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Life at Low Temperatures

Cryobiology is the study of life at temperatures too low to support life. On the surface, this statement appears to be a contradiction, however, it must be kept in mind that the study of life cannot be carried out without also studying the loss of life. This is because life is very difficult to define in precise terms; a far simpler task is to define when a living thing has expired. In cryobiology, we study the interface, fuzzy though it may be, between life and death that can be found at low temperatures. For the most part, low temperatures are those below which water freezes. Water, not DNA, is the fundamental molecule of life and it is the rare and precious qualities of liquid water that make life possible. Without an environment that contains generous quantities of liquid water, life would not exist. Living in Canada, each winter we see environments in which liquid water does not exist and yet, when spring arrives, life flourishes without any re-population from warmer climes. What is this state that some living things inhabit, where no growth occurs -- to our eyes no time appears to pass -- yet these organisms remain alive? This curious phenomenon is the essence of cryobiology. The environment for life, the surface of the earth, is constantly in flux: from the cosmic time scale in which our sun is becoming hotter to the realm of rapidly changing weather phenomena. Living things must be able to adapt to this changing environment in order to preserve the existence of life itself. It does this by a process of mutation and natural selection. Organisms can undergo random mutations (essentially random errors in chemical processes) which make them slightly different from what they were before. If these differences give them an advantage over other organisms in their environment, then they will thrive, if not, then they will dwindle. One of the paths that has proven successful is the requirement of sexual reproduction in multicellular organisms. In this evolutionary route, two organisms combine their genes (the blueprints for constructing new, identical organisms) to generate mutations at a faster rate than can be achieved with chemical errors. In order for this approach to be successful, however, it is necessary that the organisms die after creating at least two more offspring. If they were to be immortal, then the population would soon rise to a level that could not be supported by the environment (thus preventing the success of any advantageous mutations). This is the boat that we find ourselves in: after a finite time, our organs cease to function, our bodies fail us: we are programmed to die. Although we may pass our genes to our offspring, we cannot pass on our memories or our conscious selves -- those intangible qualities which make us individual entities -- thus it is not easy to accept death knowing that we will cease to be. Low temperatures provide a means of escaping this fate by suspending the aging process. The processes by which our programmed obsolescence is carried out are chemical in nature, thus the rate at which these reactions occur can be slowed dramatically by lowering the temperature. It might be as simple as this were we not principally composed of liquid water. For when we lower the temperature, water freezes: and this makes all the difference. All mammalian cells exist in an ionic environment and also maintain a similar concentration of ionic salts in the intracellular space to balance the osmotic pressure. When water freezes to form ice, the crystal is composed only of water molecules which leaves the solutes that were present before freezing to concentrate in whatever fraction of the initial solution remains unfrozen. The temperature at which water freezes is not low enough to bring the rate of chemical processes to a halt, thus the concentrated salt solutions will affect the cells chemically. It is in this temperature region -- between the freezing point of water and the temperature at which the rate of biochemical reactions becomes negligible -- that injury and death occur. The principal medical application of cryobiology is the benign storage of cells, tissues and organs (some would like to include organisms as well) for use in repairing those parts that no longer function due to disease, injury or aging. The challenge is to take these specimens through the temperature region in which damage can occur and back again without causing irreparable damage. Thus far, this challenge has only been met successfully for cell suspensions and several relatively simple tissue systems.

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Modern medicine may be said to have begun with the application of science to the field of medicine. Although medicine is only concerned with the results, the historical accomplishments of the application of the scientific method provides overwhelming support for continuing on this path. This point is often lost on the layperson, who sees basic science as an esoteric extravagance which is performed merely to satisfy the curiosity of the scientist (satisfying curiosity is the purpose of basic science; knowledge of natural phenomena, however, is neither esoteric nor extravagant), and it is frequently lost on the cryobiologist who believes that subtle alterations to existing protocols are all that are necessary to achieve success with tissues and organs. Trial and error is not necessarily unscientific. Indeed, guessing is necessary for the scientific method; however, the success of the method relies on building a foundation of guesses that are all supported by the evidence of experiments. In this way, further guesses are supported by the edifice that is the body of scientific knowledge (this is the collection of results of controlled experiments which are summarized by theoretical constructs -- usually mathematical in nature). In this brief course, we will try to set out the scientific groundwork and the body of knowledge that make up the field of cryobiology.

