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Mini-review on the possible importance of an intracellular circulation
Life Sciences, Vol. 36, pp. 299-307 Printe~ i n the U.S.A.
ON
THE
POSSIBLE
Oerzamon Press
MINI-RE'VIEW IMPORTANCE OF AN INTRACELLULAR
CIRCULATION
Denys N. Wheatley Cell ])athology Laboratory, Department of Pathology, University Medical Buildings, Foresterhill, Aberdeen, AB9 2ZD, Scotland, U.K.
Sunmary Since ultrastructural, biophysical and other studies continue to demonstrate that the internum of the cell is highly structured, the question raised and discussed in this review is whether an intracellular circulatory system is essential for the maintenance of active metabolism. Although cytoplasmic streaming is evident in large animal and plant cells, it is argued that it probably occurs in all cells irrespective of size, and is of particular importance in bringing together interacting molecules fast enough for metabolic processes to occur which would otherwise be far too slow if diffusion were the only form of motion. A cc~mon intracellular system would suffice for most metabolic processes and would also help to dissipate waste products. Interruption of this internal circulation would result in the inhibition of metabolic functioning, including for example protein turnover, for which evidence is presented to substantiate this hypothesis. Many significant advances have been made in biological sciences through the surge of new technology, and in particularbecause of the rapid progress in molecular biology in the last twenty or so years. Although this has led to a far better understanding of biochemical aspects of life, the awesome complexity and dynamic nature of organisms means that d~eir physiology in terms of the integration of individual biochemical reactions remains obscure. The gulf between what is known about an enzyme in vitro and how it operates and is regulated in situ within some compartment of the living cell is still very wide Many studies on enzymes involve highly purified preparations observed under specifically tailored laboratory conditions which may give data on substrate preferences, initial reaction rates, etc., in accordance with the laws of mass action, but probably tell us little about their role when embedded in some particular metabolic pathway on some membrane surface inside the cell (I). The 'watery' cell Now that the oversia~lifications and artificiality of most in vitro biochemical analyses are becoming better appreciated, the internal operations of a cell are less likely to be compared with a jumble of chemical interactions taking place in bulk aqueous phase in which the laws of mass action apply (2). While a number of reactions undoubtedly take place in the bulk aqueous phase, the question is whether they represent the majority or a very small minority of the total, with most proceeding according to laws of surface chemistry (3). Recent experiments with dehydrated cells suggest the latter (4,5), and many }~m~nlc 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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have long been convinced that life is mostly a surface-based operation: "Although it is fluid and watery, most of the cell is not a true solution. A drop of true solution, of hmmogenous liquid, could not 'live'. It is ren~)te from 'organization'. In the cell there are heterogeneous solutions. The great molecules of protein and aggregated particles are suspended not dissolved. A surface is a field for chemical and physical action. The interior of a pure solution has no surfaces. But the aggregate of surface in these foamy colloids which are in the cell mounts up to something large. The 'internal surface' of the cell is enormous. The cell gives chemical results which in the laboratory are to be obtained only by temperatures and pressures far in excess of those of the living body. Part of the secret of life is the i~nense internal surface of the cell." (Sherrington, ref. 6). Regulation of metabolic rate The rate at which simple reactions proceed, assuming that the conditions are appropriate, conducive and -- for ease of analysis -- constant, is governed by the accessibility of the substrates (precursors/nutrients) to the catalytic surfaces, their concentrations (up to a certain level), and the rate of removal of products. Small changes in conditions in living systems can quickly result in a reaction being reversed, inhibited, accelerated or diverted along some other pathway. Assuming that products will be rapidly utilised in a growing cell type with a relatively fast turnover time for protein, the rate of many reactions will be determined by the rate at which the reactants can be delivered to the catalytic sites (it is implicit t]~at in the absence of enzymes, the vast majority of metabolic processes would be far too slow to sustain life). The principle to which Coulson et al. (7,8) have rightly drawn attention from the gross physiological viewpoint offers an explanation for the widely differing rates of metabolism found in the cells of different species of manuals, i.e. that these may depend to a large extent on the rates at which precursor molecules can be delivered to their individual cells. This, it is argued, can impose severe limitations on the relative metabolic rates of a whale co,oared with a shrew, which may differ by as much as three orders of magnitude in their metabolic activity. The flow constant of essential nutrients will have very different values in the shrew than the whale because the sheer size of the latter imposes a large time delay in getting substances from source (e.g. gut or lungs) to sink (the individual cells of the body). Actual delivery rates will not just depend on circuit time, but also on differences in concentrations between arterioles and extracellular space, between the latter and the internum of the cells, and also on the relative volume of the delivery medium (blood for the most part) and its circulation time. Provided with similar medium in a tissue culture vessel, it is suggested that little difference would be found between shrew cells and those of the whale because the delivery rate of nutrients would be equal. Although this concept offers a simple explanation for divergent metabolic rates, there will be a number of other factors governing intracellular activity, one of which is the ability of the cell to 'take on board' the supplies being delivered by the circulatory system. Holley (9) suggested that the persistent proliferation of cancer cells was due to their inability to restrict amino acid transport across the pla~namembrane. His hypothesis was based on the improbable assumption that, because precursors would continue to be made freely available inside the cell, the ribosomal machinery is obliged to sustain a high rate of protein synthesis,
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thereby necessitating repeated division of cells. This concept is inconsistent with amino acid uptake data in both normal and tumour cells (10), but it does raise the question of whether the delivery rate of precursors to the intern~n of the cell is of as much importance as delivery to the locality of the cell by the main circulatory ~jstem. It is certainly true that much of cellular functioning is regulated at the plasmamembrane, including ultimately the regulation of division (11). Thus it may be pertinent to suggest that delivery rates of precursors to the immediate vicinity of the cell, e.g. sugars for energy metak~lism, may determine the potential metabolic rate under those conditions (the coarse control), while the cell membrane transport mechanisms modulate the actual accessibility of these precursors to the internal machinery (the fine control). The broad implication of Coulson et al. (7) is that the metabolic machinery in the whale cell is capable of working faster in situ, but that the major limiting factor here is the rate at which precursors arrive; just as a fire burns brighter when it is fanned, the whale cell would metabolise faster if the delivery of essential nutrients could be accelerated. It is suggested that within the confines of the intracellular regulatory factors just mentioned, this principle should at least be taken