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Session 1: Origins of Life: Protocells and Non-Enzymatic Template-Directed RNA Synthesis

Transcript of Part 2: Protocell Membranes

00:00:00.00		My name is Jack Szostak,
00:00:02.10		I'm a Professor Genetics at Harvard Medical School,
00:00:05.22		and an Investigator at Massachusetts General Hospital,
00:00:08.22		where my laboratories are.
00:00:10.21		I'm also an Investigator of the Howard Hughes Medical Institute.
00:00:14.07		And in this lecture, I would like to tell you about some of our

00:00:18.21		recent work relevant to the origin of life on Earth,

00:00:22.12		and we're going to focus this time on the membrane component of simple protocells,

00:00:30.03		how those membranes assemble,

00:00:32.03		what their properties are,

00:00:34.03		how those properties are relevant for early forms of life,

00:00:38.08		and how we imagine them gradually changing

00:00:42.27		as a result of the emergence of Darwinian evolutionary processes.

00:00:48.28		So, let's begin by having a look at our conceptual model

00:00:55.26		of what an early protocell looks like.

00:00:59.09		So, again, we think of protocells having two parts:

00:01:03.24		A membrane boundary that closes up into a spherical vesicle

00:01:11.25		that can encapsulate molecules in the internal, aqueous compartment...

00:01:16.21		and the most important of those would be genetic molecules,

00:01:21.25		possibly RNA or DNA or maybe some related kind of nucleic acid.

00:01:27.04		And what we would like to understand is how,

00:01:33.07		in the absence of highly evolved genetic material,

00:01:37.18		this membrane compartment could first grow

00:01:41.22		and subsequently divide into daughter cells.

00:01:47.10		And because we're thinking about this at a very early time,

00:01:53.14		prior to the emergence of Darwinian evolution

00:01:56.16		and advanced, highly evolved cellular machinery,

00:01:59.17		we have to look to simple chemical and physical processes

00:02:04.15		to drive these transformations.

00:02:08.11		So, to think about that,

00:02:10.22		we need to think about the molecules that are going to be used

00:02:14.11		to build these membranes, and our favorite molecules

00:02:18.28		in this regard are the fatty acids.

00:02:22.01		So, these range from short-chain, saturated fatty acids,

00:02:26.20		like capric acid, which are the kinds of molecules

00:02:29.05		that we think could have been easily made in different scenarios

00:02:33.01		on the early Earth.

00:02:35.05		Many times in our laboratory work,

00:02:37.07		we use more modern fatty acids

00:02:40.03		(myristoleic acid or oleic acid) simply for convenience,

00:02:45.01		but in the end, we always come back to more prebiotically reasonable systems.

00:02:52.12		So what are the biophysical properties of these closed-up membranes,

00:03:00.08		in the form of vesicles or liposomes?

00:03:04.12		So, the most important aspect,

00:03:08.13		and something which is quite different from modern membranes

00:03:12.21		that are based on phospholipids, is that fatty acid vesicles...

00:03:19.20		basically, their formation and existence is controlled

00:03:23.08		by a series of phase transitions.

00:03:26.29		And the most important of those depends on the pH of the environment.

00:03:31.21		So, at fairly alkaline pHs,

00:03:35.17		the carboxylates of fatty acids are basically fully ionized,

00:03:40.24		and then charge repulsion wants to keep those groups

00:03:45.00		as far apart from each other as possible,

00:03:48.07		while the hydrophobic forces want to keep the hydrophobic tail

00:03:52.18		of the molecule as close together as possible.

00:03:55.26		And the result is the formation of micelles,

00:03:59.14		which are very small aggregates, a few nanometers in size,

00:04:05.08		and composed of roughly 10 to 100 fatty acid molecules.

00:04:09.20		Now as the pH in the environment gradually

00:04:12.00		drops towards roughly 8.5,

00:04:16.26		which is the point at which half of these carboxylates

00:04:19.21		will be ionized and half-protonated,

00:04:22.06		there's a phase transition that results in the spontaneous

00:04:25.14		assembly of bilayer membranes, as you can see here.

00:04:29.21		And because the curvature is much reduced in these much larger aggregates,

00:04:36.00		these are actually stabilized by hydrogen bonding

00:04:41.19		interactions between adjacent charged and ionized carboxylates.

00:04:47.07		Now, as the pH drops even further into the acidic range,

00:04:51.07		all of the carboxylates will become protonated eventually,

00:04:55.05		and then these structures collapse into oil droplets.

00:04:59.01		So the existence of these bilayer membranes

00:05:02.27		is only possible at an intermediate pH range, close to the pKa.

00:05:08.23		You'll notice this pKa of 8.5 is considerably higher

00:05:12.11		than the pKa of individual fatty acid molecules in solution,

00:05:17.24		which is closer to 4.5.