What is Temperature?
The temperature of some object is a number that tells us how hot or cold that object is. We have various scales of temperature (e.g. Celcius, Farenheit, Rankine, Kelvin) that relate the measurable numbers to some physical events (phase transitions of water, hot and cold days in Denmark, etc.). The numbers that we measure come from observing a physical quantity that changes when it gets hot or cold (such as the expansion of liquids). These measurements can then be tied to some less tangible quantity like energy. We usually say that the temperature is proportional to the average kinetic energy of a system. So if we measure the temperature of the air, we're really measuring the average speed of the air molecules. The hotter the air, the faster the molecules are moving. When all the molecules of a particular object (or system) have the same mean kinetic energy, then we say that the system is in thermal equilibrium (no heat (kinetic energy) is passed from one part of the object (system) to another). The physicists, however, say a system is in thermal equilibrium at temperature T if the probability of it being found in any state of energy E is proportional to exp(-E/kT). Here k is Boltzmann's constant. There is another earlier definition involving entropy, but it is equivalent, and it's likely that the concept of entropy is apt to confuse rather than clarify things for most people who are still working on the concept of temperature. Certainly thinking about entropy is useful to figure out why the above definition is "the right one", or at least a very good one and the standard one. Let me throw in two related remarks. We may ask if it is really true that temperature is not defined for systems that are not in thermal equilibrium. This is certainly true according to the above definition, and indeed most of textbook thermodynamics should really be called "thermostatics", since it only deals with situations that are in thermal equilibrium or can be adequately approximated as such. The last 7 words are crucial. Nothing that we'll ever come across in this universe is really exactly in thermal equilibrium. When applying "thermostatics" to the real world we always need to think about whether treating our system as if it were in thermal equilibrium is a good approximation. Often it is; sometimes it's not. For example: stricly speaking, flowing water is not in thermal equilibrium and does not have a well-defined temperature according to the textbook definition. But in practice, as long as the relative velocity of nearby water molecules due to the flowing of the water is much less than their relative velocity due to random thermal motion, we can often get away with pretending the water is in thermal equilibrium. For example, you can dissolve just about as much salt in a pot of still water as in a stirred pot of water at the same, ahem, temperature. Probably not exactly the same amount: the turbulence of the stirred water should have some small effect on what those sodium and chloride ions do. But unless you are stirring the water insanely fast (in

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which case the, ahem, temperature goes up) the effect is negligible. Of course, the stirring of the water can have a big effect on dynamical rather than static questions, like how fast salt will dissolve when you throw it in! (I write "ahem" in front of "temperature" above because the temperature is not precisely well-defined according to the textbook definition, and yet we have no trouble measuring it with our thermometers.) There is a subject called nonequilibrium statistical mechanics, and Prigogine won a Nobel prize in it, so maybe he knows what to do in situations where pretending you are in thermal equilibrium is a really bad approximation. However, my hunch, backed up by a few short peeks at the nasty-looking equations in his books (the serious ones, not his pop books), is that there aren't any good substitute for the definition of "temperature" that handles these situations. Stuff about intuitive notion of temperature and heat flow...realsoonow

What is Life? Where Does Life Come From?

