In Search of the Protocell - The Work of Jack Szostak

The origin of life problem is perhaps the most important question to ever have been the focus of scientific scrutiny. The only other question that I think rates of similar importance is the origin of the universe. Both questions have special obstacles to overcome before any answers are within sight. 

The Miller-Urey experiment of the 50s was a lightningrod for research into this question, but the euphoria caused by the viewpoint that the answers were near quickly receded once the scope of the problem was realized and it was decades before the excitement was rekindled in the scientific world. It's no surprise that the search is a difficult one. Remember, researchers are trying to compress the millions of years that undoubtedly were required for nature to give life a kick start into the lifespan of humans. Couple this with only a limited knowledge of what the conditions were at the time life started except in the grossest terms with the possibility that trace elements may be essential for the synthesis of life greatly compounds the issue. Anyone thinking that if life arose through naturalistic processes means it should be both easy, and that a mere 50 years of research should have resulted in the creation of protolife really doesn't have a good grasp of the scope of the problem. 

The molecules of life in the prebiotic world were all over the place, and not just on this planet. We know this not only from the Miller-Urey experiment itself, though we now think were the conditions at the time were somewhat different (which does not change the conclusions drawn from that experiment, or from similar ones which simulated what we now think the conditions at the time were), but meteorites have been found with complex organic molecules which could have seeded a barren Earth with the raw materials for the synthesis of life. Amphiphilic molecules (molecules possessing both water-loving (hydrophilic) and water-hating (hydrophobic) regions) have been generated by a variety of means simulating conditions found naturally: ultraviolet radiation of ice particles in the vacuum of space and at hydrothermal vents. Such molecules would provide the first cell membranes. 

A plan for synthesizing life was put forward by Szostak in 2001. It is based on a heterotophic model (cell structure first) rather than on an autotrophic one (metabolism first). First, create a spontaneously-replicating membrane through which small molecules can diffuse but bar larger molecules synthesized from these precursors from escaping. Next, create a replicase - a molecule mediating polymerization of a second molecule - a template containing protogenetic information to be copied.  The template could be RNA complimentary in sequence to the replicase or an unfolded replicase. RNA molecules can be encapsulated in vesicles and the whole cell self-assemble. This compartmentation inevitably results in the replicase component being subject to variation and natural selection.

Under the right conditions, amphiphilic molecules in solution can form micelles, or vesicles. This is similar to what soap, another amphiphilic class of molecules, does. Soap molecules (in the correct range of concentrations) cling together to form balls with the water-loving heads facing outward. In the case of vesicles, the molecules stand tail-to-tail with their hydrophilic heads facing outward from both the inner and outer surfaces of the ball. 

These vesicles would provide microenvironments for retaining and protecting primitive oligonucleotides (short sequences of RNA or DNA, typically of less than 20 bases). It is unlikely that early cell membranes would be made up of the same types of molecules which make up those in modern cells: phospholipids. Membranes made up of phospholipids are far too efficient at keeping out negatively charged ribonucleotides. Modern cells have evolved specific transport proteins to take in nutrients, but the earliest cells would have had no such mechanism available to them. 

Rather, the earliest cells would have used less efficient amphiphilic molecules, such as fatty acids, through which small molecules like ribonucleotides (such as uridine monophosphate, which make up RNA) could pass accross by simple diffusion. One hypothesis for both vesicle formation and RNA synthesis is respectively the interaction of fatty acids and ribonucleotides with clays. There is a growing body of evidence that this is a viable mechanism by which both of these process could happen. The clay montmorillonite has long been known to be able to catalyze RNA from activated ribonucleotides, but it can also greatly increase the rate of formation of fatty acid vesicles. The clay has a positively charged surface which attracts and concentrates the negatively charged fatty acids and thus facilitates their formation. Fatty acid membranes are also permeable to magnesium, a divalent cation necessary in many biochemical reactions and itself increases membrane permeablilty to negatively charged ribonucleotides. 

The surprise is that vesicles created in the presence of montmorillonite will also incorporate clay particles! It was immediately obvious to Szostak that this provides not only a mechanism for vesicle formation, but a method of synthesizing RNA oligonucleotides from ribonucleotides which diffuse through the membrane. Oligonucleotides formed within the vesicle are unable to escape the interior and are trapped. (As an aside, it also provides an explanation as to why L- rather than D-amino acids are utilized in protein synthesis. D- and L-amino acids are non-superimposable versions of each other, rather like the mirror image of your hand is not superimposable on your physical hand. Amino acids synthesized in an isotropic medium would be an equal (racemic) mixture of both optical isomers. These optical isomers have exactly the same physical properties bar one - each rotates the plane of polarized light in opposite directions. However, catalysis by a surface breaks the symmetry and one optical isomer would be selected over the other. It just so happens that L-amino acids were the ones selected. For sugars like glucose, it is the D-optical isomer that is used in biochemical reactions.)

