Bioscience Reports, Vol. 11, No. 6, 1991
Biochemical Topology" From Vectorial
Metabolism to Morphogenesis
Franklin M. Harold
In living cells, many biochemical processes are spatially organized: they have a location, and often a
direction, in cellular space. In the hands of Peter Mitchell and Jennifer Moyle, the chemiosmotic
formulation of this principle proved to be the key to understanding biological energy transduction and
related aspects of cellular physiology. For H. E. Huxley and A. F. Huxley, it provided the basis for
unravelling the mechanism of muscle contraction; and vectorial biochemistry continues to reverberate
through research on cytoplasmic transport, motility and organization. The spatial deployment of
biochemical processes serves here as a point of departure for an inquiry into morphogenesis and
self-organization during the apical growth of fungal hyphae.
KEY WORDS: Chemiosmotic theory; spatial order; vectorial metabolism; proton circulation;
cytoskeleton; calcium; apical growth; ion currents.
INTRODUCTION: O R G A N I Z E D COMPLEXITY
Biochemists, E r w i n C h a r g a f f o n c e wrote, are peculiar people; they would take a
fine Swiss watch, grind it up in a m o r t a r , and meticulously e x a m i n e the debris
hoping to learn h o w the watch w o r k e d ! To be sure, when Chargaff p e n n e d this
c o m m e n t in the mid-sixties, the time was long past w h e n we t h o u g h t o f a cell as a
bag of e n z y m e s and dismissed c o m p a r t m e n t s as the last refuge of an i n c o m p e t e n t
enzymologist. B u t there was and remains m o r e than a little justice in Chargaff's
taunt. B i o c h e m i s t r y is still m o r e c o m f o r t a b l e with clean e n z y m e s than with
c o m p l e x ( " d i r t y " ) systems, and the first step in o u r research m e t h o d o l o g y
c o m m o n l y is to grind the intricate architecture of the living cell into a
homogenate.
Still, w h e n c o r n e r e d , biochemists will c o n c e d e that as long as they are alive,
cells and organisms are elaborately organized systems. T o m a k e a cell, the
molecules we find on o u r gels must be d e p l o y e d in a characteristic spatial o r d e r
that has functional meaning. I n d e e d , terms such as o r d e r , complexity and
organization are w o v e n into o u r intuitive sense o f what is m e a n t by "life" and
"living" (Simon, 1962; Polanyi, 1968; Riedl, 1978; M a y n a r d - S m i t h , 1986).
Department of Biochemistry, Colorado State University, Fort Collins, CO 80523, U.S.A.
347
0144-8463/91/1200-0347506.50/0 ~) 1991 Plenum Publishing Corporation
348
Harold
Organization poses a raft of problems, for the experimental scientist no less than
the philosopher, for if we hope to understand how living things function we
cannot ignore the higher levels of order. How do cells coordinate the activities of
thousands of genes and tens of thousands of proteins into purposeful activities
such as movement or digestion? How does structural and functional organization
arise, in the growth of an individual and in the evolution of a species? How is the
pattern of spatial organization transmitted from one generation to the next, and
maintained for milennia with minimal variation? How can we reconcile the
progressive elaboration of biological organization during evolution with the bleak
dictates of the second law of thermodynamics? Do laws of order exist and do they
apply to living things?
Biological research today is almost wholly dominated by analytical methods
and attitudes. The power of this approach is undeniable, but I am surely not
alone in suspecting that most of the important questions amenable to it have, in
fact, been answered. The conceptual frontiers of the foreseeable future run
through the problems of growth, morphogenesis, behaviour and development.
Progress in these areas will not automatically spring from the ever more detailed
scrutiny of the underlying molecular mechanisms, but will demand that we
reconstitute living systems from their molecular elements, at least in the mind's
eye. For the next generation of biological scientists, the central issues will be
defined by Warren Weaver's (1948) phrase, "organized complexity".
The reader may well wonder what this homily on the future direction of
biological inquiry has to do with the present celebration of proton circuits and
vectorial chemistry. We are accustomed to think of the chemiosmotic theory in
the context of energy transduction, an association constantly reinforced by the
textbooks. But we can also claim the chemiosmotic theory as one of the first
milestones in the exploration of supramolecular organization: vectorial reactions,
membranes and ion fluxes are conspicuous links between biochemistry and
physiology.
The subject of this article is the organization of biochemical and physiological processes in space; the title adopts the Oxford English Dictionary's usage of
topology as the science of space. It is an essay, not a comprehensive review, and
its purpose is to clarify (for myself, as much as for the reader) what a research
programme on biological organization at the cellular level should seek to
discover. Understanding an organization is not the same as unravelling a
mechanism: the focus must be, not on the parts but on how they contribute to the
operation of the whole. The relevance of vectorial chemistry and ionic currents to
cellular organization was plain to Mitchell from the beginning (Mitchell, 1962),
but it remains a road little travelled by others. So I have taken the chemiosmotic
revolution as the point of departure for an exploration of molecular parts and
functional wholes, particularly of the connections that knit the parts into a whole.
THE D O M A I N OF VECTORIAL BIOCHEMISTRY
Protein molecules are regular and shapely bodies, not blobs; they come with
clefts, cavities and sometimes channels, undergo ordered changes of conforma-
Biochemical Topology
349
tion and possess intrinsic asymmetry or polarity. This is a commonplace today,
colourfully illustrated in every textbook, but in the 'fifties it was a speculative and
pregnant notion. Mitchell and Moyle (1958a, b) recognized its profound implications for enzymology: enzyme-catalyzed group transfer reactions proceed along a
definite spatial trajectory within the protein molecule. The directionality of
chemical processes has no particular consequences for enzymes in solution, but
may acquire biological significance if the enzyme becomes part of a larger
structural complex.
When enzymes are inserted into a membrane in such a manner that the
reaction pathway crosses the barrier, chemical transformation of the substrate
may be accompanied by the translocation o f a chemical group or of the reaction
product(s) across the membrane. Mitchell coined the terms "group translocation"
and "vectorial metabolism" to describe chemical reactions that have a direction in
space, and proposed that such reactions underlie many, if not all, biological
transport processes (Mitchell, 1963, 1967, 1972). One of the first fruits of his
insight was the chemiosmotic hypothesis of energy transduction in oxidative and
photosynthetic phosphorylation (Mitchell, 1961), whose experimental basis was
then elaborated by Mitchell and Moyle (Weber, 1991). Nowadays, if vectorial
metabolism appears in the index of a textbook of biochemistry or cell biology, it
is invariably in the context of ATP generation. But the implications of anisotropic
protein structure and directional chemistry extend far beyond energy transduction: they are the chemical foundations upon which rests the spatial organization
of biological activities.
Membranes act both as barriers and as links: lipid bilayers define topological
compartments, while proteins that span the barrier mark channels for the passage
of matter, energy and information. Early on, Mitchell (1967) defined two classes
of transport systems, primary and secondary. The former are those in which a
chemical reaction is coupled at the molecular level to the concurrent translocation
of a chemical group, an ion or of electrons across the membrane. These catalysts,
endowed with both a chemical and an osmotic aspect, are the chemiosmotic
enzymes that lent the hypothesis their name; they are now designated osmoenzymes (Mitchell, 1977, 1981, 1987). Actions catalysed by osmoenzymes are
by definition vectorial. Secondary transport systems, or porters, involve only the
exchange of secondary bonds, not of covalent ones. They mediate transport of
solutes in either direction, to a first approximation isotropically, the direction of
net flow is then determined by the prevailing gradients of solute concentration
and/or of electric potential difference between the domains in which the solutes
are dissolved. These will concern us later.
Familiar representatives of the class osmoenzymes include the protontranslocating catalysts of electron transport in respiration and photosynthesis, the
proton-translocating ATPases and the Na+/K+-ATPase of animal-cell plasma
membranes. But the class as a whole is larger: we now recognize a variety of
catalysts that couple a chemical reaction of some sort to the translocation of an
unrelated substrate; their usual function is the accumulation of nutrients (Harold,
1986). The uptake of sugars by vectorial phosphorylation, commonly seen among
both gram-positive and gram-negative bacteria (Meadow et al., 1990), remains
the prime example of group translocation in the original sense of the term.
350
Harold
Bacteria also produce a suite of high-affinity transport systems consisting of a
periplasmic binding protein and a trio of membrane proteins, one of which
hydrolyzes ATP (Ames, 1986; Ames and Joshi, 1990). Other kinds of ATPases
mediate the accumulation of K § ions and the expulsion of Na § ions and of Ca 2+
ions (Walderhaug et al., 1987; Skulachev, 1987; Lynn and Rosen, 1987).
The biosynthesis of cell wall polysaccharides by bacteria, fungi, algae and
higher plants requires a vectorial pathway that spans the plasma membrane,
because the precursors are formed in the cytosol while the product is discharged
to the exterior; Mitchell (1963) described this kind of process as vectorial
biosynthesis. The chitin synthase of Saccharomyces cerevisiae is a case in point
(Cabib et al., 1983), the cellulose synthase of Acetobacter xylinum another. The
molecular mechanisms of translocation and work performance are quite unknown, and the designation of those synthases as osmoenzymes is questionable,
but from the present viewpoint molecular categories are of secondary importance.
The same applies to the secretion of proteins across the bacterial plasma
membrane, or into the lumen of the endoplasmic reticulum. These too represent
vectorial biochemistry in the functional sense, even though the biosynthetic
function of the ribosome is only loosely connected to that of the translocation
apparatus.
Passing mention, at least, must be made of the many transmembrane
signalling pathways, by which the binding of a messenger molecule to a
cell-surface receptor initiates a chemical reaction at the cytosolic face. The
transducing proteins of bacterial chemotaxis are well-studied representatives of
this class (Koshland, 1981, 1988; Hazelbauer, 1988), the epinephrine-activated
adenylate cyclase another (Limbird, 1981; Gilman, 1987). The object of these
vectorial systems is the transfer, not of matter or energy, but of information: the
equivalent of the command "now!" or "go!". Pharmacologists elaborating the
diphosphoinositide pathway, cell biologists enmeshed in growth factors and
oncogenes and physicians puzzling over the respiratory burst of neutrophils, are
all thinking about signals. Many of them have never heard of Mitchell and Moyle,
and they practise vectorial biochemistry without a licence, but they are colleagues
of ours all the same.
