Exocyst Mediated Recycling of Primary Cilia

April 20, 2021

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Yale Cancer Center Grand Rounds | April 20, 2021

Derek Toomre, PhD

ID6520

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00:00Our second speaker today is Derrick Tom Ray,
00:03who's professor of cell biology and
00:05director of the Yale Cinema Microscopy Lab,
00:08received his PhD from University.
00:10California is an expert on
00:12quantitative live cell imaging,
00:13and he specializes in innovative
00:15approaches to microscopy,
00:16including fluorescent probes,
00:17data analysis, and spatial mapping.
00:19Received an NIH director's New
00:21Innovator Award and a coffee fellow,
00:23and today is there talk
00:25about his recent work,
00:26which is primarily focused on spatial
00:29control of membrane trafficking
00:30during cell morphogenesis migration.
00:32In cilia formation.
00:33So Derek, the floor is yours.
00:49So.
00:52Derek, your music. Yeah
00:55OK, great. Let me go back a slide or two.
01:03Great, so I'll be talking bout cilia which
01:05have some relevance to cancer and signaling.
01:08I'll be talking basically about a new
01:11pathway that we're discovering and tell
01:13you about the ramifications of this.
01:16I not talking anybody work on SARS?
01:18Kobe 2 but I do have some work on that and
01:22my conflict of interest is declared there.
01:25So is Sherman sort of a primary?
01:28Let me just tell you the main message in
01:31the main message is that we're proposing
01:34and characterizing a novel pathway.
01:36Of cilia delivery to the surface
01:38and I will sort of tell you what.
01:40Basically the central paradigm is
01:41and then go on to sort of describe to
01:44you that I think that that basically
01:46our data suggests that we have
01:48an alternate pathway here.
01:51OK. Just. OK, it's clicking differently,
01:56so we first start off with a quick
01:59primer here about primary cilia so you
02:02know really sort of two aspects here.
02:05What are they?
02:07And I suppose sort of related here,
02:10why do you care? Why should we care?
02:13So in terms of the first part,
02:16that's relatively easy there
02:18long slender cellular antenna.
02:20They're basically present on
02:21nearly all cells. There's a.
02:24The exceptions being red
02:25blood cells and T cells.
02:28If you go by electron microscopy.
02:31End both sort of light microscopy.
02:34You see their long and slender.
02:36The width is maybe 152 nanometers and
02:38they can extend about 10 microns long.
02:41There's solid Terry organelles their
02:43primary cilia and let me tell you a little
02:47bit about some of the known functions.
02:51So you can sort of think of them
02:54as a specialized sensory antenna,
02:56so you have a adaption of this
02:59for vision of your rod cells.
03:01I guess relevant to right now and and
03:04coronavirus they also important for smell.
03:07And there is a loss of cilia which
03:10is causing the Syrian dysfunction of
03:12that and as well as signaling aspects.
03:15And we sort of mentioned a little
03:18bit more on that because it's
03:20really relevant to this audience.
03:23Especially in regards to cancer.
03:26So primary slip, so why?
03:29Why do you care in this?
03:31And the easiest way of sort of
03:34thinking of this is thinking
03:36of in terms of ciliopathies.
03:38OK,
03:39there's a number of diseases that
03:41are basically dependent was actually
03:43sort of discovered retractively
03:45these diseases have been known
03:47for quite a long time,
03:49and then only in the last couple
03:52of decades was it really realized,
03:54in part from pioneering work at Yale.
03:57About the sort of link between
04:00these diseases and disruptive
04:02ciliary function so you can see this
04:04in sort of the Joubert syndrome
04:07or liver cysts kidneys.
04:09It's quite multi multi organal.
04:12But again, for this audience,
04:14I think it was particularly relevant
04:16is in the context of of cancer,
04:18and you can sort of think of it
04:21in terms of sort of two aspects,
04:24one aberrant signaling,
04:25and then your cilia being part of that
04:28rule that would turn things on and off.
04:30It gets a little more complex here
04:32because you actually can release vesicles
04:34from a cell which can transmit signals,
04:37so that's one aspect of it, but you can
04:40sort of think of it in a simple way.
