SEX AND DEATHE Pluribus Unum
When looking at my face in the mirror, i sometimes wonder what has lived within me all the numbered years of my life. While the surface features of my face and body change imperceptibly day by day, over the decades a remarkable change in appearance is obvious. Is the me at age seven the same as the me at seventeen? Or the same as me now? As i puzzle over who or what has maintained the continuity between younger and elder face, my wonder is enhanced by a thought delivered by the esteemed biologist Albert Claude in his 1974 address upon receiving a share of the Nobel Prize for Physiology or Medicine.
After a lifetime of work, Claude was honored for his achievement in cell physiology-- fathoming the mystery of respiratory pigments in eukaryotic cells and localizing their presence to the mitochondrion. In his address, Claude presented a personal view, emphasizing the awe and wonder he experienced during the mid-twentieth century as knowledge of cell biology slowly accumulated through the hard work of many investigators. In his seniority, Claude was not shy about expressing his respect for the living cell:
"Man, like other organisms, is so perfectly coordinated that he may easily forget, whether awake or asleep, that he is a colony of cells in action, and that it is the cells which achieve, through him, what he has the illusion of accomplishing himself. It is the cells which create and maintain in us, during the span of our lives, our will to live and survive, to search and experiment, and to struggle." [1]
Indeed, each of us is a magnificent manifestation of an integrated colony of cells-- trillions of them! Every cell in one's own colony is descended from a single cell, initially an ovum within the mother colony until fertilized by a single-celled sperm from the father's colony, then nourished within the mother's tissue. As the single fertile cell and its immediate descendants divide repeatedly (2, 4, 8, 16, 32, 64...), they eventually produce a mature colony capably of surviving in its own right. From a single cell, i am a colony of cells.
The mammalian ovum had itself descended from an ancestral series of fertilized ova over hundreds of millions of years as the vertebrate lineage evolved. Moreover, the tens or hundreds of mitochondria inhabiting each of the trillions of cells in any one human's colony are directly descended from the ancestral mitochondria carried forward by each ovum in that unbroken chain of being unique to one's own person. The living lineage reaches back to the time before integrated cell colonies had evolved, back to the solitary ancestral cell, the first eukaryote with a nucleus and organelles, the first complex cell which developed a symbiotic relationship with a bacterium-- the primitive mitochondrion-- which thrived within the eukaryote's protective membrane.
Although the mitochondrion was identified as a cellular "organelle" under the microscope as early as 1894, no one knew what this organelle did or what it was until the work of Claude and many others demonstrated that mitochondria produce 94% of the energy used by the eukaryotic cell which hosts them. We could hardly raise a finger or think a thought without the empowerment of mitochondrial energy. Our cell colony walks and talks with the motive force of mitochondria.
Early on, Claude surmised that the mitochondrion is like a bacterium, with its own DNA and the obligate ability to reproduce through binary fission. Mitochondria are multiplying within our cells, hour by hour. Each cell in our body contains from one to many hundreds of mitochondria, depending upon its size and energy needs. Our liver cells, for example, each typically contain from 1000-2000 hardworking mitochondria. While other investigators had only suggested a symbiotic role for this energy-producing creature, in the late 1960s and early 1970s a biologist at the University of Massachusetts in Amherst, Lynn Margulis, argued the case conclusively to demonstrate that the mitochondrion-- as well as its cousin, the plant-empowering chloroplast (a.k.a. plastid)-- is indeed an endosymbiont descended from a bacterium. (The term 'endosymbiosis' names the relationship in which two organisms, one living within another, contribute significantly to each other's metabolic process.) Margulis argued that the ancestral bacterium, the proto-mitochondrion, originally insinuated itself into (although others suppose it initially was engulfed by) our ancestral unicellular eukaryote. There it stayed, according to hypothesis, to generate a lineage within our lineage. We'll never know how it actually happened but it did-- some 1.5 billion years ago.