one stage further. We propose that in addition to the delivery of nutrients to the cell environment, it is important (possibly essential) to have a circulatory system within the cell, without which most biochemical pathways would be unable to operate at rates sufficient to sustain even modest metabolic activity. The asstmption being challenged is that when molecules are delivered to the vicinity of the cell, diffusion -regardless of whether it is assisted in some way or not across the plasmamembrane itself -- takes over from this point. This proposal may have little direct evidence to support it at the present moment, but it does present an alternative to a generally accepted, but probably erroneous, assumption that simple diffusion of free molecules is the mechanism by which nutrients move from the inner aspect of the cell membrane to all parts of the cell. It is worth exploring this idea a little further because it seems to offer a framework upon which many observations relating to cell functioning can be more easily explained. Circulation and metabolic rate Harvey's great contribution to science (12) was in recognising that the flow of blood within the vessels of the body described a one-way, continuous circulation as opposed to an ebb-and-flow motion. It is interesting that this process by which all nutrients reach the cells of the body has only just begun to be appreciated in terms of its relationship with metabolic rate (7,8), some three hundred and fifty years later. The vital importance of a continuous circulation in an actively metabolising body, such as that of man, can be readily demonstrated by the fact that its interruption for more than a few minutes leads to death. This can only be postponed if metabolic activity has been reduced beforehand, as can be shown with smaller m ~ i s which enter torpid states. Creatures without elaborate circulatory systems are obliged to be small and highly mobile or long, slender, delicate and sessile, living in places where the environment constantly flushes over them. Even under these conditions, it is all too readily assumed that life processes are sustained by the simple diffusion of essential nutrients from the environment into the cells, and of waste products out again by the same process in reverse. Cells of many organisms contain about 70% water and about 20% protein. Some are even richer in protein; for example, the human erythrocyte can contain 35% protein, with haemoglobin so concentrated that it crystallises out when cells are suddenly lysed (13). Some pure protein crystals contain much less protein than cells because of a large amount of hydration--tropomyosin
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crystals are more than 95% water (14). Presumably the admixture of proteins with many other molecu±es as aggregates and on surfaces prevents proteins from normally appearirLg as crystals within cells, although they can be found in some storage organelles (e.g. peroxisomes, 15), and can sometimes appear in liver mitochondria under certain pathological conditions. If all the protein in a cell was in simple solution, this would present enormous resistance to the movement of any but the smallest and most mobile molecules and ions. Proteins also attract and adsorb smaller molecules, which will further restrict their movement. It would seem probable therefore that substances bigger than, e.g., urea would experience considerable difficulty moving through cytoplasm. Indeed, cc~parisons are now being made with gelatin reference phases within the cytoplasm of oocytes to explore the relative rates of diffusion of small molecules such as amino acids (16) and ions (17), and their differential distribution in the cell, as opposed to a 14% gelatin reference phase. Certainly the movement of very large molecules within the cell (e.g. most proteins) would be far too slow to sustain metabolic rates found in warn~blooded animals, especially during periods of prolonged and vigorous activity. How then d o m o s t molecules move around inside the cell? We are left with two main alternatives: (i) random movements, at best assisted by the ebb-and-flow created by the overall activity of the cell, analogous to the pre-Harveian concept of the movement of blood, (ii) organisedor structured movement of a 'cytocirculation' or 'endocirculation' occurring in defined, if labile and transient, channels within the cell, and for the most part describing a unidirectional pathway. Cytoplasmic streaming as a general phenomenon Cytoplasmic streaming has been observed for well over a century in large animal cells (e.g. Amoeba) and in plant cells (e.g. Nitella), especially those of the latter group having very large central vacuoles around which streaming describes an obvious circular motion, giving us the expression 'cyclosis'. Streaming of the soluble aqueous phase inside cells remains an unproven phenomenon since it can only be inferred from the behaviour of particles wafted along by underlying currents. Furthermore, this type of movement is not random because it is seen in distinct channels. It must be distinguished, however, from the precise , highly non-random movements seen with,e.g., pigment granules in chromatophore cells, whose yo-yo-like sorties are probably due to their being attached to specific contractile elements (18). Huxley (19) recognised the importance of the flow within large plant cells as a means of distribution of materials, and remarked that "if only we had ears to hear it, the roar of a tropical forest" would drown that of a railway station. It is quite conceivable that the circuitous motion of an intracellular soluble phase -- which may become correctly termed the cytosol in future -- seen in such paradigms as Nitella and Chara, is required in these cases, primarily because of their size. This rationalisation persists today and is not incorrect, except possibly in its inference, viz. that small cells would presumably not require it. It is equally valid to argue that the former examples are simply the more obvious examples of a general phenomenon occurring in all cells, and there is considerable evidence that streaming and organised movement of fluid phases does occur in many kinds of cell irrespective of their size, a notion which is supported by the observations of Allen and his coworkers (40, 41). To develop this argument a little further, it is worthwhile considering current developments in our understanding of cytoarchitecture,
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while reme~nbering that the interpretation of intriguing electronmicrographs of it which have recently been taken may not necessarily be an entirely accurate representation of the real situation in the living cell. There is now an increasing awareness that the 'spongioplasm' suggested by Sch~fer in 1898 may exist as an elaborate macrc~olecular reticulum, referred to as the m i c r o t r ~ u l a r lattice (21; see Fig. I). Careful centrifugation and stratification of intact Euqlena cells by Kempner and Miller (22) led to separation of organelles frcm the macromolecular matrix and from the aqueous phase of the cytoplasm. Their data is interesting because the last mentioned layer contained little protein and was virtually devoid of enzyme activity, suggesting that very few of the proteins in the highly protein-rich cytoplasm were in true solution in the living cell (cf.6). Recent analysis of data on protein turnover in HeLa cell cytoplasm has led to our estimating that the aqueous phase of the cell contains as little as 0.01% protein, end would therefore approximate the properties of water rather than a viscous macromolecular solution (23). The sudden disruption of the internal integrity of the cytoarehitecture when a cell blebs as a result of an osmotic shock results in a very distinct change in the movement of intracellular particles, which becc~e very active and show Brownian movement. The almost ccmplete absence of such activity in the undamaged cell by itself strongly indicates that the internum is highly structured. In agreement with Schifer's idea of a sponge-like matrix, life would seem to depend on perfusion occurring in a non-random, partially polarised manner through a matrix of insoluble protein.
