Dr. Theriot explains how tiny, nanometer sized actin molecules can self-assemble into filaments that are hundreds of microns in length. These actin filaments are constantly growing and shrinking, and this dynamic behavior allows a network of actin to generate enough force for cell motility. The intracellular bacterial pathogen Listeria monocytogenes uses actin polymerization to propel itself through the cytoplasm and to invade other cells. Many years of studies using Listeria have allowed Theriot and others to dissect the regulation of actin network growth in Listeria “comet tails” and at the leading edge of crawling cells.
00:09.1 My name is Julie Theriot
00:10.2 and I'm a professor at Stanford University.
00:12.1 I'm delighted to be taking part in this iBio Seminar series
00:15.1 and in the first part of my presentation today
00:17.2 I would like to talk about protein polymers,
00:19.1 crawling cells,
00:20.3 and comet tails.
00:23.2 One of the things that I find absolutely fascinating
00:25.1 about biological systems
00:27.0 is how the enormous complexity
00:28.2 that we see in living cells and living organisms
00:30.2 can arise by the assembly
00:32.3 of relatively small and simple subunits or parts.
00:35.3 For example, the protein structure
00:38.1 that's shown here
00:40.1 is one of the most abundant proteins in eukaryotic cells,
00:42.2 called actin.
00:43.1 It also happens to be my favorite protein.
00:45.2 And it's a small, globular protein,
00:47.2 not particularly distinguished in terms of its overall shape.
00:50.0 It has the capacity to bind and hydrolyze ATP.
00:54.1 However, it also has the capacity to self-assemble,
00:57.1 to make filaments made up of many copies,
00:59.1 many identical copies,
01:00.2 of the same protein,
01:02.1 and by that self-assembly process
01:04.0 actin and other cytoskeletal proteins like it
01:06.1 are able to elaborate tremendously complicated structures
01:09.0 inside of living cells,
01:10.1 giving rise to shapes
01:12.1 like the beautiful Purkinje cell
01:13.2 that's shown here in this classic drawing
01:15.2 by Santiago RamÃƒÂ³n y Cajal.
01:17.2 So, to me this is one of the most profound problems
01:20.2 facing our understanding of cell biology,
01:22.2 is to try to figure out what are the rules
01:24.2 by which these tiny nanometer-scale proteins
01:27.3 can come together and organize themselves
01:29.2 in order to make structures that are many orders of magnitude larger
01:33.0 - 10^4 or 10^5 orders of magnitude larger.
01:36.2 And that question becomes even more profound, I think,
01:39.0 and even more puzzling,
01:40.1 when we realize that in the context of biological structures
01:42.2 everything is dynamic.
01:44.0 This is certainly true of the cytoskeletal elements
01:46.1 and I think that's really beautifully illustrated
01:47.2 by this classic movie of a neutrophil
01:50.1 chasing down bacteria.
01:51.3 This movie was made by David Rogers at Vanderbilt University
01:54.2 in the 1950s,
01:55.3 and what we're looking at here is a blood smear,
01:58.0 where the little round dark objects
02:00.2 are red blood cells,
02:02.0 the white blood cell is labeled there in the middle,
02:04.1 and you'll see right at the front end of the white blood cell
02:06.2 is a tiny little pair of bacteria.
02:09.1 Now, when the movie starts to play,
02:12.0 you can appreciate that the white blood cell
02:14.1 is very well organized
02:15.2 -- it has a definite front and a definite back --
02:17.1 but it's also very active,
02:18.3 so it's chasing after that bacterium.
02:20.2 As the bacterium is bouncing around,
02:22.1 because of fluid motion in the background,
02:24.1 the neutrophil is able to change direction
02:27.0 and keep going after the little bacterium,
02:29.1 until finally it's going to catch up
02:31.2 and then engulf it,
02:32.2 which is its biological function.
02:34.2 Now, that happens to be a human neutrophil,
02:37.0 probably taken from David Rogers' own blood,
02:39.1 but if we look at other types of eukaryotic cells
02:42.2 we can find examples like this amoeba.
02:44.1 This is Acanthamoeba castellanii,
02:45.3 which lives in the soil,
02:47.1 and it also makes a living by crawling around
02:49.0 and eating bacteria.
02:50.1 And when we watch it move under the microscope,
02:52.1 it looks actually, remarkably similar
02:54.2 to the movement of the human neutrophil.
02:55.3 It would really take an expert to be able
02:57.2 to tell the difference between these two different cells.
02:59.3 And yet these two cell types
03:01.1 have been separated by about two billion years of evolution.
03:04.3 No, we appreciate today that not only
03:07.1 is the overall appearance of motility quite similar
03:09.1 between these two different cells,
03:10.2 but also the fundamental molecular machinery
03:12.3 that drives the motion
03:14.1 has also been conserved all of this time.