From Fission to Meiosis
The ancestral mitochondrion, like all cells in the beginning of evolutionary history, was initially a simple prokaryote. In other words, it was a tiny bacterium composed of a single circular chromosome of naked DNA producing a few dozen types of protein to enact its metabolism within a sheltering membrane. This particular prokaryote, however, eventually evolved symbiotically within its evolving eukaryotic host cell to perform a highly specialized function-- energy production. Briefly put, the host cell breaks down carbohydrates (e.g. sugar) to feed electron pairs and small carbohydrate fragments into the mitochondrion. The endosymbiont then converts the fragments into additional electron pairs, harnesses all these electrons via enzymes and respiratory pigments to pump protons across a membrane, and finally channels the protons through wee rotary motors (ATP Synthases) which transform the energy into small molecular packets (of ATP) to serve as the power source for cellular activity. [2]
In greek, karyos denotes a kernal or nucleus. Simple cells without a nucleus are called prokaryotes. A larger, more complex cell with a nucleus is called a eukaryote. Prokaryotes keep their DNA (a string of genes, the genome) in a large loop, spread akimbo throughout their cell body. Such a simple genome needs no nuclear seculsion and is continually transcribed and translated into proteins-- many of which work enzymatically-- amidst the hustle-bustle of molecular activity in the cytoplasm. Prokaryotes are commonly known as bacteria; they have evolved great variety in lifestyle and habitat. Unless they incur starvation, predation, or accidental death, prokaryotes never age and are immortal. A live prokaryote simply grows when nutrients are available until it doubles in size, then replicates its DNA and splits in two by fission while ensuring that each daughter cell has a copy of the genome as well as a complement of proteins to facilitate genomic metabolism within a snug membane. Whatever was alive in the parent cell is still alive as it splits into two daughter cells. The bacterium alive atop the skin behind your ear or in your kitchen sink is a living fraction of its ancestor, which in turn has kept something of its ancestor alive since time immemorial.
Like bacteria, the many mitochondria alive in each of your body's cells are likewise an extension of an ancient lifeform. The few carried in the single-celled ovum from which your colony arose divided by fission and were apportioned to each daughter cell produced by mitosis from that ovum, and so on to populate the trillions of cells now in your body. Aside from starvation or severe stress, descendants from those initial mitochondria only die when the cell they inhabit within your body dies-- yet their sisters live within the remaining cells of your colony. Ultimately, a mitochondrial lineage must die with its multicellular eukaryotic host-- i.e., when your colony dies--, unless passed on through a gamete to the host's offspring. In mammals, this transference of mitochondria to the next generation only happens through the ovum. That is to say, our human cells have inherited their initial mitochondria only from our mother.
While wild bacteria may live forever in their fissioned fashion, aging and death seem only to have arisen at some time after a solitary eukaryote evolved from a lineage of prokaryotes. From a long line of cells without a nucleus, a descendant arose with a nucleus-- the first eukaryote. As the initial lineage of eukaryotes generated a larger cell body and a larger genome enclosed by a sheltering nuclear membrane, then chanced upon proteins (via duplication and mutation of genes for proteins dedicated to related tasks) able to organize its genome into two or more chromosomes (separate packages of DNA), its nucleus grew in size and complexity. Likewise, as the lineage evolved, the cell organized by that nucleus grew in size and complexity, chanced upon a relationship with symbiotic mitochondria, and thus empowered was poised to make evolutionary history. The eukaryote concomitantly developed a method for genetic recombination that required a mode of reproduction more complex than simple fission.
Recombination of genetic elements was not an innovation of eukaryotes: it was common before eukaryotes evolved. Early in evolutionary history, prokaryotes developed ways to exchange bits of their genome with other prokaryotic lineages, at random and occasionally. One prokaryotic cell every now and then contributed a copy of some portion of its DNA to another. The random genetic recombination of prokaryotes was sufficient, along with the occasional mutation intrinsic to every genome, to generate varied phenotypes (variants with unique traits and behaviors) which environmental conditions tested. Some died without heir, some reproduced. Gradually, successfully reproducing prokaryotic species evolved to fill most every ecological niche available to creatures of their miniscule size. They alone evolved to fill the world with life, albeit a tiny form of life, for the first several billion years of evolutionary history and their ancestors persist today in magnificent abundance. Yet if genetic recombination is the mainspring of evolution and if we imagine the genome as a deck of cards, prokaryotes are only able to draw one card on rare occasion from the deck of another cell's lineage.
Eukaryotes chanced upon a method to exchange many cards. Sometime after the second billionth year of evolutionary history, a unicellular eukaryotic lineage slowly accumulated genetic material in multiple chromosomes and the means for mitotic cell division, enabling a parent cell to perfectly replicate and partition its chromosomes-- to insure that each daughter is given an identical set of chromosomes. Aligned with that ability, the lineage also accumulated the wherewithal for any individual to mate with a member of its species. Moreover, additional mutations in a subsequent lineage enabled a modification of the mitotic process so that, temporarily and prior to mating, the replicated genome was randomly shuffled before being divided into half by a double round of modified mitosis-- meiosis-- and subsequently exchanged with a mate. As a result, mating conjugation routinely exchanged half the deck after shuffling the cards to maximize randomization before each exchange.
During the third billion years of life's history on earth, an amazing variety of single-celled eukaryotes evolved from this magnificent randomness as the ever-changing environment fueled the adaptable lineages and failed the inadept. At least one path (or perhaps several paths, some of which became dead-ends) of this meiotic eukaryotic environmental exploration led beyond single-celled organisms. Somehow, a eukaryote evolved the means to bind with its sister after mitosis and this opened the way for two to become four, four to become eight, and so on. Gene by gene, the lineage slowly evolved multicellular creatures-- integrated colonies-- some 700 million years ago, 2800 million years after prokaryotes initiated life and 1000 million years after solitary eukaryotes began experimenting.