Fig. I
High-voltage electron micrograph of the cytoplasmic ground substance or matrix of an L-929 cell, showing its microtrabecular nature after freeze substitution (from ref.5 through courtesy of Dr. K.R. Porter, see ref. 21). The bar indicates 0.1bm (negative image).
Because of the small size of the channels (see Fig. I), some pumping action would be necessary to initiate and presumably sustain flow. Contractile actomyosin is present in almost all protoplasmic material obtained from living organisms, and these may be responsible for pt~nping or pulsating, or as Malone suggests (24), the aqueous phase may be moved by a process akin to syneresis following the rhythmical contraction and relaxation of (helical) proteins. It is also noteworthy that a little energy is required to initiate proteolysis in cells (see 25 for a review), but it remains controversial as to how much and where this energy investment is made. Since pumping mechanisms utilises energy, there is good reason to believe that this could be an energy
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requirement associated with conti~uous turnover of proteins inside most types of cell. Other mechanisms will also operate which can cause transloeation of molecules, such as movements associated with the polymerisation and depolymerisation of microtubules (see refs. 40 and 41 for this and other possibilities) . There is little doubt that cytoplasmic streaming can be created by these means. To create a true circulation rather than simple ebb-and-flow motion, a co-ordinating centre would have to be envisaged, but as yet there has been no clear evidence that a structure analogous to the heart (in whole body terms) exists, even in large cells with very clear channels of streaming, such as Amoeba. The fact that directional streaming can be thrown into reverse in this organism must also be considered. The channels of a cytoplasmic circulatory system will not have the rigidity, the inflexibility or presumably the elaborate valvular control systems found in the blood system of the whole organism. It seems improbable that electron microscopists will reveal a miniaturised form of it at the intracellular level. But movements within cells can be highly non-random, as shown by the figure-of-eight route taken by food vacuoles in Paramecium, despite the absence of a detectable intracytoplasmic equivalent of an alimentary canal. Temperature, ~orotein turnover and ~rtop__lasmic streaming It cannot be concluded that only a circuitous flow of the aqueous phase occurs in all cells; beth the abovementioned alternatives may be involved to different degrees at the same time, i.e. a main flow could be accompanied by ebb-and-flow into peripheral pockets. It is suggested, however, that once a stream has been set in motion, the course of least resistance is for further flow to continue in the same direction until some event occurs which leads to its interruption or reversal. The cytoplasm and nucleus of the cell constitute a macrc~olecular labyrinth in which movement is probably highly restricting for the average protein (or similar) macromolecule, much as a jungle is restricting to the movement of man unless pathways are cleared through it. Blocking the pathways, or preventing traffic flowing along them by inhibiting the motive force, could have dire consequences on normal functioning. One particular facet which has recently been observed in our laboratory is the effect of heat on protein turnover. Synthesis of proteins is very sensitive to temperatures above 42°C, and falls to about 6% at 45°C (23). Degradation, being a largely exothermic reaction, was expected to increase with elevated temperature, and greater turnover of proteins was also anticipated because more aberrant and denaturation of proteins would arise. After a cell has been hemogenised, its hydrolytic enzymes can be shown to be more active at 45 ° than at 37°C, which establishes that the enzymes involved in proteolysis are not themselves destroyed. In the intact cell, however, raising the temperature to 45°C leads to the cc~plete abolition of protein turnover. The most notable change during short-term treatment of cells at the elevated temperature was their i~nediate cessation of all motile activity, saltation of particles, streaming, ruffling of advancing membranes, An~! p~eudopodial extension. The arrest of activities dependent upon actomyosin proteins and ATP-utilising systems appears to include the intracellular circulatory system. This reduces the flow constant of proteins past the proteases on internal surfaces or membranes in the cytoplasm, and thereby halts their hydrolytic activity, since the only interactions which can occur will require free diffusion of these large molecules in a highly restrictive environment. This now seems to be a plausible explanation for what od~er~ise might appear as a paradoxical situation (see ref. 23). A cell contains proteins appropriate to its environmental circumstances. It must be adaptable, malleable and quickly responsive to this environment to
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survive. And yet its general make-up involves throughout the whole of nature a similar set of proteins and other molecules which came together to create a thixotropic gel with r ~ k a b l e strength (25). It would seem that it continually makes proteins of a reasonably wide s p e c ~ (26-68), frcm which it retains those commensurate with prevailing conditions (29). Surpluses are cleared away by proteolysis. The cell is unlikely to utilise with 100% efficiency all the proteins that it synthesises. Indeed, the sorting and turnover of proteins at source involves very intense proteolysis which has hitherto been grossly underestimated (29) . The mechanism also indicates that proteins which are made but have no stabilising site or function to perform will be poorly retained and may not in fact appear to be synthesised at all (30,31). If there is a sudden change in the environment, e.g. the introduction of a substrate for a particular enzyme (31,32), such a protein may be stabilised and beccme evident by its a c ~ u l a t i o n within the cell. Equally, if the substrate is removed again, the enzyme