Molecular Motor Proteins

Transcript of Part 3: Regulation of Mammalian Dynein

00:00:15.10	Hello.
00:00:16.07	I'm Ron Vale from UCSF,
00:00:18.20	and in this part of my iBiology talk
00:00:20.04	I'd like to discuss
00:00:21.23	the regulation of mammalian dynein,
00:00:24.21	research that's come from out of our lab
00:00:26.28	as well as others in the field.
00:00:29.26	So, we began
00:00:32.00	to do our research on dynein
00:00:34.15	using yeast cytoplasmic dynein
00:00:36.27	and there are many reasons for this.
00:00:39.01	It was easy to express
00:00:41.28	large quantities of yeast dynein
00:00:44.23	and we can also modify it
00:00:46.25	to create different types of recombinant protein,
00:00:49.13	and furthermore when we tested
00:00:51.13	yeast cytoplasmic dynein
00:00:53.13	for single molecule motility,
00:00:55.22	we got this type of fantastic motion
00:00:57.27	that you see here.
00:00:59.27	Again, all of these individual green spots
00:01:02.00	are single molecules of dynein
00:01:04.09	that are moving along microtubule tracks,
00:01:06.29	shown here in blue.
00:01:09.04	So, later we wanted
00:01:11.02	to extend our work
00:01:12.26	to mammalian dynein...
00:01:16.05	this was work that Rick McKenney did,
00:01:18.00	and we thought it was going to be
00:01:19.28	fairly straightforward,
00:01:21.07	we'd just do all of the same things
00:01:22.28	that we did with yeast
00:01:24.13	and apply it to dynein from a different species.
00:01:27.07	But unfortunately,
00:01:28.19	the initial results were rather disappointing.
00:01:31.26	So we first tried to make
00:01:33.24	recombinant human dynein,
00:01:35.24	very much like the type of recombinant dynein
00:01:38.03	that we made with yeast,
00:01:40.27	and the dyneins here are in red in this case,
00:01:43.14	but we saw very little motility.
00:01:45.17	They would bind to the microtubule
00:01:47.08	but did not move.
00:01:48.29	So we took, then, a step back
00:01:51.15	and decided to purify dynein
00:01:53.26	from a native tissue, from rat brain.
00:01:56.15	So this is the normal dynein from rat brain,
00:02:00.10	and again we purified it,
00:02:02.02	we fluorescently labeled it,
00:02:03.22	but again found fairly disappointing results.
00:02:06.12	We saw dynein binding to the microtubule,
00:02:08.19	but no movement.
00:02:11.05	However, these dyneins
00:02:13.14	weren't completely inactive
00:02:15.02	because we then tested them
00:02:17.04	in another kind of motility assay
00:02:19.17	and saw that they could produce movement,
00:02:22.24	and this is what's called a microtubule gliding assay.
00:02:24.28	In this case, the dynein
00:02:27.17	is coated and attached
00:02:29.21	onto a glass cover slip.
00:02:31.09	It then grabs hold of a microtubule
00:02:33.04	and then it can move it across the surface.
00:02:35.12	So, in this case, however,
00:02:37.04	this is not single molecule motility;
00:02:39.07	there are many dynein molecules
00:02:40.24	underneath that microtubule.
00:02:42.24	And these are the microtubules, here.
00:02:44.20	When you give it ATP,
00:02:46.24	they move very nicely across the glass.
00:02:49.20	So, the conundrum was:
00:02:51.21	Why didn't we see any single molecule motility,
00:02:55.02	processive motility of the dynein?
00:02:58.05	We saw it in yeast,
00:02:59.28	but we didn't see it in mammalian cells,
00:03:01.23	and we expected the mammalian dynein
00:03:04.00	also to be processive,
00:03:05.20	especially if it's going to carry cargo
00:03:08.07	for long distances inside of cells.
00:03:11.25	So when you get a negative result like this,
00:03:14.12	one often has to go back to the drawing board
00:03:16.23	and figure out,
00:03:18.09	well, maybe something
00:03:20.01	wasn't right about the experiment.