As we witness the results today, the multicellular eukaryotic lineage founded three great kingdoms-- fungus, plant, and animal. Each kingdom includes some members which reproduce asexually, by fission or cloning. Yet in most multicellular lineages, individual creatures thrive as colonies which designate certain of their cells meiotic to produce gametes for sexual reproduction and all the rest of the cells within the colony are mortal. Even the gametes, by and large, prove mortal-- except for the female gamete (e.g., an ovum) when fertilized. Certain unicellular eukaryotes, on the other hand, retain an ability to perpetuate without requisite mortality. Such unicellular creatures, for example the protozoa discussed below, stand at the evolutionary border between immortality and death as we know it.
The transition from simple fission's immortality to the complexity of mitotic cell division and sexual recombination required many small steps, each a fresh accident of genetic mutation. Each random mutation only worked in its first generation and in every subsequent generation insofor as it was amenable to and an enhancement of the genetic expression already developed by the lineage, accumulated and alive in a single cell. Solitary eukaryotes living today express genes acquired early in their lineage-- as do we multicellular creatures-- and some manifest a lifestyle very much like that of their most primitive ancestors. We may study them for insight into the transition from prokaryotic fission to eukaryotic mitosis and meiosis or, more poignantly, the transition from perpetual life to the practicalities of death.
Procreation Breeds Mortality
The fancy term for growing old and feeble is senescence. In search of clues about the evolutionary origin of senescence prior to the rise of multicellular creatures, William R. Clark-- in A MEANS TO AN END: The Biological Basis of Aging and Death (Oxford University Press, 1999)-- considered the role of sex in one of its primitive expressions, a protozoan. Protozoa live as single-celled eukaryotes, various species of which have evolved different methods for sexual reproduction. Some species of protozoa live with a haploid genome (n chromosomes, where n may equal any small number), others with a diploid genome (2n chromosomes), and all manifest a haploid genome at some point during their lifecycle. For example, the ciliated protozoan Tetrahymena always carries 10 chromosomes in its diploid genome but meiotically segregates them into haploids, each with 5 chromosomes, prior to sexual conjugation. The diploid genome-- composed of a haploid from "mother" and a haploid from "father"-- contains two representatives of each chromosome (for the Tetrahymena, 2 x 5) with homologous but not necessarily identical genes. The working diploid set thus includes two representatives (alleles) of every gene vital to the species in case one allele, through mutation or environmental change, doesn't work so well as the other. Or more optimistically, in case one works to do more than ever before.
Like other scientists concerned with death and aging, Clark focused upon a protozoan (in his case, the Paramecium) which manages its diploidy in a somewhat-- nowadays, compared to multicelluar creatures-- unusual fashion. A rather large unicellular eukaryote, the ciliated paramecium offers a clue into the mystery of death by the way in which it employs its diploid genome. Like certain other protozoa including Tetrahymena, the paramecium requires two separate nuclei-- a micronucleus and a macronucleus. The micronucleus of the paramecium carries the cell's diploid set of chromosomes in quiet seclusion within the cell body. The DNA in its macronucleus is derived from the DNA in its micronucleus and, once created at a location within the cell body distinct from the micronucleus, the macronuclear DNA does all the nuclear work of the cell. In other words, the macronucleus manifests all the active genes which generate all the working proteins that give the organism its life.
The paramecium cell-cycle promotes its reproduction through either of two methods, asexual fission or sexual conjugation. When on the path to fission, the parent cell simply replicates the DNA in both micronucleus and macronucleus then partitions itself so that an equivalent set of nuclei is provided to both sides as the cell divides. Prior to division, the micronucleus is partitioned mitotically while the macronucleus is simply elongated and fissioned after its DNA has been replicated and randomly distributed throughout the macronucleus. Each of the two daughter cells resulting from the parent cell's division then uses its two nuclei in the same fashion as the parent did; the macronucleus does the work while the micronucleus sits idle. From an evolutionary perspective, only the genes at work in the macronucleus are tested by the environment and the genome-- indeed, the cell itself-- only survives as these macronuclear genes prove efficient. Many generations of fissioned paramecia may thrive through their replica of the ancestral macronucleus (i.e., a temporarily 'immortal' lineage) until, at last, a descendant paramecium 'decides' it is ready for sex.
In this image, two mating Tetrahymena thermophila cells have just completed their second meiosis. Their macronulei show as large bluish blobs and the smaller blobs are pronuclei.
