Session 5: Cell Motility

00:07.3 Hello.
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 Ramón 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
04:33.2 #NAME?
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,
05:25.1 hydrolyze,
05:26.2 disassemble,
05:27.2 and keep this going in a treadmilling-type cycle
05:29.2 indefinitely.
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
08:01.2 #NAME?
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
08:13.1 C-crit.
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
08:48.1 #NAME?
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, δ,
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.