03:17.1 And one of the most important elements of that fundamental molecular machinery
03:20.2 is the actin that self-assembles into filaments,
03:23.2 as I just told you about.
03:26.2 If we look at the leading edge of a crawling cell,
03:28.3 and in this particular case
03:30.3 the image on the bottom here
03:32.1 is a fixed cytoskeleton from a cell called a keratocyte,
03:35.1 and we can label the actin filaments in that cell
03:37.2 and see their overall distribution.
03:38.2 In this particular case,
03:39.3 they are very much focused towards the front,
03:43.0 towards the leading edge.
03:44.1 And in this beautiful electron micrograph
03:46.0 by Tanya Svitkina
03:47.2 you can see at the ultrastructural level
03:50.1 exactly how abundant
03:52.2 and how densely intertwined and crosslinked
03:54.1 these actin filaments actually are.
03:56.0 The top shows a relatively low magnification
03:58.2 and then the bottom part here
04:00.1 shows individual boxes
04:02.2 magnified from the marked portions in the top
04:05.2 where you can see the actin filaments
04:07.2 are not only crosslinked to each other,
04:09.1 but also actually branching off one another
04:11.2 to make this large structure
04:13.0 that is able to push the cell forward.
04:16.0 Now, when we think about how
04:18.1 these tiny little nanometer-scale proteins
04:19.2 are able to assemble
04:22.1 tnto such an enormous structure that's able to perform
04:24.2 this vast amount of physical work,
04:26.0 of pushing a whole cell forward,
04:27.2 the most important place to start
04:29.1 is just with the filament itself.
04:30.2 As I mentioned,
04:32.1 actin is a globular protein
04:35.0 is shown here at the top --
04:36.3 and this image gives you a sense
04:39.0 at the structural level
04:40.2 of how all of those monomers are able to come together
04:42.1 in order to make a filament.
04:44.0 Now, this textbook diagram over here
04:46.2 emphasizes another very, very important aspect
04:48.2 of actin behavior,
04:49.2 which is critical for understanding motility,
04:51.2 and that's the fact that it's able to bind and hydrolyze nucleotide.
04:54.3 And although this diagram is somewhat oversimplified,
04:57.0 the point is that
04:59.1 when actin is bound to ATP
05:01.1 it has a propensity to assemble into filaments.
05:03.3 Once it enters the filament,
05:05.1 the hydrolytic activity of actin is activated,
05:08.0 and so within the filament
05:10.0 the ATP is converted to ADP.
05:12.1 Once that has happened,
05:13.2 the ADP-containing filaments
05:15.1 are relatively less stable,
05:16.3 and those monomers are able to come off again.
05:19.1 Now, the consequence of this is,
05:20.3 as long as there's free ATP present,
05:22.1 actin will bind ATP,
05:24.0 assemble into a filament,
05:27.2 and keep this going in a treadmilling-type cycle
05:30.3 And it's that assembly and disassembly
05:33.0 that actually drives a lot of the dynamic movements of cells
05:35.2 that we'll be talking about.
05:36.3 A few other important things to bear in mind about actin
05:39.1 when we're thinking about how it works inside of a living cell
05:41.2 is that in order to form a filament
05:44.1 it's nucleation that's the rate-limiting step.
05:46.0 So, there's lot of actin monomers
05:47.2 floating around all the time,
05:49.0 some of them are bound to other proteins
05:51.1 that modulate their propensity to make filaments,
05:53.2 but when three of them come together,
05:55.1 or when they're catalyzed to come together
05:57.1 by a specialized nucleating protein,
05:59.1 that's the initiating event
06:01.0 that will allow filament elongation to begin.
06:03.3 Because of the constant hydrolysis of ATP
06:06.0 in this process of assembly and disassembly,
06:08.2 equilibrium is never reached inside of a cell.
06:10.2 All of the actin filaments
06:12.0 are always constantly turning over.
06:13.3 This is obviously true in something like the neutrophil
06:15.3 that has to rearrange all its actin filaments
06:17.2 so fast in order to crawl,
06:19.0 but it's also true,
06:21.1 although to a somewhat lesser extent,
06:22.3 in cells like skeletal muscle or cardiac muscle,
06:25.1 where the actin filaments are relatively static.
06:28.0 Always, there's ATP hydrolysis
06:29.2 and always there's some kind of turnover
06:31.1 associated with the filament structures.
06:33.3 In the kinds of cells we're going to be focusing on,
06:36.1 so, relatively rapidly moving cells,
06:37.3 the average half-life of the filaments
06:39.1 is on the order of a few tens of seconds
06:40.2 or maybe a few minutes at most,
06:42.2 and they're organized in order to make
06:45.1 these large-scale structures that can span
06:46.2 throughout the entire cell and govern its behavior
06:48.3 by literally hundreds of different kinds of
06:51.2 actin binding molecules
06:53.0 that bind along the filament,
06:54.2 regulate their assembly and disassembly,
06:57.0 crosslink them, sever them, bundle them together...