(Image courtesy of Kathleen Stuart and Eric Cole)
http://www.uga.edu/protozoa/
When the sexually ready paramecium meets a likely mate, a portion of each cell-body becomes temporarily fused with its mate along a special perforated junction. Then each paremecium prepares itself for an exchange of genes by shuffling its own deck. In effect, the diploid micronucleus of each paramecium undergoes meiosis to generate four haploid pronuclei, each containing a randomized set of alleles. Within each paramecium, one pronucleus is randomly selected for transmission and the other three are disassembled for recycling; the remaining pronucleus then undergoes mitosis to generate two identical haploid pronuclei-- one to keep and one to trade with the conjugal partner. Finally, each paramecium exchanges a haploid pronucleus across the perforated junction with its mate and, after a short period of post-coital bliss, the genetically refreshed creatures separate to pursue their own evolutionary destinies.
The replacement macronucleus is formed as the fresh diploid pronucleus undergoes two mitotic divisions to produce four pronuclei, each an exact diploid replica of the other. Two of these pronuclei fuse to become the new macronucleus, one becomes the new micronucleus, and the fourth is simply broken down for recycling. All of these operations are directed from the old macronucleus which, as the new macronucleus is completed, also generates a set of 'suicide' enzymes (working proteins) which immediately go to work deconstructing the original macronucleus. In other words, the still living old macronucleus destroys itself to make way for the new.
The paramecium's set of suicide enzymes includes a signaling system to coordinate their activity which centers around an endonuclease, an enzyme inherited from prokaryotic ancestors which breaks down DNA. [3] There are several related endonucleases at work in a cell (e.g., during the recycling events mentioned above) but, by virtue of its ultimate role, this one is called the DNase. Its task is to chop the old macronuclear DNA into fragments while the DNA is still active, alive. Other suicide enzymes work to condense the old (and chopped up) chromosome fragments in preparation for recycling. As this process accelerates, a signal is sent to the new macronucleus which then begins to generate its own enzymes, some of which go to work digesting the old macronucleus and recycling its component parts. Soon thereafter, the old macronucleus is resorbed and the paramecium resumes its normal lifestyle with a fresh set of genes in its new macronucleus to test against the vicissitudes of protozoan life.
As far as we can tell, this protozoan style of quasi-suicide is necessitated by its unusual reproductive style; and, its DNase evolved to serve that purpose. This paramecium's process appears to be the oldest form of autonomous cell 'death' in the eukaryotic world, although there were hints of it in the more ancient prokaryotic realm.
We Die to Live
As the protozoan example suggests, autonomous cell 'death' has a regulated role to play in life's most basic process and cell biologists have had their eyes on its mysterious ways for quite some time. The possible role of regulated cell death in organismal development was initially surmised from fixed preparations of frog embryo neural tissue under the microscope in 1842. Although likely manifestations of regulated cell death were frequently observed with some interest over the following century, efforts to elucidate the nature of the beast only gained steam after the 1950s as more sophisticated investigative tools were developed. Biologists studying multicellular creatures in the mid-twentieth century obtained increasing evidence that any complex embryonic development is only possible through genetically programmed cell death. Over the following decades, they studied many interesting cases and began itemizing the role of regulated cell death in life's process. [4]
The embryo of our own species, for example, can only grow a hand through an elongated mass of tissue composed of sister cells which differentiate into fingers only as interdigital cells commit suicide. As another example, vertebrate nervous systems grow interconnected by sending out large numbers of neurons growing more or less blindly toward contact with any cells (e.g., other neurons or cells in muscles, skin, and organs) in need of innervation. Cells which require innervation send out small signal molecules called neurotrophins, e.g., nerve growth factor. Neurons are born to commit suicide within a specified timeframe if they fail to receive sufficient neurotrophins-- survival signals-- to override their own internal suicide program. As one consequence of this simple mode of development for neural connectivity, human adolescence is shadowed by the death of cortical neurons by the millions, so prolific is childhood in the generation of neurons looking for a purpose in the brain.
Beyond its morphogenic role in growth and development, regulated cell death is also a vital tool for the maintenance of health and cell-system integrity in mature multicellular organisms. The trio of investigators (John Kerr, Andrew Wyllie, and A.R. Currie) who generalized the idea of programmed cell death, according to a review article, "coined the term 'apoptosis' to focus attention on the yin-yang relationship of death to birth (that is, homeostasis is not maintained [in a mature organism] unless the loss of cells equals the birth of cells). The three argued that the ritualistic nature of cell death implied an organized and conserved mechanism: cell death or apoptosis was an aspect of life like any other. In other words, cell death was as much a part of cell biology as mitosis, extension of an axon, the enzymatic sequence of glycolysis, or secretion." [5]
The authors of a recent review article on the subject offered this prelude: "In a [mature] human about a hundred thousand cells are produced every second by mitosis, and a similar number dies by a physiological suicide process known as apoptosis. Most of the cells produced during mammalian embryonic development undergo physiological cell death before the end of the perinatal period. During our life span, over 99.9% of our cells undergo the same fate." [6]
The most vivid examples of apoptosis are manifest via the vertebrate immune system. As large and complex as the nervous system, the mammalian immune system depends upon a variety of cells numbering in the billions which often operate in a solitary fashion, many of which travel amoeba-like throughout the body in search of trouble, then intercommunicate and cooperate to eliminate the trouble when they find it. Some types of immune cell often reside within or regularly visit lymph tissue and respond to distant signals of inflammation like so many firefighters and emergency medical technicians. B-cells and T-cells are the most prominent, each in its own way keeping track of antigens-- any molecular structure foreign to the cell colony. The colony is, of course, the commonwealth-- the body politic in need of defense.