00:05:19.20		And the reason for that is that these charged head groups

00:05:23.15		are much closer together in this aggregate structure.

00:05:29.16		Now, when you do form these membranes,

00:05:32.14		they have very interesting properties,

00:05:34.18		which again are very different from the

00:05:37.01		properties of phospholipid-based membranes.

00:05:40.27		And one of the things that is very important to us

00:05:44.08		is the dynamic exchange processes that occur in these membranes.

00:05:49.02		So fatty acid molecules in a fatty acid membrane

00:05:52.25		flip-flop back and forth, from the outer leaflet to the inner leaflet,

00:05:57.28		very rapidly, sub-second timescale.

00:06:01.00		They also exchange in and out of the membrane,

00:06:06.03		so into solution, where they can exchange with molecules in micelles,

00:06:10.25		and again this is a very rapid process,

00:06:14.03		so that the molecules that make up any given vesicle

00:06:17.08		are constantly in flux and exchanging with the molecules in other vesicles,

00:06:22.01		again on the second timescale.

00:06:27.13		Another very interesting aspect of these membranes is that

00:06:34.24		they can be quite heterogeneous at a molecule level.

00:06:40.02		And so these membranes can incorporate not just fatty acids

00:06:46.14		but also their glycerol esters, as you see here,

00:06:50.01		and even lyso-phosphatidic acids,

00:06:53.10		so an ester with phosphoglycerate.

00:06:56.12		They can incorporate alkanes and

00:07:00.02		polycyclic aromatic hydrocarbons as well.

00:07:04.00		Now, what happens when you make membranes

00:07:07.00		with these kinds of mixed compositions?

00:07:10.09		It's very interesting that, in many cases,

00:07:14.15		this results in membranes that are much more stable,

00:07:18.15		they're more physically stable, more thermostable, and also,

00:07:22.25		often more permeable to polar nutrients that we would like to

00:07:27.04		bring in from the outside environment.

00:07:30.27		So this slide simply illustrates an experiment

00:07:35.05		to look at the thermostability of some vesicles

00:07:39.18		made from a pure fatty acid, as opposed to mixtures.

00:07:43.01		So in this experiment, what we've done is to encapsulate

00:07:47.18		a short DNA oligonucleotide within vesicles

00:07:52.08		and watch it leak out over the course of about an hour.

00:07:57.07		And now if the membrane is composed purely of myristoleic acid,

00:08:02.23		this 14-carbon, singly unsaturated fatty acid,

00:08:06.11		you can see that up to about 50 degrees,

00:08:09.04		nothing leaks out over an hour.

00:08:11.11		But as the temperature goes up to about 80,

00:08:14.20		they become more leaky, and then at 90 degrees,

00:08:18.09		everything is leaked out over an hour.

00:08:21.00		Now, if you add some of the corresponding alcohol,

00:08:23.26		you can see significant stabilization,

00:08:27.10		but the most dramatic effect is when you add a fraction of the

00:08:31.27		glycerol ester of that fatty acid.

00:08:34.04		And now, you can essentially boil these vesicles for an hour,

00:08:39.00		and none of the contents leak out,

00:08:41.13		at least these oligonucleotide contents are trapped inside.

00:08:46.02		So that's really exactly what we want.

00:08:49.00		We want the genetic material, long oligonucleotides,

00:08:53.18		to stay inside indefinitely,

00:08:56.24		while small molecules (nutrients)

00:08:59.00		can get across the membrane very rapidly.

00:09:03.06		And again, as the temperature goes up,

00:09:05.07		the permeability to small molecules increases dramatically.

00:09:11.02		So, we got into this area about 10 years ago,

00:09:17.16		and started doing some very simple experiments

00:09:21.20		based on the pioneering work of people like Dave Deamer

00:09:26.14		and Pier Luigi Luisi.

00:09:28.14		Now, the Luisi Lab in particular had shown already

00:09:32.05		that you can make vesicles grow simply by

00:09:35.24		adding more fatty acids in the form of alkaline micelles.

00:09:40.05		When the micelles go into a buffered solution,

00:09:42.21		they become a thermodynamically unstable phase,

00:09:45.21		so there's an energetic driving force for those molecules

00:09:49.04		to assemble into new membranes or

00:09:51.21		to integrate into preexisting membranes.

00:09:56.09		And at the time,

00:09:57.27		Martin Hanczyc was a postdoc in the lab,

00:09:59.29		Shelly Fujikawa was a student...

00:10:03.01		they basically repeated those experiments,

00:10:06.01		but using real-time methods of analysis,

00:10:09.23		so that we could watch the process of vesicle growth in real time,

00:10:14.19		after the addition of food molecules.