In order to get some insight into what makes an organism, it might be helpful to consider the most likely scenario for abiogenesis (the self-organizing formation of life from non-living components). Let's consider a primitive planet that's far from equilibrium (lots of energy sources--solar, nuclear, chemical) and has liquid water present as well as a terrestrial component. Solar variation (day/night) will cause evaporation and waves, so we'll get puddles of water on the land. Chemicals will also be dissolved in the water, so we'll have pockets of chemical soup. One of the interesting things about chemistry is the fact of catalysis--a compound that isn't changed by a chemical reaction can lower the activation energy for that reaction by stabilizing an intermediate form. This means that the presence of one chemical can drive the equilibrium for a reaction in a particular direction. One of the interesting things about carbon compounds (and carbon-nitrogen compounds in particular) is how adept they are at catalysis--especially the catalysis of reactions between other organic (carbon compounds) molecules. Thus we can imagine our pocket of chemical soup to begin collecting catalysts. Let's say our pocket has a molecule that converts methane to ethane. There's lots of methane spewing out of volcanoes and such, so our pocket begins to become concentrated with ethane. Perhaps there's another chemical that catalyzes the formation of ethanol from ethane. We can imagine whole chains of reactions occurring from the primitive chemicals that are present in the atmosphere, building ever more complex molecules which themselves serve as catalysts. Eventually, as the complexity of our pocket of soup increases, there will come a point at which a reaction chain will feed back on itself--a product of the reaction chain becomes a catalyst for an earlier reaction in the chain. At some point of complexity, all reactions in the chain are catalysed by products or intermediates from other points in the chain. This is called an autocatalytic set and it represents the most primitive form of metabolism. In a sense, our chemical soup is now alive. If lipids invade our pool, then they spontaneously form micelles (spherical bags of water). Should they encapsulate the autocatalytic set, then we have a primitive cell. Now you're probably saying, "Fine, but how does this bag of water reproduce? What about the genetic material?" Well, with the accumulation of lipids, the micelle becomes unstable (think of a big water drop, stretching and wobbling about) and will eventually divide. Micelles are huge compared to molecules, so if you have an autocatalytic set (where the reaction chain leads to the production of all of the components of the reaction chain, so the micelle is full of everything that's required) then it's likely that both daughter micelles will also contain the complete autocatalytic set. With a lipid micelle that contains an autocatalytic set, we have a living organism (if you think this claim is outlandish, the strong hypothesis of artificial life claims that living organisms can be created in silico). The rest of what we see as life on earth is just a bit of tinkering for efficiency.

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Sugars and nucleotides are among the simple organic compounds that could easily be part of such an autocatalytic set and can easily polymerize into ribonucleic acid (RNA). It turns out that RNA is extremely versatile as a catalyst for organic molecules as long as you have a few nucleotide bases for variation (4 seems to be sufficient). RNA will also form a self-catalyst--it will form a molecule that catalyzes the polymerization of RNA. So for me at least, it seems a small jump from the micelle contained autocatalytic set to a cell with a genetic template. Natural selection will ensure that any improvement in efficiency will not only surpass the predecessor, but starve it of its resources leading to extinction (so once the 4 bases of RNA were chosen, we got locked in to the standard). Bacteria are the most primitive organisms that we have today, and are essentially just micelles with a bunch of chemicals inside that form an autocatalytic set, the genetic template being part of that set. It's important to realize that the DNA is not sufficient to define an organism, there is a crucial collection of chemicals that are also required and that are passed on during reproduction in addition to the DNA.

The Fundamental Unit of Life

Cells are basically sacs of water surrounded by an oil membrane. The membrane components have a hydrophilic end (dissolves in water) and a hydrophobic end (oil soluble), so when you put them in water, they line up back to back with the hydrophobic ends against each other and the hydrophilic ends extending into the water to make a bilayer. The edge energy of this bilayer can be eliminated by forming a spherical shell, although this introduces a surface energy due to the curvature. Once formed, such micelles (spherical shells of lipid bilayers) are stable, but not in equilibrium (you can pop them like soap bubbles). The membrane limits water movement between the inside and outside because water is poorly soluble in the hydrophobic interior of the bilayer. It's not insoluble, though, so water can move by a solubility-diffusion mechanism. If you add salt to the outside, water will leave the micelle to try to equilibrate osmotic pressure. The interior of cells is also filled with smaller vesicles, each containing a different chemical milieu in its interior than that of the bulk cell interior. Also, the amino acids that are used to construct proteins (20 of them in humans) come in varying degrees of hydrophobicity. So by varying the sequence of amino acids in a protein, you can make part of it hydrophobic and part hydrophilic. Sometimes the hydrophobic regions get turned in on each other to minimize the surface energy of the 3D structure of the protein and sometimes the hydrophobic regions get stuck into the lipid bilayer. The latter phenomenon creates membrane bound proteins, usually with a domain that is inside the cell (or vesicle) and a domain that is outside the cell (connected by the transmembrane portion). A lot of transmembrane proteins are constructed as hollow cylinders; the protein winds up and down through the membrane (with about 7 transmembrane segments) to form a cylinder within the bilayer (if you looked down on the bilayer, you'd see a small hole in the center of the cylinder). The center of the hole is hydrophilic, so it is an aqueous channel through the membrane. These proteins have essentially 2 degrees of freedom (translation in the plane of the membrane and spinning), so any chemical reactions that provide heat (e.g., the hydrolysis of ATP (the body's energy currency)) will cause them to spin. Since they have amino acids with charged groups situated around the cylinder, the spin creates a strong magnetic field through the membrane. Ions with a magnetic moment (that are small enough to fit through the pore) are then "pumped" from one side of the membrane to the other, creating chemical and electrical gradients. These gradients can be used to drive chemical reactions, or changes in the gradients can be used to signal events occuring outside the cell to the computational center of the cell. Inside the cell is the nucleus (bacteria don't have a nucleus, their DNA just sits inside the cytoplasm), with the DNA enclosed in a membrane (not a lipid membrane, it has large pores connecting it to the cytoplasm). This is where genes are read to create proteins. A human has around 10^5 genes, comprising about 1% of the total DNA (the other 99% is often called "junk" DNA, an indication of just how arrogant biologists can get). An individual cell will only transcribe a small subset of the genome because most cells are terminally