04:43Simplified way of controlling
04:46this signaling on or off.
04:49Now,
04:49just to sort of just highlighting
04:52that complexity was sort of a review,
04:54that sort of indicates that it
04:56actually can go either way.
04:58You could have a certain degree
05:00of activation you could turn on
05:02hedgehog by activation of this
05:04receptor level and sort of pathway B.
05:06Or you could actually do it by
05:09going independent that.
05:10So if you look at cancer is summer
05:13activated by the presence of silly,
05:15and some are by the repression.
05:17OK, but if we sort of.
05:19Go beyond that aspect and say well,
05:22what is sort of the normal paradigm.
05:24Well, the normal paradigm here is
05:27that cilia occur once per cell cycle.
05:29OK, so you generate them and during
05:32G and G1G0 there sort of present
05:34throughout and then they have
05:36to be assembled during division.
05:38So in that sense,
05:40if you think of it from that lens,
05:43you're really not controlling except
05:45for to the point of cell division.
05:49And maybe any delays on whether you
05:51would have slid Genesis or not,
05:53and I'd like to say,
05:55change that paradigm view.
05:57So remind you again.
05:59I do a lot of imaging as you
06:01sort of heard in the intro,
06:03and this is just one example of
06:05super resolution image Ng where we
06:07can actually see this very long
06:09structure of cilia, which is,
06:11like I said,
06:12it could be you know 10 microns or
06:14so long with the resolution down
06:16to sort of 20 nanometers where you
06:18could actually see it as a tube.
06:21So that's one sort of technique,
06:22but I'm going to show you another
06:24source of imaging techniques,
06:26and in short of.
06:27Exemplify that can provide new insight.
06:30Here again,
06:31this is sort of the structural
06:33aspects of this accident.
06:35Sort of in the side, which is,
06:38it's just relatively relevant,
06:39is that they can present with this
06:42pocket Macy put the laser pointer
06:45on and in the ciliary pocket.
06:47Here,
06:48and this is the sort of shown
06:50extra short here,
06:52but this can be a very deep imagination.
06:55In fact it can almost be.
06:58Nearly entirely evaginated.
06:59OK,
06:59So what is sort of the key process
07:02of how they how they form?
07:04So the general paradigm,
07:05in fact there's there's one
07:07for epithelial cells,
07:08which I will not sort of discuss,
07:10but for most of the cells.
07:12In fact the majority,
07:14if they start with this silly
07:16or vesicle which forms,
07:17and then you have this double membrane
07:19membrane which should have bends in.
07:21And then you actually have this,
07:23really think of it as a super large vesicle.
07:26It's a vesicle.
07:28This vesicle could be 7 microns long,
07:31OK,
07:31and then as a an event it actually has
07:35to go infuse the plasma membrane produce.
07:40This is a, you know,
07:41this is a standard paradigm.
07:43This happens once per cell cycle
07:46would be the standard paradigm.
07:48Now I want to bring in one other
07:52player here and this is one that
07:55we've worked with quite a bit,
07:58which is called the exocyst complexes,
08:00the tethering complex.
08:02It was first discovered actually
08:04at Yale in yeast,
08:06and basically it is known to basically
08:09drive the upstream monsters near
08:12Fusion machinery to allow spatial
08:14temporal control of vesicle exocytosis.
08:17This is you can sort of see in.
08:21This review has been thought to
08:23for quite some while play a role
08:25in cilia and mainly ciliogenesis
08:27and and stabilization by targeting
08:29the vesicles right here where
08:31they would
08:32fuse and then basically drive let's
08:35say control within the accident
08:36that is the standard paradigm.
08:39And while I don't want to say that is wrong,
08:42I think it's actually missing.
08:44Let's say 80% of the picture OK.
08:49So we sort of go back to,
08:51you know why we have this paradigm and and
08:53and what sort of the underlying underpinning?
08:56Well, is it? You know I want to
08:58basically first say that is Dre.
09:00It's based on indirect evidence.