00:10:17.15		The assay that we used was a fluorescence assay

00:10:21.21		based on energy transfer from donor to acceptor dyes.

00:10:27.02		It's basically a measure of how far apart the dyes are on average.

00:10:31.15		And as the membrane grows by incorporating new molecules,

00:10:34.28		the dyes are diluted, they get further apart,

00:10:37.14		and energy transfer efficiency decreases.

00:10:40.18		So you can use this as a very effective assay for

00:10:43.05		surface area in real time, so we can watch things grow very easily

00:10:48.27		by this simple fluorescence assay.

00:10:52.17		And the essential result from that is that, if you add the food slowly,

00:10:57.02		almost all of it gets incorporated into preexisting membranes;

00:11:00.13		if you add it very quickly, only a fraction, maybe half,

00:11:04.26		will get incorporated, and the rest will form new vesicles.

00:11:08.00		So that takes care of growth at a basic level.

00:11:11.22		What about division?

00:11:13.24		So in the early days, we didn't really have any good idea

00:11:17.27		of how to drive protocell division through environmental fluctuations.

00:11:23.25		So essentially, in desperation,

00:11:26.04		we resorted to a brute force approach of forcing big vesicles

00:11:31.12		through small pores in a filter,

00:11:33.23		and little vesicles come out the other side.

00:11:37.00		So, on the one hand,

00:11:39.00		it's satisfying because it's a proof of principle that

00:11:41.26		cell division could be driven by a physical, environmental force.

00:11:47.00		On the other hand, it's not very satisfying because

00:11:49.22		we didn't think there was any realistic analogue

00:11:53.26		to that process on the early Earth.

00:11:56.07		And furthermore, when you take a large vesicle

00:11:59.16		and physically divide it into smaller spherical vesicles,

00:12:03.18		you necessarily lose some of the contents,

00:12:06.16		which is also not very satisfying.

00:12:09.15		Nonetheless, that process could be turned into a cycle,

00:12:13.19		where we could do growth and division

00:12:15.28		and growth and division, again and again.

00:12:20.04		So then the problem really became:

00:12:22.15		How could we find other pathways that might have

00:12:27.15		been more physically realistic, more robust,

00:12:30.24		and more plausible for the early Earth?

00:12:34.29		So, the first advance in this area came from

00:12:41.21		thinking about an alternative growth pathway.

00:12:46.03		And so this is a pathway of growth that,

00:12:48.08		instead of relying on the environmental influx of new molecules,

00:12:54.23		occurs in a different way,

00:12:56.04		by competition between protocells in a population.

00:13:01.13		So some of are going to grow at the expense of others.

00:13:04.15		And this was work done by Irene Chen when she was a student in my lab,

00:13:09.09		and initiated by discussions with Rich Roberts,

00:13:12.12		when he was a postdoc in the lab.

00:13:14.14		So the basic idea was that,

00:13:17.08		if you think of our basic protocell model,

00:13:21.17		we have genetic molecules trapped inside a semipermeable membrane.

00:13:27.19		Now, large polymers contribute very little to the

00:13:31.06		overall internal osmotic pressure in a system like this.

00:13:36.13		Most of the osmotic pressure actually results from

00:13:40.11		counter-ions that have to be there to neutralize the charge

00:13:44.11		on the polyanionic genetic molecule.

00:13:47.29		And so this is the classical Donnan effect:

00:13:51.25		These ions contribute to an internal osmotic pressure,

00:13:56.03		and we thought that, as a result of this physical phenomenon,

00:14:01.05		this physical effect,

00:14:02.28		vesicles that had more RNA inside of them

00:14:06.01		should have a higher osmotic pressure,

00:14:09.12		and maybe that could actually drive growth competitively.

00:14:14.21		So, the way that we looked at that experimentally

00:14:19.08		was using the same fluorescence assay that we had been using

00:14:24.17		before to monitor membrane growth following

00:14:28.11		the addition of new membrane molecules,

00:14:31.13		but in this case, the idea is that we're going to take

00:14:37.05		osmotically swollen vesicles that have,

00:14:39.28		for example, a lot of RNA inside,

00:14:42.16		and we'll monitor the surface area of those vesicles following

00:14:47.22		mixing with vesicles that are osmotically relaxed,

00:14:51.25		and we'll ask what happens to the surface area.

00:14:56.14		So here's the experiment,

00:14:58.23		where we're monitoring the surface area of the swollen vesicles,

00:15:03.10		these are vesicles that have a lot of RNA on the inside,

00:15:06.28		and by themselves, they're perfectly stable, nothing happens,

00:15:11.02		same thing for the relaxed vesicles, nothing happens.

00:15:13.24		As soon as you mix the swollen vesicles with relaxed vesicles,

00:15:18.02		you can see that their surface area starts to increase

00:15:22.05		over a period of minutes.