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differentiated and carry out specific tasks (the cells in the thymus may transcribe the whole genome, since that's the place where the immune system learns what not to attack. Just a guess). Many of the genes that are transcribed are regulatory in nature (i.e., they allow control over the expression of functional genes). The cytoplasm is often thought of as a bag of water with a bunch of chemicals floating around, although there is actually exquisite structure. Small molecules diffuse through the cytoplasm at rates comparable to that in bulk water and some even move faster. There is a cytoskeleton composed of protein filaments that connect the nucleus to the outer membrane, as well as other structures within the cell. Only fat cells and red cells have a smooth outer membrane, most cells have thousands of microvilli protruding from the surface and these move in and out quickly. Cells are usually only round balls when they're suspended in liquid. If they're allowed to attach to a plate, or a biological matrix (all the stuff that isn't part of cells), then they take on a variety of shapes and can move (almost all human cells can move). Some nerve cells grow up to a meter in length and have little motors to transport chemicals along the cytoskeleton. When attached to a matrix, cells can become polar (e.g., have different proteins in the membrane on one side compared with the other). All cells descend from the original zygote, so obviously the potential to become any type of cell is latent in the germ cells. The process of differentiation (specialization) that occurs during development, as well as within the adult, often is accompanied by irreversible changes. A terminally differentiated cell usually won't have the potential to become a different terminally differentiated cell, although it can become further differentiated (e.g., a neoplastic transformation where a cell becomes cancerous). This is often called de-differentiation because the cell takes on the phenotype of less differentiated cells. Any terminally differentiated cell has to have strict control on its growth and proliferation, otherwise we'd become too large for the system to work properly. The natural state for a cell is to grow constitutively (no regulation) as with bacteria and cell death by means other than necrosis (by natural causes, e.g. oxygen starvation) is also completely unnatural for our ancestors. In order to construct a multicellular, differentiated organism, however, these controls must be developed. A lot of cell communication occurs to coordinate this regulation.

What do Living Things Do?

Now we come to one of the fundamental laws of biology. Expressed simply, it says that every act of cooperation provides an opportunity for parasitism. The autocatalytic nature of an organism can be considered to be an act of cooperation (because of the positive feedback in the system) so naturally, we can expect parasites to act on bacteria. In fact, they do. Phage are viruses that infect bacteria, injecting their DNA into the bacterium so that the replication and translation machinery creates new phage, destroying the bacterium in the process. Bacteria that are separated spatially, may also be subjected to different environments, causing them to adapt toward a more efficient lifestyle. If two different bacteria then come into proximity in an environment in which the waste products of one can be used to the benefit of the other, then another opportunity for cooperation is created. At one such instance, one of the bacteria involved in such cooperation actually engulfed the other bacterium. This appears to be how the eukaryotes were derived-mitochondria being derived from the archaebacteria. The eukaryotes developed further specialization (nucleus, endoplasmic reticulum, etc.) that allowed them to develop a larger genome (the longer your DNA, the more fragile it becomes) but at the expense of diversity. They also duplicated their chromosomes which led to crossing-over during replication, thus they soon found (lucky accident) that cooperation and specialization allowed them to exploit many of the diverse ecological niches that were previously only host to the microbes. The multicellular organisms now required a means for morphogenesis (growth from a single cell to the adult stage), coordination (each cell has to know what it's supposed to do), and death (selection acts on the organism). These tasks require communication between cells. Perhaps we get some of this organization for free, though.