Whether the antigen source is an invading parasite, bacterium, virus, or mutant protein produced by a native member of the colony, the health of the whole depends upon antigen recognition by native B- and T-cells. These cells only recognize antigens through an evolved mechanism which harnesses chance. Each B- or T-cell is born from an immune system stem cell via mitosis and cell division. Soon after its 'birth', the B- or T-cell manifests multiple copies of a unique antigen receptor on its cell surface. Each receptor is formed by a specific protein expressed from a gene assembled through a carefully orchestrated randomization process. In effect, the immature B/T cell randomizes that particular part of its genome which specifies the shape of its antigen receptor protein. That is to say, the immature cell shuffles its own deck! Once its antigen-receptor genes are shuffled, that cell's expressed receptor gene is unique and fixed in that cell's lineage, should it live and divide to produce exact clones of itself.
Once the unique gene is expressed through antigen-receptors on the cell's surface, the receptor may or may not lock onto a molecule presented within its colonial environment which fits its unique shape.
If the antigen receptor of an immature B/T cell locks onto a molecule normally expressed by any other cell which is a member of the colony or if it fails to lock onto any molecule at all, within a short time after its birth the immature B/T cell undergoes apoptosis without heir. But if the immature B/T cell's receptor locks onto any other molecule-- which is presumed foreign by default--- then that B- or T-cell matures and proliferates, generating many clones of itself through mitosis. In other words, the immune system's B/T cell clonal selection process is a microcosm of Darwinian evolution-- only the successful reproduce. Most of the first generation clones of a successful B/T cell become immediately active and circulate as their receptors search for their uniquely specified foreign antigens throughout the greater colony. However, some of these clones stand aside as idle 'memory cells' on guard for the next invasion of antigens unique to their receptors and, if subsequently activated, they will clone themselves prolifically to generate regiments of identical cells for defense. Stem cells in a healthy immune system continually produce immature B- and T-cells to provide the colony with a never-ending array of novel antigen receptors which are, in their turn, tested against exigent circumstance.
Mature B-cells secrete small antibodies identical to their parent cell's receptors; these antibodies float independently through the bloodstream and tissue fluids to entrap antigen (foreign molecular structures). Upon finding an antigen, an antibody signals scavenger cells in the immune system to attach and degrade the foreign structure. If the antigen is present upon a bacterium or parasite, the antibody signals the attack-dog cells of the immune system-- the neutrophil, eosinophil, and macrophage. These scavanger cells may also find and attack invaders without antibody signaling. The macrophage is so voracious and amoeba-like that, with a few small genetic changes, it could probably thrive all by itself in the wilds of garden or pond grazing upon bacteria and protozoa. That is to say, the macrophage is a vestige of our most ancient unicellular heritage. Tens of thousands crawl about within us, serving our commonwealth by eating invaders.
Mature T-cells travel throughout the colony on search-and-destroy missions. Unlike the phages, T-cells monitor only citizens of the colony. They directly attach to native cells which present alien antigen on special proteins which the natives express on their surface. If the antigen source is a malfunctional (e.g. cancerous) cell presenting a fragment of mutant protein or an infected cell presenting a viral fragment, the T-cell is empowered to induce the errant or disabled citizen's apoptosis, i.e., to trigger the citizen's suicide program. In its deadly role as guardian, the T-cell is often assisted by its cousin, the NK (natural killer) cell, which frequently works autonomously and does not necessarily wait for apoptosis but rather can murder the errant cell outright.
Beyond these particular examples of apoptosis, each and every cell in the colony contains a separate internal pathway which monitors its own cell-cycle and triggers apoptosis when critical variables run amuck. Aside from neural cells, citizen cells in the colony are perpetually progressing from 'birth' through maturation through mitosis and another round of cell division. Each step in this cycle is internally monitored at critical checkpoints. The most crucial checkpoint is prior to cell division, after the nucleus generates a replicate copy of its DNA but prior to the initiation of mitosis. If the DNA is miscopied, broken, or scrambled beyond repair, the cell-cycle is halted and the pathway for apoptosis is initiated internally-- by the troubled cell itself. Cancer arises if malignant cells, through mutation, have aborted this pathway to escape apoptosis, and proliferate with mutated DNA. They become, in effect, immortal (at least, until the host body dies)! Unless the immune system can kill them, malignant cells establish aberrant rebel colonies and/or traveling terrorists antagonistic to their former siblings. Such unauthorized cells may destroy the entire legitimate commonwealth by absorbing nutrient resources and severely disrupting normal commerce.