00:15:24.09		And in control experiments,

00:15:25.29		where we mix with buffer or other swollen vesicles,

00:15:29.21		there's very little change.

00:15:31.28		So it seems like the swollen vesicles are growing

00:15:35.19		by absorbing molecules from their relaxed neighbors.

00:15:40.11		We can of course do the converse experiment,

00:15:43.11		and monitor the surface area of the relaxed vesicles,

00:15:47.12		and when they are mixed with swollen vesicles,

00:15:49.25		their surface area declines, so molecules are leaving

00:15:53.05		those vesicles and going into the swollen vesicles.

00:15:56.10		Again, control experiments where relaxed vesicles

00:15:58.17		are mixed with more relaxed vesicles, not much happens.

00:16:03.06		So what's going here is that,

00:16:07.09		as soon as we mix these populations of vesicles,

00:16:09.20		the swollen ones are growing, the relaxed ones are shrinking,

00:16:13.28		and the reason that's happening is that this osmotic pressure

00:16:17.10		creates a tension in the membrane,

00:16:20.20		which is only relaxed by the incorporation of more molecules,

00:16:24.21		which allows the surface area to increase.

00:16:28.04		So the exciting implication of that physical process

00:16:32.17		is that it's a potential link between genome replication

00:16:37.09		and faster membrane growth.

00:16:39.08		So, any process that results in faster replication

00:16:43.15		of the genetic material inside a vesicle will automatically,

00:16:48.28		as a result of the Donnan effect,

00:16:51.05		lead to faster growth of the membrane compartment.

00:16:54.07		So it's a coupling between genome replication

00:16:57.01		and cell growth as a whole.

00:16:59.16		Now, that could be particularly interesting if genome replication

00:17:04.22		is an autocatalytic process driven by RNA-catalyzed RNA replication.

00:17:10.11		So now, any mutation that leads to faster,

00:17:14.01		more efficient replication of the genetic material

00:17:17.18		will automatically go along with faster growth

00:17:20.12		of the cell as a whole,

00:17:22.04		and it's a competitive form of growth

00:17:24.10		that results from these cells essentially eating their neighbors.

00:17:30.06		So that was a very exciting conceptual advance,

00:17:34.17		but there was always this nagging problem.

00:17:38.09		That here, we're looking at growth driven by osmotic forces,

00:17:43.15		which means that these are growing as spherical vesicles.

00:17:48.09		And dividing a spherical vesicle,

00:17:50.13		especially one that's osmotically swollen,

00:17:52.25		is something that's actually quite difficult to do.

00:17:55.08		It takes a lot of energy,

00:17:57.10		and as I said in the case of the extrusion experiments,

00:18:00.18		results in loss of some of the contents,

00:18:02.27		which is not very satisfying.

00:18:05.04		So, we were left thinking for a long time

00:18:08.23		about possible alternative membranes

00:18:11.17		and alternative division processes,

00:18:15.07		and that's something we'll come back to later.

00:18:19.28		So, let's think about more natural systems.

00:18:27.24		Everything that we've looked at up till now

00:18:30.26		has been highly constrained, very artificial laboratory system

00:18:38.16		designed so that we can follow what's happening

00:18:42.29		analytically in a very clear and simple way.

00:18:46.12		But of course, the vesicles that form naturally

00:18:51.21		aren't so nicely constrained.

00:18:55.04		They're extremely heterogeneous,

00:18:58.19		and that generates a system which is much harder to study experimentally.

00:19:03.26		So that's illustrated by this image,

00:19:07.24		which shows the range in the diversity of vesicles that are formed

00:19:11.16		when you just shake up oleic acid with some salt and buffer.

00:19:15.18		You get these huge vesicles, tiny vesicles,

00:19:18.29		compound vesicles, it's a terrible mixture,

00:19:23.27		and if you imagine adding some more oleic acid to

00:19:27.08		this in the form of micelles,

00:19:28.25		so that everything grew a little bit,

00:19:31.22		you couldn't tell that anything had happened.

00:19:34.27		So we need new analytical ways of looking at this,

00:19:38.14		or new ways of preparing vesicles so that we can actually

00:19:42.13		watch what's happening.

00:19:44.08		And our major advance in that area came from the

00:19:48.06		work of a new graduate student, Ting Zhu,

00:19:51.28		who had a background in mechanical engineering

00:19:54.14		and proposed a very simple and elegant way of

00:19:57.25		preparing more or less uniform populations of vesicles

00:20:01.23		that were large enough to just look at in the microscope.

00:20:06.04		So for the first time, we were able to do experiments

00:20:09.08		where we take vesicles, add food, and just watch how they grow.