Nucleic acids and proteins are linear molecules, topologically speaking, a
feature expressed in their form and biosynthesis. Nucleic acids always grow by the
addition of nucleoside 5'-triphosphates to the 3' end of the elongating chain, and
proteins grow from the amino terminus to the carboxyl end. Ribosomes read the
nascent messenger R N A in the direction of the latter's own elongation. D N A
replication is elaborately polarized: the 3'---~5' strand can be replicated in a
continuous manner to make the 5'---~ 3' copy, but the complementary strand of
the template is copied in short segments with the help of R N A primers. Until
recently, none of this appeared to have any relationship to cellular vectors for the
polymerases were thought to be soluble enzymes, free to track the template
without constraints. But there is growing evidence to support an alternative and
much more structured view of replication and transcription. Polymers appear to
be fixed to the nuclear matrix (to the cell wall in bacteria), and the templates pass
vectorially through the copying machinery (Cook, 1989). If this proves to be
Biochemical Topology
351
correct, the basic processes of gene replication and expression would be vectorial
in the physiological sense as well as the chemical.
Could it be that, in the living cell, vectorial biochemistry is the rule rather
than the exception? Microtubules and microfilaments, bacterial flagella and
myosin filaments are polarized structures thanks to the intrinsic asymmetry of
each monomer. In vivo, all are anchored, commonly to a membrane, and now
molecular polarity confers physiological direction. Microtubules elongate (and
shrink) preferentially at the "plus" end, the free one (Kirchner and Mitchison,
1986). Microfilaments grow by the addition to subunits to the "barbed" end, the
anchored one (Pollard and Cooper, 1986). And bacterial flagella quite astoundingly manage to pass subunits through the central channel up to the tip (Macnab
and Aizawa, 1984). The most familiar instance of vectorial biochemistry outside
the membrane feld is undoubtedly the contraction of muscle. Actin and myosin
filaments run antiparallel; the myosin heads, cycling in a directional manner,
exert mechanical force upon the actin filaments; and, since the actin filaments are
anchored at their ends, the structure as a whole telescopes together. The beating
of cilia is likewise a vectorial process, and the scope of the field has expanded
greatly with the discovery of kinesin, dynein and many new "motor" proteins yet
to be characterized (Vallee and Shpetner, 1990).
To a cell biologist, the structural side of biochemistry is no less vital than the
dynamic one; and structure often grows out of the shapeliness of proteins. If
there is a single principle of morphogenesis that is universally acknowledged, it is
"self-assembly". Macromolecules often associate spontaneously into structures of
higher order, with little need for additional energy or information; and the form
of the product is wholly (or at least very largely) determined by the subunits
themselves. Viruses are the prime example (Butler and Klug, 1978; Kellenberger,
1990), but many more are to be found among the standard parts of cells.
Microtubules assemble spontaneously from tubulin monomers, microfilaments
from actin. Myosin molecules aggregate into bipolar structures with the heads
clustered at either end, just like the thick filaments of muscle. Bacterial flagella,
ribosomes, nuclear membrane pores, clathrin cages, the glycoprotein subunits of
algal cell walls and the histones of nucleosomes supply further examples.
Astonishingly, the eukaryotic nucleus itself can be reconstituted from its
dissociated elements and must be considered capable of self-assembly (Newport,
1987). And that most basic of biological shapes, the phospholipid bilayer vesicle,
arises by self-association in the absence of informational macromolecules.
There is no need to belabour the general point. A cell is a structure, a
material pattern in space and simultaneously the snapshot of a continuing
process. Whenever we inquire how the anisotropy of a particular function or
structure comes about, we are led back to anisotropic molecules, proteins in
particular. And they in turn can be traced to a linear, vectorial array of symbols
on a D N A chain. Some would argue that, therefore, all forms and vectors are
ultimately determined by that "alphabet [in which] can be written all the diversity
of structures and performances the biosphere contains" (Monod, 1971, p. 104). I
am not so sure about that, and shall return to the genetic specification of
biochemical topology in a later section. But of the proximal cause there is no
352
Harold
doubt: the directionality of all biological processes is ultimately built upon
molecular anisotropy.
COMPARATIVE
CHEMIOSMOTICS
The chemiosmotic hypothesis was initially presented (Mitchell, 1961) as a
novel mechanism to explain the coupling of electron transport to phosphorylation. The essential principles (with the polarity revised; Mitchell, 1966) are
illustrated in Fig. 1. The hypothesis then proposed that the electron transport
chain is arranged within and across the mitochondrial inner membrane, so that
when electrons pass from the substrate to oxygen, protons are expelled from the
matrix. The membrane is relatively impermeant to protons; consequently, proton
translocation generates a difference in electrochemical potential, with the matrix
side electronegative and alkaline. The proton potential gradient is the driving
force for the phosphorylation of ADP to ATP, mediated by a protontranslocating ATP synthase. This enzyme provides a path for the protons to
return to the matrix, and harnesses the free energy of the proton flux to ATP
synthesis (Fig. 1). The proton circulation was also assigned several ancillary
functions, notably to drive the transport of diverse metabolites across the
membrane, either inward or out, with the aid of a suite of proton-coupled
symporters and antiporters (Mitchell, 1963, 1966).
This scheme has become so familiar that it takes a mental effort to recall
that, at the time, its principal postulates were seen as radical, even alien. The
heart of the matter is the proposition that, thanks to intrinsic anisotropy of
protein molecules, certain enzyme-catalyzed reactions have a direction in space;
this becomes manifest when the enzymes are inlaid into a membrane in an
H§
\
\
\
\
\
4+
+
I
/
/
Acid
/
/
/
H*
Fig. 1. Chemiosmotic coupling of respiration to ATP
production.
Biochemical Topology
353
oriented manner. Whenever two such osmoenzymes, that share a common
translocation substrate, are housed in the same vesicle, their activities will
become coupled so that one reaction can drive the other. The linkage usually
involves a "coupling ion" (though in principle any substrate would do), and
depends on the electrochemical potential gradient generated by one reaction and
consumed by the other. Finally, note that incorporation of the protein catalysts of
vectorial action into a membrane-bound vesicle creates a functional unit orders of
magnitude larger than its constituent proteins.
Some of the postulates that underlay the chemiosmotic hypothesis had been
foreshadowed in the writings of earlier investigators, notably E. J. Conway, R. E.
Crane, R. E. Davies, E. Lund, H. Lundegardh and R. N. Robertson (for
historical overviews see Robertson, 1970; Crane, 1977; Weber, 1991). But it was
the chemiosmotic hypothesis that brought them together and spelled out the
implications of spatially structured biochemistry. A function as basic as oxidative
phosphorylation appears from Fig. 1 as the emergent property of a system
composed of independent elements catalysing vectorial actions, arranged within a
topologically closed vesicle and linked by an ion current. That we are here
dealing, not with a particular mechanism but with a broad organizational
principle, is demonstrated by its diverse uses in cell physiology: comparative
chemiosmotics is now a flourishing discipline (Harold, 1986; Skulachev, 1988).
Figure 2 illustrates the canonical view of bacterial energetics, as it appeared
to me in the late 'seventies (Harold, 1978). The unifying thread is the circulation
of protons. Bacteria expel protons by one of several alternative mechanisms
(aerobic and anaerobic respiratory chains, light-driven electron transport and the
ATP synthase itself), generating the proton potential A # H +. The contributions of
electric membrane potential and pH gradient are variable, but the polarity is
uniformly such that the cytoplasm is alkaline and electronegative. Protons are
drawn back into the cytoplasm, returning through diverse pathways that harness
the proton potential to the performance of useful work: symporters and
antiporters, the ATP synthase, transhydrogenase, even the rotary motor at the
base of the bacterial flagellum. All this remains true, but it is no longer the whole
truth. We now recognize a host of variations upon this theme, to the extent that
our perception of the theme itself has shifted significantly.
The marine bacterium Vibrio alginolyticus operates with a sodium circulation: it features a sodium-translocating respiratory chain, a sodium-translocating
ATP synthase, sodium-coupled porters, a sodium-driven flagellar motor and a
sodium potential as the integrative parameter in cellular energetics (Fig. 3a).
There is also an ancillary proton circulation, based on a proton-translocating
segment of the respiratory chain. Both proton and sodium fluxes can support
ATP synthesis; it is unclear whether the ATP synthase accepts both H + and Na +,
or two ion-specific enzymes are present (Tokuda and Unemoto, 1982; Skulachev,
1989; Krumholz et aL, 1990).
In Propionigenium modestum and in several other bacteria (chiefly anaerobes), the decarboxylation of methylmalonate and succinate is effected by
osmoenzymes that mediate the concurrent extrusion of sodium ions. ATP is
produced by a sodium-translocating ATPase that accepts protons at a pinch (Fig.
354
Harold
H't" H'I"
-I-
"t-
H
a
" t
"a,
/
c
d~ ~
/
+
S
H
§
; "a
H +
H+
AnH§
e
f
g
Fig. 2. The bacterial proton circulation, in principle. The upper register shows three
proton-extruding pathways: (a) respiratory chain; (b) proton-trans-locating ATPase; (c) cyclic
electron transport in photosynthesis. All three generate a proton potential difference (middle
register) that tends to pull protons back into the cytoplasm. The bottom register shows the
utilization of the proton circulation to perform useful work: (e) nutrient transport; (f) ATP
production; (g) transhydrogenation of pyridine nucleotides. After Harold (1978) with permission from Academic Press.