Pandora's Box
In recent years, cell biologists have focused upon apoptosis at the molecular level and isolated several of the apoptotic molecular pathways common to a variety of eukaryotic species. Much investigative work remains to done but, for the sake of our story, it should be noted that most, if not all, apoptotic pathways seem to center upon the humble mitochondrion. Early investigations of cell death found evidence that, under stress, a mitochondrion will release one of its respiratory pigments-- cytochrome C-- in sufficient quantity to disrupt the host cytoplasm and cause cell death. Although Albert Claude might not have imagined such a powerful role for one of his respiratory pigments, the release of cytochrome C into the eukaryotic cytoplasm is now known to be crucial for the initiation of a cascade of signals which, if unchecked, leads to apoptosis. In a minireview of the mitochondrial role in cell death, the authors suggest that "it is intellectually appealing to speculate that the mitochondrial control of cell death is an evolutionary relict of endosymbiosis and that the sophisticated control of mammalian cell death has been built up on a primitive program in which mitochondrial membrane permeabilization kills the cell." [7]
It is not hard to imagine a primitive cell of eukaryotic lineage, healthy except for an endosymbiont mitochondrion which was ailing to the extent that its membrane sprung a few leaks. Respiratory pigments like cytochrome C traffic in electrons and, when run amuck in the host cytoplasm, severely disrupt orderly metabolic processes. Moreover, like a leaky car battery, the mitochondrial power plant contains acidic substances (in the form of naked protons), among other things, which can wreak havoc on exposed flesh. The primitive eukaryotic lineage which evolved proteins to contain such damage might thrive; and, the multicellular lineage from that root which further evolved proteins to programmatically control or release such deadly power might thrive even more.
While cytochrome C was the first mitochondrial molecule noted for its role in apoptosis, the list of potent molecules grows as investigation of the mitochondrial role continues. Much of the work is focused upon the permeability transition pore complex (PTPC), an ensemble of proteins which forms a pore in the mitochondrial membrane. Normally closed when the mitochondrion is healthy, these pores can be manipulated by gate-keeping proteins generated by the host cell. Known as the Bcl-2 family of proteins [see note 6], both pro- and anti-apoptotic members have been characterized as gate-keepers. By controlling the PTPC with these gate-keepers, the host cell can determine when its own death should occur. While only a few have been fully characterized, it seems that more than fifty different mitochondrial proteins are released when the PTPC is opened. "In a way, the mitochondrion thus may be conceived as a Pandora's box from which catabolic enzymes and their activators [are] released upon apoptosis induction." [8]
The most potent type of catabolic enzyme is known as a cysteine-aspartate protease or, in the short-form of the name, a caspase. [9] When on the prowl, these enzymes cleave targeted proteins which possess an amino acid sequence that terminates with a cysteine followed by aspartate. A gene family of related caspases is constitutively expressed in a healthy cell and its members are named simply in order of their discovery as caspase-1, caspase-2, and so on through caspase-13. They are expressed as procaspases which become active when cleaved by another caspase, except in the case of procaspase-9 which becomes inactivated by cleavage. [10] Procaspases 2, 3, and 9 are installed within the mitochondrial membrane and released through the PTPC. [11] Once released, procaspase-9 forms a multiprotein complex known as the apoptosome with cytochrome C and an adaptor protein (Apaf-1). This complex recruits procaspase-3, then cleaves it to initiate a cascade of caspase activity which enacts apoptosis. [12]
Caspase-3 triggers downstream procaspases by cleavage and these, as activated caspases (e.g. caspase-6), begin chopping key proteins throughout the cell. While the count rises as research continues, so far over 100 types of protein are known as caspase targets, each of which otherwise plays a role in keeping the cell alive. Proteins targeted by caspases include those vital to the cytoskeleton, nuclear membrane integrity, and protein activation, as well as DNA replication and transcription. The critical caspase in this cascade activates a latent form of DNase, so the latter enzyme is named the caspase activated DNase or CAD. [13] Once caspase orchestrated apoptosis gets underway, the deconstructed cell is easily ingested and resorbed by immune system phages.
As mentioned previously, a cell's apoptosis may be initiated externally by morphogenic or immune system signals, as well as autonomously. We'll consider the mitochondrial connection for each in turn.
