Diffusion, perfusion and the exclusion principles in the structural and functional organization of the living cell: reappraisal of the properties of the `ground substance'
Department of Cell Pathology, University of Aberdeen, 581 King Street, Aberdeen AB24 5UA, UK
e-mail: wheatley{at}abdn.ac.uk
Accepted 15 January 2003
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Summary |
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Key words: intracellular, molecular movement, transport, diffusion, perfusion, survival, cytomatrix, cytosol
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Introduction |
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Diffusion, an old paradigm on which molecular interactions were based |
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Complex systems such as a living cell cannot be reduced to simple equations |
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`To be sure, when the number of factors coming into play in a phenomenological complex is too large, scientific method in most cases fails us...Occurrences in this domain are beyond the reach of exact prediction because of the variety of factors in operation, not because of any lack of order in nature.'
While it would be nice to think that equations for cellular functioning
based on the physical laws of diffusion adopted and adapted by Fick
(1855) from Fourier's analysis
of heat transference in metal bars
(Fourier, 1828
; see
Freeman, 1878
for a
translation) would apply, and although they might seem largely capable of
representing the situation in a living system, the evidence has piled up to
show that there is no way in which diffusion alone can make a major
contribution to much of cellular physiology (reviewed in
Agutter et al., 2000
), and it
does not provide the `connection of profound generality' required of a concept
that Einstein mentioned in the continuation of the above statement:
`We have penetrated far less deeply into the regularities obtaining within the realm of living things...What is still lacking here is a grasp of connections of profound generality, but not a knowledge of order in itself.'
However, despite all that we have written in defence of the notion that diffusion is indeed largely irrelevant in living systems, we cannot avoid the fact that it nevertheless occurs and will naturally (and without apparent additional energy expenditure) contribute to such activities as nutrient uptake by cells. An important issue concerns these `energy' requirements, and three points that rarely get aired ought to be considered.
First, in living systems, most molecules do not generally move, but are
moved, when we consider what would happen if everything depended upon Brownian
motion and the law of mass action
(Wheatley, 1993b). The most
pertinent comment in this regard was made by Johnson
(1983
), who recognised a grey
area at the molecular level when considering the movement of molecules within
living cells:
`This is the region of scale where flow and diffusion are not clearly separated; where the concepts of temperature and molecular movement overlap; where it is not clear whether molecules move or are moved; where the ideas of active and passive lose their meaning'.
They are moved by other molecules or indirectly by energy liberated from
their interactions with other molecules [the seminal work of Einstein
(1905) and von Smoluchowski
(1908
) dealt with `Brownian
motion', which is not diffusion per se].
This leads to the second point: diffusion ought to connote its full (and
precise) scientific meaning and not the vernacular used in the quotation by
Darnell et al. (1986; see
below). It has to imply that there is a tendency of molecules, through their
thermal agitation and jostling with others (Brownian movement), to move down
their physical gradients, from regions of higher concentration to regions of
lower concentration. `Diffusion' of molecules, meaning the random jostling of
Brownian movement, is not a correct use of the word in a scientific context,
and such a connotation has to be strongly resisted. It is this problem that
almost certainly led in the early days of (cell) biology to diffusion being
seen as the obvious way in which molecules `sorted themselves out'. The
assumption is so ingrained that almost everyone falls into the trap until he
or she has repeatedly been shown its inadequacy and their attention has been
drawn to rational alternatives. But the real issue is that if diffusion does
assist in such activities as the uptake of nutrients or release of waste
substances then gradients do need to exist. Without them, i.e. without `sinks'
to effectively help create them, diffusion is of little or no relevance (use).
The discontinuous gradients that exist at cell membranes are not created
without considerable energy input. Therefore, the energy that needs to be
expended to make diffusion of any value to a cell, e.g. in nutrient uptake, is
mostly spent in manufacturing and sustaining gradients that give the system
the unlevel playing field or step on which diffusion can effectively provide
the molecules to move in a particular direction. It is this vectorial aspect
that becomes the truly important issue in cell biology, but diffusion is not
always (often?) an efficient means of vectorially transferring molecules.