Biochemical Topology
355
Vibrio alginolyticus
Na + (H +)
donyl
/~t) Na
I +
m
CoA
Na +
Propionyl CoA
+
~ ' ~
C02
1
ATP
Cl-
Propionigenium modestum
O X -"
K+
Halobactetium halobium
"
q/
y
CO 2
--
l
Form
H+
ATP
Oxalobacter formigenes
Fig. 3. Chemiosmotic diversity. Ion currents and their functions in various bacteria. (a) Vibrio
alginolyticus ; (b)Propionigenium m o d e s t u m ; (c)Halobacterium halobium ; (d)Oxalobacter
formigenes.
3b; Dimroth, 1987; Laubinger and Dimroth, 1989). And halobacteria, in addition
to the familiar proton pump bacteriorhodopsin, feature a light-driven chloride
pump called halorhodopsin (Fig. 3c). Chloride ions are pumped into the cell,
augmenting the negative membrane potential. The physiological function may be
to drive the accumulation of potassium ions (Lanyi, 1990).
The anaerobic bacterium Oxalobacter formigenes manages to generate a
356
Harold
proton potential without ever pumping protons (Anantharam et al., 1989).
Instead, the coupled exchange of the divalent anion oxalate for the monovalent
anion formate (itself the product of oxalate decarboxylation) leaves the cytoplasm
electronegative (Fig. 3d). There are variations on this theme, too: in L e u c o n o s t o c
oenos, which transduces energy by the fermentation of malate to lactate, A/IH +
arises by the efflux of lactate with two protons; and A/IH +, in turn, supports ATP
synthesis (Cox and Henick-Kling, 1989). All these must clearly be accommodated
in the chemiosmotic scheme of things, and the scheme broadened accordingly.
Osmoenzymes and their uses vary, not only between classes of organisms but
also as a function of environmental circumstances. A striking illustration comes
from research on streptococci (recently renamed enterococci), whose metabolic
simplicity drew me into the chemiosmotic arena more than two decades ago.
Enterococci lack respiratory electron transport and oxidative phosphorylation;
they generate ATP by glycolysis and use the proton-translocating ATPase to
expel protons. The proton potential gradient then serves as the driving force for
the uptake of sugars, amino acids and potassium ions; in motile strains it also
drives the flagellar motor (Harold and Baarda, 1968; Harold, 1978, 1986). There
ATP
TP
K+
a
Na+
~
+
H + ~ K+
Medium low in
Medium high in Na §
Na § or acidic
or alkaline
Fig. 4. Pathwaysof cation transport in Enterococcus faecalis. (a) In low-sodium
media; (b) Under conditions that render the H+-ATPase inoperative. After
Harold and Kakinuma (1985) and Kakinuma and Igarashi (1989).
Biochemical Topology
357
is also a Na+/H + antiporter to expel Na + (Fig. 4a). These quite conventional
mechanisms apply in "normal" media, i.e. in the presence of low to moderate
sodium levels and at a neutral or acidic pH. But at elevated sodium levels, at
alkaline pH and in the presence of certain inhibitors, an alternative set of
osmoenzymes comes into play (Fig. 4b). The proton-translocating ATPase,
though present, is inoperative. Instead, the cells extrude Na + ions by means of a
primary ATPase that apparently exchanges Na + for K + (Harold and Kakinurna,
1985; Kakinuma and Igarashi, 1989). There is also evidence for an altogether
novel ATPase that expels K + in exchange for H +, and may play a role in
regulating the cytoplasmic pH (Kakinuma and Igarashi, 1988).
It seems appropriate, then, to ask what the chemiosmotic theory stands for
thirty years after its initial formulation. From the beginning, Mitchell saw his
proposed mechanism of oxidative and photosynthetic phosphorylation as one
instance of a more general class of physiological phenomena. Whenever two
transport systems, located within the same topologically closed structure, share a
common substrate (be it no more than an electron!) their activities will become
linked through the thermodynamic parameters of substrate concentration and
electrical potential. That is the essence of chemiosmotic coupling, all the rest is
illustrations and commentary. It just so happens that this is the foundation for
much of biological energy transduction, and a first stage along the road of
biological self-organization. But then the elements of chemiosmotic coupling, the
osmoenzymes and porters, took on lives of their own; and they are busy
reassorting themselves independently. Now, everyone is a chemiosmoticist--at
least to the extent of invoking proton pumps or antiporters. I cannot deny a
twinge of regret at the blurring of the line that once divided the elect from the
unshriven; but there is much satisfaction in the thought that what seemed
outlandish in 1961 has become a cornerstone of cell biology.
COMPARTMENTS A N D CONNECTIONS IN
EUKARYOTIC CELLS
A bacterial cell corresponds in its entirety to the membrane-bound vesicle of
chemiosmotic logic. By contrast, when one examines an atlas of eukaryotic cell
ultrastructure, it is the profusion of intracellular membrane-bound compartments
that catches the eye. Some make up familiar and discrete organelles: nuclei,
mitochondria, chloroplasts. Others are elements of the endomembrane system
that ramify throughout the cell: vacuoles, lysosomes, endoplasmic reticulum,
Golgi stacks. The extensive researches on isolated organelles have given us a
thorough understanding of the barrier functions of biological membranes and of
their roles in energy transduction. But from the organizational perspective, a
eukaryotic cell appears as an intricate and unified system of membrane-bounded
spaces; we are just beginning to appreciate the role of membranes in integrating
cellular space.
Figure 5 depicts a fungal hypha as a chemiosmoticist sees it. Fungal hyphae,
like bacterial cells, use a proton circulation to perform work; but in these
eukaryotic organisms three essentially independent proton circulations coexist
358
Harold
H+ S
I.j+ ATP
Ca
~\
-Ira
~,~,
ATP
+
Fig. 5. Multipleproton circulations in a fungal hypha, showing a mitochondrion (right), a vacuole
(left) and the plasma membrane.
(and in plants, four), each associated with a discrete membrane system. The
compartments recall the evolutionary origins of eukaryotic cells as consortia, with
mitochondria and chloroplasts almost certainly derived from eubacterial
endosymbionts.
Mitochondria drive a proton circulation in the bacterial manner, based on a
respiratory chain and FoF1 ATP synthase; ATP produced by oxidative phosphorylation is exported to the cytosol. Vacuoles also pump protons but drive
them into the lumen, which becomes relatively acidic and electropositive. It is
noteworthy that the vacuolar proton-translocating ATPases form a distinct family
that split away from the FoF1 lineage very early in cellular evolution. The family
includes the ATPase of archaebacterial plasma membranes; this is one of the lines
of evidence suggesting that the protoeukaryotic cells that hosted the eubacterial
endosymbionts were themselves related to the archaebacteria (Gogarten et al.,
1989; Woese et al., 1990). In fungi, as in plants, the vacuolar membrane
(tonoplast) is a major transport organ. The vacuole serves as the storage site for
K § Na § Ca +§ Mg ++, cationic amino acids and inorganic polyphosphate, which
are accumulated with the help of H+/cation antiporters. The vacuole(s) occupy
much of the cell's volume and contain much of its complement of osmolites; most
of the turgor pressure that drives the extension of fungal and plant cells is
generated there (Klionsky et al., 1990).
The plasma membrane, which encloses and defines the eukaryotic cell, drives
yet a third proton circulation. In fungi, and also in algae and in higher plants, the
enzyme responsible is an ATPase of the EaE2 family; the designation refers to the
characteristic shift in configuration and orientation of the active site, brought
about by phosphorylation of the enzyme in the course of the catalytic cycle. The
ATPase expels protons, probably one per cycle, and thus generates a proton
potential which, in turn, energizes the movements of ions and metabolites
mediated by a suite of symporters and antiporters (Fig. 5; Harold, 1986; Bowman
and Bowman, 1986; Slayman, 1987). It should at least be mentioned in passing
that proton circulations are characteristic of fungi, algae and higher plants; animal
Biochemical Topology
359
cells, by contrast, drive a sodium circulation with the aid of another EIE2
osmoenzyme, the familiar Na+/K+-ATPase. This undoubtedly has to do with the
natural habitat of animal cells, namely serum and other Na+-rich fluids; unlike
walled plants and fungi, the unwalled animal cells employ sodium extrusion for
osmotic stability and volume regulation.
The role of proton and sodium currents in energy transduction is common to
prokaryotes and eukaryotes, but only the latter characteristically employ a
calcium circulation to transmit signals (Hepler and Wayne, 1985; Harold, 1986;
Carafoli, 1987). All cells extrude calcium ions from the cytosol; that is necessary
lest calcium accumulation in response to the electric membrane potential interfere
with metabolic reactions based on phosphate. Eukaryotic cells have made a virtue
of necessity (Fig. 6). Cytosolic Ca 2+ levels are maintained in the vicinity of 0.1 M
by continuous calcium expulsion, mediated by a calcium ATPase. In response to
environmental stimuli, recognized by receptors that control channel proteins,
calcium ions rush down the electrochemical potential gradient into the cytosol,
either from the external medium or from intracellular calcium stores. The rise of
cytosolic calcium, often detected with the aid of specialized calcium-binding
proteins such as calmodulin, activates the cell's response. In other words, it
commonly functions as a trigger: in some cases, a subtly modulated one (Berridge
and Irvine, 1989). As a rule the cytosolic calcium level peaks quickly and then
returns to baseline as the excess calcium is expelled or sequestered. There are
literally dozens of cellular reponses that are set off in this manner, including
contraction and glycogen breakdown in muscle, the closure of guard-cell stomata
in plants, the reversal of swimming direction in ciliates and flagellates, possibly
2H~
H* or Na+
"~
Stimulus i
~
C a 2+
C a 2+
w
f
Ca2*
Calcium transient
+
Caleium-~
binding ~
S
Transport
Secretion
Kinases
proteins~
Motility . ~
+
[Ca]i --=0.1 pM
[Ca]o --- 1 mM
,~'
Fig. 6. The calcium paradigm. A stimulus, acting upon a receptor, elicits a transient rise in the
cytosolic calcium level; this in turn triggers diverse cellular responses.
360
Harold
even mitosis. The calcium signal is, of course, a chemiosmotic phenomenon; it
represents a special kind of work, informational work.