09:01We've actually not seen the
09:03vesicles their fusing there,
09:04nor actually seen much of the Exorcist.
09:06And I would say that you're missing something
09:08that's really important and it's incomplete.
09:11But let's sort of get beyond that and
09:13show you what I may be talking about.
09:15But first I have to tell you.
09:18Why, if we're claiming to
09:20see something different,
09:21why and how are we able to do so when people
09:25been looking at Syria for quite awhile?
09:28And so there are a number of
09:31technical aspects to the solution.
09:33The first one is we're using a technique
09:36called total internal reflection for us.
09:38Since microscopy,
09:39let's say axial superresolution
09:41technique and it allows us to image
09:44just the lower surface of the cell and
09:47actually see silly's emergence OK.
09:49But that actually even
09:51without superresolution,
09:51technique is not enough
09:53to be unequivocal there.
09:54The distance between cilia and
09:56the surface at times is very low,
09:59and it's actually not clear if they
10:01are inside the cell or actually have
10:04emerged in or outside and think of it
10:07again like your antenna, your antenna.
10:09If you sort of.
10:10Just give an analogy,
10:12is going to respond to signals quite
10:14differently if it's actually outside
10:16your car truck, what, whatever it be.
10:19Versus pulled it inside where
10:20it cannot receive the signals.
10:23OK, at least not the same signals.
10:25The other technical aspect is is we
10:27used a a McLeod clever pH switching
10:30to identify when cilia are in or out.
10:33We're using this as an impulse way and
10:35we do molecular replacement of the Exorcist.
10:38The latter was important because for a long
10:42time people were image in the Exorcist.
10:45Or just simply overexpressing it and they
10:47would sort of see localizations like
10:49this everywhere or some accumulations,
10:51but when we did this replacement
10:54strategy we could see it in these
10:56discrete punkte OK and this is now
10:58this is going into HeLa and other
11:01types of cells looking at vesicle
11:03excess cytosis and we could see in
11:05these kind of graphs distinct events
11:07and I just show you one trace where
11:10vesicle has arrived as we see with the
11:13Exorcist and then with another Reporter.
11:15And this is a.
11:16Any Reporter here which is a floor in
11:19with the pH sensor we can actually
11:22unequivocally identify the Fusion event,
11:24so this is sort of a constitutive pathway.
11:27We know it's coming from recycling vesicles
11:30and we can identify and study that.
11:33OK,
11:34and we can tell the events about
11:35when The
11:36Exorcist appears and when
11:37you have the Fusion fit.
11:38This is relevant to the how
11:40we're going to be looking
11:41at things with the cilia.
11:43So what is it that we see?
11:46In I'm really just.
11:53OK, good. OK, so here's
11:55a short movie of what we're seeing
11:58in terms of cilia,
12:00sort of called the Biogenesis aspects,
12:02where we see basically
12:03exorcist recruited to cilia.
12:05So that's sort of standard that itself
12:08is is basically showing it going
12:10through the base of a long 80 cilia,
12:13but there's actually another phenomenon
12:16that we observed which is quite different
12:19and so now you actually see this cilia
12:22with this is the Reporter here is.
12:24Smooth and flooring or we can also
12:27use smooth and GFP so that looks
12:29to sort of characteristic curve.
12:31Linear silly and this would be in
12:34the dimensions several microns long.
12:36And what I'd like you to note,
12:38and I'm going to play this more than once,
12:42is that we actually see the red signal
12:45getting recruited to this silly boom,
12:47right there off on again off
12:49again have several times.
12:51This is the time one last time.
12:55Is in hours OK, so there's no signal there.
12:58It appears it disappears.
13:00It appears again over the course
13:03of in this case of this movie,
13:05which is basically 4 hours.
13:07So in appearing there in this
13:09sort of the minutes range,
13:11this does not fit with what you
13:14would expect of sort of the well.
13:17A couple of things.
13:19One is, it's along the entire cilia,
13:21not just the base.
13:23Two is it appears there.
13:25Goes there and then vanish
13:26is and then comes back.
13:28You know an hour or two later again.