What Makes an Organism?

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Is self-organization responsible for some of the order that we see in complex organisms? Consider a network of light bulbs, in which connections exist between each bulb. As an example, we'll consider just three bulbs, each wired to the other two. Each light bulb then has a rule telling it whether it should change state or not (the two states being on and off) depending on the inputs that it receives from the other light bulbs that it's wired to. Say that each bulb will OR its two inputs so that it will only go on when at least one of its neighbors is on. This is a boolean network. We can start it off in any random configuration (there are 8 possible states that the network can be in) and see how it evolves dynamically. In the case of the OR rule, there are three possible state cycles (we'll use 0 for off and 1 for on). 000 -> remains 000 001 <-> 010 constantly flipping back and forth 100, 110, or 101 -> 011 -> 111 -> remains 111 So there are two frozen state cycles (where all the lights stay on or off) and one cycling state cycle. We call these state cycles "attractors" and the initial states that lead into them their "basins of attraction". Now let's consider something more complex. Let's say we have a network of 100 000 light bulbs and we wire each light bulb up to 2 other light bulbs (at random), assigning each light bulb a boolean rule for its two inputs (also at random). Such a network has a state space of 2^100 000, much too large to ever be traversed in the lifetime of the universe (assuming a time slice of a femtosecond, say (pretty fast switching!)). But if you start this network in any random configuration, it will settle down to a state cycle that consists of a mere 317 states!!! The number of attractors will also be on the order of 317!!! (100 000 light bulbs suddenly go on if you know where I'm going with this). The human genome has about 100 000 genes that are transcribed. All genes code for proteins, some of these are structural (they form part of the organism or enzymes that assist in its operation) and some are regulatory (they bind to DNA to control the transcription of other genes) and some are involved in both. What we have, in essence is a boolean network, where the transcription of each gene can affect the transcription of a couple of other genes. A human has about 256 cell types (attractors) and the cell cycle usually takes between about 5-50 hours. Genes are turned on and off with a time scale of about 1-10 minutes so traversing a state cycle with 317 states will take about 5-50 hours! These relationships scale precisely as expected for other organisms with radically different structures and genome sizes. In short, an organism may just be the chemical manifestation of a huge boolean network. Anyone wishing to pursue such wonderful (but obviously crackpot) ideas should read Stuart Kauffman's The Origins of Order, probably the most important book in biology since the Origin of Species. In a less glamorous light, an organism is made of organs (functionally and anatomically distinct, these are made of at least two tissues and perform at least one function that is necessary for the organism) which are made of tissues (aggregation of cells of common embryological origin that work together to perform a single function) which are made of cells. Each cell type uses a very small subset of the entire genome (with the possible exception of cells in the thymus) but carries the entire genome (some cells, notably those of the immune system, corrupt the genome through somatic cell (non-germ cells) mutations--this allows them to generate the diversity required to build antibodies to any possible invading organism). And despite the charm of getting organization for free, there still is an enormous amount of communication between cells.

Communication Between Cells

The business of biologists often seems to be to make the field more obscure (as opposed to physicists who search for unifying laws). Cell-to-cell communication is currently a huge area of research (it goes under the name 'molecular biology'), but the major effort right now is simply to find out what happens and, to a lesser extent, how to jimmy it to do something slightly differently. I think Lovelock hit the nail on the head with the