The apoptotic pathway active in morphogenesis is mediated by molecular signals generically known by two names-- growth factors (e.g. epidermal growth factor) or trophins (e.g. neurotrophin). Cells in developing tissue send out trophins to establish concentration gradients, the intensity of which varies depending upon the cell's position within the tissue and stage of development. Growing and dividing cells express cell-surface receptors keyed to specific trophins. Receptors which receive an adequate supply of their specified trophin transmit the signal internally to activate a protein (kinase) which tags an effector protein with a small molecule (usually, a phosphoryl group from an ATP), causing the effector's reconformation and thus moderating its function in the apoptotic pathway. For example, in some cell types subject to trophic regulation, a protein known as Bad (Bcl-2 associated protein) holds the key to survival. Unless the cell receives sufficient trophin to prompt continual phosphorylation of Bad, molecules of unphosphorylated Bad will interact with Bcl-2 proteins in the PTPC and trigger the caspase cascade to initiate apoptosis. [14] But the caspase cascade is only part of the story. For example, a caspase independent molecule known as AIF (apoptosis-inducing factor) has recently been characterized and localized to the mitochondrial intermembrane. While a likely role for AIF has been demonstrated in morphogenesis, its trophic regulation and downstream mechanism await further investigation. [15]
The immune system exercises its apoptotic authority over citizen cells of the body politic with a signaling molecule known as FasL. Healthy citizens express multiple copies of a cell-surface receptor called Fas which spans their membrane and presents a domain within the cytoplasm able to latch onto procaspase-8. If at least three of these Fas receptors simultaneously receive a FasL molecule from a T-cell (activated to send this signal when its antigen receptor locks onto a foreign molecule presented by the citizen cell), the Fas receptors trimerize such that their cytoplasmic domains cooperate to recruit multiple molecules of procaspase-8. Brought into close contact, these procaspases cleave each other to become activated, then circulate to initiate an apoptotic cascade along one of two pathways. Caspase-8 can activate caspase-3 which then proceeds to activate downstream events or, caspase-8 can cleave Bid, a proapoptotic member of the Bcl-2 family which then opens the PTPC floodgate for the complete mitochondrial cascade. [16]
Autonomous apoptosis is generated internally in response to a signal generated at a cell-cycle checkpoint-- for example, when the cell's DNA is damaged or its replication is fouled-- which leads to an assessment of the problem and a decision. There are a number of checkpoint proteins (e.g. Chk2), most of which signal their discovery of trouble by activating a transcription factor named p53. (The 'p' is for protein and the number represents its molecular weight of 53 Kilodaltons.) A modest quantity of p53 is normally present in the cytoplasm of a healthy cell but remains inactive until signaled by checkpoint proteins which monitor DNA integrity. Once activated, p53 is in turn able to enter the nucleus and activate genes for proteins which temporarily halt the cell-cycle (e.g. p21) and attempt emergency repair (e.g. p53R2) or, if all else fails, activate genes for proteins which trigger the caspase cascade. Several putative p53 targets are under investigation (e.g. Bax, PIG, p53AIP1) and the most potent, according to the initial evidence, is named the p53 upregulated modulator of apoptosis (PUMA). A close cousin to the Bcl-2 family of proteins, the PUMA gene is subject to p53 activation and, after generation of the gene into protein, PUMA apparently translocates directly to mitochondria. Once there, PUMA interacts with Bcl-2 proteins on the PTPC to trigger the complete caspase cascade. [17]
Molecular biologists have recently discovered several homologues of p53, namely p63 and p73, which like p53 are known primarily because they are disabled by mutation in cells which become cancerous. Once these initiators of the autonomous apoptotic pathway are malformed through mutation to become inoperative, a cancer cell is on its way toward immortality-- at the expense of the greater body politic-- unless the immune system's cells can destroy it one way or another. [18]
Sex and Death
Until the details are more fully known, we may suppose that not all apoptosis is mediated through mitochondria. But most is and we must pay homage to our precious endosymbionts for the control they enable in our multicellular development as well as the energy they provide us, second by second. We may further suppose that, while not all apoptosis is caspase dependent, most apoptotic pathways culminate with the activation of a DNase. Although the gene sequences for the various DNases manifest in eukaryotic species are not yet mapped out, it may be the case that all are at least functionally homologous and perhaps many are even genetically homologous. In other words, there is some reason to suspect that our DNase is like the more ancient form which may have evolved to solve an early problem in sexual recombination. Although the evolutionary progression from solitary eukaryotes to multi-cellular bodies is hardly understood, it is probably the case that nothing complex could have evolved without both sex and death as hand-maidens.
The initiation of sexual conjugation may always and forever require a bit of luck. Shuffling the deck of chromosomes and exchanging haploid genomes is chancey. Death, however, follows a schedule more strict. We humans pray for appropriate apoptosis during embryogenesis-- when a fetus is under development-- and we pray again that our adolescent children lose their lame neurons while retaining those better connected. Ultimately, we pray when one of the cells in our own mature colony goes haywire from viral infection or appears cancerous. Let it die, that 'I' shall live. Yet if too many cells within a vital organ grow so feeble with old age that they fail their cell-cycle checkpoints, their apoptosis is mourned by the whole colony. If only we were prokaryotes, we might live forever.