00:03:21.19	Maybe the conditions for the assay
00:03:24.05	weren't quite right,
00:03:25.20	whatever buffers we were using
00:03:27.09	or other elements of the assay,
00:03:29.14	or maybe we're just
00:03:31.13	entirely missing some critical component
00:03:33.19	that dynein needs to move
00:03:35.12	at a single molecule level.
00:03:37.14	So, we began to test this latter possibility,
00:03:40.28	and a good candidate for that missing factor
00:03:43.21	was a protein complex
00:03:46.00	that was known as dynactin.
00:03:48.01	Now, dynactin was discovered
00:03:50.09	many years ago
00:03:51.28	by Schroer and colleagues
00:03:53.23	as a cofactor
00:03:55.21	that would stimulate organelle transport by dynein.
00:03:58.17	And subsequently
00:04:00.10	it's been found that dynactin
00:04:01.27	is required for many functions
00:04:03.21	of cytoplasmic dynein,
00:04:05.00	effectively a knockout of dynactin
00:04:07.14	produces a very similar phenotype
00:04:09.15	to knocking out dynein itself.
00:04:12.10	So it seems to be involved
00:04:13.29	in many kinds of dynein functions,
00:04:16.12	although the exact role of dynactin
00:04:18.25	has not been entirely clear.
00:04:21.19	So, this is what dynactin looks like
00:04:24.25	and it's a monstrous protein
00:04:26.18	of comparable size to dynein itself.
00:04:29.02	It has about 23 proteins
00:04:31.09	in one dynactin complex
00:04:33.07	and it's about a megaDalton in size.
00:04:36.02	And it has a very interesting
00:04:38.11	and unusual architecture.
00:04:40.02	The core of dynactin
00:04:41.19	is composed of a short filament
00:04:44.04	of actin-related proteins
00:04:45.23	that are capped
00:04:47.26	at both ends by a different group
00:04:51.03	of special capping proteins,
00:04:53.00	and in the middle
00:04:54.18	there's another group of proteins
00:04:56.22	that create a type of shoulder
00:04:59.24	on one part of the dynactin molecule,
00:05:03.05	and there's a long coiled-coil
00:05:06.20	that extends from this core structure,
00:05:09.06	called p150,
00:05:10.23	that also contains
00:05:12.13	a microtubule binding domain at the end.
00:05:14.29	Now, recently, there's also been
00:05:17.02	some exciting developments
00:05:19.19	because Andrew Carter's label
00:05:21.08	recently got a cryo-EM structure
00:05:24.10	of dynactin,
00:05:25.26	so we now know the organization
00:05:27.21	of all of these subunits
00:05:30.02	through their work
00:05:31.29	at near atomic resolution.
00:05:34.12	So... but the conundrum is that
00:05:37.23	for many years, although it was known
00:05:39.23	that dynein and dynactin
00:05:41.19	are involved in cellular activities together,
00:05:44.08	it was very hard to demonstrate
00:05:46.01	a biochemical interaction
00:05:47.27	between dynein and dynactin.
00:05:52.12	But this mystery was resolved
00:05:56.00	when another protein came on the scene
00:05:58.25	called Bicaudal-D or BicD.
00:06:01.25	Now, BicD is a much smaller protein
00:06:04.00	and it's composed of several coiled-coils, here.
00:06:07.13	It was originally discovered
00:06:10.24	in a Drosophila genetic screen
00:06:13.08	by Eric Wieschaus
00:06:15.11	and Christiane Nüsslein-Volhard,
00:06:18.00	and subsequently it was shown
00:06:20.13	that this big BicD is involved
00:06:22.24	in recruiting dynein
00:06:24.21	to membrane organelles.
00:06:28.02	And an important piece of work
00:06:29.24	was done in this paper
00:06:31.25	by Splinter and colleagues
00:06:33.25	where they showed that this BicD
00:06:35.19	can join dynein and dynactin, now,
00:06:38.13	into a stable interacting complex,
00:06:41.03	and this was shown in this sucrose gradient,
00:06:44.05	here, where all three of these components
00:06:46.12	are combined,
00:06:47.27	and they comigrate together
00:06:49.20	in a very large molecular weight complex...