disappears with considerable rapidity. To have an autc~atic control of this nature, protein turnover in the cell must be due to the operation of a continuous and wide-scale operation, of the type described as the basal system in previous work (33). The stabilisation of proteins is based on their utilisation within the cellular matrix and as part of this structure (29) ; if they do not integrate into the functional bicmass, they will be turned over very rapidly. This applies to newly-synthesised proteins and to those which through their activity are moving from an integrated conformation to one which destabilises them. The half-life of nascent proteins, and by inference of these destabilised proteins, in the growing HeLa cell is about 53 min (29). For individual proteins, some may be turned over even faster. This has to be compared with stabilised proteins which have entered the bicmass, and which now have an average half-life of about 24-30 h, i.e. sc~e 20-30 times longer (34). The essential point is that when proteins are not integrated, they remain at high risk of degradation, which follows first order kinetics, i.e. it is a randcm process (29). The rapid removal of proteins, which keeps the 'decks clear' of surplus, spent, error or spontaneously denatured molecules (33), would approach zero if diffusion alone wore the only mechanism by which these protein substrates met intracellular proteases. Requirement for an intracellular circulatory system What has been said for degradation is but one part of the whole scenario of cellular functioning based on a requirement for the delivery of molecules for both anabolic and catabolic function at many different sites throughout the cytoplasm and nucleus, for it will be as readily appreciated that the distribution of new proteins to their sites of operation could take far too long in a rich protein solution. It has been shown that large protein molecules move very rapidly through the cytoplasm, e.g. fluorescent-tagged protein introduced into a cell by microinjection is seen to move to all parts of the cell within the time it takes to cQmplete the injection (a matter of a second, see 35). Calculation of the time taken for a protein molecule to move across the average diameter of a HeLa suspension culture cell (14-16 ~m) can be made frcm the data of Wojcieszyn et al. (36), and this is about 26-27 rain at 20°C, because of the obstacles and the viscosity of the cytoplasmic matrix. The same molecule would move about 70 times faster through a simple aqueous buffer solution, and therefore take about 22-24 sec to traverse the cell. Both these estimates are irreconcilable with the observed rate ~.t which tagged proteins can be dispersed throughout the entire cytoplasm, even the latter estimate being more than an order of magnitude too slow (35) . A perfusion system or intracellular circulation is seen as an efficient means of sustaining active metabolism within the cell, but it does not exclude
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ebb-and-flow. Irrespective of which of these two processes may predominate at any time in any particular type of cell, the essential point is that conducted diffusion (perfusion) is probably vital to life processes. Put another way, life is highly dependent on a hydrodynamically driven endocellular perfusion or circulation system. The validity of this hypothesis, as of the proverbial pudding, lies in the eating, for it is readily testable. Evidence from glucose metabolism (4), amino acid uptake (37,38) and protein metabolism (39) already indicate that these diverse processes are dependent upon a sustained flow over internal surfaces and membranes upon which most reactions take place within the cytoplasm (6). It is possible that other means of translocation of molecules for metabolism exist, but it seems inconceivable that every substrate or product of every metabolic interaction has some guiding mechanismwhich directs its particular movements from one place to another inside such a complicated milieu as the internum of the cell. A con~non distribution system bringing in essential nutrients and removing waste products would therefore seem to be a reasonable proposition at the cellular level for the reasons discussed above, just as our blood circulatory system operates effectively to supply our entire body. Acknowledgements I wish to thank The Wellcome Trust Drs. P.C. Malone, J.S. Clegg and R.P.C. discussions, Mrs. Marget Inglis for her Martin for help with the preparation of
for supporting this work, Johnson for helpful conments and assistance throughout, and Miss Sandra the manuscript.
References I. H. KACSER and J.A. BURNS, Symp.soc.exp.biol. 27 65-104 (1973). 2. M.J. TAIT and F. FRANKS, Nature 230 91-94 (1971). 3. S.L. WOLFE, 'Biology of the Cell', Wadworth, Belmont, Calif., pps. 67-68 (1972). 4. J.L. MANSELL and J.S. CLECgS, Cryobiology 20 591-612 (1983). 5. J.S. CLEGG, Amer.J.Physiol. (in press). 6. C. SHERRINGTON, 'Man on his Nature', Gifford Lectures 1937/8 Penguin Books, Harmondsworth, Middlesex, pps. 69-98 (1940). 7. R.A. COUI~ON, T. HERNANDEZ and J. HERBERT, Comp.Biochem.Physiol. 56A 251-262 (1977). 8. R.A. COULSON and J. HERBERT, Cc~p.Bioohem.Physiol. 7_~ 125-132 (1982). 9. R.W. HOLLEY, Proc.nat.acad.sci., U.S.A., 69 2840-2841 (1972). 10. D.N. WHEATLEY, New Scientist 87 294-296 (1980). 11. A.B. PARDEE, In vitro 7 95-104 (1971). 12. W. HARVEY, 'De Motu Cordis', (Movement of the heart and blood in animals. Translated from the latin), Blackwell, Oxford (1957). 13. S. GRISOLIA, J. CARRERAS, D. DIEDERICH and J. CHARACHE, in 'Red Cell Metabolism and Function' (Ed. E. Brewer), Pergamon Press, Oxford pps. 39-55 (1970). 14. C. COHEN, D.F.D. CASPAR, J.P. JOHNSON, K. NAUSS, S.S. MARGOSSIAN and and D.A.D. PARRY, Cold Spring Harbor Syrup. 37 287-297 (1972). 15. S.E. FREDERICK and E.H. NEWCOMB, J.cell biol. 43 343-353 (1969). 16. S.B. HOROWITZ, P.L. PAINE, L. TLUCZEK and J.K. REYNHOUT, Biophys. j. 25 33-44 (1970). 17. P.L. PAINE, T.W. PEARSON, L.J.M. TLUCZEK and S.B. HOROWITZ, Nature 291 258-261 (1981). 18. K.R. PORTER and M.A. McNIVEN, Cell 29 23-32 (1982) . 19. T.H. HUXLEY, 'Collected E ssa~
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23. 24. 25. 26.