06:59.2 anything you can imagine that a protein can do to these filamentous structures,
07:02.2 there's probably some protein out there that does it.
07:05.1 And one of the things that I particularly want to focus on
07:07.1 in our discussion today
07:08.2 is this last fact down here at the bottom here,
07:10.2 that polymerization of actin,
07:12.2 and also actually of tubulin into microtubules,
07:15.1 can actually generate physical force,
07:17.2 very similar to the way that a molecular motor
07:19.2 can generate physical force.
07:21.1 And in the context of cell motility,
07:23.0 like those movies that I just showed you,
07:24.2 it's actually the assembly of actin
07:26.1 at the leading edge
07:27.3 that is thought to be one of the major drivers
07:29.1 of that particular kind of motion.
07:32.3 So, let's take a step back for a minute
07:34.3 and think about how it is that
07:36.2 polymerization of a protein
07:38.1 can actually lead to physical force.
07:39.3 One way I find helpful to think about it
07:42.1 is to imagine the protein polymerization reaction
07:43.3 simply as a biochemical binding reaction,
07:45.3 where on the left-hand side
07:48.0 we have a small subunit that's four subunits long
07:51.1 and a free subunit.
07:53.1 Those are going to be the reactants.
07:55.3 When they bind to each other
07:57.1 with some on rate designated by k-on,
07:59.0 they'll make a filament that's one subunit longer
08:03.3 And because this is just a binding reaction,
08:05.1 it can go to equilibrium,
08:06.2 and the ratio between the k-on and the k-off
08:08.2 is going to give you some equilibrium constant,
08:10.2 which for cytoskeletal proteins we call
08:14.1 That stands for critical concentration.
08:16.1 Now, if we think about a filament like this
08:20.2 that is growing not at equilibrium,
08:22.2 but rather is growing
08:24.1 with some sort of non-equilibrium situation
08:26.1 in its environment,
08:28.0 we can end up with a situation like
08:30.1 what's illustrated at the top here.
08:31.2 If we have excess monomers present,
08:33.2 so that is, you know,
08:34.3 just one little filament and lots of monomers,
08:36.1 but more than would be present at equilibrium,
08:38.2 then those filaments will tend to polymerize.
08:41.0 Because we have excess monomers,
08:42.3 that will put us in a regime
08:44.1 where polymerization is favored thermodynamically
08:49.3 is gonna be negative overall.
08:52.2 Now, if we imagine taking one of those little filaments
08:54.2 and putting it between,
08:56.2 on the one side, a rigid barrier like a wall,
08:59.1 and on the other side
09:01.0 a smaller barrier that can move back and forth,
09:03.1 as long as the free energy
09:06.2 that is released by the polymerization reaction
09:09.1 is greater than or equal to
09:11.2 the amount of work
09:14.1 that it takes to move that little black barrier
09:16.2 through that distance delta,
09:17.3 then the overall polymerization reaction
09:19.1 will still be favored
09:20.2 and the barrier will be pushed forward.
09:23.0 So, using that kind of very fundamental
09:26.0 thermodynamic, energetic argument,
09:27.2 Terrell Hill and Marc Kirschner in 1982
09:29.2 wrote this fabulous 125 page manifesto
09:32.3 describing all the different ways that protein polymerization
09:34.3 and depolymerization
09:36.1 can be used to generate force.
09:37.1 And from their calculations
09:39.0 they came up with this simple rule
09:41.0 about the max amount of force
09:43.0 that can be generated by polymerization of a filament.
09:45.1 That depends on the size of the monomer, ÃŽÂ´,
09:48.0 and depends on the concentration of free monomers
09:50.1 in solution,
09:51.2 relative to this critical concentration,
09:53.2 or the equilibrium binding constant.
09:56.2 So, if you calculate for actin inside of a living cell
09:59.2 what those numbers actually should turn out to be,
10:01.1 we estimate that the amount of force that
10:03.1 you should be able to get from a single actin filament
10:04.2 is on the order of actually 5-10 picoNewtons,
10:08.0 which surprisingly is just about the same amount of force
10:10.2 that you get from a real molecular motor
10:12.1 like myosin or kinesin.
10:14.0 So, although this kind of polymerization-driven motion
10:16.1 is maybe not as familiar
10:17.2 and not as intuitive,
10:18.3 it's actually a perfectly good motor
10:20.1 and it can generate just as much force
10:21.3 as something like myosin.
10:24.2 Now, thinking about how fast processes
10:27.1 can be driven in this way
10:29.0 is a little bit different from thinking about the energetics.
10:30.2 In order to understand how fast things go,
10:32.2 we have to have some sort of kinetic model,
10:34.1 and in particular we're thinking about a filament
10:36.2 that's polymerizing up against a barrier...