00:06:51.25	this top band is dynein,
00:06:53.18	this is the p150 subunit of dynactin,
00:06:57.11	and this lower band is this BicD.
00:07:00.18	So, they all comigrate together
00:07:03.09	on this sucrose gradient,
00:07:04.21	so we they're interacting
00:07:07.12	all together in one large complex,
00:07:09.08	and in fact with the recent cryo-EM work,
00:07:12.04	we actually have some idea
00:07:14.05	why and how BicD
00:07:15.29	can join dynein and dynactin together.
00:07:18.01	It basically... this is work from Andrew Carter's lab...
00:07:21.02	sits in between them.
00:07:22.21	It first of all...
00:07:24.01	the coiled-coil of BicD
00:07:25.21	falls along a groove
00:07:27.11	in the actin filament of dynactin,
00:07:29.14	but it also makes contact
00:07:31.12	with dynein as well,
00:07:32.28	so it kind of serves as a glue
00:07:34.17	to bring these two proteins together
00:07:36.12	and sits in the middle.
00:07:39.01	And also, work from Chowdury et al,
00:07:41.28	from another laboratory,
00:07:44.07	imaged this dynein-dynactin-BicD complex
00:07:48.07	on the microtubule,
00:07:50.03	and you can see the dynein motor domain here,
00:07:52.10	in this case j
00:07:54.21	ust kind of colored in yellow,
00:07:56.16	carrying this huge entourage behind it,
00:07:59.04	which is this complex of dynactin and BicD.
00:08:03.01	So, it's really an amazingly large
00:08:06.05	collection of proteins, here,
00:08:07.25	that are connected to the dynein motor domains.
00:08:11.06	Well, going back in time now
00:08:13.09	to 2013,
00:08:15.10	we were interested in whether this complex
00:08:18.26	would convert dynein
00:08:20.24	from that nonprocessive motor
00:08:22.13	that I showed you
00:08:24.02	to a processive motor.
00:08:25.24	So, Rick McKenney purified this complex.
00:08:29.01	He also tagged the BicD
00:08:32.02	with a GFP
00:08:33.28	and in this case we just used the N-terminus of BicD,
00:08:36.29	which is sufficient
00:08:39.14	to combine all these proteins together.
00:08:42.01	And to our great satisfaction
00:08:44.28	there was tremendously good processive movement
00:08:47.16	of this entire complex.
00:08:49.06	So here, all these, again,
00:08:51.04	all these individual green dots
00:08:53.12	are the BicD,
00:08:54.27	which are traveling along the microtubules
00:08:56.17	due to the interaction with dynein and dynactin.
00:08:59.10	In fact, this is one of the
00:09:02.04	most processive motors
00:09:04.09	that we've studied.
00:09:05.22	In fact, it's about five to ten times
00:09:07.19	more processive
00:09:09.20	than the kinesin motor.
00:09:11.12	So this converted this motor
00:09:13.08	from this very poor motor
00:09:15.19	for single molecule motility
00:09:17.14	into a real champion processive motor, here.
00:09:23.01	So, this same observation
00:09:25.08	was also made
00:09:29.02	by Andrew Carter's lab, independently,
00:09:31.08	specifically by Max Schlager.
00:09:35.00	So, we now also
00:09:38.03	wanted to be sure
00:09:40.19	that all these protein components
00:09:42.07	were indeed interacting
00:09:43.16	and traveling together on the microtubule,
00:09:45.22	so we then did this experiment
00:09:47.21	where we labeled each of the components
00:09:49.13	with a different fluorescent dye.
00:09:52.03	So, GFP was tagged
00:09:54.16	with a green dye,
00:09:56.00	BicD a blue dye,
00:09:57.19	and dynactin with a red dye,
00:09:59.20	and you can see when you superimpose
00:10:02.08	all of these colors
00:10:03.22	you get these really nice multicolored spots over here.
00:10:07.02	And I'll feature this for one spot in detail,
00:10:10.01	where you can see all three components
00:10:13.01	moving together along a microtubule.