27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.
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D.N. WHEATLEY, Exp.cell res. (in press). P.C. MALONE, Med. hypotheses 7 1477-1489 (1981). S. GRISOLIA and D.N. WHEATLEY, Life chem. ~Teps. 2 257-297 (198'i). K. FINK and E. Z ~ , in 'Ce__llReproduction, in Honor of Daniel Mazia', (eds. E.R. Dickson, D.M. Prescott and C.F. Fox) Academic Press, New York pps. 103-110 (1978). R. BRAVO and J. CELIS, J.cell biol. 84 795-802 (1980). C. MII2AREK and Z. HA~{N, J.cell biol. 79 833-838 (1978). D.N. WHEATLEY, S. GRISOLIA and J. H E ~ E Z - Y A G O , J.theoret.biol. 98 283-302 (1982). D.N. WHEATLEY, K.S. BAIRD and M.S. INGLIS, Mol.physiol. (in press). A. RON and D.N. WHEATLEY, Exp.cell res. 153 158-166 (1984). S. GRISOLI'A, Physiol.rev. 64 657-712 (1964). D.N. WHEATLEY, J.theoret.biol. 107 127-149 (1984). D.N. WHEATLEY, M.R. GIDDINGS and M.S. INGLIS, Cell biol.int.reps. 4 1081-1090 (1980). D.W. STACEY and V.G. AT,F,FREY, J.cell biol. 75 807-817 (1977). J.W. WOJCIESZYN, R.A. SCHIZGEL, E-S. WU and K.A. JACOBSON, Proc.nat.acad. sci., U.S.A. 78 4407-4410 (1981). D.N. WHEATLEY,~M.S. INGLIS and J.S. ~ , ICRS Med.sci. 11 594-595 (1983). D.N. WHEATLEY and M.S. INGLIS, J.theoret.biol. 83 437-445 (1980). D.N. WHEATLEY, M.S. INGLIS and J.C. ROBERTSON, Mol.physiol. 6 163-181 (1984). R.D. AT.LEN, J.L. TRAVIS, J.H. HAYDEN, N.S. AT,T.EN, A.C. BREUER and L.J. LEWIS, Cold Spring Harbor Syrup. quant.biol. 46 85-87 (1981). J.H. HAYDEN, R.D. AT.T£N and R.D. GOI~)MAN, Cell motil. 3 1-19 (1983).
ON
THE
POSSIBLE
Oerzamon Press
MINI-RE'VIEW IMPORTANCE OF AN INTRACELLULAR
CIRCULATION
Denys N. Wheatley Cell ])athology Laboratory, Department of Pathology, University Medical Buildings, Foresterhill, Aberdeen, AB9 2ZD, Scotland, U.K.
Sunmary Since ultrastructural, biophysical and other studies continue to demonstrate that the internum of the cell is highly structured, the question raised and discussed in this review is whether an intracellular circulatory system is essential for the maintenance of active metabolism. Although cytoplasmic streaming is evident in large animal and plant cells, it is argued that it probably occurs in all cells irrespective of size, and is of particular importance in bringing together interacting molecules fast enough for metabolic processes to occur which would otherwise be far too slow if diffusion were the only form of motion. A cc~mon intracellular system would suffice for most metabolic processes and would also help to dissipate waste products. Interruption of this internal circulation would result in the inhibition of metabolic functioning, including for example protein turnover, for which evidence is presented to substantiate this hypothesis. Many significant advances have been made in biological sciences through the surge of new technology, and in particularbecause of the rapid progress in molecular biology in the last twenty or so years. Although this has led to a far better understanding of biochemical aspects of life, the awesome complexity and dynamic nature of organisms means that d~eir physiology in terms of the integration of individual biochemical reactions remains obscure. The gulf between what is known about an enzyme in vitro and how it operates and is regulated in situ within some compartment of the living cell is still very wide Many studies on enzymes involve highly purified preparations observed under specifically tailored laboratory conditions which may give data on substrate preferences, initial reaction rates, etc., in accordance with the laws of mass action, but probably tell us little about their role when embedded in some particular metabolic pathway on some membrane surface inside the cell (I). The 'watery' cell Now that the oversia~lifications and artificiality of most in vitro biochemical analyses are becoming better appreciated, the internal operations of a cell are less likely to be compared with a jumble of chemical interactions taking place in bulk aqueous phase in which the laws of mass action apply (2). While a number of reactions undoubtedly take place in the bulk aqueous phase, the question is whether they represent the majority or a very small minority of the total, with most proceeding according to laws of surface chemistry (3). Recent experiments with dehydrated cells suggest the latter (4,5), and many }~m~nlc 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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have long been convinced that life is mostly a surface-based operation: "Although it is fluid and watery, most of the cell is not a true solution. A drop of true solution, of hmmogenous liquid, could not 'live'. It is ren~)te from 'organization'. In the cell there are heterogeneous solutions. The great molecules of protein and aggregated particles are suspended not dissolved. A surface is a field for chemical and physical action. The interior of a pure solution has no surfaces. But the aggregate of surface in these foamy colloids which are in the cell mounts up to something large. The 'internal surface' of the cell is enormous. The cell gives chemical results which in the laboratory are to be obtained only by temperatures and pressures far in excess of those of the living body. Part of the secret of life is the i~nense internal surface of the cell." (Sherrington, ref. 6). Regulation of metabolic rate The rate at which simple reactions proceed, assuming that the conditions are appropriate, conducive and -- for ease of analysis -- constant, is governed by the accessibility of the substrates (precursors/nutrients) to the catalytic surfaces, their concentrations (up to a certain level), and the rate of removal of products. Small changes in conditions in living systems can quickly result in a reaction being reversed, inhibited, accelerated or diverted along some other pathway. Assuming that products will be rapidly utilised in a growing cell type with a relatively fast turnover time for protein, the rate of many reactions will be determined by the rate at which the reactants can be delivered to the catalytic sites (it is implicit t]~at in the absence of enzymes, the vast majority of metabolic processes would be far too slow to sustain life). The principle to which Coulson et al. (7,8) have rightly drawn attention from the gross physiological viewpoint offers an explanation for the widely differing rates of metabolism found in the cells of different species of manuals, i.e. that these may depend to a large extent on the rates at which precursor molecules can be delivered to their individual cells. This, it is argued, can impose severe limitations on the relative metabolic rates of a whale co,oared with a shrew, which may differ by as much as three orders of magnitude in their metabolic activity. The flow constant of essential nutrients will have very different values in the shrew than the whale because the sheer size of the latter imposes a large time delay in getting substances from source (e.g. gut or lungs) to sink (the individual cells of the body). Actual delivery rates will not just depend on circuit time, but also on differences in concentrations between arterioles and extracellular space, between the latter and the internum of the cells, and also on the relative volume of the delivery medium (blood for the most part) and its circulation time. Provided with similar medium in a tissue culture vessel, it is suggested that little difference would be found between shrew cells and those of the whale because the delivery rate of nutrients would be equal. Although this concept offers a simple explanation for divergent metabolic rates, there will be a number of other factors governing intracellular activity, one of which is the ability of the cell to 'take on board' the supplies being delivered by the circulatory system. Holley (9) suggested that the persistent proliferation of cancer cells was due to their inability to restrict amino acid transport across the pla~namembrane. His hypothesis was based on the improbable assumption that, because precursors would continue to be made freely available inside the cell, the ribosomal machinery is obliged to sustain a high rate of protein synthesis,