_________________________________________________________
FOOTNOTES
NR:MCB = Nature Reviews: Molecular Cell Biology, Nature Publishing Group, London
[1] from Albert Claude's 1974 Nobel Address, reprinted in SCIENCE 189:433-5.
[2] For a glimpse into just one of the intricacies apparent in endosymbiosis, see Tsuneyoshi Kuroiwa, "The primitive red algae Cyanidium caldarium and Cyanidioschyzon merolae as model system for investigating the dividing apparatus of mitochondria and plastids." BioEssays 20:344-354 (John Wiley & Sons, Inc.)
For an overview of the mitochondrion proton motor, see Masauke Yoshida, et al., "ATP synthase-- a marvellous rotary engine of the cell." NR:MCB 2:669-677.
[3] Even a simple bacterium needs an endonuclease enzyme since, when ingesting all or part of another bacterium upon which it has preyed, the predator does not want to confuse its prey's DNA with its own. Moreover, every cell is constantly recycling its own RNA and broken bits of DNA. For more details of DNA breakdown in paramecia, see Solomon Mpoke and Jason Wolfe, "DNA Digestion and Chromatin Condensation during Nuclear Death in Tetrahymena." Exp. Cell Research 225:357-365 (1996)
For more details of paramecium conjugation, see Maria C. Davis, et al., "Programmed Nuclear Death: Apoptotic-like Degradation of Specific Nuclei in Conjugating Tetrahymena." Developmental Biology 154:419-432 (1992)
[4] Richard A. Lockshin and Zahra Zakeri, "Programmed cell death and apoptosis: origins of the theory." NR:MCB, 2:545-550, p546 (2001). (See also CELL 96:245.)
[5] Lockshin and Zakeri, Op. cit. p545.
[6] David Vaux and Stanley Korsmeyer, "Cell Death In Development." CELL 96:245-254 (1999), p245. It was Vaux who discovered in 1988 the first component of an apoptotic mechanism, a human protein named Bcl-2 (derived from investigations of B-cell lymphoma). It inhibits a caspase in the apoptosis pathway and, in 1992, he transfered the human bcl-2 gene into the genome of a nematode-- a microscopic 'worm' that normally lives wild in the soil. The human gene worked in the nematode to block apoptosis, thus demonstrating that the apoptotic process is highly conserved up and down the eukaryotic family tree. See text for further details.
[7] Markus Loeffler and Guido Kroemer, "The Mitochondrion in Cell Death Control: Certainties and incognita." Exp. Cell Research 256:19-26 (2000), p20.
[8] Loeffler and Kroemer, Op. cit. p23. (See figure in NR:MCB 2:69 for schema of pore!)
[9] The formal title is actually "cysteinyl-aspartate-cleaving protease." (NR:MCB 2:549) Both cysteine and aspartate are amino acids, small molecules employed as building blocks which are bonded together in the assembly of proteins. The protease is an enzyme which breaks apart a specific protein.
[10] Raluca Gagescu, "Shifting the fat." NR:MCB 2:234 (2001).
[11] Loeffler and Kroemer, Op. cit. p19.
[12] Martin Holcik and Robert G. Korneluk, "XIAP, the guardian angel." NR:MCB 2:550-556 (2001), see figures p551 and p555.
[13] Shigekazu Nagata, "Apoptotic DNA Fragmentation." Exp. Cell Research 256:12-18 (2000), p16. The proCAD is complexed with a chaperone, ICAD, which is cleaved by caspase-3. (Op. cit. p13.) The DNA sequences for mouse and human CAD are well-conserved but show no homology with other DNases! (Op. cit. p13.)
[14] Harvey Lodish, et al, *Molecular Cell Biology*. W.H. Freeman and Co. New York. Fourth edition (2000), p1048.
[15] Alison Mitchell, "Death shapes life." NR:MCB 2:322 (2001). Marcel Leist and Marga Jaattela, "Four Deaths and a Funeral: From Caspases to Alternative Mechanisms." NR:MCB 2:589-598 (2001), p594.
[16] Nagata, Op. cit. p12-13.
[17] For Chk2 see NR:MCB 2:877 (2001).
For Bax, PIG, and p53AIP1 see Katsutoshi Oda, et al., "p53AIP1, a Potential Mediator of p53-Dependent Apoptosis, and Its Regulation by Ser-46-Phosphorylated p53." Cell 102:849-862 (2000), p849.
For Bax/Bak see NR:MCB 2:6, 2:63, and 2:406 (2001).
For PUMA see NR:MCB 2:319 (2001).
[18] Annie Young and Frank McKeon, "p63 and p73: p53 mimics, menaces and more." NR:MCB 1:199-207 (2000).