracts of particulate fractions of D. discoideum [21]. A different, and perhaps more direct approach, to the study of the macromolecular species responsible for cell cohesion was initiated by the observation that, as six species of slime mold develop aggregation competence, there appear in crude extracts of the cohesive cells proteins which are potent agglutinins of erythrocytes [22]. These agglutinins are multivalent carbohydrate binding proteins which have distinct sugar specificity in each species [22,23] even though they are similar in subunit molecular weights, amino acid compositions and, in some cases, their tryptic peptide fingerprints [23]. The relevance of these lectins to the cell adhesion process has been investigated in detail in the two species D. discoideum and Polysphondelium pallidurn. The lectin activity of D. discoideum cells accumulates during differentiation with a time course very similar to the development of cohesiveness [ 151 and the appearance of contact sites A [19]. The activity has been purified using Sepharose 4-B, a galactose-containing polymer, as an affinity adsorbent [24,25]. The purified protein was found to contain two lectins, both with a native molecular weight near 100,000. The major lectin, designated discoidin I, has a subunit molecular weight
of 26,000 and the minor lectin, called discoidin II, has a subunit weight of 25,000 [25]. The two lectins are also distinguishable on the basis of their isoelectric points, amino acid compositions, tryptic peptide maps, the types of red cells which they agglutinate, the simple saccharides which inhibit their agglutination activity, and their time course of appearance during differentiation [25]. Lactose is the best inhibitor of discoidin II and N-acetyl-D-galactosamine is the best inhibitor of discoidin I. Glucose and mannose do not inhibit either lectin [25]. The developmentally regulated lectin activity of P. pallidurn (called pallidin) has been purified by affinity adsorbtion on formalinized (fixed) human type 0 and rabbit erythrocytes [23,26,27]. The native lectin aggregates extensively forming decamers of about 250,000 mol. wt and higher aggregates. The proteins purified with either type of red cells have a subunit molecular weight of 25,000 [23,27]. However, the lectin purified with rabbit erythrocytes appears to consist primarily of subunits with a slightly basic isoelectric point while that purified with type 0 erythrocytes consists of species with acidic isoelectric points. Furthermore, the two products have slightly different sugar specificities, although both are very sensitive to inhibition by lactose and melibiose. While the structural differences of these two forms of pallidin are as yet unclear, it does appear that at least two distinct types of subunits (with identical molecular weights) are made. There also appears to be a carbohydrate binding protein, a dimer of 18,000dalton subunits, which binds N-acetyl-Dglucosamine present in P. pallidurn cells. It is a very poor red cell agglutinin, but does appear to function in some aspect of cell adhesion. The presence of lectins on the surface of D. discoideum has been inferred in two ways. First, cohesive cells form mixed aggregates or rosettes with erythrocytes and these are specifically dissociated by the sugars that inhibit the agglutination activity of the purified lectins. This aggregation is not due to lectin leaking from ruptured slime mold cells [22]. Secondly, if rabbit antibody prepared against purilied discoidin I and II is reacted with cells followed by fluorescein of ferritin labeled anti-rabbit IgG, labeling of the surface of cohesive, but not vegetative, cells is found. If the cells are in suspension at room temperature or 37” C, this surface label forms patches, and later, caps [28]. Evidence for the involvement of this surface lectin in cell adhesion is found in the observation that sugars which inhibit the red cell agglutination of purified lectin also inhibit the endogenous cohesiveness of
