Exocyst Mediated Recycling of Primary Cilia

Transcript

• 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.