10:38.1 we have to have some idea in mind
10:40.3 about how that space can be opened up
10:42.2 for another monomer to come and sneak
10:44.2 onto the end of the filament,
10:46.1 and extend the filament,
10:47.1 and then push the barrier.
10:48.2 And George Oster and his colleagues
10:49.2 have done a lot of interesting calculations,
10:51.2 assuming different kinds of thermal flexibility
10:53.2 and different components of the system.
10:55.2 For example, you can imagine the barrier
10:57.3 is able to move back and forth,
10:59.0 or you could imagine the filament
11:00.1 is able to flex up and down a little bit,
11:01.3 or you could imagine the crosslinks between the filaments
11:04.2 maybe can move their angles.
11:06.1 All of those things can give you
11:09.1 enough space to allow actin monomers to come in
11:10.2 and continue the polymerization reaction
11:12.0 and generate force to
11:14.0 physically move the barrier forward,
11:16.1 and doing some reasonable calculations
11:17.2 about how fast those kinds of things
11:19.1 should be able to happen in the context of a living cell,
11:21.2 they've been able to estimate that this very non-intuitive reaction
11:24.3 is enough,
11:26.1 is fast enough to drive processes
11:27.2 even like the extension of that neutrophil
11:29.1 as it's chasing a bacterium.
11:33.1 So, those are calculations...
11:34.1 of course, it's always very satisfying
11:35.2 to be able to see an experimental result
11:37.3 that confirms the models
11:40.1 that have been put forth,
11:41.2 and a few years ago
11:43.2 Matt Footer in my lab,
11:44.2 together with our collaborators
11:46.2 Marileen Dogterom and Jacob Kerssemakers
11:48.1 in Amsterdam,
11:49.1 were able to actually measure the force of actin polymerization directly.
11:53.1 And the way that they did this was
11:55.2 to prepare a very stiff,
11:58.1 rigid actin bundle
12:00.1 from the sperm of the horseshoe crab
12:01.1 -- it's called an acrosome --
12:02.2 and those acrosomes
12:04.0 can grow actin filaments off of one tip.
12:07.0 So, after preparing the acrosomes
12:08.1 and making sure they were able to polymerize actin,
12:11.1 Matt and Jacob put those acrosomes
12:13.1 into a special kind of optical trap
12:15.2 that had been invented in Marileen's lab,
12:17.2 in order to pull this structure,
12:19.3 where here is the filament bundle
12:23.0 and then there's a bead that's being held
12:25.0 onto by the optical trap,
12:26.3 and bring it up against a microfabricated wall.
12:30.0 In that configuration, then,
12:31.2 when the bead is pushing up against the wall,
12:35.2 when we add actin monomers to that mixture,
12:37.2 the actin monomers,
12:39.0 given the thermal fluctuations of the bead and the trap,
12:41.3 are able to sneak in between the end of the acrosome and the wall
12:45.3 and actually extend that bundle of actin filaments
12:48.2 and push the bead out of the trap.
12:51.1 We're able to measure, very precisely,
12:53.1 exactly how far the bead has moved out of the trap
12:55.3 and use that to measure the amount of force
12:57.3 that's generated in this experiment,
12:59.0 and what they were able to find
13:00.2 was that the actin filament
13:02.0 will continue to grow,
13:03.1 as you see in this trace,
13:05.0 until it reaches a stall,
13:06.2 where the force is exactly balanced
13:09.2 by the pushing against the wall.
13:11.1 And the force that they can measure
13:13.1 for these small bundles of actin filaments
13:15.0 is on the order of a few picoNewtons,
13:16.2 1 or 2 picoNewtons,
13:17.3 which is very close to what
13:20.0 that thermodynamic prediction
13:21.1 would have guessed
13:23.1 under the conditions that we used.
13:26.1 So, it's very satisfying to see here
13:27.3 that we've got one filament
13:28.3 that is able to actually directly generate a force
13:31.0 that we can measure,
13:32.1 but if we want to think about how this works in the context
13:34.1 of cell motility,
13:35.1 we have to actually think about many filaments working together,
13:38.1 and this kind of experiment
13:39.2 is not really going to help us understand
13:41.2 what the rules are
13:43.1 that govern the cooperation
13:45.0 among a whole gang of filaments
13:46.2 that are operating over a very large area of space.
13:50.2 In order to get some mechanistic insight
13:52.2 into this question of
13:54.2 What happens when many actin filaments are trying to work together?,
13:56.2 we've actually been able to learn a tremendous amount
13:58.3 from a completely different area of biology
14:01.1 and that's actually the field of bacterial pathogenesis.
14:04.3 So, it turns out that infectious bacteria,
14:07.1 and also viruses,
14:08.2 that live inside of mammalian hosts
14:11.1 and exploit the resources
14:13.0 of their mammalian hosts' body
14:14.1 in order to replicate and in order to spread,
14:16.3 actually turn out to be excellent cell biologists.