Not only will these vesicles form, they have been shown to be able to spontaneously grow and divide in a series of elegant experiments. It was found that if the high vesicle concentration decreased by slowly adding a dilute solution of fatty acids, the vesicles would actually grow rather than just form new micelles. Vesicle division can be accomplished by extruding them through a polycarbonate filter. This likely happens by elongating the micelles so that they are no longer spherical and resealing after being pinched-off. As confirmation of this, vesicles preloaded with fluorescent dye were run through a filter released the dye into the medium in amounts only slightly greater than what was predicted for this mechanism of division. Had complete membrane disruption and reformation of vesicles occurred, the entire contents of the micelles would have been dumped into the medium. Vesicle division thus strongly resembles cellular division via budding and their formation, growth and division require no complex machinery at all, only raw physical forces. This is consistent with our current hypotheses on how early cell membranes must have formed. It even supplies a means for the first genetic material to have been generated through ribonucleotide uptake and mineral-catalyzed oligonucleotide formation.

Now our good friend Darwin steps in. Vesicles under osmotic stress due to their encapsulated contents need to decrease osmotic pressure by increasing their volume (and hence their surface area) by capturing fatty acids. Either that, or explode, dumping their contents. They do this by stealing fatty acids from other vesicles. But this is not a random process. The encapsulated contents have something to say about how well a vesicle will relieve the stress. Thus, we have what may have been the first example of biological competition! In the paper which covers this research (Chen, 2004), however, the competition was purely for stealing fatty acids from isotonic micelles (that is, vesicles not under osmotic stress). In other words, they feed. Once a truly replicating protocell is synthesized, a goal not yet reached, natural selection will become paramount in importance. The replicase can easily mutate through random mutation (since there are no error correcting mechanisms yet) and those which replicate better than others, eat other vesicles more efficiently and, as a consequence, divide more often will become more prevalent. Sounds like evolution to me.

So, when vesicles divide, how can the genetic material split into two as well? This is a Holy Grail in abiogenesis research. Some RNA can act like enzymes (another tantalizing clue to the origin of bioactive molecules). Such RNA molecules are known as ribozymes. Hammerhead ribozymes, which can catalyze cleavage and ligation of RNA molecules, are thought to be important in an RNA world and allow a mechanism for self-replication in the presence of magnesium. Encapsulated hammerhead ribozymes perform this self-cleavage as well, a necessary first step in this line of study. Research continues in developing a truly self-replicating protocell, and the results to date are highly encouraging. Activated nucleotides permeating across amphiphilic membranes have been shown to non-enzymatically replicate - this is key - encapsulated DNA templates. It just remains to fill in the lines.

When all is said and done, is this going to show us how abiogenesis occurred? Maybe. Note the language that Szostak uses: "model protocell vesicles", "prebiotically plausible membrane", etc. It's very careful language. What these experiments and others give us is a possible pathway, not necessarily the pathway. Perhaps autotrophic and heterotrophic abiogenesis are not either/or propositions and both are possible but only one historically occurred. Unless someone invents a time machine that can take us back to that point in time (current theoretical designs can only take us - well, actually only particles, not us - back in time to the point at which the machine was turned on), it is unlikely that we will be at all confident in having found the pathway. But this is not the point. The point is to find a plausible mechanism whereby abiogenesis could have occurred naturally, and we are well on our way there.  

Sometimes the journey is more important than the destination.

References:

Szostak JW, Bartel DP, Luisi PL, Synthesizing Life, Nature 409387-390 (2001)

Hanczyc MM, Fujkiawa SM, Szostak JW, Experimental Models of Primitive Cellular Compartments: Encapsulation, Growth, and Division. Science 302:618-622 (2003)

Chen IA, Roberts RW, Szostak JW, The Emergence of Competition Between Model Protocells. Science 305:1474-1476 (2004)

Chen IA, Salehi-Ashtiani K, Szostak JW, RNA Catalysis in Model Protocell Vesicles, JACS 127:13213-13219 (2005)

Mansy SS, Schrum JP, Krishnamurthy M, Tobe S, Treco DA, Szostak JW, Template-directed Synthesis of a Genetic Polymer in a Model Protocell, Nature [Epub ahead of print] (2008)

The Biology of Sexual Orientation - Cristian C A Bodo

Reposted from the American Sexuality magazine online.