00:10:16.20	And the reason why these colors
00:10:18.27	appear separated
00:10:20.08	is that the microscope is
00:10:23.01	first taking a snapshot in green
00:10:25.03	and then another one in red
00:10:26.14	and another one in blue,
00:10:28.06	and in between taking these
00:10:30.18	different colored snapshots
00:10:31.27	of course the dynein continues to move along the microtubule,
00:10:35.04	which is why these colors
00:10:36.22	appear to be separated,
00:10:39.07	but that's just due to the way
00:10:41.00	that these images were acquired.
00:10:43.20	So, the next thing we wanted to know was
00:10:46.08	whether this was some unique mechanism
00:10:48.16	of BicD,
00:10:50.17	or did this reflect
00:10:52.18	a more general mechanism
00:10:54.12	of how you activate dynein processivity
00:10:56.16	inside of cells.
00:11:01.02	So, to answer this question,
00:11:02.18	I need to give you a bit of more explanation
00:11:05.23	of BicD and how it interacts
00:11:08.11	with other molecules in the cell.
00:11:10.08	So, I just described to you
00:11:11.28	how the N-terminus of BicD
00:11:13.25	can join dynactin and dynein
00:11:15.21	into this one protein complex,
00:11:17.27	but BicD also has
00:11:20.00	another coiled-coil at the C-terminus,
00:11:23.16	and this C-terminal part of the coiled-coil
00:11:26.13	interacts with another protein,
00:11:28.11	which is a Rab GTPase,
00:11:31.08	and this Rab GTPase
00:11:33.02	is docked onto the surface
00:11:35.14	of a membrane vesicle.
00:11:37.16	So this BicD
00:11:39.14	really acts as an adapter protein.
00:11:41.06	At one end, it's connected to a cargo,
00:11:44.09	in this case this Rab6 protein
00:11:46.21	from the Golgi.
00:11:48.09	At the other end,
00:11:49.22	it's interacting
00:11:51.19	with the dynein motor and dynactin,
00:11:53.23	so it's really bridging
00:11:56.07	and bringing the dynein-dynactin complex
00:11:59.12	normally in the cell
00:12:01.10	to a specific cargo.
00:12:02.26	Now, in the dynein literature,
00:12:04.06	it was known that there were
00:12:05.27	other types of adapter proteins
00:12:07.14	that served other functions.
00:12:09.00	Some seemed to be linking dynein onto endosomes,
00:12:13.01	the Spindly protein
00:12:15.08	brings dynein to kinetochores...
00:12:17.10	so what we wanted to know was:
00:12:18.26	Do all these other adapter proteins
00:12:20.19	described in the literature...
00:12:22.09	could they also convert
00:12:24.18	dynein and dynactin
00:12:26.03	into a processive motor?
00:12:27.26	So we purified these other adapters
00:12:29.15	and tested them in the single molecule assay
00:12:32.01	and again found
00:12:34.06	this very gratifying, beautiful movement
00:12:37.11	where all of these adapter proteins
00:12:39.07	can form a complex with dynein-dynactin
00:12:43.26	and induce highly processive dynein motility.
00:12:48.03	Now, in addition to regulating dynein,
00:12:50.29	it also appears that this BicD
00:12:52.17	can regulate dynactin as well.
00:12:55.08	Remember that I said
00:12:57.19	at the end of this little antenna subunit here,
00:13:00.03	p150,
00:13:01.26	there's a microtubule binding domain.
00:13:04.01	Well, what we found with single molecules studies
00:13:06.12	was that if we looked at the microtubule binding
00:13:08.27	by dynactin alone,
00:13:11.15	or even dynactin with dynein,
00:13:14.03	we found that dynactin really
00:13:16.09	had very, very low binding
00:13:18.06	to the microtubule,
00:13:19.20	suggesting that this microtubule binding domain
00:13:22.12	is somehow repressed.
00:13:24.22	But when it's combined
00:13:26.27	with BicD and dynein,
00:13:30.01	the microtubule binding
00:13:32.29	of this dynactin component increases,
00:13:37.04	so, as I'll explain more later on in this talk...
00:13:39.21	so, it appears as though
00:13:41.19	this complex formation
00:13:43.17	also is able to convert
00:13:45.27	the microtubule binding activity of dynactin
00:13:49.00	from a repressed to an active form.