00:20:14.09		And so what you can see in this image is a field of vesicles,

00:20:18.00		they're all roughly four microns in diameter,

00:20:21.24		so big enough to image optically,

00:20:25.29		and what you're actually seeing here is a fluorescent dye

00:20:29.24		encapsulated in the aqueous internal space of each vesicle.

00:20:35.05		So now, what you'll see next is a time-lapse video

00:20:41.01		of how these things actually grow following the addition

00:20:44.17		of more fatty acids.

00:20:47.04		What we expected was simply that these spherical vesicles

00:20:51.26		would swell up a little bit, we'd just watch them get bigger.

00:20:56.19		We thought that, if the surface area grew faster than the volume,

00:21:01.11		they might become somewhat elongated in shape.

00:21:05.24		But we did not expect what actually happened,

00:21:09.05		which is quite dramatic.

00:21:12.16		So, what you see here is that, initially,

00:21:15.24		a faint filament emerging from the parental spherical vesicle,

00:21:20.06		and over time that filament grows and absorbs

00:21:23.21		all of the material in the starting spherical vesicle,

00:21:27.21		which has now grown into a long, thin, branched,

00:21:32.03		filamentous morphology.

00:21:35.13		So, this was a very surprising observation.

00:21:41.05		It immediately raised a host of mechanistic questions,

00:21:45.16		which we're still investigating.

00:21:48.01		But it, very importantly, immediately solved the problem

00:21:52.27		of how cell division might happen in a manner

00:21:56.29		driven by environmental fluctuations.

00:21:59.28		As I said, spherical vesicles are hard to divide,

00:22:02.23		it takes a lot of energy.

00:22:04.15		But these structures are incredibly fragile,

00:22:07.17		and all you have to do it shake them up.

00:22:11.21		Gentle agitation is sufficient to cause them to divide,

00:22:16.09		and you can see that process in action in the next video.

00:22:21.13		So here you see the pearling instability:

00:22:25.07		The filament forms a set of beads,

00:22:28.16		and then they snap apart and generate fragments,

00:22:32.27		which eventually round up into spherical daughter vesicles.

00:22:36.25		And so this process is driven by very gentle perturbations

00:22:43.01		in the fluid, very mild shear forces.

00:22:46.08		You can easily imagine the shear forces generated by

00:22:50.15		wave action on a shallow pond leading to cell division

00:22:54.06		in this kind of process.

00:22:56.27		Now, this process of growth followed by shear-induced division

00:23:03.10		makes a cycle that can be iterated indefinitely.

00:23:07.09		So you can start off with a spherical vesicle,

00:23:09.20		grow it into a filament, divide it into spherical daughter vesicles,

00:23:14.00		which can grow again.

00:23:16.03		If they sit around, the volume increases,

00:23:18.20		they round up, then you can grow again, divide, grow.

00:23:23.22		This cycle of growth and division can be carried on indefinitely.

00:23:29.19		So, let's think for a minute about some of the

00:23:32.29		mechanistic processes that are involved.

00:23:35.10		We don't understand everything that's going on,

00:23:37.13		but at least we have some experiments that tell us

00:23:41.21		what some of the critical factors are.

00:23:45.11		So, what we think is going on,

00:23:47.21		and what the important factors are,

00:23:49.26		is that surface growth is happening much faster than volume growth,

00:23:57.07		and so that makes a shape change inevitable.

00:24:01.15		Volume is conserved on the timescale of growth

00:24:04.16		because most of the solvents are only very slowly permeable

00:24:08.05		across these membranes,

00:24:10.05		and the solvent can be anything from the rather artificial buffer that we use,

00:24:16.13		to a mixture of amino acids,

00:24:18.12		such as you would get from Miller-Urey-type syntheses.

00:24:22.00		The other and somewhat surprising factor that's critical is the

00:24:26.07		multilamellar structure of these vesicles.

00:24:29.03		So these large vesicles tend to have several bilayers closely apposed,

00:24:35.29		as their sort of outer structure.

00:24:38.25		What we think happens is that when we add food molecules,

00:24:41.23		they first integrate into the outermost bilayer,

00:24:46.28		there's very little space between it and the next bilayer,

00:24:51.00		and so that extra surface area is extruded as a

00:24:55.01		thin, initial filament, which then subsequently elongates

00:25:01.27		into this long, tubular, filamentous structure.

00:25:05.21		We really don't understand the physical forces

00:25:08.04		that are involved in those later stages,

00:25:11.25		but we have some ideas about the initiation phases.

00:25:16.04		So I want to show you first an experiment that shows

00:25:19.25		that volume conservation is very important.

00:25:22.23		If you use a buffer that is very, very rapidly permeable,

00:25:27.24		then you don't see this kind of process at all.

00:25:30.22		Instead, what happens is that the outermost bilayer just balloons up.