The Biology of Sexual Orientation

Insight from animal research about what turns us on

By Cristian C A Bodo

What determines sexual orientation? What makes a person gay, bisexual, or straight? These sort of questions hold an undeniable interest to the general public and the answers are still hotly debated both by the experts in the field and by society at large. There are some powerful reasons for this universal appeal: First, the vast majority of us have experienced in the course of our lives some sort of sexual attraction toward other human beings, and this attraction in turn exerts a powerful influence in our mood, our behavior, our social interactions, and on the image that we have of ourselves. Since this plays such a key role in our lives, it is only natural that we would be interested in knowing at some point about its origins, and why we are oriented only toward people with certain characteristics and not others.

The examination of sexual orientation also holds relevance to specific social policies. Whether sexual orientation is the result of a conscious choice by the individual, as opposed to just another trait that comes "built-in" in our system, helps determine if it should be categorized as a "moral problem" or not. This seems to matter a lot in shaping out attitudes toward sexual minorities. Specifically (and for better or worse), the public appears to be more sympathetic to variations from the norm, in this case strict heterosexuality, if they are convinced that the individual has no "say" on this departure since it is the product of biological determination. On the other hand, sexual minorities have traditionally regarded this argument with suspicion. They fear that scientific research may open the door to treating these variations as little more than a disease and that efforts will be made to reduce or eliminate incidences of homosexuality in human populations.

Still, as it often happens with contentious issues that have such an impact for everyday life, society has turned to scientific research in order to get some answers. Despite the many occasions in which it has been proved otherwise, science still holds in the public conciousness the image of an unbiased actor whose answers are based solely in the pure application of a rational methodology and are therefore beyond the usual "contaminations" introduced by those who have specific interests in directing the public opinion toward their side of the field.

The question of sexual orientation has received special attention from biologists from early on. In part, this is due to the relevance that this trait is supposed to have for the survival of animal species with two (or more) separate sexes: If reproduction depends on successful mating with a member of another sex, then being attracted and actively attempting to interact with them would seem to be important to ensure that the genes of one generation be well represented in the next.

Plenty of experimental work has been done using lab animals to try to figure out how this is established, and how they develop an attraction for potential mates belonging to a sex other than their own. Most of it has been carried out in rodents (rats, mice, hamsters) for the simple reason that they breed well and adapt easily to a laboratory environment. Their proverbial capacity to deliver plenty of litters in a short time also comes handy at the time of doing an experiment.

The evidence derived from lab animals points directly to hormones derived from the gonads (testes in males, ovaries in females), specifically testosterone, in the determination of sexual orientation. When male pups are castrated at birth, they no longer seek the company of females after they have gone through puberty. Conversely, when females are injected with testosterone early in life, they later show an attraction toward other females, just like a male.

This is often referred to as the "organizational" effect of hormones, meaning that hormones trigger changes in the brain circuit, so that the brain develops in a particular way making animals predisposed to seek the company of one sex over another after reaching sexual maturity. In addition, the levels of hormones that they have in adulthood are very important to maintain this preference: If the gonads are removed, the preference quickly disappears, no matter how strong it may have been before the surgery. So the evidence is strong for a "built-in" mechanism in the determination of sexual orientation in rodents. Whether they will be attracted to females or to males when they grow up seems to be largely determined by the presence of functional testes or ovaries early in life (or even before they are actually delivered by the mother). The million-dollar question, the one that continues to generate heated debates both within and outside the scientific community, is whether this can be extrapolated at all to humans. And the answer is far from being trivial, since there are indeed powerful reasons that call for caution when doing so.

Sexual behavior in rodents is strictly associated with reproductive function, to the point that females will normally accept to mate with a male only during a particular stage of the estrous cycle: immediately after ovulation. Attempts by the male to initiate copulation during any other stage of the cycle are generally met with rejection, and this can turn into downright aggression. It is easy to see why this should be so. By limiting sexual activity to the period in which the female is actually fertile, the waste of energy that mating not resulting in pregnancy represents is actually avoided. Not surprisingly, gonadal hormones control the coordination between these two events (ovulation and sexual receptivity). Cycling ovaries release estradiol and progesterone to the bloodstream, which triggers ovulation and sexual receptivity.