That a calcium transient can convey the message "now!" is well established;
can it also say "here!"? Potentially, yes. It has been recognized for many years
that calcium ions diffuse very slowly through cytoplasmic space. This is not due to
any peculiarity of calcium ions as such, but results from their tight association
with cytoplasmic macromolecules and their sequestration into vesicles. Consequently, calcium ions are especially well suited to convey spatially localized
signals, and there is growing evidence that cytosolic calcium levels do fluctuate
considerably in space and in time. The functional significance of these fluctuations
is still under discussion (Jaffe and Nuccitelli, 1977; Picton and Steer, 1982; Hepler
and Wayne, 1985; Berridge and Irvine, 1989; Harold and Caldwell, 1990; Harold,
1990; Tsien and Tsien, 1990).
In the hierarchy of biological order, vectorial chemical reactions and energy
transduction by ion currents represent the first level. The next is the nonrandom,
ordered distribution of proteins within membranes, recently emphasized by
Williams (1988) as well as by Harold and Caldwell (1990). Every micrograph of
cellular ultrastructure suggests how many levels of order lie beyond. In this
respect, Figs. 5 and 6 are misleading: they imply autonomous organelles bathed in
a homogeneous cytosol and linked by the diffusion of substrates and of regulatory
molecules. Closer inspection suggests a more structured conception of cellular
space, with implications for cellular metabolism and energetics.
By tradition, ATP has been regarded as the diffusible energy carrier of the
cytosol, produced by numerous small mitochondria. But it now appears that in
many cells mitochondria are spatially extended structures that ramify throughout
the cytoplasm. A case in point is the unicellular alga Polytomella, whose single
but extensively fenestrated mitochondrion occupies much of the cell's volume; a
more familiar instance is provided by the mitochondria of muscle. The physiological consequences of this ultrastructural organization have been carefully explored
by Skulachev and his colleagues. The membrane potential, they point out,
spreads almost instantaneously along the membrane's surface; a large ramifying
structure can supply ATP and other metabolites locally, bypassing ATP diffusion
(Skulachev, 1990). That reticular mitochondria constitute a functional unit was
demonstrated by experiments in which localized laser damage blocked the uptake
of a fluorescent cation throughout the reticulum (Amchenkova et al., 1988).
Reticular mitochondria are presumably one mechanism that supports functional
coordination on the cellular scale.
The time-honoured presumption that intracellular organelles can be thought
of as discrete, membrane-bounded compartments, breaks down altogether when
we turn to the pathway of protein export. Proteins travel from the perinuclear
region to the surface via a succession of interconnected cisternae, passages and
mobile vesicles aptly called the endomembrane system (Fig. 7; Mellman et al.,
1986; Burgess and Kelly, 1987; Morr6, 1990). The Golgi functions as a central
switching station where nascent proteins are packaged, addressed and directed to
their ultimate destination. There are many vectorial reactions in this pathway,
including the translocation of nascent proteins into the lumen of the endoplasmic
BiochemicalTopology
Secretiong r a n u l e ~
361
" ~ "~,:~.~ ~
~/
~
~__,~.,/~._~___~Smoothendoplasmic
L~"~- - J ) / reticuium
Fig. 7. The endomembranesystemof a eukaryoticcell. From: A Guided Tour of the Living Cell, by
C. de Duve, with permissionfrom W. H. Freeman & Co.
reticulum, and the acidification of intra-vesicular space by proton-translocating
ATPases. And there is an overall directional flow of proteins and of lipids from
the perinuclear region to the cell surface and back. This is a higher level of spatial
order, appropriately described by the term "vectorial physiology."
FILAMENTS, SCAFFOLDS AND TRACKS
The spatial dimension entered twice into biochemistry, once through
biomembranes and again through sliding filaments. By the mid-nineteen sixties,
the principles of muscle contraction were well understood (Fig. 8), and presented
as a prime example of vectorial biochemistry (Huxley, 1969, 1973). Polarized
actin filaments run from both Z lines in opposite directions into the central zone.
The bipolar myosin filaments hydrolyze ATP, and confer spatial direction and
coherence upon the free energy released thereby; in consequence, as the myosin
heads pass through parallel mechanochemical cycles, each head exerts upon the
actin filament a force pushing the latter toward the central zone. It is a design to
gladden the heart of a mechanical engineer.
It was not long before eukaryotic cilia and flagella were recognized as a
second instance of the conversion of scalar free energy into vectorial mechanical
force. Most axonemes are built on the standard 9 + 2 pattern: two central
microtubules plus nine microtubule pairs set in a circle and connected by "arms"
that contain dynein. ATP hydrolysis by the dynein arms exerts mechanical force
upon the adjacent microtubule pair, pushing it outward into the cilium's tip. The
characteristic lashing motion of the cilium as a whole requires a second vector,
362
Harold
Actin
Myosin
b
<
Actin
<
Myosin
Fig. 8. Muscle contraction as an example of vectorial biochemistry. Active changes (shown in a and b) in the angle of
attachment of myosin crossbridges to actin filaments produce
relative sliding force. From Huxley (1973), with permission from
Nature.
the activation of microtubule arms in succession around the circumference (Satir,
1974; Gibbons, 1981; Porter and Johnson, 1989).
Both microfilaments and microtubules are increasingly prominent in the
structured--indeed, mechanized--conception of eukaryotic cytoplasm that has
emerged over the past two decades. The architecture of the various filament
systems displays a global spatial order that is characteristic of the cell in question,
and underlies much of its structural and functional organization. A familiar
pattern, that of many animal cells in culture, features cytoplasmic microtubules
that grow out from the cell centre towards the periphery. Microtubules usually
(probably always) arise from specialized regions called microtubule organizing
centres. They have an intrinsic polarity such that the "plus" end (the rapidly
assembling one) is distal to the organizing centre; in animal cells this is the
centrosome, external and adjacent to the cell nucleus. The microtubule array
provides mechanical support (cells round up if the microtubule array is
BiochemicalTopology
363
disorganized), as well as a framework upon which organelle such as endoplasmic
reticulum and Golgi are pegged out. The microtubule array also provides tracks
for the movement of vesicles from the endoplasmic reticulum to the plasma
membrane. The motor proteins thought to mediate these movements have their
own intrinsic vectoriality: in nerve axons, for example, kinesin is the motor for
anterograde movement of vesicles (from the cell body towards the growth cone)
whereas dynein drives retrograde movement (Vale, 1987; Vallee and Shpetner,
1990).
Microfilaments serve as an alternative set of tracks for the movement of
vesicles and organelles, at least in giant algae and certain microorganisms (Allen
and Allen, 1978; Schliwa, 1984; Warrick and Spudich, 1987). In this instance the
motive power is supplied by a special class of myosin molecules, and the
unidirectional translocation of organelles may be what drives cytoplasmic
streaming. Suddenly, we are well advanced along the biological scale-bar: the
stately streaming of algal cytoplasm, following a helical path up one side of an
internodal cell and down the other, is one of the most spectacular displays of
vectorial physiology. It is available to anyone with access to a pond and a
microscope. And microfilaments, like microtubules, serve as dynamic scaffolds,
particularly in moving cells. In animal cells, the hexagonal configuration of active
filaments is often strikingly beautiful and suggestive of Buckminster Fuller's
geodesic architecture. It goes without saying that constructions of this kind rely,
not upon actin alone, but upon its association with one or more of the numerous
actin-binding proteins.
Where do these higher levels of structural order come from? Biochemistry
has traditionally looked for answers to the self-assembly of protein monomers
into supramolecular structures, as illustrated by the production of virus particles.
In principle, this is a spontaneous process, moving towards equilibrium, that
requires no input of energy or information beyond that supplied by the
monomers. In practice, however, even the assembly of bacteriophage heads is a
rather more subtle affair (KeUenberger, 1990), and self-assembly seems only
marginally relevant to the construction of (say) the microtubule cytoskeleton. To
be sure, tubulin monomers do assemble spontaneously into microtubules, given
the proper ionic conditions; the association of microtubules with kinesin and
other proteins involves the usual forces that allow macromolecules to recognize
and bind each other; and the nucleation of microtubule outgrowth must be a
physicochemical process that can be reproduced and studied in vitro. But answers
at the level of molecular mechanism do not quite resolve the central problem.
Microtubules can assume a variety of configurations in different cells, or even in
any one particular cell, all of which must be compatible with the physicochemical
requirements of microtubule assembly. There must be a network of local signals
and influences, characteristic of a particular cell, that determines such things as
the spatial disposition of nucleating sites; the propensity of any particular
microtubule to elongate or shrink; and the selection of particular tubules for
long-term stabilization (for an illuminating discussion see Kirchner and Mitchison, 1986). Each such operation will, of course, have a molecular mechanism, but
the mechanism should be seen as executing the spatial pattern rather than
364
Harold
specifying it. The forces that generate the higher levels of order are not well
understood, but are likely to fall chiefly into the province of cellular mechanics
(Albrecht-Buehler, 1990; Heidemann, 1990). This is why it is essential to have
the physiologist's top-down view as well as the biochemist's bottom-up view; the
two perspectives are complementary, not antagonistic.
APICAL GROWTH
We have now reached the point where we must confront the spatial
organization of real organisms, as represented by their outward form and internal
anatomy. Even the simplest cells, the bacteria, display regular shapes and
localized growth regions. But it is the eukaryotic microorganisms--the algae,
fungi and protozoa--that challenge the imagination with their diversity of forms
and intricate architecture. A good focus is to ask how each organism generates its
particular spatial order during growth.
One organizational feature that is all but ubiquitous in the eukaryotic world
is polarity. The term implies, not only that the two ends of an organism differ, but
that various features of its anatomy, physiology and behaviour are arrayed along
an axis or vector. Organisms that lack polarity exist, the actinopoda for example,
but polarity is the norm. It underlies directional responses to environmental cues,
allows organisms to concentrate resources at a particular locus, and is a natural
concomitant of the tendency to maximize the surface area for a given cell volume.
Polarity is especially marked in the fungi, whose archetypic form is the hypha.