00:25:36.25		As surface area increases, volume keeps up,

00:25:40.10		the volume expands, and so you end up with

00:25:43.18		most of the bilayers almost unchanged,

00:25:48.12		and trapped within a much larger unilamellar vesicle.

00:25:53.26		Now, the second important aspect is how

00:25:58.18		multilamellar vesicles grow in contrast to how unilamellar vesicles grow.

00:26:04.10		So when you have these multiple bilayers, as I suggested,

00:26:08.17		it looks like, from these confocal images,

00:26:11.00		that it's the outermost bilayer that grows first,

00:26:14.13		and that extra surface area is extruded as a thin tubule.

00:26:18.21		Eventually, you get a long filament,

00:26:21.04		and when you divide that by mild shear forces,

00:26:25.11		you get back small, multilamellar vesicles,

00:26:29.05		and then you can repeat the cycle.

00:26:31.11		Now, something very different happens if you go to the

00:26:33.22		trouble of making a unilamellar initial vesicle.

00:26:38.16		This grows the way we intuitively thought it would grow:

00:26:41.08		It just expands, becomes somewhat elongated.

00:26:45.25		But now these structures are extremely delicate and unstable,

00:26:50.17		and when they're subjected to shear force fluctuations,

00:26:54.19		they tend to rip apart, the contents spill out,

00:26:57.25		and remarkably, they reassemble into multilamellar structures.

00:27:03.19		So, to summarize this,

00:27:05.23		the two important aspects of the overall process

00:27:09.04		are beginning with multilamellar vesicles

00:27:11.28		and having something in the surrounding solution

00:27:16.08		which can only permeate slowly across the membrane,

00:27:20.02		resulting in slow volume increase

00:27:23.26		compared to rapid surface area increase.

00:27:26.19		And then environmental perturbations lead to cell division

00:27:29.25		in a very simple and natural way.

00:27:34.02		So, to summarize this, in a series of stages,

00:27:38.22		we've gone from a very artificial laboratory simulation,

00:27:43.19		to a process that looks simple and robust enough

00:27:47.06		to have occurred on early Earth environments.

00:27:53.04		Now, more recently, we've come back to the idea of competition

00:27:59.17		between protocells as an alternative mechanism of driving growth.

00:28:05.18		And this is particularly interesting in terms

00:28:09.11		of a way that this could emerge from Darwinian processes.

00:28:16.20		We think that one of the first ribozymes to emerge,

00:28:20.10		perhaps THE first, was actually a ribozyme

00:28:23.21		that could catalyze the synthesis of phospholipids.

00:28:28.15		And so why do we think this?

00:28:30.08		This came out of considering the series of evolutionary transitions

00:28:35.24		that must have occurred to get from

00:28:39.20		primitive membranes based on fatty acids,

00:28:42.05		with the appropriate dynamic and exchange processes

00:28:46.13		for a primitive cell,

00:28:48.01		up to modern membranes, which are based on phospholipids,

00:28:52.18		which are good barriers and which rely on

00:28:55.15		highly evolved biochemical machinery to mediate

00:28:59.07		growth and division and the transport of molecules

00:29:03.20		across the membrane.

00:29:04.27		So you couldn't go directly from the primitive state

00:29:08.20		to the modern state.

00:29:10.10		That would be suicidal,

00:29:11.21		because now a cell that depended on the influx

00:29:14.27		of nutrients from the environment

00:29:16.18		would no longer have access to those nutrients

00:29:18.29		because these membranes are so impermeable.

00:29:22.12		So presumably, the transition had to occur in a series of stages,

00:29:27.02		and what would an intermediate stage look like?

00:29:31.19		Probably it would look like a fatty acid membrane

00:29:35.09		with a small amount of phospholipid doped into the structure.

00:29:41.02		So if you think of a protocell containing genetic molecules,

00:29:46.21		if one of those sequences happens to be a simple

00:29:49.26		acyltransfer ribozyme that could make phospholipids

00:29:54.11		(after all, it's a simple, single, one-step acyltransfer reaction),

00:29:59.05		you could imagine making just a little bit of

00:30:02.09		phospholipid as an initial step in this transformation.

00:30:07.14		The thing that puzzled us for a long time is

00:30:11.11		what possible selective advantage could there be to

00:30:16.11		the presence of a small amount of phospholipid

00:30:19.16		in a fatty acid background?

00:30:22.24		It was very hard to imagine any reason why that would be advantageous.

00:30:27.21		It didn't really seem that a little bit of phospholipid

00:30:31.05		would change the physical properties of the membrane

00:30:33.18		in any particularly useful way,

00:30:37.06		and so the answer to this conundrum

00:30:39.29		came from doing experiments,

00:30:42.10		and this is all work done by Itay Budin,

00:30:45.06		who's a graduate student in the lab.