13:30So what's going on there
13:32and what do we you know?
13:33How can we sort of probe into that with?
13:36I guess you can intend to punt.
13:39So we do this by using.
13:42Again,
13:42we're using this exorcist and
13:45we look after after stimulation.
13:48See something interesting
13:49happening here and you actually
13:51see it quite really in the movie.
13:54Let me just play it again and you see Pam.
13:57Let me just.
13:58I realize it's hard to catch.
14:00Bam you see that green object?
14:03That's the vesicle flying off.
14:04OK, so we see the release of
14:07the vesicle happening as well.
14:09Now that's actually you know
14:10that part is known,
14:12but later we actually see
14:14then the Exorcist here.
14:15OK, after that we dropped off that signal.
14:19So basically the SEK 8 which is an
14:21extra quarter decorate psyllium after
14:23serum stimulation in vesicle release OK.
14:26And by the way I mean you know we
14:29artificially to generate cilia
14:31interesting culture, starve them.
14:33Of course the normal situation
14:36would be in syrup.
14:38OK, so.
14:41You know, per that sort of model,
14:43well, is there any evidence for this?
14:45Yes, there have been some some
14:47papers here and you called it called.
14:49The word.
14:50Is it called decapitation OK,
14:52where it plays off that vesicle?
14:54We actually think that the mechanism
14:56is going to happen is is is different?
14:58OK they are releasing it,
15:00but you'll see in our cartoon in
15:02the end we're thinking that it's
15:04happening by a different mechanism.
15:06You can obviously see sort of
15:07the importance of that on signal
15:09transduction as I was.
15:11Early, basically indicating earlier so.
15:17What do we happens to our RXS
15:20reporters after we add cereal?
15:22In this case, the FBS.
15:25And and So what we can see here,
15:27and you're sort of the overview slide.
15:30We're using X-70 as as our Our Exorcist
15:33Reporter I MP5 E is a silly Reporter
15:35and a couple of different things.
15:37One is now I should mention
15:39for soul this is endogenous.
15:41*** is no longer replaced.
15:43OK so this is sort of native conditions
15:46and what you can see is you can very
15:48clearly see it localising to cilia,
15:51but if you look carefully and I
15:53think you can see in this case here.
15:56And in this case here it actually.
15:59It localizes there but also forms these
16:02additional tubes which are pulling
16:04about look to pee in most movies
16:06would know are pulling out of it.
16:08So what's going on there?
16:11I will propose to you and submit to
16:14you is that this silly is actually
16:17inside the cell and is actually being
16:21remodeled by the X assist to pull off
16:24other membranes and remodel it through.
16:27Actually have the role of Exorcist being
16:30to tether and help fuse this monster huge,
16:33you know 510 Micron long vesicle.
16:38We we you know we basically see this
16:41in here sort of another view of that.
16:44And again there would be flooring
16:46smooth in there as well.
16:48So you smooth in IPP in 5E and these
16:51large tubules that are pulling off now.
16:53Does this happen when you
16:55stimulate with steering?
16:56And the answer is yes and if you
16:59look at percent acilia you had
17:01serum they they drop down a bit.
17:04That's that's that's expected.
17:06What is particularly interesting
17:08and exciting to us?
17:09Is that the colocalization with extra 70,
17:12which is usually quite low,
17:15goes up dramatically by adding
17:17serum OK so serum is is,
17:20you know going to turn over this.
17:23Also with the cell cycle.
17:26But the important thing is that's
17:29actually driving that recruitment.
17:31Now we do some controls here.
17:33If we knocked out the other
17:36exocyst complex members OK.
17:38Set an RX-70.
17:39We do not see that another way of
17:42saying this is if we destabilize
17:44The Exorcist so you don't have your
17:47entire 8 protein complex and we
17:50no longer see that localization.
17:51OK, so that's sort of a control experiment.
17:54Then you might well ask.
17:56Well, OK, so it's recruited.
17:58But where is? Where is it being recruited?
18:01Relative to the Exorcist relative
18:03to the cilia?