14:19.3 They know all the little details,
14:21.1 all the little ins and outs of exactly how mammalian cells work,
14:25.1 exactly what the most important aspects are
14:27.1 for things like their replication or their motion,
14:28.3 so that the pathogens can take advantage
14:31.2 of those properties of their hosts' cells
14:33.2 in order to perform whatever it is the pathogen
14:35.1 wishes to perform.
14:37.2 One of the examples of a pathogen,
14:39.3 a bacterial pathogen,
14:41.1 that's turned out to be a really outstanding cell biologist
14:43.1 is this organism here, Listeria monocytogenes.
14:46.3 Now, Listeria is a common soil organism
14:49.1 and all of us eat a little bit of it every day
14:52.0 when we eat a salad or something,
14:53.3 but when we consume food
14:56.1 that is very heavily contaminated with Listeria
14:58.2 -- this could be something like a cantaloupe,
15:00.1 or a chicken,
15:01.2 or sometimes ice cream --
15:03.0 then the bacteria are actually able
15:07.1 to invade the mammalian host cells
15:09.0 that line our intestines.
15:11.0 And for most healthy adults,
15:13.1 this infection is self-limiting,
15:14.1 you'd never even know you had it,
15:15.2 but for people who are immunocompromised
15:17.1 or for pregnant women,
15:18.2 the infection can actually spread throughout the entire body.
15:21.1 And what we're looking at here
15:23.3 in the micrograph
15:25.1 is a single mammalian host cell
15:26.3 that has been grown in tissue culture
15:29.1 and that was infected
15:31.0 with probably one bacterium
15:32.2 about five hours before this movie was made.
15:34.2 And you can see the bacteria have been replicating in the cytoplasm
15:38.2 -- they're each of these little dark
15:40.0 bullet-shaped objects that you see --
15:42.0 and what I hope that you'll appreciate
15:43.2 when I start to play the movie
15:44.3 is that these bacteria are able
15:46.2 to not only replicate in the most cell cytoplasm,
15:48.1 but they're also able to do this absolutely extraordinary thing,
15:50.3 which is they can cruise around
15:53.1 like little speedboats.
15:55.2 And in fact the motion of these bacteria
15:58.1 inside the cytoplasm of the host cell
16:00.0 is driven by the assembly of host cell actin filaments.
16:04.3 So, behind each of the little bacteria that's moving
16:07.2 you can see a phase-dense streak,
16:09.0 and that's actually what's called a comet tail,
16:11.3 that is the actin left behind the bacterium as it moved.
16:17.0 Now, we can see for example in this fluorescence micrograph
16:19.2 that those filaments,
16:21.1 those comet tails that are made of host cell actin,
16:24.0 where here the bacteria have been labeled red with a fluorophore
16:26.3 and then the actin filaments of the host cell
16:28.2 have been labeled in green.
16:30.0 And you can see every bacterium
16:31.2 that's inside the cell
16:33.1 is associated either with a little cloud
16:34.2 or else with one of these comet tails
16:36.1 of actin filaments.
16:39.1 And looking a little more closely
16:40.2 at the level of the electron microscope,
16:42.1 you can see the way that those filaments are arranged
16:45.1 in this incredibly dense crosslinked structure
16:47.2 that traces behind the bacterium
16:50.0 and records the path it traveled over.
16:52.2 Now, having analyzed some of the dynamic behavior
16:54.3 of the actin filaments associated with bacteria comet tails,
16:58.0 what I can tell you is that the assembly of all of the filaments in this comet tail
17:01.2 took place right at the bacterial surface,
17:04.1 and it was actually the assembly of those actin filaments
17:06.2 up against that smooth cell wall of the bacterium
17:09.3 that generated the force
17:11.3 to push the bacterium through the cytoplasm of this host cell.
17:16.0 As we move further back in the comet tail,
17:18.1 the actin filaments stay crosslinked to one another
17:20.2 to make this very nice, tight,
17:22.1 characteristic columnated line,
17:24.0 and the filaments actually remain stationary in the cytoplasm,
17:26.2 like the wake behind a boat,
17:28.2 as a history of where a bacterium has moved.
17:31.2 And then, finally,
17:33.0 at the back end of the comet tail,
17:34.1 the old filaments fall apart.
17:35.2 Because their ATP has been hydrolyzed,
17:37.0 they get disassembled by accessory factors
17:39.2 in order to regenerate actin monomers
17:41.2 that can then rejoin the actin monomer pool,
17:44.1 diffuse around the cell,
17:45.2 and come back to the front,
17:46.2 where they can continue to generate force.
17:50.0 So, part of the reason that this particular organism
17:52.2 has been so appealing as a way to
17:55.3 study the cooperation among many actin filaments
17:57.2 is because it is particularly amenable
17:59.0 to both biophysical and biochemical manipulation.
18:02.3 So, for example, on the biochemical side,
18:04.2 we were able to reconstitute the movement of this bacterium
18:07.1 in cytoplasmic extracts,
18:09.1 more than 20 years ago now.