00:30:47.25		So what Itay did was simply to prepare vesicles

00:30:53.13		containing a little bit of phospholipids

00:30:56.28		doped into a fatty acid membrane structure.

00:31:01.11		And then he asked, well,

00:31:03.00		how would that affect the properties of the membrane?

00:31:05.20		And what turned out to be particularly interesting

00:31:08.12		is what happens when these vesicles are mixed with

00:31:11.18		pure fatty acid vesicles.

00:31:14.23		And it turns out, as you can see on the next slide,

00:31:18.14		that even a little bit of phospholipid confers a really dramatic ability

00:31:24.12		on that vesicle to absorb fatty acids from its

00:31:28.02		neighboring fatty acid vesicles.

00:31:31.13		So here's the experiment that Itay did:

00:31:34.11		He prepared vesicles with 10% of a phospholipid

00:31:41.11		in an oleic acid background,

00:31:43.19		and mixed them with a large excess of pure,

00:31:46.02		fatty acid, oleic acid vesicles.

00:31:48.23		So the vesicle starts out as a spherical structure,

00:31:51.13		you can see the encapsulated dye.

00:31:53.26		Within seconds, you see the protrusion of small filaments,

00:31:59.00		and over time, you can see a transformation

00:32:01.17		into a filamentous structure,

00:32:05.04		much as you saw before following the addition

00:32:08.07		of a large excess of fatty acids.

00:32:11.07		But this time the process of growth being driven purely

00:32:16.20		by the initial vesicle absorbing fatty acids

00:32:20.03		from its neighboring molecules.

00:32:21.27		It's essentially growing by eating its neighbors.

00:32:24.26		The mechanism of this phospholipid-driven growth is extremely interesting,

00:32:30.01		and you can see what we think is going on in the next slide.

00:32:34.16		It turns out there are actually two processes:

00:32:37.07		The first is a simple, entropically favored process

00:32:40.17		in which the absorption of fatty acids molecules into the mixed bilayer

00:32:46.04		dilutes the phospholipids,

00:32:50.10		which are extremely insoluble in the surrounding space,

00:32:53.19		so this is an entropically driven dilution.

00:32:58.04		But it turns out that, in addition to that factor,

00:33:01.02		there's actually a quantitatively more important factor,

00:33:05.00		which is the fact that these phospholipid molecules

00:33:09.01		change the properties of the bilayer such that

00:33:13.14		the disassociation of fatty acids from the bilayer is slowed down.

00:33:18.25		As a result, the vesicles grow because the

00:33:24.06		association rate constant is the same,

00:33:27.27		but the dissociation is slowed down,

00:33:30.27		so they hold onto more of their molecules, they grow,

00:33:34.10		and the pure fatty acid vesicles therefore shrink.

00:33:37.21		So how do we demonstrate this experimentally?

00:33:41.06		Itay took advantage of an assay that was developed by Jim Hamilton.

00:33:48.00		It's a rather indirect but very effective assay.

00:33:51.00		So if you have a fatty acid vesicle,

00:33:52.26		you can monitor the dissociation of

00:33:56.04		fatty acid molecules from that membrane, which is a slow step,

00:34:02.11		by looking at how they dissociate into solution,

00:34:07.06		integrate into the membrane of a reporter vesicle,

00:34:10.26		which is a fatty acid vesicle that contains inside it

00:34:15.20		a fluorescent dye that's pH-sensitive.

00:34:18.20		Now when the fatty acid molecules integrate into the outer leaflet,

00:34:23.05		they flip rapidly to the inner leaflet.

00:34:26.10		It's the protonated form that flips because it's neutral,

00:34:29.16		it can flip very rapidly, and then it re-equilibrates on the inside,

00:34:34.10		releasing protons, which acidify the interior.

00:34:38.05		So we have a fluorescence assay for this pH decrease,

00:34:42.16		and that gives us essentially a real-time assay for this

00:34:46.01		initial slow step of dissociation.

00:34:49.17		You can see that this assay works by comparing

00:34:53.25		the dissociation of fatty acids of different chain lengths,

00:34:58.00		and so here you see that oleic acid, which is an 18-carbon chain,

00:35:03.16		dissociates fairly slowly.

00:35:06.13		Palmitoleic acid, two carbons shorter, dissociates more rapidly.

00:35:11.19		And myristoleic acid, 14 carbons, dissociates extremely rapidly.

00:35:17.06		So it gives you a good readout for the dissociation kinetics,

00:35:21.09		and that allows you to monitor the rate of dissociation

00:35:26.21		as a function of phospholipid content of the membrane.

00:35:31.17		And so if you start off with a pure fatty acid membrane,

00:35:35.28		so a pure oleic acid membrane, dissociation is quite rapid.

