Tuesday, June 24, 2008

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.


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)

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