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thereby necessitating repeated division of cells. This concept is inconsistent with amino acid uptake data in both normal and tumour cells (10), but it does raise the question of whether the delivery rate of precursors to the intern~n of the cell is of as much importance as delivery to the locality of the cell by the main circulatory ~jstem. It is certainly true that much of cellular functioning is regulated at the plasmamembrane, including ultimately the regulation of division (11). Thus it may be pertinent to suggest that delivery rates of precursors to the immediate vicinity of the cell, e.g. sugars for energy metak~lism, may determine the potential metabolic rate under those conditions (the coarse control), while the cell membrane transport mechanisms modulate the actual accessibility of these precursors to the internal machinery (the fine control). The broad implication of Coulson et al. (7) is that the metabolic machinery in the whale cell is capable of working faster in situ, but that the major limiting factor here is the rate at which precursors arrive; just as a fire burns brighter when it is fanned, the whale cell would metabolise faster if the delivery of essential nutrients could be accelerated. It is suggested that within the confines of the intracellular regulatory factors just mentioned, this principle should at least be taken one stage further. We propose that in addition to the delivery of nutrients to the cell environment, it is important (possibly essential) to have a circulatory system within the cell, without which most biochemical pathways would be unable to operate at rates sufficient to sustain even modest metabolic activity. The asstmption being challenged is that when molecules are delivered to the vicinity of the cell, diffusion -regardless of whether it is assisted in some way or not across the plasmamembrane itself -- takes over from this point. This proposal may have little direct evidence to support it at the present moment, but it does present an alternative to a generally accepted, but probably erroneous, assumption that simple diffusion of free molecules is the mechanism by which nutrients move from the inner aspect of the cell membrane to all parts of the cell. It is worth exploring this idea a little further because it seems to offer a framework upon which many observations relating to cell functioning can be more easily explained. Circulation and metabolic rate Harvey's great contribution to science (12) was in recognising that the flow of blood within the vessels of the body described a one-way, continuous circulation as opposed to an ebb-and-flow motion. It is interesting that this process by which all nutrients reach the cells of the body has only just begun to be appreciated in terms of its relationship with metabolic rate (7,8), some three hundred and fifty years later. The vital importance of a continuous circulation in an actively metabolising body, such as that of man, can be readily demonstrated by the fact that its interruption for more than a few minutes leads to death. This can only be postponed if metabolic activity has been reduced beforehand, as can be shown with smaller m ~ i s which enter torpid states. Creatures without elaborate circulatory systems are obliged to be small and highly mobile or long, slender, delicate and sessile, living in places where the environment constantly flushes over them. Even under these conditions, it is all too readily assumed that life processes are sustained by the simple diffusion of essential nutrients from the environment into the cells, and of waste products out again by the same process in reverse. Cells of many organisms contain about 70% water and about 20% protein. Some are even richer in protein; for example, the human erythrocyte can contain 35% protein, with haemoglobin so concentrated that it crystallises out when cells are suddenly lysed (13). Some pure protein crystals contain much less protein than cells because of a large amount of hydration--tropomyosin
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crystals are more than 95% water (14). Presumably the admixture of proteins with many other molecu±es as aggregates and on surfaces prevents proteins from normally appearirLg as crystals within cells, although they can be found in some storage organelles (e.g. peroxisomes, 15), and can sometimes appear in liver mitochondria under certain pathological conditions. If all the protein in a cell was in simple solution, this would present enormous resistance to the movement of any but the smallest and most mobile molecules and ions. Proteins also attract and adsorb smaller molecules, which will further restrict their movement. It would seem probable therefore that substances bigger than, e.g., urea would experience considerable difficulty moving through cytoplasm. Indeed, cc~parisons are now being made with gelatin reference phases within the cytoplasm of oocytes to explore the relative rates of diffusion of small molecules such as amino acids (16) and ions (17), and their differential distribution in the cell, as opposed to a 14% gelatin reference phase. Certainly the movement of very large molecules within the cell (e.g. most proteins) would be far too slow to sustain metabolic rates found in warn~blooded animals, especially during periods of prolonged and vigorous activity. How then d o m o s t molecules move around inside the cell? We are left with two main alternatives: (i) random movements, at best assisted by the ebb-and-flow created by the overall activity of the cell, analogous to the pre-Harveian concept of the movement of blood, (ii) organisedor structured movement of a 'cytocirculation' or 'endocirculation' occurring in defined, if labile and transient, channels within the cell, and for the most part describing a unidirectional pathway. Cytoplasmic streaming as a general phenomenon Cytoplasmic streaming has been observed for well over a century in large animal cells (e.g. Amoeba) and in plant cells (e.g. Nitella), especially those of the latter group having very large central vacuoles around which streaming describes an obvious