18:04And I kind of reminded you early on
18:07that you have this ciliary sheath.
18:10This sort of a membrane which kind
18:13of does this put the pointer on.
18:17You know we have the membrane here and then.
18:20It's a double membrane, right?
18:21So kind of goes like that and
18:23curves all the way around.
18:25OK so or you can kind of see it here
18:28in this cartoon. And is it here?
18:30If this is there or there now?
18:33As I there's sort of different reports,
18:35there's obviously the ones that
18:36have done the base.
18:38There have been some reports
18:39of about being inside,
18:40but we actually believe it is
18:42actually here along the pocket.
18:44And how do we know that by using a
18:46super resolution imaging modality?
18:48Construction limination microscopy and I
18:52think you can sort of see it easily here.
18:55You'd have smooth and so this would
18:57be basically your cilia Reporter,
18:58which would be on the extracellular
19:00side where I should write.
19:02So would be.
19:04On the inner membrane,
19:06if you like to look at it that
19:09way and we see SEK 8 or X-70 is
19:11a wider distribution.
19:13OK,
19:13so it is on for going.
19:18Turn off my paper airplane
19:20here for going back.
19:22It would be on the sillari
19:25pocket membrane OK.
19:27Maybe switch to pen?
19:30So we we see this is tribulus emerging.
19:33As I showed you an example and we
19:36actually see it in live cell imaging
19:38as well were these tubules are
19:41dynamically pulling out of Syria?
19:43OK so we think it's actually
19:46important for the remodeling.
19:48*****
19:51It's a little funny and
19:52giving this presentation.
19:53Switching back between the pens or not,
19:56but let me just tell you to sort
19:58of focus on here on the point of
20:00the Arrowhead where we actually
20:02think of that as a Fusion event
20:04so you have this internal cilia,
20:06the cilia which is inside the cell.
20:10Should. And is is here and you
20:14have SEK eight there on it.
20:16We think it's important for them,
20:18the tethering function,
20:19and at this point right here where
20:22the membrane it looks like to expand
20:24you see that little bit of a burst.
20:26At that point, the SEK 8
20:28collapses to the base,
20:30which has been sort of reported in the
20:32literature to exist most of the time,
20:35again at a single snapshot you
20:36mainly see it at the base,
20:38but actually if you look at it live over,
20:41you know it's called longitudinal.
20:43You would actually see it.
20:48Earlier on these internal cilia,
20:49so we actually think it's
20:51driving that process out.
20:56OK.
21:00So well, how can we actually
21:02sort of prove this unequivocal?
21:04Blee and the way that we've done this is
21:07basically using a pH switching experiment,
21:10and when the cilia are outside,
21:12if we switch the pH,
21:14and we have this pH sensitive Reporter,
21:17every time we switch it, it goes on and off.
21:21OK, so we make acidify, we can turn it off,
21:25and then it goes back on again.
21:28So we basically.
21:29Switching the pH,
21:30we can tell if it's outside here,
21:32switching, going up and down,
21:34but as you can see in this context, is not.
21:37It's actually resistant.
21:38And why is it resistant?
21:40Because inside the cell.
21:41So it is basically inside inside,
21:43inside and then here you can see it
21:45switch into the purple or magenta
21:48indicating this now outside.
21:49OK, so there we can really say.
21:53Quickly that it is is
21:57actually merging overtime.
21:59And you can sort of see these
22:02experiments were switching pH,
22:03the intensity goes up and down,
22:05the second doesn't change.
22:06Here is a case where is
22:07actually not changing much,
22:09the thing fuses.
22:13Right, so it's not switching much.
22:14It fuses now start switching and then
22:17just a little bit later you see the
22:19The Exorcist is actually changing
22:21part of it and then it eventually
22:23drops off and fully into the base. OK,
22:27so we can start to identify with machinery.
22:30Is there sort of looking at who might
22:33be the players that might be engaging
22:36with The Exorcist and there is some
22:39interesting ones such as Rab 10 is a
22:42likely player and actually we see Rab
22:4610 localising there which is actually
22:48different from some of the other apps
22:52so I'd like to sort of end now and their
22:55remaining minutes with basically are.