living or heat-killed slime mold cells [22]. Recently it has been shown that the aggregation of predifferentiated P. pallidurn cells is blocked by the addition of the purilied pallidins. Furthermore, glycoproteins, and glycopeptides derived from them, which contain terminal or penultimate galactose residues inhibit this aggregation at very low concentrations (S. Rosen, unpublished observations). These glycoproteins and glycopeptides also inhibit the red cell agglutination by purified pallidins and inhibit the binding of 1251-labeled pallidin (purified on type 0 red cells) to glutaraldehyde fixed cohesive P. pallidurn cells (W. A. Frazier, unpublished). Reitherman et al. [29] demonstrated that glutaraldehyde fixation of cohesive D. discoideum and P. pallidurn cells destroys the endogenous cohesiveness of these cells. However, there remains on the surface of the cells a receptor for the lectins. In the case of D. discoideum cells the binding of discoidin I or II (each with a KU of about 1 x log M-i) to this receptor (5 x lo5 per cell) results in agglutination of the cells. Even high concentrations of discoidin I or II would not agglutinate vegetative cells. However, with increasing times of differentiation up to 9 h, the cells become progressively more readily agglutinable by both lectins. Pallidin also agglutinated 9-h D. discoideum cells better differentiated than vegetative cells, but the affinity of its binding was lower than the discoidins [29]. Interestingly, Ricinus communis agglutinin I which, like the discoidins has a specificity for galactose residues, agglutinated fixed differentiated but not vegetative D. discoideum cells. Concanavalin A agglutinated cohesive and vegetative cells equally well, and wheat germ agglutinin was a much better agglutinin of vegetative than cohesive cells. These results indicate an alteration in the distribution or composition of surface carbohydrate residues during differentiation. In contrast to the discoidins, pallidin (purified on type 0 cells) does not agglutinate fixed cohesive P. pallidurn cells. It does, however, bind to these cells (3.2 x lo5 sites per cell) with an apparent KU of 4 x log M r, and to fixed cohesive D. discoideum cells with a K, of 5 x 10s M -l, nearly an order of magnitude weaker. Both discoidin I and II bind to fixed cohesive P. pallidurn cells with a K, of 2 x lO8, ten times weaker than they bind to D. discoideum cells. The sum of these experiments indicates that there are high affinity lectin receptors which appear on the surface of slime mold cells as they develop aggregation competence [29]. Furthermore, in the species tested the interaction of the homo-specific combination of lectin and receptor is of greater affinity than any
133
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hetero-specific combination. This result suggests that the observed species specilicity of cell aggregation may be due to the similarly specific high affinity combination of cell surface lectin with a complementary cell surface receptor. Perspectives
The data summarized here strongly argue that the protein-carbohydrate interaction of lectin and its receptor mediate the specificity and provide the mechanical strength of intercellular adhesion in aggregating cellular slime molds. Is the slime mold lectin-receptor system only intercellular glue, or does this interaction have a role in the initiation of subsequent differentiative events? The fact that the induction of many developmentally regulated enzymes in D. discoideum [ 1,2] depends on actual cell contact, not just on the development of aggrega.tion competence, suggests that the interaction of membrane components may trigger a certain phase of the developmental program. The profound effects of cohesive membranes on the regulation of enzymes indicate that membrane contact may be able to turn off some enzymes, as occurs in contact inhibition, as well as to initiate the production of others [2]. A good candidate for the mediator of these contact-dependent responses is the lectin receptor interaction. Whether cell recognition and adhesion in higher organisms is mediated by protein-carbohydrate interactions remains to be seen. However, the diverse nature of recognitive processes which have been found to employ the binding of proteins to carbohydrates, is suggestive of a generalized function for this type of recognition. These processes include viral-host cell recognition [30] yeast mating factor, the interaction of a number of bacterial (diphtheria, cholera, botulinurn) and plant (abrin, ricin) toxins with mammalian cells, and the recognition and uptake of desialated serum glycoproteins, including hormones, by liver [3 11.This latter case is particularly interesting in that the binding protein in liver plasma membranes has a specificity for galactose and N-acetyl-ogalactosamine like the slime mold lectins discoidin and pallidin. When the binding protein is solubilized it appears to aggregate, and in this form it is a red cell agglutinin as are the slime mold lectins. This analogy suggests that the agglutination activity of these lectins may be due to a fortuitous aggregation of the soluble proteins and not a true reflection of the oligomerit state of the carbohydrate binding proteins as they exist at the surface of the cell membrane. This same consideration may apply to the recently identified ‘electrolectin' found in extracts of Torpedo elec-