Apical growth of fungal hyphae will serve here to illustrate directional processes
at the cellular level.
Figure 9 illustrates the organization of the apical region of the water mould
Saprolegnia ferax, an oomycete. Strictly speaking, oomycetes are not fungi but
algae in disguise (Gunderson et al., 1987), but mycologists continue to claim them
for their own because of their hyphal morphology and apical growth habit. Note
the abundance of membrane-bound compartments, each of which is a unit of
chemiosmotic energy transduction: mitochondria, vacuoles and several kinds of
"apical vesicles". Elements of the endoplasmic reticulum ramify throughout the
section but do not show up well. What concerns us here is a higher level of order,
the linear distribution of anatomical parts. The apex proper is packed with
vesicles but is otherwise devoid of organelles. Mitochondria first appear a few
micrometers behind the tip, nuclei and Golgi stacks still further behind and out of
our section. The cytoskeleton, microtubules and microfilaments, are arranged in a
generally axial manner; the tip is marked by a delicate actin cap, revealed by
staining with certain fluorescent dyes (Heath et al., 1985; McKerracher and
Heath, 1987; Heath and Kaminskyi, 1989). The cytological gradient obviously
corresponds to the polarized mode of growth: hyphae extend by continually
producing fresh tip. As a hypha forges across the substrate, it concentrates
resources from a length of protoplasm at the tip, where new cell wall and plasma
membrane are laid down.
Does the conspicuous directionality of growth and anatomy have anything to
do with vectorial chemistry? At an early stage in the development of the
Biochemical Topology
365
Fig. 9. Longitudinal section through the hyphal tip of Saprolegnia ferax. Note the aggregate
of elongated, opaque wall vesicles (wv) at the extreme apex. Mitochondria (M), vacuoles (V)
and Golgi bodies (G) are prominent behind the tip. Scale bar, 1/~m. Photomicrograph by
courtesy of I. B. Heath; reprinted with permission of Protoplasrna,
chemiosmotic theory, Mitchell (1962) bade us look upon cell growth as a grand
symphony of transport: " G r o w t h and morphogenesis represent transport processes which, in c o m m o n with the more popular membrane transport, must be
described by vector quantities, having both magnitude and direction. The spatial
asymmetry of the individual catalysts, and of the organized polymolecular systems
in which they reside, can properly be regarded as a primary cause of organized
metabolism and growth". This viewpoint is especially apt in the case of fungal
hyphae, whose polar character is so marked that, observing a living hypha, one
can all but see the protoplasm streaming forward to make new tip. All the same,
366
Harold
Mitchell's sweeping vision skated too blithely over the problem of scale. Vectorial
chemical reactions traverse molecular distances, on the order of nanometers.
Cells, by contrast, are organized on a scale three to six orders of magnitude
larger, micrometers to millimeters. Molecular asymmetry is surely the root cause
of cellular vectors, but cannot in itself supply a satisfying explanation for
polarized growth and anatomy.
How do organisms bridge the gap between the molecular scale and the
cellular one? Molecular self-assembly surely plays a major role in the formation
of the hypha's standard parts--its membranes, ribosomes, cytoskeletal filaments,
even nuclei. But a hypha is not like a jigsaw puzzle, whose form is uniquely
specified by its individual pieces: the standard parts are compatible with a host of
cellular forms. Besides, fungal cell walls are not self-assembling structures, but
more akin to a woven fabric. Hyphae, like other whole cells, must be understood
as products of large-scale, directional work functions. I like to describe such
processes as "vectorial physiology"; the term is, of course, an extension of
Mitchell's vectorial metabolism, and is intended to draw attention to the special
features of directionality on the micrometer scale.
Apical extension is best described as a process of polarized secretion. The
vesicles that fill the apex are believed to carry enzymes, and perhaps precursors,
required for the construction of new cell wall and plasma membrane. They are
produced in the endoplasmic reticulum, modified and repackaged into transport
vesicles in the Golgi, carried to the hyphal tip and exocytosed there (BartnickiGarcia, 1973; Grove, 1978; Wessels, 1986, 1990; Harold, 1990). The cell wall,
which in the oomycetes consists of interlaced fibrils of cellulose and glucans, is
produced in situ by newly secreted polymerases; nascent wall is plastic at first but
quickly rigidities as a result of crosslink formation. The shapes of tapered tip and
cylindrical trunk can be understood on this basis (Fig. 10).
It takes work to extend an apex, not just the work of biosynthesis and of
transport, but also that required to expand the surface in the face of cohesive
forces between previously assembled molecules. The driving force for surface
Cell wall
~
Apical
z vesicles
F~o 10. Apicalgrowth: localizedcompliancewith global force. Turgor supplies the driving
force for expansion;localizationis due to the local exocytosisof precursor vesicles. Nascent
wall is plastic, but hardensprogressivelyas crosslinksform. The tracks that guidevesiclesto
the apex are largelyspeculative (after Wessels, 1986, and other sources).
Biochemical Topology
367
enlargement is almost certainly turgor, more precisely the hydrostatic pressure
exerted upon the wall by the cytoplasm. Hydrostatic pressure, of course, bears
uniformly upon the surface; a growing hypha yields to it locally, by stretching and
by secretion. The principle of localized compliance with global force is quite a
general one (see Harold, 1990, for fuller discussion) that helps one to understand
morphogenesis in all manner of walled cells.
Apical growth is polarized and vectorial overall; which of the many
physiological activities that contribute to growth have a direction in cellular
space? So far as we know, the generation of ATP, proteins, lipids or nucleic acids
is not vectorial on the micrometer/millimeter scale, but at least two higher-order
candidates have been put forward. The first is exocytosis itself. Mycologists have
for years assumed, provisionally at least, that exocytosis is restricted to a small
region at the very tip, possibly by a signal of some kind. If that proves to be true
(and I am not aware of any direct evidence that either corroborates the
hypothesis or refutes it), then whatever localizes the site of fusion would make a
large contribution to the directionality of secretion and of extension. A second
possibility is the transport of precursor vesicles. We simply do not know whether
these vesicles are carried in the (bidirectional) cytoplasmic stream, or are carried
vectorially on tracks consisting of either microtubules or microfilaments. The
issue was recently brought to a head by Bartnicki-Garcia (1990; Bartnicki-Garcia
et al., 1989), who succeeded in modelling hyphal extension on the premise that
vesicles arise in an apical vesicle-supply centre that moves forward by an
unspecified mechanism. Their model generates realistic shapes while dispensing
with the traditional assumption of localized exocytosis. Finally, there must be
forward movement of the cytoplasm within the hyphal tube that it secretes,
together with the cytoplasmic organelles (McKerracher and Heath, 1987). There
is mounting evidence that mitochondria, Golgi and endoplasmic reticulum are
[inked to microtubules, whose growth is again a vectorial process. Whether
organelles can move along microtubules, or are carried passively along with the
growing cytoskeleton, is not clear at present.
One comes away with the sense that Mitchell's global transport process is
quite real, but different in nature from its molecular counterpart. Vectorial
metabolism is to vectorial physiology what a musician is to an orchestra.
Direction on the cellular level is not prescribed by the asymmetry of molecules,
but by whatever signals or forces impose spatial coherence upon the supramolecular products of assembly reactions. The heart of the matter is pattern
formation, and of this we must speak in the most tentative manner.
T R A N S C E L L U L A R ION CURRENTS
That eukaryotic cells and organisms commonly drive electric currents
through themselves has been known for nearly a century, but a whiff of
hocus-pocus still clings to the subject. Elmer Lund, the same who speculated on
vectorial transmembrane reactions in the 'twenties, pioneered the field. His book,
"Bioelectric Fields and Growth" (Lund, 1947), records many examples and
expounds his belief that electric currents are part of the mechanism by which
368
Harold
growing and developing organisms generate spatial order: "[It] is proposed that
the electrical pattern is intimately related to the morphogenetic processes and
polar or vector properties of cell and tissue functions. One function of this electric
field or pattern is to act as a directive force in laying down new s t r u c t u r e s . . . "
(italics his, p. 284).
The contemporary resurgence of research on endogenous electric currents
began when L. F. Jaffe and R. Nuccitelli invented an instrument capable of
measuring the miniscule electric fields generated by single cells in the surrounding
medium (Jaffe and Nuccitelli, 1974). The special feature of the "vibrating probe"
is a microelectrode tipped with a small ball of platinum black, 10-30 #m in
diameter; this vibrates at 200-400 Hz over a span of about 30/~m, and measures
the voltage difference between the ends of its excursion with the aid of a lock-in
amplifier. Given the resistivity of the medium, the instrument output is readily
converted from volts to current density by application of Ohm's law. The probe is
exquisitely sensitive, measuring potential differences in the nanovolt range; and
by moving the electrode around the cell one can generate a spatial map of current
distribution. Much of the tedium associated with this procedure has been
eliminated in modern versions of the vibrating probe, which determine current
vectors in two dimensions automatically.
By use of the vibrating probe, Jaffe and his disciples mapped currents around
diverse cells and small organisms, in space as well as in time: eggs and embryos,
shoots and roots, muscles and nerves, algae, amoebae, fungal hyphae, and
filaments of cyanobacteria. The results leave no doubt that transcellular electric
currents are widespread, possibly ubiquitous, among eukaryotes. Their patterns
are visibly correlated with anatomy and function, and often with subsequent
development or differentiation (for reviews see Jaffe, 1979, 1981; Jaffe and
Nuccitelli, 1977; Nuccitelli, 1983, 1988; Harold and Caldwell, 1990; Gow, 1990). I
was drawn (should I say seduced?) into the field by Jaffe's writings, and by the
hope that the study of transcellular currents might provide new clues to the
mechanisms by which living organisms generate spatial order. We elected to study
fungi, and particularly the oomycete Achlya bisexualis (closely related to
Saprolegnia), because of its large size and polarized growth habit.