00:13:53.01	So, the general model that's emerging
00:13:55.11	from these in vitro studies
00:13:57.06	is that, perhaps,
00:13:59.09	in the cytoplasm,
00:14:00.24	when dynein is not attached to a cargo
00:14:03.05	and dynactin is not attached to a cargo,
00:14:05.24	these molecules are largely inactivated.
00:14:11.06	And that the activation that these molecules
00:14:14.06	require is linked to their simultaneous
00:14:17.04	binding to a cargo.
00:14:18.29	So, in this case there's a cargo molecule
00:14:22.03	with a receptor
00:14:25.19	that can bind to an adapter protein,
00:14:27.22	in some cases we think it actually
00:14:29.18	can activate that adapter protein,
00:14:31.25	and that adapter protein
00:14:33.25	then brings dynein and dynactin
00:14:36.09	onto the surface of the cargo,
00:14:38.12	and also activates it
00:14:40.15	so that the motor is now active
00:14:42.02	and can transport that cargo
00:14:43.29	along the microtubule track.
00:14:46.10	So, I showed you, now,
00:14:49.06	how the cargo
00:14:51.13	can regulate dynein-dynactin motility.
00:14:54.02	What about the microtubules themselves?
00:14:56.25	Are they just kind of inert tracks?
00:14:59.08	Or do they have a regulatory function as well?
00:15:03.09	Well, it's known that microtubules
00:15:05.18	actually have many different types
00:15:07.16	of post-translational modifications.
00:15:11.09	These modifications are described here
00:15:14.06	and they often occur
00:15:16.11	on the very C-terminus,
00:15:20.16	these somewhat disordered peptides
00:15:22.26	that emerge from the surface of the microtubule.
00:15:25.22	And I'd like to focus on one of these
00:15:29.10	post-translational modifications,
00:15:31.14	which involves
00:15:33.21	the very C-terminal tyrosine
00:15:35.25	of the α-tubulin subunit.
00:15:38.12	So, it turns out that
00:15:40.11	the C-terminus has this tyrosine residue,
00:15:42.29	but in cells there's an enzyme,
00:15:44.27	a carboxy-peptidase,
00:15:46.28	that can specifically remove
00:15:49.10	that tyrosine residue,
00:15:51.21	and in fact, post-translationally again,
00:15:53.19	there's an enzyme,
00:15:55.16	a tubulin-tyrosine ligase,
00:15:58.09	that can shift the reaction in the opposite direction
00:16:00.19	and re-add that tyrosine
00:16:03.04	to the end of α -tubulin.
00:16:05.21	So, this modification has been known for many years,
00:16:08.15	it's not clear exactly
00:16:10.10	what it's doing in cells,
00:16:12.00	but cell biological studies have shown that
00:16:17.08	these types of microtubule tracks
00:16:19.05	in fact can be separate,
00:16:21.06	even inside of one cell.
00:16:23.06	So, for example, this just shows
00:16:25.20	an image of a fibroblast
00:16:27.24	which is migrating, it's polarized,
00:16:29.18	it's extending and moving in one direction,
00:16:32.13	and it was found that
00:16:35.01	the microtubules that are extending
00:16:37.18	in the direction of movement
00:16:39.23	are preferentially detyrosinated.
00:16:43.28	So it's been tempting to speculate
00:16:46.10	that these different types of microtubule tracks
00:16:50.08	that are tyrosinated and detyrosinated
00:16:52.27	may serve and interact differently
00:16:55.25	with different types of molecular motor proteins
00:16:58.14	in cells,
00:17:00.16	although evidence for that hasn't been entirely clear.
00:17:04.17	So we decided to test that,
00:17:06.14	whether the [tyrosine of the C-terminus]
00:17:08.22	makes a difference or not
00:17:11.24	for the processive motility
00:17:13.17	of dynein and dynactin
00:17:15.17	connected to this BicD.
00:17:17.07	So we made, first of all,
00:17:19.02	recombinant tubulin
00:17:20.23	that was absolutely identical,
00:17:22.14	except for one difference:
00:17:24.04	whether the α-tubulin had the tyrosine
00:17:26.19	or not.