22:57The working model and just walk
22:59you through that very briefly,
23:01so the working model is that.
23:03In this starves,
23:04say you have cilia with these
23:07sort of deeply emerged pockets.
23:09The Exorcist is in the facilities
23:11recycling endosomes and probably have
23:14some targeting of that success here.
23:16In the serums to stimulation,
23:18that which is actually the normal case,
23:20right?
23:20If you sort of thought about
23:23sleep in the body.
23:25There, there there actually
23:27constantly being remodled.
23:28OK,
23:28so the event of releasing the vesicles
23:31actually different than sort of was proposed.
23:34We believe of it sort of coming off from
23:38one that's fully outside this actually,
23:41as it starts to pull in,
23:43we believe that it was remodeling that can
23:47actually promote this vesicle to release.
23:49Once inside you have recruitment of
23:52the Exorcist to this large and inside.
23:55Which is again majority of this sort
23:59of internalize cilia which can remodel,
24:02pulling off tubules and consequently
24:04then can recycle back.
24:06So you have this entire pathway here
24:09that can modulate the signaling.
24:12So with sort of that this is.
24:18Switch back to automatic.
24:19Really this is really driven by senior
24:22scientist in the lab Felix Riviera Molina.
24:25Sort was reported by the people,
24:27but really he took the lead here,
24:30so that's where I'd like to end and
24:33address any comments that you might have.
24:38Thank you for very interesting talk.
24:40Are there questions from the audience?
24:42Let me put that you
24:44can just type them in.
24:47While we're waiting, yeah.
24:48So there are a lot of genetic disorders
24:51of cilia formation that have many
24:53different phenotypes or anything
24:55associated with increased cancer risk.
25:00Yes, there there have been and
25:02there were sort of where it gets
25:05complicated is it depends on sort of
25:07what cell types you're looking at it.
25:10Again, again, you know pushing things up
25:12and down and sort of mentioning earlier.
25:15So yes, there are.
25:17Haven't been sort of investigating
25:19this so much personally but but yes.
25:23Thank you.
25:27Questions for Derek.
25:30I know this zoom atmosphere
25:32makes it a little bit different.
25:34Will. So enjoy when we see
25:37people face to face again.
25:48Another question then should in.
25:52In cancer cells,
25:52which are obviously many of them
25:54often constantly proliferating,
25:56do you see abnormalities of
25:58cilia formation you could?
25:59Do they have more cilia?
26:01Do they turn over more rapidly?
26:03What happens?
26:06This is a little bit as alluding to
26:08you can kind of push it either way,
26:11so that's where that's actually where the
26:13confusion is to cancer is that you would say,
26:16well, do they have more silly?
26:18Do they have less cilia and basically
26:20the paper review had indicated that
26:22there's sort of the two sides on it,
26:25so in one case you actually hyper
26:27activate by, let's say adding
26:29smoothing and hedgehogs signaling,
26:30so the Hedgehog is obviously a key component,
26:33as same as sort of PGF would
26:35be basically a hyper activated.
26:37The other case is where you would
26:40actually activate the signaling
26:41by the absence of the cilia.
26:44So that's sort of where it's you know.
26:48In some cancers are driven by having cilia,
26:51and some are driven by the absence.
26:56Sort of given the this sort
26:58of the the funny paradox.
26:59Depending on that nature of which
27:01signaling pathway you're talking about.
27:03Is it a hedgehog sort of smoothing
27:04type of one or other signaling pathway?
27:07And what is the activation
27:08at the Basel State?
27:10So that's why I think it's given some
27:12complexity to the field because you couldn't
27:14just simply say this is only this week,
27:16but it does.
27:19Yeah, yeah.
27:21There's evidence for both there.
27:24Are there other any other
27:25questions in the audience?
27:34If not, thank you.
27:35Thank you all for coming.
27:36Thank you for two speakers.
27:38It was very interesting and
27:40everybody you have a 22 extra
27:41minutes for your day. Thank you.
27:43Thank you Dan.