Giulia BERTOLIN – Illuminating mitochondrial functions during the cell cycle using FRET – based biosensors : Mitochondria are signaling hubs which are key for cell physiology.
At the molecular level, mitochondria are perfectly integrated with other subcellular compartments and machineries. For instance, functions such as mitochondrial morphology and ATP production are known to change according to the cell cycle phase of the cell. However, our view on how the cell cycle machinery can directly shape mitochondrial activities is still fragmentary.
Although biochemical approaches are largely used to investigate mitochondrial functions, genetically-encoded Förster’s Resonance Energy Transfer (FRET) sensors are becoming convenient tools to uncover how mitochondrial activities evolve in space and time. During my talk, I will cover how we discovered the activation of a custom-made FRET biosensor of the cell cycle protein Aurora kinase A/AURKA at mitochondria1–3. I will also describe the experimental and analytical strategies we implemented to increase the dynamic range of the biosensor, and to make it compatible with multiplex FRET strategies relying on Fluorescence Lifetime Imaging Microscopy (FLIM). These strategies include alternative donor/acceptor FRET pairs, and multiple ways to calculate FRET efficiency.
Thanks to the activation of the biosensor at mitochondria, we identified novel roles of AURKA in regulating the turnover of defective organelles and ATP production2,4. Given our interest in the turnover of intracellular material –including mitochondria–we engineered the LC3B biosensor, a genetically-encoded FRET probe to follow this pathway. I will describe notonly the design of the probe, but also a broad-to-sensitive image analysis pipeline to follow clearance by autophagy with an unprecedented spatiotemporal precision. With this tool, we discovered the impact of the cell cycle in regulating turnover by autophagy5. Together, I will highlight how the use of genetically-encoded FRET biosensors pave the way for a better understanding of mitochondrial functions, and show how the cell cycle is a key element regulating the multiple activities of this organelle.
Giulia Bertolin,CNRS, Univ Rennes, IGDR(Institute of Genetics and Development of Rennes),
UMR 6290, F-35000 Rennes, France
https://igdr.univ-rennes.fr/giulia-bertolin
Um so yeah let’s change gears a little bit I’m going to um drive you into a more biology applied Journey for today and I will be telling you about how we investigate mitochondrial functions and how they’re in interplay with a cell cycle by using a lot of sensors and how
We infer information from the sensor uh so how we obtain information from the sensors to infair infair um signaling regulation Pathways so if you’re in the mitochondri field or even if you’re not you might recall this from um your previous um lectures at University they are key um organel for cell homeostasis
So they are first of all the main producers for of ATP for all sorts of metabolic reactions they can act of course as signal in platforms and their functioning metabolism and in singal signal transduction is key to the development of tissues and organs but they also um counteract uh the
Production of reactive oxygen species that can act as a source of stress and if the stress is to Extended that can also trigger programs of cell death and you might have heard that mitochondial dysfunctions are actually uh very widespread in a variety of um multifaceted diseases but from the in platforms they
Are also a huge Highway and here I uh it’s showcased actually what’s happening inside or actually even a small fraction of what is happening inside the organel but what you should keep in mind is that they are not only an internal Highway signaling to them themselves um but they
Are also signaling outside of the cell so they are in a constant interplay uh with the rest of cellular compartments and they receive decode and produce output signals um and of course from a signal in point of view this is um a source of um I would say complexity in
Trying to decipher what is happening here and how uh mitochondria signal back um and in the lab we’re trying to dissect a small proportion of this complexity by trying to understand how mitochondria interact with the cell cycle machinery and we do so um by developing um several approaches for
Instance first of all we want to sense mitochondrial activities that oscillate throughout the cell cycle and we do so uh and you will see during my presentation we do so by implementing threet sensors but we also aim to identify cell cycle proteins that act on mitochondria and last of course we are signaling
People so we want to understand the signaling choreography behind this interplay and this across three resolution scales so from Individual mitochondria up to the tissue level and in today’s presentation we will see a little bit of all of this um first of all I’m going to be telling you a story
Of um how we um detected a cell cycle protein which acts on mitochondria so this protein is called Aurora a and to do so so to um uncover the roles of this protein on mitochondria um we mixtured fret uh biosensors and also photoc conversion in cells and inv
Vivo uh and also I will be telling you about how from these uh first piece of data uh we engineered a novel fret sensor to um monitor intracellular turnover so basically uh a sensor to detect how the cell recycle its very own material through the autophagy
Pathway so um if you’re in the cancer field or if you’re in a Cancer Institute for instance you might have heard that orora a is an important protein in cancer therapies uh why because it is um actually a bona hmark of epithelial cancers so breast ovarian pancreatic cancers for instance and it’s actually
Old client since decades because we know that the overexpression of Aurora a increases the size of the mamory Grand and you can appreciate it here in this uh hology section compared to a control one but not only this um increase in the size of the mam Grand gland leads