18:10.3 And then a series of biochemists
18:13.1 started fractionating extracts
18:15.1 and trying combinations of ideas of different proteins
18:17.2 that they thought might contribute to this process,
18:20.1 until in 1999 a really heroic piece of work
18:22.2 from Marie-France Carlier's lab
18:24.1 was able to demonstrate
18:26.1 motility of these bacteria
18:27.3 using a mixture of only purified proteins
18:29.1 that had all been completely identified.
18:32.3 Now, in the meantime,
18:34.1 we were actually also able to get rid of the bacterium
18:37.0 and replace it with a polystyrene bead,
18:38.3 which you see... sorry, which you see right here.
18:42.0 That polystyrene bead is coated with
18:43.2 actually just a single protein from the bacterium,
18:46.1 which is enough to initiate this whole reaction
18:48.2 and grow this beautiful comet tail
18:50.2 that looks very much like the comet tail associated with a real bacterium.
18:53.2 And in fact when we put those beads
18:55.2 under the microscope,
18:57.0 what we can see, here with labeled actin,
18:59.1 is that those beads actually jet around
19:01.1 and in fact look exactly like the bacteria do.
19:04.1 So, this is something that be reconstituted
19:06.2 both biochemically and biophysically
19:08.0 with these artificial substrates.
19:10.2 So, based on those kind of experiments,
19:12.1 over a period of about 10 years
19:14.1 about 20 different labs
19:16.1 contributed to understanding the roles
19:18.1 of all the different proteins associated with this form of motility,
19:21.0 identifying the proteins,
19:22.1 figuring out their roles,
19:23.2 and figuring out how they all work together.
19:25.1 So, to summarize all this work,
19:27.0 we're gonna focus right on the surface of the bacterium,
19:28.2 which is where all the action takes place,
19:30.2 and the first step in motility
19:31.3 is that the bacterium has to express
19:34.0 a particular protein.
19:35.1 For Listeria monocytogenes,
19:36.2 it happens to be called ActA;
19:38.1 for other bacteria that do the same kind of trick,
19:39.2 they express other proteins
19:41.2 that interestingly enough actually evolved
19:44.0 independently from ActA,
19:45.1 so it seems like this mechanism
19:47.1 for pathogen actin-based motility
19:48.3 has actually appeared multiple times
19:50.3 in evolution
19:52.2 in apparently completely unrelated strains of bacteria.
19:56.0 When that protein is presented
19:58.0 on the surface of the bacterial cell,
19:59.1 it's able to bind to particular factors
20:01.3 in the host cell cytoplasm
20:03.2 that are critical for nucleation of actin filaments,
20:06.1 and remember that nucleation is the rate-limiting step.
20:09.2 So, for the ActA protein
20:11.3 from Listeria monocytogenes,
20:14.2 it binds directly to the nucleating complex,
20:16.1 called the Arp2/3 complex,
20:18.0 that is then able to first bind
20:20.1 to the side of a preexisting actin filament
20:22.1 that's already in the tail,
20:23.2 and then nucleate the growth of the new actin filament
20:26.2 in a branch off of the side of that old actin filament.
20:30.1 And you'll remember,
20:31.2 you saw branches like that in the electron micrographs
20:33.2 at the leading edge of a crawling cell -
20:36.1 it's thought to work by a very similar process.
20:38.2 Now, as those filament are nucleated,
20:40.1 they grow by addition of actin monomers
20:43.1 that are present in the cytoplasm,
20:45.3 just floating around,
20:47.1 and landing on the ends of those filaments
20:48.2 by diffusion.
20:50.0 And that growth, as I said,
20:52.0 pushes the bacterium through the cytoplasm
20:53.2 using this force generation mechanism
20:55.3 that I described.
20:57.2 Now, this whole thing doesn't require
20:59.2 any classical molecular motors
21:00.3 - there's no involvement of myosin
21:02.1 or any of its relatives in this process.
21:05.2 As the bacterium moves forward,
21:07.0 it leaves those actin filaments behind,
21:09.2 including leaving the branch junctions behind,
21:11.2 and those old filaments,
21:13.3 as they're ripped off the surface of the bacterium
21:16.2 based on the motion of the bacterium,
21:18.1 get capped by proteins like CapZ or gelsolin,
21:20.2 so that they don't continue to grow out of control,
21:22.3 and that keeps the comet tail in its nice,
21:24.2 characteristic narrow shape.
21:27.1 And then finally, depolymerizing proteins,
21:29.1 things like cofilin and ADF,
21:31.0 will come in and disassemble those filaments,
21:32.3 tear them apart into their native monomers,
21:36.2 so that the whole thing, again,
21:38.2 can continue indefinitely,
21:39.3 as long as the bacterium is present
21:41.2 in a cytoplasm that has these factors in there.