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following quote: "When seen from the outside, much of biology appears to be the building of databases--making the 'whole life catalog'. Sometimes, in a pensive mood, I fancy that to biologists the living world is a vast set of book collections held in interconnected libraries. In this dream, the biologists are like competent librarians who devise the most intricate classification of every new library they discover but never read the books. They sense that something is missing from their lives, and this feeling intensifies as new collections of books grow hard to find. I see the biologists expressing an almost palpable sense of relief when joined by molecular biologists who dare to start the even greater task of classifying the words the books contain." Anyway, enough fun, now on to the lesson. Molecules move within cells and between cells, therefore information transfer does occur between cells by means of molecular diffusion. Hypotheses of alternative methods of communication may be driven by attempting to analyze where the current paradigm breaks down, so I'll briefly describe something of what is known about cell-to-cell communication mediated by the exchange of molecules. Within cells, there can be communication between regions by simple diffusion. There are also molecular motors (such as kinesin) which carry vesicles along microtubules. Other intracellular communication mechanisms may rely on mechanical force transduction (e.g. stress/strain behavior of the cytoskeleton. There was a recent experiment where fibroblasts (I think) were grown in culture and then subjected to cyclic increase in pressure on the order of several atmospheres. The cells started producing different compounds in response to the pressure. This represents a volume change for the cells on the order of 1 ppm. It's simply inconceivable that cells could detect something like that (and they probably don't, I think it turned out that the cells were grown on silastic membrane that stretched with the pressure increase, so they were actually detecting something that was on the order of 1 part in 10). We don't know too much about this yet. Between cells, the most obvious communication scheme is the direct coupling of cells. Gap junctions can form between cells that have a specific membrane-bound channel when they are brought within 15 nm of each other. Two such protein channels will join together and connect the cytoplasm of the 2 cells with a 2 nm diameter aqueous channel (the channels only open when they join with another cell's channel, by a twisting motion). Small molecules (<1200 kd) will pass freely through the gap junction, allowing communication by diffusion. The most common cell-to-cell communication mechanism that has been studied is mediated by extracellular messengers (molecules who's only function is to communicate a specific message). Classically, this system is divided into the autocrine system (where the cell producing the messenger is also the cell that receives the message), the paracrine system (where a cell produces a messenger, then it diffuses through the interstitial fluid to a cell that receives it), and the endocrine system (where the messenger travels through the circulatory system before it delivers its information). Secretory cells have to synthesize the extracellular messengers and release them. Since transcription, translation, etc. takes a long time, there will usually be a stockpile on hand, with release being the part of the system subject to regulation. These cells will monitor something within the organism (e.g. glucose levels) and respond to changes (e.g. release of insulin to elevated levels of glucose). Target cells must detect the messenger at very low concentrations (as low as 10-12 mol/L) so the binding between hormone (messenger) and receptor must be of very high affinity. The principal steps involved with hormone detection are signal transduction (the process of generating a biological response after the detection of the hormone), hormone degradation (to allow the system to be used continuously), and regulation (response to feedback so that the system can be used as a part of a control system). There are many different secretory and target cells, as well as messenger molecules, but there are relatively few (known) signal transduction mechanisms and secondary messengers (messenger molecules that transmit information from a receptor molecule to the cell interior).
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Extracellular messengers are classified by their solubility (water or oil). The solubility also determines their site of action, mechanisms of action, and storage times (oil soluble molecules are more difficult to break down than water soluble (cf. detoxification of water soluble or oil soluble narcotics)). Not all messengers have specific receptors (e.g. prostaglandins), nor do they all require signal transduction (i.e. the hormone-receptor complex can have biological action). Receptors are either situated in the plasma membrane (the outermost membrane of a cell) or they can be inside the cell (less common). The receptor doesn't just bind the hormone but by doing so, the hormonereceptor complex produces a biochemical event inside the cell. Membrane bound receptors usually are coupled to a G-protein (binds GTP), have a tyrosine kinase activity (phosphorylate tyrosine residues on other proteins), or regulate a membrane-bound ion channel. Receptors that are inside cells bind steroids. Often, the receptor-hormone complex will function as an enzyme to modify some compound that's already in the cells (e.g. ADP to cAMP). These new compounds then act as secondary messengers for intracellular signalling. They can bind to an enzyme, thereby giving it an activity such as binding to DNA. Proteins bound to DNA can act to regulate gene transcription. Signal transduction can cause amplification (1 hormone-receptor complex can lead to the production of up to 104 secondary messengers) or integration (several different H-R complexes might be required to generate a biological response) of signals. Communication between cells and the matrix that they construct undoubtedly occurs, but the nature of these mechanisms has yet to be worked out in detail. When cells are put on certain surfaces, they will form attachments to that surface and can detect movement of the surface relative to those attachments (e.g., stretching of an elastic membrane). There are also strong theoretical models of how the cells/matrix/chemical milieu all interact to drive morphogenesis during development (and repair following injury, although most of this work is in amphibians (limb regeneration)). These models are made by mathematicians, and are therefore way beyond the ken of your everyday biologist, so there hasn't been a lot of experiments done to validate the models (also due to their complexity). The models are being developed very actively and this is what most people mean when they talk about theoretical biology. Essentially, it seems that cells are able to respond to mechanical and chemical changes in the matrix in which they're embedded, and these changes in turn alter the structure and behavior of that matrix. The simplest models use differential geometry to describe these interactions as a field theory, the more complicated ones use math that I've never heard of, but I take their word for it that it's difficult. There are also cells that are dedicated to the task of communication within the organism, the neurons. These work by means of diffusion of molecules as well as an excitable membrane.

