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The Origin of Life on Earth

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. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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