00:17:28.01	And second of all, then,
00:17:30.04	we labeled those two different types of microtubules
00:17:32.22	with different fluorescent dyes,
00:17:34.18	so the tyrosinated microtubules
00:17:36.26	you'll see, in the movie that I'll show you,
00:17:38.18	are labeled with a red dye,
00:17:40.18	whereas we labeled
00:17:42.25	the detyrosinated tubulin
00:17:45.12	with a blue dye.
00:17:47.00	And then we just combined these two microtubule types
00:17:49.07	together in the same cover slip
00:17:52.05	and then examined how they would interact
00:17:54.12	with different molecules.
00:17:56.29	So, first of all,
00:17:59.02	we tested just the p150 domain of dynactin,
00:18:02.03	so this is not the whole dynactin molecule,
00:18:04.05	it's basically
00:18:07.00	the microtubule binding portion of dynactin.
00:18:12.01	And this was actually known earlier
00:18:14.16	and we replicated the results,
00:18:16.09	and you can see see all these dynactin molecules
00:18:18.24	found in green
00:18:20.22	decorating these red microtubule tracks,
00:18:23.29	but these blue microtubule tracks
00:18:26.01	that don't have the tyrosine
00:18:27.21	are devoid of the p150
00:18:31.05	microtubule binding region.
00:18:33.22	We tested this also for dynein,
00:18:36.06	again labeled in green,
00:18:38.02	and remember that dynein on its own
00:18:39.21	does not really move along microtubules,
00:18:41.26	so we're just examining here
00:18:44.08	its microtubule binding properties
00:18:46.10	in this static image.
00:18:48.03	Well, you find here that
00:18:50.08	the dynein doesn't really have much of a preference
00:18:52.20	between either of these tracks;
00:18:54.08	it binds to both.
00:18:55.23	So, the question is,
00:18:57.11	if we look at this whole complex together,
00:18:59.17	would it look more like dynein
00:19:01.07	or would it look more like the dynactin molecule?
00:19:05.03	So we did this, combined the microtubules,
00:19:06.26	and did a motility assay,
00:19:08.19	and the result is quite striking.
00:19:10.08	I think you can see that there are
00:19:12.22	lots of these individual dynein-dynactin molecules
00:19:15.02	moving along the tyrosinated microtubules,
00:19:18.12	but there's very little motility
00:19:20.19	occurring on these blue microtubules
00:19:23.06	without the tyrosine.
00:19:25.13	So the motility
00:19:28.02	seems to behave much more
00:19:29.24	like the p150 binding properties
00:19:31.21	than the dynein.
00:19:34.06	So, another question is:
00:19:37.15	What is the mechanism of how
00:19:39.20	this p150-dynactin
00:19:41.27	is influencing dynein motility?
00:19:43.20	Is it necessary just to
00:19:45.12	initiate the first encounter of the motor with the track,
00:19:49.14	or is it continuously needed
00:19:52.10	to hold on to the track
00:19:53.29	during processive motility?
00:19:55.17	So we wanted to distinguish
00:19:57.03	between these two possibilities
00:19:58.18	with the following type of experiment.
00:20:01.19	We actually made a chimeric microtubule
00:20:04.26	by fusing a tyrosinated microtubule,
00:20:07.12	again in red,
00:20:08.23	with a detyrosinated microtubule
00:20:11.04	in blue,
00:20:12.18	but in this case it's a continuous track, here.
00:20:15.21	And we wanted to know...
00:20:17.03	we know the dynein-dynactin
00:20:18.20	will first bind to this tyrosinated track
00:20:21.18	and then move along it,
00:20:23.24	but what happens when it reaches the junction?
00:20:26.02	Is it going to fall off
00:20:28.01	or is it going to continue along the track?
00:20:30.22	And the result is shown here
00:20:32.27	in a real movie
00:20:34.01	and you can follow these spots
00:20:35.21	that are first binding to this red portion,
00:20:38.15	and you can see that they
00:20:41.00	move continuously from the red portion
00:20:43.10	into this blue region.