circular motion, giving us the expression 'cyclosis'. Streaming of the soluble aqueous phase inside cells remains an unproven phenomenon since it can only be inferred from the behaviour of particles wafted along by underlying currents. Furthermore, this type of movement is not random because it is seen in distinct channels. It must be distinguished, however, from the precise , highly non-random movements seen with,e.g., pigment granules in chromatophore cells, whose yo-yo-like sorties are probably due to their being attached to specific contractile elements (18). Huxley (19) recognised the importance of the flow within large plant cells as a means of distribution of materials, and remarked that "if only we had ears to hear it, the roar of a tropical forest" would drown that of a railway station. It is quite conceivable that the circuitous motion of an intracellular soluble phase -- which may become correctly termed the cytosol in future -- seen in such paradigms as Nitella and Chara, is required in these cases, primarily because of their size. This rationalisation persists today and is not incorrect, except possibly in its inference, viz. that small cells would presumably not require it. It is equally valid to argue that the former examples are simply the more obvious examples of a general phenomenon occurring in all cells, and there is considerable evidence that streaming and organised movement of fluid phases does occur in many kinds of cell irrespective of their size, a notion which is supported by the observations of Allen and his coworkers (40, 41). To develop this argument a little further, it is worthwhile considering current developments in our understanding of cytoarchitecture,
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while reme~nbering that the interpretation of intriguing electronmicrographs of it which have recently been taken may not necessarily be an entirely accurate representation of the real situation in the living cell. There is now an increasing awareness that the 'spongioplasm' suggested by Sch~fer in 1898 may exist as an elaborate macrc~olecular reticulum, referred to as the m i c r o t r ~ u l a r lattice (21; see Fig. I). Careful centrifugation and stratification of intact Euqlena cells by Kempner and Miller (22) led to separation of organelles frcm the macromolecular matrix and from the aqueous phase of the cytoplasm. Their data is interesting because the last mentioned layer contained little protein and was virtually devoid of enzyme activity, suggesting that very few of the proteins in the highly protein-rich cytoplasm were in true solution in the living cell (cf.6). Recent analysis of data on protein turnover in HeLa cell cytoplasm has led to our estimating that the aqueous phase of the cell contains as little as 0.01% protein, end would therefore approximate the properties of water rather than a viscous macromolecular solution (23). The sudden disruption of the internal integrity of the cytoarehitecture when a cell blebs as a result of an osmotic shock results in a very distinct change in the movement of intracellular particles, which becc~e very active and show Brownian movement. The almost ccmplete absence of such activity in the undamaged cell by itself strongly indicates that the internum is highly structured. In agreement with Schifer's idea of a sponge-like matrix, life would seem to depend on perfusion occurring in a non-random, partially polarised manner through a matrix of insoluble protein.
Fig. I
High-voltage electron micrograph of the cytoplasmic ground substance or matrix of an L-929 cell, showing its microtrabecular nature after freeze substitution (from ref.5 through courtesy of Dr. K.R. Porter, see ref. 21). The bar indicates 0.1bm (negative image).
Because of the small size of the channels (see Fig. I), some pumping action would be necessary to initiate and presumably sustain flow. Contractile actomyosin is present in almost all protoplasmic material obtained from living organisms, and these may be responsible for pt~nping or pulsating, or as Malone suggests (24), the aqueous phase may be moved by a process akin to syneresis following the rhythmical contraction and relaxation of (helical) proteins. It is also noteworthy that a little energy is required to initiate proteolysis in cells (see 25 for a review), but it remains controversial as to how much and where this energy investment is made. Since pumping mechanisms utilises energy, there is good reason to believe that this could be an energy
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requirement associated with conti~uous turnover of proteins inside most types of cell. Other mechanisms will also operate which can cause transloeation of molecules, such as movements associated with the polymerisation and depolymerisation of microtubules (see refs. 40 and 41 for this and other possibilities) . There is little doubt that cytoplasmic streaming can be created by these means. To create a true circulation rather than simple ebb-and-flow motion, a co-ordinating centre would have to be envisaged, but as yet there has been no clear evidence that a structure analogous to the heart (in whole body terms) exists, even in large cells with very clear channels of streaming, such as Amoeba. The fact that directional streaming can be thrown into reverse in this organism must also be considered. The channels of a cytoplasmic circulatory system will not have the rigidity, the inflexibility or presumably the elaborate valvular control systems found in the blood system of the whole organism. It seems improbable that electron microscopists will reveal a miniaturised form of it at the intracellular level. But movements within cells can be highly non-random, as shown by the figure-of-eight route taken by food vacuoles in Paramecium, despite the absence of a detectable intracytoplasmic equivalent of an alimentary canal. Temperature, ~orotein turnover and ~rtop__lasmic streaming It cannot be concluded that only a circuitous flow of the aqueous phase occurs in all cells; beth the abovementioned alternatives may be involved to different degrees at the same time, i.e. a main flow could be accompanied by ebb-and-flow into peripheral pockets. It is suggested, however, that once a stream has been set in motion, the course of least resistance is for further flow to continue in the same direction until some event occurs which leads to its interruption or reversal. The cytoplasm and nucleus of the cell constitute a macrc~olecular labyrinth in which movement is probably highly restricting for the average protein (or similar) macromolecule, much as a jungle is restricting to the movement of man unless pathways are cleared through it. Blocking the pathways, or preventing traffic flowing along them by inhibiting the motive force, could have dire consequences on normal functioning. One particular facet which has recently been observed in our laboratory is the effect of heat on protein turnover. Synthesis of proteins is very sensitive to temperatures above 42°C, and falls to about 6% at 45°C (23). Degradation, being a largely exothermic reaction, was expected to increase with elevated temperature, and greater turnover