The basic findings are illustrated in Fig. l l A . Hyphae of Achlya growing in
the standard rich medium generate a transcellular electric current such that
positive charges flow into the apical region (100-200/tm in length) and exit from
the hyphal trunk. Since in aqueous media electric charges are carried by ions, the
observations must report an aspect of the ionic traffic across the plasma
membrane. Some of these ion transport systems must be sufficiently well
segregated in space that the vibrating probe detects a net influx of cations (or an
efflux of anions) in the apical region, and a net efflux of cations (resp., influx of
anions) along the trunk (Kropf et al., 1984).
The transcellular electric current almost certainly represents a spatially
extended chemiosmotic proton circulation. We drew that conclusion from the
following observations: current flow was unaffected by the omission of any one of
the medium's ionic constituents, or even of all the ions together; the medium
adjacent to the apex turns slightly alkaline, while acid is secreted along the trunk;
Biochemical Topology
369
0.4
Out
0
In
0.4
~0__~
,.
o
DMA~2, 18hr
0.8
-~
In
~3
0.2
~~
0
1 95
1.0
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0.5
0
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Out 0.2
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DMA 32, 72 hr
I
0
I
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f
--
800
Distance behind the tip, IJm
200
400
600
Fig. 11. Transcellular electric currents in Achlya bisexual&. Upper register,
in standard growth medium; middle register, in medium lacking amino
acids; bottom register, older hyphae. For details see Schreurs and Harold
(1988) and Cho et aL (1991).
omission of amino acids from the medium abolished the current; and so did
inhibitors of cellular energy metabolism such as azide, cyanide and dinitrophenol.
Though each one observation admits of alternative interpretations, taken
together they buttress the hypothesis depicted in Fig. 12A: Protons are extruded
from the cytoplasm by the proton translocating ATPase, and return by symport
with amino acids (particularly methionine), which serve as A c h l y a ' s major
nutrients. Pumps and porters are partially segregated (Fig. 12B), so that the latter
predominate in the apical region while the former favour the trunk. Thanks to
this nonuniform distribution of transport systems, operation of this familiar
proton circuit generates a net flow of protons through the hypha, accompanied by
370
Harold
"--"-
f
H+ a a
!1
"'"o'"'o'"o"
/"//
A
" -""
A
l
8
Ill
Ii
" o'
" o"
.' o
' o io
oo
o. oo~o~o.a
o" " o oO'O'O'~%'/.eR~/'
9 o- 9 . o . o ' ~ o o ~ t ~
iil
,/
///
Fig. 12. The extended proton circulation of Achlya bisexualis hyphae.
(A) Current arises by the spatial segregationof proton pumps from amino
acid/proton symporters; (B) Segregation is partial: porters (dots) are
concentrated in the apical region, pumps (circles) predominate distally.
the electric current that the vibrating probe detects (Kropf et al., 1984; Gow et
al., 1984; Kropf, 1986; Cho et al., 1991).
The spatially extended proton circulation obviously results from the
differential incorporation (or regulation) of porters and pumps; to that extent, it
is a consequence of polarized growth. Is the transceUular ion current also part of
the mechanism that generates polarity? Apparently, the answer is No. When
grown under a variety of conditions, Achlya hyphae generate diverse electric
patterns, including one featuring outward current in the apical region (Fig.
l l B , C). Yet under all conditions growth was localized to the apex (Schreurs and
Harold, 1988; Cho et al., 1991). We must regretfully conclude that the
transcellular electric current is a manifestation of polarized extension, not its
cause, and that it plays no obligatory role in apical growth (for another view, see
Potapova et al., 1988; Skulachev, 1990). Whether the flow of protons into the tip,
which cannot be tracked directly, is involved in localizing that tip must be left in
abeyance.
There is now a number of organisms in which transcellular electric currents
can be assigned to particular ions: protons in Achlya, Neurospora and other
fungi; protons again in the algae Chara and Nitella; potassium and protons in
Pelvetia and Fucus embryos (for reviews see Harold and Caldwell, 1990; Gow,
BiochemicalTopology
371
1990). In no instance has an obligatory relationship between current and growth
localization been demonstrated. The case of fucoid embryos, where the correlation is particularly strong (Jaffe and Nuccitelli, 1977; Jaffe, 1981), is now being
re-examined (Kropf and Quatrano, 1987; D. L. Kropf, personal communication).
Until a case to the contrary is made, I must conclude that Lund was mistaken.
Transcellular electric currents are epiphenomena. They are the consequence of a
widespread tendency among eukaryotic cells and organisms to segregate transport
systems to particular locations. The reasons for this remain largely unknown, but
the resulting transcellular electric currents arid electric fields seem to serve no
obligatory function in cell physiology. Whether localized fluxes of individual ions,
calcium in particular, participate in the localization of growth remains open; we
shall return to this possibility below.
PATTERN FORMATION
The quest for an understanding of how cells shape themselves must be
conducted on two levels. The first is the physiological one, centering on the
physical and chemical processes that generate the material fabric at each stage of
growth and development. The second, deeper level is the organizational one:
here we ask, not what cells do but how they direct what is done. A key issue is
the spatial localization of growth processes, but progress along this line has been
slow. Growth, morphogenesis and development retain an aura of mystification,
largely because we find it difficult to formulate explicit and testable hypotheses to
account for pattern formation.
The problem is the appearance of organized structure where there was none
before. The germination of a spore, the outgrowth of a Fucus zygote and the
elaborate modelling of a desmid algal cell in the course of division, all entail the
spontaneous emergence of a regular spatial pattern. How this comes about still
largely passes understanding, but we can start out from the remarkable paper by
A. M. Turing (1952). Boldly entitled "The chemical basis of morphogenesis", the
paper made two seminal points. The first was the proposition that developmental
events are called forth by specialized informational molecules, the morphogens,
whose distribution in space supplies a pre-pattern for the subsequent genesis of
biological structures. In contemporary idiom, the graded distribution of morphogens constitutes a field of positional information, a kind of map, that instructs
a population of cells concerning the course of their future development. The
second was the wholly novel insight that spatial patterns of concentration can
arise spontaneously when two substances that react with one another diffuse at
different rates. Contrary to intuition, which associates diffusion with smoothing
out concentration differences, in systems that obey particular kinetic constraints,
random fluctuations arising within a homogeneous region will be spontaneously
amplified, generating stable local maxima and minima of reagent concentration.
It was this demonstration that spatial order can in principle arise ex nihilo that
brought morphogenesis within the purview of physical science.
Turing's leap of the imagination gave birth to the mathematical discipline
called reaction-diffusion kinetics (Nicolis and Prigogine, 1977; Peacocke, 1983;
372
Harold
Harrison, 1987). These investigators and others devised several reaction schemes
which, when fitted with judiciously chosen rate constants and run on a computer,
simulate the spontaneous generation of spatial patterns; some bear an uncanny
resemblance to biological ones. An important feature is that stable patterns arise
only in systems far from thermodynamic equilibrium; they are "dissipative
structures," dependent on a continuous flux of energy through the system, and
they illustrate the conversion of energy into spatial organization. Although
morphogens were invented to account for the development of multicellular
embryos, the principles are applicable to single cells; and at least one putative
morphogen, the bicoid protein of Drosophila embryos (Driever and NussleinVolhard, 1988) acts at the cellular level.
The theoretical analysis of spatial self organization has tended to revolve
around diffusible morphogens, but there is no reason to regard these as the only
possible basis for the emergence of spatial patterns. On the contrary, we can
entertain the more general proposition that self organization begins with the
establishment of a field of some kind, which provides initial spatial cues for the
later emergence of more elaborate order. The term "field", as used in biology,
designates a region of space over which some agency acts in a coordinated
manner; more generally, it is any domain of relational order (Frankel, 1982,
1989; Goodwin, 1986; O'Shea, 1988). Morphogenetic fields have special spatial
and temporal features, notably a characteristic distance over which spatial order
holds (a "wavelength" in the range of/~m, not nm) and a timeframe of minutes to
hours. Since a field consisting of diffusible molecules is likely to be perturbed by
cytoplasmic movements, we should also consider other kinds of fields that may
arise early in the course of self organization.
What fields might there be? The most familiar candidates are endogenous
electric fields, generated by transcellular electric currents, which could serve as a
localizing mechanism by the electrophoresis of organelles or membrane proteins.
This proposition, originally put forward by Lund (1947) and articulately championed by Jaffe and his associates (Jaffe et al., 1974; Jaffe and Nuccitelli, 1977;
Jaffe, 1981) has lost lustre recently because there appears to be no constant and
obligatory relationship between the polarity of the transcellular electric current
and that of growth (Harold and Caldwell, 1990; Gow, 1990; see preceding
section). A more appealing version of the idea, that ionic currents localize
morphogenetic events, identifies the spatial signal with a localized influx of
calcium ions. This hypothesis, which has found its way into textbooks (Alberts et
al., 1989), also owes much to Jaffe's advocacy and now appears to be the
dominant paradigm in cellular pattern formation. It is well known that calcium
ions regulate numerous physiological processes including contraction, exocytosis,
cytoskeleton dynamics and diverse enzyme activities. Insofar as these very
processes are the physical basis of cell shaping, calcium ions (and to a lesser
degree protons) are plausible candidates for the role of cellular morphogens. The
hypothesis to be developed and tested by application to particular cases states
that a localized flux of Ca 2§ ions, and/or a spatial steady-state gradient of
cytosolic free Ca 2§ plays a causal role in the initial establishment of spatial
patterns.
Biochemical Topology
373
Endogenous mechanical forces may seem a prosaic solution to so profound a
riddle as self organization, but this has recently become an especially promising
avenue of inquiry. It will be recalled that the plasma membrane of eukaryotic
cells is subtended by a continuous actin-rich cortex, that is mechanically coupled
to the deep cytoskeleton. There is mounting evidence that the cortex exerts
tension in a non-uniform manner, and that local stresses and strains can develop
in response to internal as well as external stimuli. The mechanical properties of
the cortex will be modulated by the degree of crosslinking, the presence of
binding proteins and the local ionic milieu, particularly the pH and calcium
concentration. The mechanical patchiness may translate into diverse kinds of
secondary consequences: a flow of actin along the gradient of tension, local
activation of stress-sensitive ion channels, or the localized accumulation of surface
enzymes physically connected to cortical actin filaments. The application of
cytomechanical principles has progressed furthest in the case of amoeboid
movement (Oster and Perelson, 1987; Bray and White, 1988; Stockem and
Klopocka, 1988; Stossel, 1990). Extrapolation to spatial localization in development and morphogenesis is just beginning (Goodwin and Trainor, 1985; Goodwin
et al., 1987; Harold, 1990).