tric organ, chick embryo pectoral muscle and L16 myoblasts [32,33] as well as a membrane glycoprotein of tibroblasts, which, when solubilized, exhibits lectin activity [34]. References I Loomis, W. F. (1975) Dictyostelium discoideum. 4 Developmental System, Academic Press, New York 2 Jacobson. A. and Lodish, H. F. (1975) Annu. Rev. Genet. 9, 145-185 3 Newell, P. C. (1971) Essays in Biochemistr_v (P. N. Campbell, F. Dickens, eds), Vol. 7, pp. 87-126, Academic Press, New York 4 Bonner, J.T. (1971) Annu. Rev. Microbial. 25. 71 97 I i-li
5 Olive, L. S. (1975) The Mycetozoans, Academic Press, New York A. W. and Raper, K. B. (1973) Am. 6 Nickerson, J. Bat. 60, 190-19.7 7 Gingold, E. B. and Ashworth, J. M. (1974) J. Gen. Microbial. 84, 70-78 8 Bonner, J.T. (1947) J. Exp. Zoo/. 106, 1-26 9 Konijn, T. M., Van de Miene, J. G.C., Bonnet’, J. T. and Barkley, D. S. (1967) Proc. Natl. Acad. Sci. U.S.A. 58, 1152-l 154 IO Rossomando, E. F. and Sussman, M. (1972) Biothem. Biophys. Res. Commun. 47, 604610 I1 Green, A. A. and Newell, P. C. (1975) Cell 6, 129136 12 Malchow, D. and Gerisch, G. (1974) Proc. Null. Acad. Sri. U.S.A. 71,2423-2427 13 Malchow, D. and Gerisch, G. (1973) Biochem. Biophys. Res. Commun. 55. 20@-204 14 Beug, H. and Gerisch, G. (1972) J. Immunol. Methods 2, 49-57 I5 Rosen, S.. Kafka, J. A., Simpson, D. L. and Barondes, S. H. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 25542557 16 Mato, J.M. and Konijn, T.M. (1975) Biochim. Biophys. Acta 385, 173-179 17 Raper. K.B. and Thorn, C. (1941) Am. J. Bat. 28, 69
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J. T. and Adams, M. S. (1958) J. &~?hrj~ol. Erp. Morphol. 6, 346 l9 Beug. H., Gerisch, G., Kempff. S.. Riedel, V. and Cremer. G. (1970) Exp. Crll Re.\. 63. 147- I58 lo Beug, H., Katz, F. E. and Gerisch, G. (1973) J. Cell Biol. 56, 647-658 21 Huesgen, A. and Gerisch. G. (1975) FEBS Let/. 56, 46-49 R. W. and Barondes. 22 Rosen S. D., Reitherman, S. H. (1975) E.up. Cell Rex 95. IW 166 R. W. 23 Frazier. W.A., Rosen, S. D., Reitherman. and Barondes, S. H. (1976) in Cell Surfircr Receptors (Bradshaw, R.A., ed.), Plenum Press, New York (in the press) 24 Simpson, D. L., Rosen, S. D. and Barondes, S. H. (I 974) Biochemisrry 13, 3487.-3493 25 Frazier, W.A.. Rosen, S. D., Reitherman, R. W. and Barondes. S. H. (1975) J. Eiol. C/rem. 250. 7714-7721 R. W., Rosen, S. D. and Barondes, 26 Reitherman, S. H. (1974) Nuture 248, 594-600 27 Simpson, P. L., Rosen, S. D. and Barondes, S. H. (1975) Biochim. Biophys. Acfa 412, 109-l 14 R. W., Rosen, S.D. 28 Chang, C-M., Reitherman, and Barondes, S.H. (1975) Exp. Cell Res. 95, 136142 R. W., Rosen, S.D.. Frazier. W.A. 29 Reitherman, and Barondes, S. H. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3541-3545 3. Schulze, I.T. (1973) Adv. Virus Rex 18, l-55 Bradshaw, R.A., ed. (1976) Cell Surfbce Recep31 tors, Plenum Press, New York (in the press) 32 Teichberg, V.I., Silman, I., Beitsch, D. D. and Resheff, G. (1975) Proc. Natl. Acud. Sci. U.S.A. 72. 1383. 1387 33 Nowak, T.P., Haywood, P.L. and Barondes. S. H. (1976) Biochem. Biophys. Res. Commun. (in the press) K. M., Yamada, S.S. and Pastan, 1. 34 Yamada, (1975) Proc. Null. Acad. Sri. U.S.A. 72. 315% 3162
Kinetics of protein synthesis in higher organisms in vivo Audrey
E. V. Haschemeyer
New methods have been developed for kinetic analysis of protein synthesis in animal tissues in vivo. Quantitative rate data can be obtained and the effects qf regulatory agents on initiation and elongation stages of pol_vpeptide chain synthesis studied independently. Protein Synthesis represents the last stage in genetic information transfer within a cell and, in’a quantitative sense, plays a significant role in the overall metabolism of animal tissues. Although the protein synthetic system has been the object of intense investigation for many years, most of our information is still of a qualitative nature, and certain basic questions remain unanswered. For example, what is the normal protein synthetic rate in animal tisHunter College of the City University of New York, 695 Park Avenue, New York, N.Y. 10021, U.S.A.
sues, relative to growth or other normal developmental processes? What role do changes in protein synthetic rate play in higher the variety of adaptations organisms make to perturbations of their environments? external internal or Answers to these questions have been difficult to come by, primarily because of poor experimental methodology for study in vivo and confusion generated by the extrapolation of protein synthesis results in vitro to the living animal. Before reviewing the new techniques developed recently for the study of protein synthesis in vivo, I