of proteins was also anticipated because more aberrant and denaturation of proteins would arise. After a cell has been hemogenised, its hydrolytic enzymes can be shown to be more active at 45 ° than at 37°C, which establishes that the enzymes involved in proteolysis are not themselves destroyed. In the intact cell, however, raising the temperature to 45°C leads to the cc~plete abolition of protein turnover. The most notable change during short-term treatment of cells at the elevated temperature was their i~nediate cessation of all motile activity, saltation of particles, streaming, ruffling of advancing membranes, An~! p~eudopodial extension. The arrest of activities dependent upon actomyosin proteins and ATP-utilising systems appears to include the intracellular circulatory system. This reduces the flow constant of proteins past the proteases on internal surfaces or membranes in the cytoplasm, and thereby halts their hydrolytic activity, since the only interactions which can occur will require free diffusion of these large molecules in a highly restrictive environment. This now seems to be a plausible explanation for what od~er~ise might appear as a paradoxical situation (see ref. 23). A cell contains proteins appropriate to its environmental circumstances. It must be adaptable, malleable and quickly responsive to this environment to
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survive. And yet its general make-up involves throughout the whole of nature a similar set of proteins and other molecules which came together to create a thixotropic gel with r ~ k a b l e strength (25). It would seem that it continually makes proteins of a reasonably wide s p e c ~ (26-68), frcm which it retains those commensurate with prevailing conditions (29). Surpluses are cleared away by proteolysis. The cell is unlikely to utilise with 100% efficiency all the proteins that it synthesises. Indeed, the sorting and turnover of proteins at source involves very intense proteolysis which has hitherto been grossly underestimated (29) . The mechanism also indicates that proteins which are made but have no stabilising site or function to perform will be poorly retained and may not in fact appear to be synthesised at all (30,31). If there is a sudden change in the environment, e.g. the introduction of a substrate for a particular enzyme (31,32), such a protein may be stabilised and beccme evident by its a c ~ u l a t i o n within the cell. Equally, if the substrate is removed again, the enzyme disappears with considerable rapidity. To have an autc~atic control of this nature, protein turnover in the cell must be due to the operation of a continuous and wide-scale operation, of the type described as the basal system in previous work (33). The stabilisation of proteins is based on their utilisation within the cellular matrix and as part of this structure (29) ; if they do not integrate into the functional bicmass, they will be turned over very rapidly. This applies to newly-synthesised proteins and to those which through their activity are moving from an integrated conformation to one which destabilises them. The half-life of nascent proteins, and by inference of these destabilised proteins, in the growing HeLa cell is about 53 min (29). For individual proteins, some may be turned over even faster. This has to be compared with stabilised proteins which have entered the bicmass, and which now have an average half-life of about 24-30 h, i.e. sc~e 20-30 times longer (34). The essential point is that when proteins are not integrated, they remain at high risk of degradation, which follows first order kinetics, i.e. it is a randcm process (29). The rapid removal of proteins, which keeps the 'decks clear' of surplus, spent, error or spontaneously denatured molecules (33), would approach zero if diffusion alone wore the only mechanism by which these protein substrates met intracellular proteases. Requirement for an intracellular circulatory system What has been said for degradation is but one part of the whole scenario of cellular functioning based on a requirement for the delivery of molecules for both anabolic and catabolic function at many different sites throughout the cytoplasm and nucleus, for it will be as readily appreciated that the distribution of new proteins to their sites of operation could take far too long in a rich protein solution. It has been shown that large protein molecules move very rapidly through the cytoplasm, e.g. fluorescent-tagged protein introduced into a cell by microinjection is seen to move to all parts of the cell within the time it takes to cQmplete the injection (a matter of a second, see 35). Calculation of the time taken for a protein molecule to move across the average diameter of a HeLa suspension culture cell (14-16 ~m) can be made frcm the data of Wojcieszyn et al. (36), and this is about 26-27 rain at 20°C, because of the obstacles and the viscosity of the cytoplasmic matrix. The same molecule would move about 70 times faster through a simple aqueous buffer solution, and therefore take about 22-24 sec to traverse the cell. Both these estimates are irreconcilable with the observed rate ~.t which tagged proteins can be dispersed throughout the entire cytoplasm, even the latter estimate being more than an order of magnitude too slow (35) . A perfusion system or intracellular circulation is seen as an efficient means of sustaining active metabolism within the cell, but it does not exclude
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ebb-and-flow. Irrespective of which of these two processes may predominate at any time in any particular type of cell, the essential point is that conducted diffusion (perfusion) is probably vital to life processes. Put another way, life is highly dependent on a hydrodynamically driven endocellular perfusion or circulation system. The validity of this hypothesis, as of the proverbial pudding, lies in the eating, for it is readily testable. Evidence from glucose metabolism (4), amino acid uptake (37,38) and protein metabolism (39) already indicate that these diverse processes are dependent upon a sustained flow over internal surfaces and membranes upon which most reactions take place within the cytoplasm (6). It is possible that other means of translocation of molecules for metabolism exist, but it seems inconceivable that every substrate or product of every metabolic interaction has some guiding mechanismwhich directs its particular movements from one place to another inside such a complicated milieu as the internum of the cell. A con~non distribution system bringing in essential nutrients and removing waste products would therefore seem to be a reasonable proposition at the cellular level for the reasons discussed above, just as our blood circulatory system operates effectively to supply our entire body. Acknowledgements I wish to thank The Wellcome Trust Drs. P.C. Malone, J.S. Clegg and R.P.C. discussions, Mrs. Marget Inglis for her Martin for help with the preparation of
for supporting this work, Johnson for helpful conments and assistance throughout, and Miss Sandra the manuscript.
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