To make this discussion of biological pattern formation a little more
concrete, let us focus once again on fungal hyphae. There are three stages that
require the characteristic pattern to be established de n o v o : germination of a cyst
or spore; the extension of a growing hypha (because the tip is ever made afresh);
and the initiation of a branch. All three share two common features. One is the
role of turgor pressure as the driving force for surface expansion. Another is the
role of secretory vesicles as the carriers of enzymes and precursors to the site of
action. Vesicles accumulate at the apex, and also at the presumptive sites of
germ-tube and branch emergence (Mullins and Ellis, 1974; Grove, 1978). We do
not know just what these vesicles contain, but since the dissolution of existing
wall is a prerequisite for the emergence of a new tip, lytic enzymes are likely to
be needed at the site of outgrowth. Emergence of a germ-tube or a branch may
thus begin with a local herniation, driven by pressure and localized by secretion.
The case of an established apex is somewhat different, since nascent wall is plastic
and lytic enzymes should not be required; but precursor vesicles must still be
targeted to the tip. This cellular targeting must be a crucial step in self
organization; if we knew how it is achieved, apical growth would be much less
mysterious. At present our understanding is rudimentary at best, but the terms of
discussion are those introduced above: ionic currents, calcium, actin and other
cytoskeletal fibres, electric fields, cytomechanics.
I was originally drawn into the study of fungal growth by the proposition that
transcellular electric currents and/or electric fields may play a role in localization.
I no longer find this a plausible hypothesis, for the reasons outlined in the
preceding section, but a localized flux of Ca 2§ ions or even protons remains in
contention. The idea was explicitly formulated by Brawley and Robinson (1985)
with reference to the germinating zygote of the brown alga Pelvetia. Extrapolated
to fungal hyphae, it states that one class of Golgi vesicles carries calcium
channels; when incorporated into the plasma membrane by exocytosis, these
Harold
374
Fig. 13. Localizationof the tip by a calcium flux: a speculative model,
adapted from Brawleyand Robinson(1985). Calciumchannelscarried in
Golgi vesicles are inserted by exocytosisinto the apical membrane: they
mediate a short-lived,local calciumfluxthat sustainsa cytosolicgradient
of calciumion concentration.
mediate a local influx of calcium ions that stimulates further exocytosis and
conveys the message "here!". The channels must be short-lived, closing spontaneously as soon as they fall behind the extending apex (Fig. 13). It is well
known that A c h l y a hyphae, like other tip-growing organisms, require Ca 2§ ions
for growth; but in the short term, it appears that extension can proceed in the
absence of extracellular Ca 2§ (Kropf et al., 1984; Schreurs and Harold, 1988;
Jackson and Heath, 1989). The observations argue against the model, but need
not be fatal to it. A variant that is receiving much attention at present proposes
that a cytoplasmic gradient of calcium ions, possibly derived from internal stores,
acts as a morphogen in the classical sense (Jaffe et al., 1974; Jaffe and Nuccitelli,
1977; Hepler and Wayne, 1985; Brownlee and Pulsford, 1988; Speksnuder et al.,
1989). The experimental evidence p r o and con is equivocal.
Does the cytoskeleton play a fundamental role in the early stages of
localization? Microtubules are popular with both plant and animal cell biologists,
and they were implicated a decade ago in the localization of the budding site of
yeast. Recently, however, the balance of the evidence has tilted decisively:
studies with mutants and inhibitors strongly suggest that microtubules do not
determine the locus of budding or of vesicle secretion (Quinlan et al., 1980;
Oakley and Rinehart, 1985; Huffaker et al., 1988; Jacobs et al., 1988). By
contrast, there is a strong association between actin polymerization and growth
localization: localized assembly of F-actin seems to be an early stage in localized
secretion (Kilmartin and Adams, 1984; Novick and Botstein, 1985; McKerracher
BiochemicalTopology
375
and Heath, 1987; Hoch et al., 1988; Drubin et al., 1988; Kobori et al., 1989;
Schreurs et al., 1989). At present we are uncertain what this means, leaving us
with several speculative schemes (Harold, 1990; Steer, 1990). But it is at least
apparent that students of tip growth have more to learn from their colleagues in
amoeboid motility than either had expected. Just where this faint trail will take us
remains to be seen; a fresh breeze to blow away the mist would be welcome!
FROM ONE DIMENSION TO THREE
The forms of organisms, like their anatomy and the changes that both
undergo in the course of the life cycle, are quite strictly inherited. We generally
interpret this to mean that spatial organization, like other facets of structure and
physiology, is encoded in the genome. No one claims that forms or dimensions
are directly specified by genes, but we do expect the genome to contain
instructions for making the organism. This conception shapes almost all contemporary research in developmental biology, and is implied whenever one invokes a
"genetic programme," or proposes that a given gene "controls" or "determines"
a particular function.
It ought to be possible to learn something about the manner in which a linear
sequence of codons in DNA specifies cellular architecture in three dimensions, by
examining mutants with altered form or organization--called "morphogenetic
mutants" in the trade. Yeasts have proved especially amenable to this approach,
beginning with L. H. Hartwell's isolation two decades ago of the first mutants
defective in the cell division cycle. The characteristic feature of these
temperature-sensitive mutants is that, at the restrictive temperature, growth is
arrested at a particular morphological stage; some accumulate at a normal step of
the division cycle, others generate grossly aberrant shapes. In recent years, the
biochemical nature of about two dozen of these lesions has been determined.
They fall into three broad classes. Some are blocked in the biosynthesis of an
essential macromolecule: DNA, chitin or the constituent proteins of so-called
10 nm filaments. Several, quite mysteriously, are deficient in glycolytic enzymes,
e.g. phosphoglucose isomerase and pyruvate kinase. And quite a number are
defective in enzymes that catalyze reactions at metabolic branch points: adenylate
cyclase, a protein kinase, a putative calcium-binding protein (Pringle and
Hartwell, 1981; Harold, 1990).
While it is gratifying to be able to pinpoint biochemical defects that give rise
to regular but abnormal forms, the outcome is strangely disappointing. It seems
that when we have at long last identified the lesion in chemical terms, we are little
closer to understanding either the normal morphology or the mutant's aberrant
one! We must not shirk the obvious conclusion, familiar to developmental
geneticists since Waddington spelled it out thirty-five years ago (Waddington,
1957). The relationship of genes to cell form is not like that of genes to proteins.
Morphology and organization, like other complex phenotypes, are the outcome
of interactions among multiple genes; more precisely, multiple gene products,
many of them enzymes. Lesions in certain genes have reproducible morphological
consequences, not because that gene controls or determines the phenotype, but
376
Harold
because its product participates in an intricate, braided web of causes and effects;
the mutant phenotype is due as much to the actions of the normal gene products
as of the defective one. There seem to be few if any true "morphogenes"--genes
dedicated to the task of cell shaping, nor can one find a set of genes activated in
concert with the cell cycle that could be fairly described as a programme.
Genes do, of course, specify products that play critical roles in morphogenesis; they may, for instance, affect the initiation, rate or duration of an
essential biosynthetic process. But these genes do only what genes normally do:
they encode the primary structure of macromolecules and receive signals that
regulate macromolecule production. Since the primary sequence of proteins and
nucleic acids determines the pattern of folding (given the "correct" ionic milieu),
that is the stage at which linear information is translated into three-dimensional
form: molecular morphogenesis is directly gene-controlled. By contrast, in
cellular growth and morphogenesis the interesting events take place at the
physiological, epigenetic level. The contemporary preoccupation with the minutiae of gene expression distracts attention from the real mysteries.
One of these mysteries is the invariant reproduction of organismic forms,
generation after generation. If one grants that form and spatial order are nowhere
spelled out in the genetic blueprint, but arise interactively at the physiological
level, should one not expect morphology to vary with every perturbation of the
environment? The causes of the observed stability need to be spelled out; this
cannot be done at present, but part of the answer must be that organisms never
arise (like a jigsaw puzzle) by joining together performed parts. On the contrary,
omni cellula ex cellula. A growing or dividing cell models itself upon itself; and
sometimes, at least, pre-existing structures demonstrably guide the correct
placement of new gene products. Spatial memory comes in diverse modes, some
simple and some elaborate. A familiar instance is the correct positioning of
integral membrane proteins: the primary structure defines the manner of
association with the bilayer, but proper orientation depends on delivery of the
precursor to the correct side. Spectacular instances of spatial memory come from
the ciliates, which position new ciliary units in the cortex by reference to
pre-existing ones (Beisson and Sonneborn, 1965). Indeed, the spatial organization
of the entire cortex is clonally transmitted with the aid of informational fields
whose nature remains unknown (Frankel, 1982, 1989).
Spatial memory is, I believe, an important and deplorably neglected aspect
of cellular inheritance. But it must be added that many, probably all, organisms
own an astonishing capacity for self organization even in the absence of visible
spatial cues. It happens whenever protoplasts regenerate, whenever a spherically
symmetrical egg or spore initiates outgrowth. The product comes to have the
right form, the expected one, and I find that baffling. In the end, it remains a
mystery that living forms exist at all. We take it for granted that the familiar
forms are products of natural selection, optimized for particular functions. But it
has been pointed out repeatedly that the theory of evolution by natural selection
explains the progressive modification of living forms, but not their origins
(Webster and Goodwin, 1981). Are there physical mechanisms that spontaneously generate forms for selection to work on? If so, the root cause of spatial
Biochemical Topology
377
organization may be found in dissipative structures, those m o d e s of order that
arise spontaneously in systems subjected to a sufficient flux of energy (Nicolis and
Prigogine, 1977; Peacocke, 1983). If there is anything to this notion, then spatial
organization and even f o r m can be envisaged as intrinsic, t h e r m o d y n a m i c
consequences of metabolism; and we m a y come to think of genes and gene
products as selecting or stabilizing a particular f o r m out of m a n y that are
compatible with a given t h e r m o d y n a m i c regimen.