00:35:40.21		But the more phospholipid that there is in the membrane,

00:35:44.25		the slower the off-rate of the fatty acid is.

00:35:51.17		So, as a result of these two processes,

00:35:56.24		this entropically driven dilution aspect

00:36:00.19		and this off-rate-driven aspect,

00:36:03.11		the presence of even a little bit of phospholipid will drive growth

00:36:08.29		by essentially extracting or absorbing fatty acid molecules

00:36:13.09		from neighboring vesicles that have either no phospholipid

00:36:17.25		or even just less phospholipid.

00:36:20.24		So what would the implications of that process be

00:36:23.15		in an evolutionary scenario?

00:36:27.15		We begin with this idea that phospholipids drive growth.

00:36:31.12		As a result, of course there would be a very strong selection

00:36:35.08		for a ribozyme that has the catalytic ability

00:36:40.08		to make phospholipids, so a simple acyltransferase ribozyme.

00:36:48.07		That kind of primitive cell would rapidly take over

00:36:51.24		the population because it has such a strong selective advantage,

00:36:55.23		but that would lead to an evolutionary arms race.

00:36:59.11		Because now, in order to grow by eating your neighbors,

00:37:03.15		you have to make more phospholipids than they do.

00:37:07.11		So there'd be an evolutionary race to make

00:37:11.29		more and more and more phospholipids.

00:37:14.27		And so now the phospholipid abundance in the membrane

00:37:19.13		would start to increase, you'd have more and more phospholipids,

00:37:23.05		and eventually that would lead to new selective pressures

00:37:26.25		favoring the emergence of metabolism,

00:37:29.09		because now it's actually useful to have internal metabolic reactions

00:37:33.24		because you get to keep the molecules you make

00:37:36.00		instead of having them just leak out,

00:37:38.20		since the phospholipid makes the membrane less permeable.

00:37:42.09		At the same time, there would be selective pressures

00:37:45.03		for the emergence of transport machinery,

00:37:48.23		which would help you absorb molecules that you still want

00:37:52.05		to have access to from the environment,

00:37:54.00		or release molecules that you want to get rid of,

00:37:56.22		so waste products.

00:37:58.15		So we think that this physical process,

00:38:01.26		this unexpected physical process of phospholipid-driven membrane growth,

00:38:06.05		would set in place a whole cascade of events eventually

00:38:11.20		leading to selective forces for much more complicated cells,

00:38:17.25		where we see the emergence of metabolism and

00:38:20.09		membrane transport machinery,

00:38:22.11		and eventually, we think that could drive the emergence of

00:38:26.10		protein synthesis, initially uncoded but eventually coded protein synthesis,

00:38:32.08		and be a major factor in the emergence of modern biochemistry.

00:38:39.25		So from all of these experiments, just to sum up,

00:38:42.23		what can we say that we've learned that's really relevant

00:38:46.03		to the origin of life on Earth?

00:38:47.22		And I think at at general level,

00:38:49.03		there are two things I'd like to point out:

00:38:52.01		One is that, from doing very, very simple laboratory experiments,

00:38:56.14		again and again we've uncovered a surprising new

00:38:59.08		physical phenomenon, things that you would have a hard time

00:39:03.05		thinking up a priori, but which emerge

00:39:07.16		naturally from doing laboratory experiments

00:39:10.08		where you're trying to build systems and see how they work.

00:39:13.08		The second general lesson, I think,

00:39:16.19		is that in our early experiments,

00:39:20.08		we tend to use very highly constrained artificial systems

00:39:24.23		because they're analytically tractable.

00:39:27.20		But they often don't exhibit the properties

00:39:30.28		that we really want to see that are relevant to the prebiotic system.

00:39:35.24		So it's very important to relax those experimental constraints

00:39:41.20		and gradually develop the ability to look at "messier"

00:39:45.00		and more natural systems that give you insight

00:39:48.01		into physical processes that really could explain some of the

00:39:52.21		apparently hard parts in the process by which life emerged

00:39:57.17		from the chemistry of the early planet.

00:40:01.02		So, again, all of this work was done by many very talented

00:40:04.08		students and postdocs.

00:40:06.26		I've mentioned in particular the work of Itay Budin and Ting Zhu,

00:40:10.28		and the early work of Irene Chen, Shelly Fujikawa, Martin Hanczyc.

00:40:17.09		Many people have played a role in developing these systems

00:40:21.09		and gradually giving us the ability to understand what's going on

00:40:26.13		in these model systems and some insight into the origin of life.

00:40:31.02		Thank you.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. MCB-1052331. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speaker and do not necessarily represent the views of iBiology, the National Science Foundation, the National Institutes of Health, or other iBiology funders.

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