21:46.0 So, thinking about this mechanism,
21:47.3 which we understand in a lot of molecular detail,
21:49.3 there are several things about it that I still find absolutely astonishing.
21:53.2 So, one is just that it works so incredibly fast,
21:57.0 and to put numbers on that,
21:58.2 here's an image of Listeria monocytogenes,
22:00.3 this organism about 2 microns long,
22:03.2 and its typical speed is about 0.2 microns/second,
22:07.1 and it's moving at that rate
22:09.0 by piling up all these tiny little actin monomers
22:10.3 that are only about 4 nanometers in diameter.
22:13.2 Now, it's hard to really judge...
22:15.3 is that fast, is that slow?
22:17.1 So let's compare it to some macroscopic thing
22:20.0 for which we have some physical intuition
22:21.2 about what's fast or slow.
22:23.2 And my former student Fred Soo
22:25.3 pointed out that the geometry of the Listeria moving
22:28.3 with its comet tail
22:30.0 is actually very similar to the geometry
22:31.2 of the Ohio class nuclear submarine.
22:33.2 Just like the bacterium, it's a cylinder
22:36.2 that's capped on two ends with hemispheres.
22:38.0 As it moves through its medium,
22:39.3 it leaves behind these characteristics curving patterns behind it.
22:43.1 And with the nuclear submarine,
22:45.1 we know that the length of that
22:47.1 is about 560 feet
22:49.0 and its typical cruising speed
22:50.2 is about 30 feet/second.
22:52.0 So, what would it mean if we scaled up Listeria
22:54.3 to be as big as the submarine.
22:56.3 Well, in order to do that,
22:58.2 we'd have to have the actin monomers go from being 4 nanometers
23:01.1 to being about the size of a basketball,
23:02.2 or about a foot across.
23:04.1 And if we do that scaling
23:05.3 and then see how does that apply
23:07.1 to the other numbers I've shown you
23:10.0 here for length and speed,
23:11.1 it turns out that if actin monomers
23:13.1 were the size of a basketball,
23:14.2 then the length of the bacterium
23:16.1 would be about 500 feet,
23:17.2 so very comparable to the submarine,
23:19.2 and its speed would be about 50 feet/second.
23:22.1 So, it can just as fast as the submarine.
23:24.1 And the astonishing thing about this fact is...
23:27.1 what it means is if you were in a satellite
23:30.0 watching submarines cruising around
23:32.1 on the surface of the ocean,
23:34.1 the movies of that would look exactly identical
23:35.3 to the movie that I showed you before of Listeria motility.
23:38.2 It's really just the same thing,
23:40.0 just scaled up massively.
23:42.1 But the thing that's most amazing
23:43.3 is the submarine is just moving through water.
23:46.3 But the bacterium is moving through cytoplasm,
23:50.1 and cytoplasm is dense,
23:52.0 cytoplasm is filled with all of these cytoskeletal filaments,
23:55.1 it's chock full of organelles,
23:57.0 there's all sorts of things in the way,
23:58.2 and if you actually try to calculate what the equivalent would be,
24:00.2 it's not like a submarine moving through water.
24:03.3 It's actually a lot closer to a submarine
24:06.0 moving through concrete.
24:08.1 So, it must be the case that
24:10.3 the bacterium is generating a tremendous amount of force
24:13.1 with this coordinated assembly of the actin filaments
24:16.1 in its comet tail.
24:18.1 Okay, again,
24:19.2 it would be nice to see some experimental data
24:21.0 that can tell us something about
24:22.2 how much force is actually being generated.
24:24.1 Well, one way you can get a bit of an intuitive feel for that
24:27.1 is by looking at these beautiful movies
24:28.3 made by my former student, Catherine Lacayo,
24:31.0 where she has labeled the mitochondria
24:33.1 in a host cell with a red dye,
24:36.0 and the bacteria are expressing GFP,
24:37.3 so you see them in green.
24:39.3 And as the movie plays,
24:41.1 we can watch the bacteria move around
24:42.2 and can see, actually,
24:44.2 what happens when the run into a mitochondria,
24:46.2 and remember, mitochondria are about the same size as bacteria.
24:49.2 Long in the distant past,
24:51.0 they actually used to be bacteria,
24:52.2 so this is a pretty equal battle.
24:54.2 And if we zoom in
24:57.2 and you see the bacterium is gonna...
24:59.1 as the movie loops back, you'll see it come in from the top... h
25:02.0 ere it comes,
25:04.1 and it just slices its way through that big pile of mitochondria.
25:05.3 It shoves them aside without really
25:07.2 even slowing down at all.
25:09.0 So, this has got to be a lot of force
25:10.2 to be able to just push all the organelles in the cell
25:12.3 out of the way.