ENVOI
The late Sir Peter M e d a w a r , in a famous epigram, described science as " t h e
art of the soluble". Consequently, the best scientific problems are those that are
difficult enough to be challenging but not so difficult as to be intractable. A t
present, localization, self organization and spatial order at the cellular level lie
beyond the " M e d a w a r z o n e " , but that may not always be so. Time was when
energy coupling to phosphorylation and transport was equally mystifying, for
want of the p r o p e r conceptual framework. This, I suspect, is what we need to
begin to understand growth, d e v e l o p m e n t and morphogensis: not only m o r e data
but a new paradigm, a fresh pair of spectacles. So, I say to those who would
rather blaze trails than pave roads, the frontier is not all settled but it has shifted:
today's frontier runs through the problems of organized complexity.
ACKNOWLEDGEMENTS
I am indebted to Willie Schreurs for preparing most of the illustrations and
to L e o n a W o e b e r for typing the manuscript. Original work f r o m this laboratory
has been supported in part by grants from the National Institutes of Health
(A103568) and the National Science Foundation ( D C B 8996200).
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DISCUSSION
A. Kotyk: My question: In describing the "vectorial" growth of a hyphal tip you
did not make any reference to the possible involvement of cytoskeletal elements
in the process. Is this irrelevant in this context? I suggest it is highly relevant.
However, the use of micro-tubule-blocking agents (colchicine?) showed that
microtubules are not the answer. Rather the microfilaments (actin) might be
involved.
F. M. Harold: The cytoskeleton is highly relevant, but we do not understand how
it fits in. Recent data from work with filamentous fungi, yeast and algae indicate
that microtubules are not essential for extension or localization of the apex. The
findings strongly implicate microfilaments, but leave their precise function
undetermined.
C. A. Pasternak: Isn't the distinction between vectorial chem&try and vectorial
p h y s i o l o g y too restrictive? A muscle fibril, after all, spans lengths of hundreds of
/~m, and fulfils its function (of contraction and relaxation over these distances) as
a result of a series of binding and release events that are exactly analogous to
those of the F1 complex. A n d chromosomal spindles, that also span almost the
entire length of a cell during mitosis, also function as a result of local chemical
events.
F. M. Harold: The distinction between vectorial chemistry and vectorial physiology i s a matter of degree, not of principle. I find it useful to employ a special term
to designate complex spatially extended processes that have a direction in cellular
space such as secretion or muscle contraction. To be sure, the physiological level
is built up from individual chemical reactions that are vectorial at the molecular
BiochemicalTopology
383
level; but the directionality of the molecular events is insufficient to determine
that of the higher-order process.
V. P. Skulachev: What do you think about the possibility that the plasma
membrane of hyphae is used for the lateral transmission of power in the form of
A/2H+?
F. M. Harold: I like it in principle, but (at least, as far as Achlya is concerned) it
is contradicted by our data (Fig. 11). However, it may be pertinent that Achlya
has no septa.
P. B. Garland: Your proposed mechanism whereby the fungal hypha knows its tip
from its trunk requires an ion porter at the tip and the respective ion translocating
ATPase in the trunk. Both of these proteins are in the same membrane, and
unless their translational diffusion is restrained then their distribution will
randomise, destroying the tip/trunk signal. However, localizingthese proteins in
their specificrespective regions requires that the cell can already tellits tip from
its trunk. So these proteins and their activitiesare unlikely to be the cell'sprimary
source of information for tellingtip from trunk. Can you comment please?
F. M. Harold: I quite agree with your argument. W h e n we began our studies on
transcelIular ionic currents, I hoped that they might be part of the chain of
causality by which a growing hypha imposes direction upon itself.It now appears
that this is not the case. But even if it were, the hypha would stillhave to "know"
how to localize its pumps and porters.
B, H. Weber: With regard to your comment about the differences of scale of
vectorial metabolism and vectorial physiology, we know that these are nonlinear,
autocatalytic chemical systems, such as the BZ reaction, which have no vectorial
chemistry, yet which show spatial and temporal asymmetries involving say 1022
molecules. Have you considered if a nonlinear, autocatalytic vectorial chemical
system might be able to account for the phenomena encompassed by vectorial
physiology?
F. M. Harold: It is quite unclear to me whether there is any connection between
self-organization at the chemical and biological levels. By the same token, I am
intrigued by the notion that dissipative structures are the foundation of biological
forms, but feel very uncertain about it. If such links do exist, I would expect that
living things have evolved mechanisms to select and stabilize the particular mode
of organization that each organism favours. I doubt whether physical chemistry in
itself will suffice to account for any of the biological systems.
A. Kieinzeller: You raised the possibility of the role of actin in the phenomenology of vectorial physiology of the alga. Our experiments with eukaryotic cells
would be consistent with such a view: hypotonic shock produces a major
(volume-independent, but reversible) change in the F-actin organization at the
membrane.
F. M. Harold: Thank you. Conversations about actin always remind me of Mark
Twain's comments about the weather--everyone talks about it, but no one does
anything about it!
384
Harold
N. Sone: You showed a figure with a Na+-translocating respiratory system in V.
alginolyticus. Is there any H+-translocating Q cycle in a bcl complex in these
bacteria? (Dr. Harold transferred this question to Dr Skulachev who said No.)
A. Jagendorf: Shouldn't we consider the cell wall geometry and structure as an
important parameter in determining the polarity of hyphal growth? Its rigidity on
the sides and flexibility when first laid down at the tip, must be important in
maintaining tip growth.
F. hi. Harold: The structure and mechanical properties of the cell wall are very
important in determining the shape of hyphal tips. In fact, by incorporating these
parameters into certain equations one can calculate the shape that tips ought to
have, and come out with reasonable results (for discussions and references see
Harold, To shape a cell: an inquiry into the causes of morphogenesis in
microorganisms. Microbiol. Revs. 54:381-431, 1990). But I suspect that the
relationship goes deeper. The cell wall at the apex is plastic, and that in itself may
favour the exocytosis of vesicles in that region and may thus perpetuate the tip.
Unfortunately, we have not been able to devise good experimental tests of the
"soft-spot hypothesis".
W. N. Konings: You demonstrated that the pattern of ion currents can vary
drastically under different conditions. That in itself is quite interesting. My
questions are: whether these changes can occur in one and the same hyphae and
whether there is any information about the ion-translocating mechanism involved.
F. M. Harold: It is technically not possible to measure current patterns in a single
hypha over long periods of time; the effects of nutritional changes must therefore
be inferred from experiments on populations of hyphae. The mechanisms that
underlie changes in the current patterns are unknown; we speculate that they
involve relatively minor shifts in the longitudinal distribution of the protonmotive
ATPase and proton-coupled porters.
M. C. Sorgato: Have you considered the possible involvement of stretchactivated calcium channels, which are found in a variety of plasma membranes?
This involvement could be possible because these channels are pressure activated,
and in Achlya you stressed that osmotic pressure can indeed develop, and
secondly because it has been postulated that there could be a connection between
these channels and the cytoskeleton.
F. M. Harold: Turgor pressure is exerted uniformly all over the wall, so an
additional control mechanism would be required.
M. Kogut: On the question of how a developing cell "knows" the "geographical"
starting point--such as the apex--and how it can establish gradients (of
chemicals) along its axes, I have two, very general comments.
The first concerns the incorporation of specificity, and hence information into
apparently uniform (and hence non-specific) structures, such as the peptidoglycan
layer, or other repetitive molecular assemblies. I am recalling Ogston's "threepoint-attachment" hypothesis to solve the paradox of how a symmetrical
molecule (citrate) could account for an asymmetrical carbon incorporation.
BiochemicalTopology
385
The answer to the question posed by Professor Harold could lie in the
formation of "receptors", which can result from juxtapositions of various
chemical groupings or radicals, to produce very specific configurations. These in
turn could account for gradients of ligands along a seemingly homogeneous
surface.
Could the difficulty of "deciding" the location of the apex, perhaps be
confined to our imagination? Could the location of the apex (or other growing
point) of a cell be quite random, but the function as the initiating trigger for an
ordered sequence of events and transformations, via the constitution of specific
receptors by molecular juxtapositions and interactions? Alternatively, the location of the apex could be determined in (geographical) relation to a certain gene,
i.e. genetically, and then initiate the ordered sequence of cellular events involved
in directional growth.
F. M. Harold: I do not believe that the localization of an apex can be truly
random, because the spatial position of the growing apex perpetuates itself.
Branch emergence, also, can be deterministic: in Achlya, at least, we can elicit
branching at a locus of our own choice by applying amino acids locally with a
micropipette (Schreurs et al., J. Gen. Microbiol. 135:2519-2528, 1989). As for
genes, there are many good reasons for believing that they determine molecular
structures but not geographic location. So your comment, Dr. Kogut, is all too
apt: it is our (my) imagination that is deficient!
H. Bantu: Is there not a possibility that the sharp curvature at the tip of a
growing hypha might create a micro-environment favouring continuation of
elongation in that direction? (I have in mind the fact that the separation of
functions on a stacked thylakoid membrane is partially determined by the
geometry of the stacks).
F. M. Harold: I like the notion that curvature is a part of the localizing
mechanism: it is one that had not occurred to me before. Thank you.
A. Jagendorf: Can the Ca 2+ stored in the cell wall take the place of Ca + depleted
from the medium?
F. M. Harold: Yes, wall calcium could come off and flow into the cytoplasm
via a localized calcium channel. But it should be depleted by treating the hyphae
with EGTA, yet this does not immediately arrest extension.