These considerations also bring home the third point that seldom gets
voiced: diffusive activity within cells, far from being set up to assist
metabolic functions as the norm, will more usually be acting
counterproductively, i.e. as a destructive force that constantly dissipates
gradients in which much energy had been expended in establishing them for one
species or other of a particular molecule. This explains in many cases why
cells have to be unceasingly active (`unresting', in the words of
Gerard, 1940). The difficulty
here is for us to quantify diffusion in terms of its global activity in a cell
and then to apportion this to constructive and destructive actions. If we only
see or measure the former then we ignore the latter, which may be n
times more prevalent. Exactly what their relative contributions are would be
difficult to estimate, but one other factor cannot be ignored at this time:
the extent to which the cell is an organized and `stable' structure; i.e. it
is not a pool in which chaotic behaviour rules.
Looking down the microscope, it is hard not to accept that cells are
relatively stable structures. However, once the level of resolution is
increased enormously, one starts to appreciate how fast a cell has to run just
to stay on the same spot. To give a more descriptive analogy, it is like
looking down on traffic from an aeroplane at 35 000 feet. It hardly seems to
be moving. Then, as you come into land, you pass over roads at about 100 feet
and the traffic is zooming along, seemingly as fast as you are. This can be
seen in cells along various transient tracks, but it takes exceptionally good
instrumentation to follow it in small cells
(Wheatley et al., 1991) and
the resolution of equipment needed to quantify it has to improve by 12
orders of magnitude. But the intact living cell is not jostling like one sees
Brownian movement in a cytoplasmic bleb caused by injury. So, while the cell
internum is very active, thermal motion of most of its molecules is
greatly restrained and Brownian movement becomes barely perceptible. This
alone should be a convincing argument that the cell is well organized and,
especially, that the milieu is not necessarily of a simple aqueous
consistency, and calculations ought not to automatically assume that this is
so (Mastro and Keith, 1984
).
The structure we see in cells is indeed a physical representation of its
metabolic processes; in brief, the cell is the functioning entity in a
holistic sense and should not be seen as a structure within
which function occurs. So, if we have largely ruled out the jostling
expected from Brownian movement in a fluid phase, we need to know more about
the cell in terms of its organization and especially about the milieu in which
everything takes place. The milieu is aqueous without a doubt, but the
question concerns its state and how it interacts with other molecules to
create living substance.
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Superb organization: the essence of the living state |
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`Extreme order has to be reconciled with a fluid anatomy...This cannot be done adequately without borrowing concepts from physiology, and especially from neurology. The cell must be considered as a reflex entity, structurally organized so far as even its chemistry is concerned, with chains of chemical substances acting as it were as reflex arcs...Our (sensitive) mosaic may radiate its effects throughout the cell. It is perfectly possible to appreciate how a co-ordinated structure may be maintained in a medium which is apparently liquid. This theory is all that is needed to enable us to understand how substances can reach a special site in the cell. Between the chains of molecules, fixed by their radiating webs, there will exist paths from the external to the internal surface, the capillaries of the cell' [My emphasis].
In other words, since as many as 4000 reactions may be occurring
simultaneously in a quiet cell, without the little crucible boiling away as
vapour by the heat dissipated, each and every one has to be harmoniously
controlled. There is no factory on earth that comes anywhere near this
complexity and, at the same time, gives the fidelity or replicative
performances while remaining flexible and adaptable to its environment. But
then life has spent 35 billion years perfecting this act. Such a
contention is totally against the notion that everything in a cell happens by
the sorting out of molecules from one another based on random movements
(Berg, 1993; see
Agutter and Wheatley, 2000
),
i.e. by a process that has wrongly been called diffusion. It has taken nearly
70 years to move away from the diffusion paradigm, from the fatally flawed
statements in some of the first editions of our current chosen textbooks on
cell biology (although new editions have made amends to some extent). Darnell
et al. (1986
) in their first
edition wrote:
`Diffusion is the entropy-driven process by which molecules distribute themselves in whatever volume is available to them...Because a cell coordinates its metabolic activities by diffusion alone, the rate at which molecules diffuse throughout the cell limits the typical cell's size to between 30 and 50 µm in diameter. More specifically, in most cells, whatever their shape, no metabolically active interior region is more than 15 to 25 µm from the cell surface.'