In humans, on the other hand, the situation is radically different. Despite several attemps that have been made over the years to measure variations in sexual desire in women during the cycle, there is virtually no evidence to support such a claim. In humans, and other selected mammalian species, the willingness to engage in sexual activities seems to be dissociated from the hormonal status, and therefore not limited to be a mere prerequisite for succesful reproduction. On the contrary, we are all familiar with the multiple roles that sex plays in human societies, ranging from the expression of affection to the validation of social status. Even in non-human primates we can see some clear evidence of this emancipation of sex from its primitive role: Pygmy chimpanzees (or bonobos) are famous for using sexual intercourse to regulate many aspects of their social interactions, including greeting each other, resolving conflicts between members of the same clan, and exchanging food and other commodities.

Does this diminished role of gonadal steroids in the regulation of sexual activities also translate to the determination of sexual orientation in humans? With perhaps a single exception, researchers have in general failed to find a link between this characteristic and exposure to gonadal hormones at any point during the life of the individual. The exception is a study that showed women affected by congenital adrenal hyperplasia (CAH), a syndrome that caused their adrenal glands to excrete an excesive amount of sex steroids during their development, exhibited a higher proportion of individuals reporting same-sex sexual orientation compared to the general population. Notice, however, that this is not the same as saying that all the women exposed to high hormonal levels as a consequence of their syndrome became homosexual, which suggests that if hormones are playing a role here, they have to be doing it in combination with other factors. On the other hand, there is not a single study to this date that has conclusively proved that gay men are exposed to subnormal levels of testosterone or other sex hormones during development.

So, the evidence for gonadal hormones as the determining factor for sexual orientation in humans seems to be much less abundant than does the evidence in animal models. Perhaps, in part, this is because in the former case studying the phenomenon under strict experimental conditions is impossible, and thus researchers have to rely almost exclusively in the so-called "experiments of nature," clinical syndromes such as CAH in which it is particularly difficult to control for other variables—such as psychosexual history, genetic and social background—that may affect the outcome.

Another reasonable consideration is that with the evolution of higher cognitive functions, our sexuality became a much more complex phenomenon, with multiple purposes beyond mere reproduction and also with multiple variables affecting its different aspects, and this includes of course sexual orientation. And yet, it is difficult to shake off the feeling that some deeply ingrained biological root exists that determines who we feel sexually attracted to. When asked, most people declare having no recollection of making a conscious choice about this issue at any point in their lives. Instead, there is a strong feeling of having been "made" in certain way, which implies an underlying biological cause that overrules any attempts to modify it by conscious decision. (This has been and often still is the cause of a heavy psychological burden for gay/lesbian individuals raised in an environment that does not tolerate their sexual orientation and blames them for it.) But regardless of whether improved experimental methodology and more advanced technology would allow us one day to shed some light on the elusive biological factors that determine sexual orientation in humans, it is important to ask ourselves if we are interested in finding an answer to it, and thus if it should continue to be the object of scientific enquiry. As mentioned at the beginning, sexual minorities have repeatedly expressed concerns about this, since they fear that it may actually increase discrimination practices against them.

It is easy to sympathize with this point of view, especially considering the many instances in which supposedly neutral scientific knowledge was used in the past to justify racist policies or to deny women their civil rights. But at the same time it is perfectly reasonable to wonder if such an attitude is not putting the blame in the wrong place. Instead of making researchers scapegoats, we should ask why society at large would use the tools they create to enforce discriminative policies.

There is a wide amount of variation in human traits, ranging from some that have an obvious external manifestation (eye, hair, or skin color) to others that are virtually impossible to recognize without resorting to specific test tools (blood type), and they are known in many cases to have an evident genetic component. Even though human societies seem to have the unfortunate tendency to use these variations to discriminate, we have made remarkable progress in exposing this tendency as irrational and as the cause of much suffering, so that what was once universally accepted and justified is today relegated to the fringes. There is no reason to believe that this would not also be achieved in the case of our attitudes toward sexual orientation, even if an agreement on its biological causes is eventually reached as a consequence of further scientific research on the subject. By embracing too quickly the other option, seeking to prevent scientists from looking for the causes because of fear of what we may do with such knowledge, we may indeed find ourselves sharing our views with very strange bedfellows.

Cristian C A Bodo was born in Buenos Aires, Argentina. He received his Ph.D. in neuroscience from the University of Virginia in August 2007. The topic of his dissertation research was the role of gonadal steroids in the sexual differentiation of the mouse brain.