25:15.1 Now, Dan Fletcher,
25:16.2 when he was a postdoc in my lab
25:18.0 -- he's now a professor at UC Berkeley --
25:19.2 he worked out this really very clever technique
25:22.0 for measuring large forces
25:24.2 using this kind of actin polymerization geometry,
25:27.2 and what he did was he took advantage of the fact
25:29.2 that we could reconstitute motility
25:31.2 on those little polystyrene beads
25:33.2 and then just took one of those little ActA-coated polystyrene beads
25:36.2 and stuck it on the end of an AFM cantilever.
25:39.1 And what this meant was he could then
25:41.2 bring that cantilever down to a glass surface,
25:44.1 allow the actin polymerization
25:46.1 to occur in between the tip of the cantilever and the glass,
25:48.1 and that would push the cantilever upward
25:50.2 in a way that he could measure the displacement
25:52.1 and therefore also measure the force.
25:54.2 And looking at these traces,
25:56.2 there's a couple of things that are very astonishing about them.
25:58.3 The most astonishing one actually being the stall force,
26:01.2 which you see up here,
26:03.2 for a bead attached to the end of a cantilever,
26:05.2 is on the order of about 300 nanoNewtons.
26:09.0 Now remember, before we were talking about picoNewtons,
26:10.3 so it's really clear that when you get
26:12.2 a whole bunch of actin filaments together,
26:14.1 they're able to cooperate with one another
26:15.2 in order to generate tremendous amounts of force.
26:18.3 And based on estimates that Dan's been able to do
26:21.0 of the density of the actin filaments in that gel,
26:23.1 if you then estimate
26:25.2 how much force comes from a single actin filament,
26:27.0 then again we get down to numbers
26:29.0 that are on the order of about
26:30.2 a few picoNewtons per filament,
26:32.3 which is again what was calculated
26:35.0 originally from the thermodynamic argument,
26:36.3 what we measured for single filaments,
26:38.2 and what also seems to be fulfilled, now,
26:40.1 in the context of this branching growth
26:42.1 of a network
26:43.3 against the surface of, in this case, a cantilever,
26:46.0 or in the cell against the surface of a bacterium.
26:50.0 So, overall,
26:51.3 what I've told you about
26:53.2 is a set of processes
26:55.1 that are essentially based simply on the self-organization
26:57.2 and self-assembly
26:59.0 of tiny little nanometer-scale protein subunits
27:00.3 in order to make an ensemble
27:03.0 that is much, much greater than the sum of its parts,
27:05.1 in terms of its ability to do real physical work,
27:07.3 such as pushing a bacterium around inside of a cell.
27:10.1 And part of the reason that this is interesting
27:13.2 is because it applies not only to the context
27:15.1 of the bacterium moving around,
27:16.2 but also to what's happening actually
27:18.2 at the leading edge of a crawling cell.
27:21.1 Basically, you can think of the bacterium
27:22.2 as imitating a little fragment
27:25.1 of a crawling cell's plasma membrane,
27:27.3 and the growth of these branching actin filaments
27:30.1 up against that membrane
27:31.2 are pushing the edge of the cell forward
27:33.2 in the same way that they push the bacteria around
27:36.2 inside of the cells or in extracts.
27:39.1 Now, there's obviously a lot more to say
27:41.1 about how all these ideas apply
27:42.2 to the actual problem of cell motility,
27:44.0 and if you'd like to hear more about that,
27:46.0 please come back for part 2.
27:47.3 Finally, I'd like to end
27:49.3 by acknowledging the absolutely amazing team of colleagues
27:53.1 that I've had working on this project, now, for many years.
27:55.1 I've listed here the members of my group
27:57.2 who have participated in various different aspects
27:59.2 of characterization of the cell biology and biophysics of Listeria,
28:03.1 and in particular today I showed experiments
28:04.2 that were done by Matthew Footer,
28:06.1 by Lisa Cameron,
28:08.0 by Catherine Lacayo,
28:11.0 and also by Dan Fletcher.
28:13.1 And we've also had the privilege
28:15.0 of just absolutely wonderful collaborators.
28:16.2 The most important one I want to draw attention to
28:19.0 is Dan Portnoy at UC Berkeley,
28:20.1 who's been a close collaborator for almost 25 years now
28:23.3 on all of these processes associated with Listeria motility.
28:27.0 Thank you very much for your attention.
In her seminar, Dr. Theriot describes a thermodynamic model that proposes a mechanism by which the energy associated with a polymerizing cytoskeletal filament can be converted to force for cell movements. What predictions of the model would you measure in order to test the idea that the proposed mechanism is used to propel a Listeria bacterium through cytoplasm in infected cells and how might this illuminate the mechanism of neutrophil motility in the blood?
Julie Theriot attended college at the Massachusetts Institute of Technology, graduating with degrees in Physics and Biology. She pursued graduate training at the University of California, San Francisco, earning her Ph.D. in Cell Biology in 1993. After four years as a Fellow at the Whitehead Institute for Biomedical Research, Theriot moved to Stanford University School… Continue Reading