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Choice of the main scenarios |
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The first scheme referred to the prevailing scenario for many years in the
20th century, that the cell was a bag of enzymes in relatively simple
solution. They can work like this in vitro, so why should they not
work like this in vivo? The other model that was proposed dealt more
with a gel-like or structured system through which molecules are directed with
great precision so that there exist thousands of microdomains in which careful
pathways are followed by the appropriate molecules, leading to the desired
products and responses in the right place at the right time. Acceptance of
this state of affairs is long overdue, but it is probable that today those
adhering to the first scenario are probably a small minority to whom few are
likely to pay attention in the future. But science demands that we test
hypotheses and gain consensus only when adequate experimental evidence is
available for us to make proper judgement. Just as we have called on those
adhering to the notion that `diffusion suffices' to prove their case by
experimental demonstration of evidence for their claim (and not just the
extrapolations; Krogh, 1941;
but see Hoofd, 1992
), those
adhering now to the second scenario are in just the same position. It has to
be shown that the dynamics (kinetics) involved are either by directed movement
or, at the least, that they could not have been accounted for by
diffusion, i.e. random molecular walks, alone. [In this discussion, all
mention to processes such as facilitated diffusion and other similarly
sounding processes are excluded, because assisted diffusion cannot
technically be diffusion at all, where that is meant to imply only these
random walks of molecules within gradients.]
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Back to basics: reappraisal of the `ground substance' |
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Having agreed that the cell contains definite organelles, from the obvious ones, such as the nucleus, mitochondria and lysosomes, to the many small ones, such as synaptic vesicles, Golgi cisterna and cell membrane, that actually exist and are not artefacts of preparation for examination, it is not too much for the structuralist to now include some large macromolecular complexes, such as ribosomes, microtubules and proteasomes, as miniature organelles, even if they are not bounded by membranes. Accepting that the nucleus is tethered in a cell (which can be proved by a variety of techniques and follows from the common sense observation that if it was not then it would lie hard against the membrane at the `bottom' of each cell after centrifugation), it follows that many other organelles have a non-random distribution.
The changes in distribution of organelles with, for example, the division
cycle is a subject of considerable topical interest; for example, if we watch
the fate of the nuclear envelope and its components
(Salina et al., 2001). I only
wish to extend this one stage further here by suggesting that the
mini-organelles mentioned above, and perhaps even free macromolecules and
macromolecular complexes, are probably not randomly distributed in the cell.
This argument simply reduces to what I said another way round above; that the
cell is superbly organised and there is no reason to suppose that it is not
similarly organized at all hierarchical levels. Having covered everything from
the nucleus down to large receptor molecules being rather precisely located,
e.g. usually on the cell membrane in the case of the latter, the question is
what is there left to discuss? And here the history must be read, for it comes
down to the nature of the milieu in which all these structures exist and all
the functions of the cell occur; and what happens to it and within it that
makes for `the living state'. We know that Szent-György
(1971
) referred to this
water-base as the mother liquor without which life would be impossible
(Harold, 2001
). This being the
case, then water is the essence of life the molecules `dancing to
the tune of the solids', according to Szent-György.
Latterly, more and more investigators are concerned with the nature and
state of the water inside cells and, especially, the properties it assumes
when it meets any kind of surface or conditions within cells (e.g. vicinal
water; Drost-Hansen and Singleton,
1992) or special kinds of molecular surfaces (primarily proteins)
such that it becomes more gel-like
(Pollack, 2001
). There is a
world of difference between these two extremes, and this is perhaps the most
crucial message that has to be imparted in this articlethat we need to
know exactly how the state of water inside the living cell differs from that
in a beaker of tap water, as depicted by Wheatley
(1993b
).