Cells That Form a Communication Channel

There are cells that carry information within an organism by electrical means, using ion flows. These cells are said to have an 'excitable membrane' as they respond to electrical stimuli that exceed a threshold value. Nerve cells, muscles cells, and many specialized transducer cells (e.g. retinal cells, cochlear (inner ear) cells) all have excitable membranes, with their 'firing' response (more about this later) being triggered by particular stimuli (or by changing their membrane potential by external means (i.e. during an experiment)). All cells maintain ion gradients across their plasma membrane, although the magnitude of these gradients can differ appreciably between cell types. The gradients are maintained by differential permeabilities and active pumping of ions across the membrane. The pumping is probably done by an electromagnetic pump (a charged cylinder, embedded in the membrane, is made to spin by the hydrolysis of ATP, creating a magnetic field that accelerates ions through the membrane in a given direction) although most biologists picture a little lever that sort of acts like a spring-loaded pushing device (lord knows why they picture this, but they do). The major ion pump that creates the membrane resting potential is called sodium-potassium ATPase (meaning it's an enzyme that hydrolyzes ATP). ATP is the principal energy currency used by the eukaryotes (organisms with

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non-circular DNA that sits inside a nucleus). The full name is adenosine triphosphate. The principal reactions of energy metabolism (both anaerobic and aerobic) drive the conversion of ADP to ATP so that the energy given off by, e.g. the oxidation of glucose can be converted to potential energy in the form of a phosphate bond (often called a 'high energy bond', although it isn't spectacularly energetic). Enzymes that drive endothermic chemical reactions (e.g. anabolic metabolism) have evolved to hydrolyze ATP to ADP, thereby liberating the energy of the phosphate bond and use it to do chemical work. Lipid bilayer membranes have very low permeability to ionic species, although the protein component of cell membranes allows control of the permeability to specific ions by creating channels (thereby increasing the permeability to certain ions). In terms of K+ and Na+, the ions that are pumped to create the resting potential, the membrane is much more permeable to K+ than it is to Na+, so the membrane can effectively be considered as a leaky capacitor, where K+ flux is the primary contributor to the potential. The concentrations of the principal ionic components in frog muscle cells (as an example) is (in mMol/L):
ion Na+ K+ Clintracellular 12 155 4 extracellular 145 4 120

And the resting potential across the membrane can be calculated by modifying the Nernst equation and assuming a constant electric field across the membrane:
RT P_K[K_o] + P_Na[Na_o] + P_Cl[Cl_i] E = -- ln{----------------------------------} F P_K[K_i] + P_Na[Na_i] + P_Cl[Cl_o]