00:20:45.00	So this result indicates that
00:20:48.01	the dynactin
00:20:50.00	is needed to initiate the motility,
00:20:51.15	but once it starts
00:20:53.17	it can then function
00:20:55.13	without p150 interaction
00:20:58.18	with the tyrosinated tubulin.
00:21:02.13	So, in summary,
00:21:03.29	these experiments provided
00:21:05.18	some clues as to
00:21:07.11	how dynactin might be able
00:21:09.13	to activate dynein motility.
00:21:11.15	So, as I just described,
00:21:13.21	this microtubule binding domain
00:21:15.09	at p150
00:21:17.03	seems to play, initially,
00:21:18.25	an important role
00:21:20.22	in delivering dynein and dynactin
00:21:23.01	to tyrosinated microtubules.
00:21:25.22	But once this interaction is made
00:21:27.23	and the dynein starts going,
00:21:29.27	the motility doesn't seem to continue
00:21:32.14	to need that microtubule binding interaction.
00:21:36.14	So... but we also think
00:21:38.21	that dynactin is still needed
00:21:41.01	for the processive motion,
00:21:42.24	for the continued processive motion,
00:21:44.11	and that may occur
00:21:46.05	through some allosteric activation
00:21:49.03	of dynein by dynactin.
00:21:51.14	And there's some hints
00:21:53.02	of this from the beautiful cryo-EM work
00:21:56.26	that came from Andrew Carter's lab,
00:21:58.19	but we still really don't understand
00:22:01.29	very much of the details
00:22:03.29	about that allosteric activation mechanism.
00:22:07.06	In addition, there are plenty of remaining
00:22:09.23	open questions in this field.
00:22:12.26	So, what I showed you was some
00:22:15.22	in vitro experiments
00:22:18.08	showing that the tyrosinated state of a microtubule
00:22:22.08	plays an important role in dynein transport in vitro,
00:22:25.03	but what about in living cells?
00:22:27.12	Are tracks that are detyrosinated...
00:22:32.26	are they poor tracks for dynein motility
00:22:35.08	and perhaps select
00:22:37.18	kinesin-based motility?
00:22:39.14	So, we don't know the answers
00:22:41.29	to these questions
00:22:43.14	about how this cycle of tyrosination and detyrosination
00:22:47.07	is used to regulate
00:22:49.27	microtubule-based transport
00:22:51.18	between dynein and kinesin motors
00:22:54.01	in living cells.
00:22:56.10	We also know that in living cells
00:22:58.03	there are other types of dynein regulators,
00:23:01.05	ones that I haven't had a chance
00:23:03.07	to describe in this talk,
00:23:04.22	but other examples of dynein regulators
00:23:06.26	include this protein called LisI,
00:23:11.09	which was found as a mutation
00:23:13.18	in a disease called lissencephaly.
00:23:15.22	There's also another factor called NudE/L.
00:23:19.29	And what we don't know is
00:23:21.24	how all of these factors are working together
00:23:24.02	as a system to regulate dynactin motility in cells,
00:23:27.22	so there's still a tremendous amount of work to do
00:23:31.11	to understand the regulation
00:23:33.25	of dynein in cells.
00:23:36.20	And I'd like to just
00:23:39.02	with acknowledging the people
00:23:41.01	that did the work that I just described.
00:23:43.15	I already mentioned Rick McKenney,
00:23:46.06	who initiated this whole project.
00:23:48.13	He's now about to start
00:23:50.19	his own lab at the UC Davis.
00:23:53.02	Walter Huynh is a graduate student
00:23:55.00	that did a lot of the work
00:23:57.00	on the other dynein-dynactin
00:23:58.27	adapter proteins.
00:24:01.01	And Minhaj Sirajuddin,
00:24:04.13	a former postdoc in the lab,
00:24:06.26	he now has his own lab at the NCBS in Bangalore,
00:24:10.11	and he did a lot of the work
00:24:12.21	on the tubulin that I described,
00:24:14.20	and pioneered a system
00:24:16.10	for making recombinant tubulin.
00:24:18.13	And with that I'd also like to thank you
00:24:20.16	for your attention.