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Redefining the `ground substance' |
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Water, ions and molecules, big and small |
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Exclusion principles, adaptation and survival |
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Some other microdomains to consider |
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So, diffusive processes are clearly operative in these microdomains,
although it is extraordinarily difficult to verify this claim, just as it is
only surmise that acetylcholine liberated at the presynaptic membrane
diffuses across that short gap to the post-synaptic membrane at nerve
junctions (Wheatley, 1998).
This does not exclude, therefore, the possibility that molecules go down their
concentration gradients by other means, such as by flow of the milieu, the
pumping action of other microstructures or some expulsive or quick capture
process. What it says is that molecules can still move relatively freely in
these domains if and when squeezing and pumping do not operate. We need to
know how a substrate in a tightly coupled pathway leaves the first enzyme as
its product and becomes the substrate of the next enzyme to act upon it. Does
it diffuse within a nanodomain such that it has a small interval of `freedom',
or is it passed on without ever losing its `tethering' in much the way that
Miles et al. (1999
) have
indicated from their work on tryptophan synthesis?
Molecular complexes may be designed either way. If the proteasome takes in
a faulty protein, it will be acted upon by a sequence of enzymes, but it may
have to roll around inside the barrel of the proteasome between each enzyme
reactionwhich is why it would seem plausible, in teleological terms
(Agutter and Wheatley, 1997,
1999
), to have such a
structure. Oster (2002
)
recently put forward the notion that myosin might be tethered by one foot to
actin but that it requires diffusion-like freedom to pivot and wobble until it
strikes the point where its other head can meet the next actin molecule. What
is much less likely is that a molecule rotationally showing maximum motion
within a small domain might also be translationally moved within the compass
of reactive sites (assuming that domain is approximately 20 nm) very quickly,
but its chances of getting right across the cell would be infinitely smaller
if it had to negotiate unassisted thousands of curtains of cytomatrix, the
received wisdom of the `crowded cytoplasm'. Again, we return to the scientific
definition of diffusion, and rotational motion through thermal agitation is
not diffusion. But within the microtrabecular lattice, we probably
have domains of approximately 20nm in diameter in which molecules spin around
making many contacts with the vast surface of the matrix on which most
metabolism takes place (also a hypothesis with strong supportive evidence that
is now so generally accepted). The freedom required here is for small
molecules that can be highly mobile (
500 Da) and need only cover a few
nanometres to be of use, but even bigger protein molecules would gyrate with
sufficient speed to enter many interactions.
Bringing molecules to these sites and maintaining the gradient through
their utilisation at these sites probably involves more than simple diffusion,
and here we must think not of the resting cell, in which action can be quite
leisurely, but when the organismal demands on a cell are at a maximum and
there is a need to deliver supplies fast. Nevertheless, one can still ask the
pertinent question of whether life can be sustained on diffusion alone,
however `leisurely' or resting we try to make the circumstances. This is where
Coulson (1986) has made the
greatest contribution, although indirectly, to this debate. For at the level
of general physiology, his principle simple states that delivery is usually
the rate-limiting factor determining how fast a tissue or organ can work.
Inside the cell, the same principle should apply, and we have considered that
delivery by perfusing enzyme beds can be used to speed up reactions as well as
effectively regulate the metabolic rate
(Clegg and Wheatley, 1991
;
Wheatley and Clegg, 1994
).
[For more information on this aspect, refer to Wheatley
(1999
).] For much of the
metabolic activity that takes place within a cell, there is now a groundswell
of opinion that little is left to chance (Brownian motion) and that perfusion
of enzyme-studded surfaces is the order of the day. While this is not
exclusively the caseone of the major points that has been
emphasised throughout this articleit is clear from the work and
carefully considered arguments of Hochachka
(1999
) that he had arrived at
this same conclusion, one which ought to be heeded for its explicit handling
of the confusing data in this field. The `capillaries of the cell' reported by
Peters (1930
) refer to a
nanocirculation (if the term `microcirculation' is reserved for the capillary
beds in tissues first seen by Malpighi). Within the cell, the nanocirculation
would indeed exist mostly as transient channels suffusing the matrix, where
catalytic activity and most functional activity is concentrated (e.g.
Getzenberg, 1997
).
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Concluding remarks |
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Acknowledgments |
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