where T = abs temp, R = gas const, F = Faraday const, P = permeability, K = concentration (_i inside the cell, _o outside the cell). E = -85.3 mV Which is very close to what is measured in muscle cells. The factors affecting the flow of ions across the membrane are then, 1. Diffusion gradients, 2. Inwardly directed electric field, 3. Membrane permeability, 4. Active pumping of ions across the membrane. The membrane permeabilities are affected by the electric field across the membrane, so that the whole equation can be changed by altering the electric field. The effect can be compared to any sort of avalanche device (e.g. geiger counter, fluroescent light, etc.) where positive feedback occurs once a threshold is reached. In the case of the membrane, as the electric field lessens, the permeability to Na+ increases, causing a further decrease in the electric field, and then a further increase in the permeability to Na+, etc. to saturation. Hodgkin and Huxley used an equivalent electrical circuit that I'll attempt to draw to derive a series of differential equations to describe the dynamics of membrane depolarization.
outside o-----/\/\/-----------o------------/\/\/-----o | -------o---------o--------o-------| | | | | / / / | \var var\ \ | / / / --\ \ \ --| Na+ K+ | Cl-| | ----| --| | | |

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-------o----------o-------o-------o | o-----/\/\/-----------o------------/\/\/-----o inside

Where the variable resistors correspond to the change in permeability (or conductance) of Na+ and K+, the batteries correspond to the effect of the pumps (and for Cl-, the effect of the electric field), the Cl- resistor is the static Cl- conductance, and the capacitor represents the membrane charge separation. You can see the equations in J. Physiol 1952 117:500. If you stick an electrode into the cell at a certain point (usually, you use very fine glass needles with a salt solution inside), you can start to pull the transmembrane potential down. When you reach a certain point (about 20 mV more positive), the process runs away so that the membrane potential goes up to about +20 mV (the potential would go to about +60 mV except the increase in Na+ conductance is time dependent and shuts down fairly quickly). The changing potential at this point will induce the same depolarization at the points on either side of the site, and the depolarization sweeps outward along the membrane in a wave. If the cell is a long cylinder (like a nerve cell), then the wave of depolarization will travel along this cylinder until it gets to the end of the cell. The effect of positive polarization of the membrane is to open up the K+ conductance, thereby allowing the membrane to become repolarized (it actually overshoots). This is followed by a refractory period where the Na+ and K+ conductances cannot be induced to change (this is to allow the pumps to re-establish the concentration gradients, otherwise the cell would soon be completely exhausted). The whole event is called an action potential. Now, the question remains as to how these cells become depolarized (locally) in the first place. For the transducer cells, it's caused by the physical coupling to whatever it is they measure. The cochlear cells become depolarized when the hair-like projections that extend into the fluid of the inner ear are bent over by motion of the fluid. Retinal cells become depolarized when they absorb light. Nerve cells are depolarized by chemical means. A certain chemical messenger binds to a receptor and the resulting complex acts to increase the conductance of Na+, initiating an action potential. This is also how nerve cells pass on the electrical signal from the end of one cell to the next cell; the depolarization, when it reaches the end of a nerve cell, causes vesicles filled with neurotransmitters (chemical messengers) to fuse with the plasma membrane, thereby releasing their contents to the extracellular space. The neurotransmitters then diffuse across the gap and induce an action potential when they reach the neighboring nerve cell. Nerve cells can branch and terminate on more than one other nerve cell and they can also have 'connections' with many other nerve cells for incoming signals. In muscle cells, the action potential causes the release of Ca++ ions within the muscle fiber. This increase in [Ca++] causes the biochemical events that drive muscle contraction. more later...

Colligative Properties of Life

We may ask what is the point of all this communication? Why should living things evolve such elaborate means of developing order and structure between living units? Is there some force that leads to an inevitable increase in complexity (not at the expense of simple life forms, but in addition to them)? Perhaps these questions are best approached by considering the collective, or colligative, properties exhibited by living things. Colligative properties are those emergent phenomena that are exhibited by an assembly but not the individual components of a system. For example, a solution has a freezing point, below which a liquid to solid phase transition occurs. A water molecule, by itself, has no such property nor does it undergo a phase transition. Although it must be possible to predict the collective behavior from the properties of the individual components, in practice, this is five times harder than impossible.

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One of the most striking colligative properties exhibited by living things is the tendency for homeostasis--the maintenance of some environmental conditions close to a steady state (and often this state is far from equilibrium). At all scales and with all living organisms, organs, tissues, and cells, we see examples of cooperative efforts to maintain homeostasis. [home] [previous] [next] Document last updated Jan. 28, 1999. Copyright 1997, Ken Muldrew.

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