Actually to tumor formation both in mice and in humans with very poor um survival actually and of course if you have such good good in a sort of a way client um the uh you want to have a look at the molecular activities of this protein so
We know uh that Aurora a is a kyes it is a seran 3on inyes that actually is playing lots and lots of roles in the cell uh it has non-tic functions and these functions take place throughout all the phases of the interface in the interface so from g0 to S going from DNA
Replication to neate extension to transcription activation for instance but um Aurora a is mainly known to be a mitotic protein so um it’s uh abundance so its levels Peak at the g2m uh phase of the cell cycle and throughout mitosis um it actually orchestrates centrosome maturation here but also it’s acting as
A timer for the mitotic phase and last it uh controls the construction and the stability of the bipolar spindle so the structure actually drives the separation of the chromosomes in the two daughter cells but so far I haven’t mentioned mitochondria so how did we link mitochondria with Aurora a uh so
Actually the evidence would pretty scarse when we started this project um so we know that epithelial cancers so um as I was saying breast ovarian and pancreatic cancers they re they rely a lot on mitochondria for ATP Supply so they are not glycolytic they don’t use the glycolysis for ATP they use this
Tiny little things um and we know that in these very same cancers Aurora a is uh hugely overexpressed um of course this was rather indirect evidence but a seminal paper um came out in 2012 uh at least indirectly linking Aurora a with mitochondrial segregation and mitosis so with the capacity of the
Mitochondria pool to be partitioned into the two daughter cells and Aurora a does so as I would say indirectly because actually it’s helping the relocalization of two substrates R A and R bp1 add m Andria you might say okay but actually we are signaling people we are microscopy people and I was really
Frustrated to uh I mean I was really interested in trying to understand how this relocation is happening uh both in space and time so basically how is Aurora a mechanistically doing that and if you believe some to a certain extent to the principle of aams Razor so that U
The simplest explanation is sometimes um the correct one uh we started to um hypothesize that maybe Aurora a is doing this function at mitochondria because it is there because we can find it there and so um narrowing down the distance between something that is potentially somewhere else in the cell but bringing
It close to mitochondria uh so the first question question that we asked was okay is it a mitochondrial protein and I promise this is the only Western blot I’m going to be showing you today but sometimes you’re obliged to do them um so if you’re in the mitochondria field there is a
Classical assay that you do to establish if a prot is and mitochondria or not and these assays are called trips in digestion assays so basically if you’re not familiar with those you take your mitochondria from cells you extract the mitochondrial population and you treat it with Trin to get rid of the outer
Mitochondrial membrane first or uh you treat them with tripin and detergents to get access to the inner mitochondrial membrane so here there invaginations um of the inner mitochondrial membrane um and basically you compare uh this is how it looks like only blood as I was saying you compare compare um the uh
Degradation pattern the digestion pattern of your um favorite protein so in our case it was Aurora a with the U digestion patterns of proteins that are known to be at mitochondria for so for instance we selected Tom 70 for the automat Coral membrane cytochrom C for the intermembrane space and pmpcb for
The um Matrix side so the innermost side and so you treat your mitochondrial fractions here you have the total ones for control uh but you treat your conal fractions either uh with Trin for increasing amounts of time alone or with DET detergents so Digitonin or Triton
And you just compare um and if you compare the uh degradation so the digestion patterns you will appreciate that Aurora a is getting digested very very late so you need tripsin and you need detergents to get rid of this protein from mitochondria um and this is
A very similar pattern um to compared to the one of PN PCB so Aurora is getting the digested late as a matrix protein is doing so we were very happy with this uh because not only it is a mitochondrial protein but it’s imported and integrated into mitochondria as uh mitochondrial
Matrix protein do but of course this is just the localization um we started to wonder okay is it playing any role there and since it is the kise uh of course the first thing you would look at is okay is it active at this compartment and to answer this question um we took
Advantage of our favorite technique in the team which is Fred By Flame microscopy uh and so uh this is just going to be a brief reminder because Yim and others um very nicely introduced this uh so in Fred by flim you rely on an intrinsic property of the fluoresence
Which is the lifetime uh which is the time that it takes for one molecule of any fluorophor in our case a Doner to go back to its ground state and this is like a fingerprint so it’s a very very stable uh property of fluorescence um that actually can change Its Behavior
When a donor and an acceptor fluorophor such as gfp and M Cherry here are in close proximity so less than 10 nanometers in this case uh an excitation of the donor would lead to will lead to an energy transfer uh towards the acceptor and in terms of Lifetime you
Will observe a shortening of the lifetime so compared to a condition when you have where you have no interaction if you have a protein protein interaction you would see a decrease in the lifetime okay but how do we apply this to Aurora a and to whether it is a
Mitochondrial active kindness or not well we created what um we can consider a biosensor so we actually performed threet with just one protein um we took the full length sequence OFA here and we flanked it with a donor and an acceptor floro for so in this configuration is
Gfp and mcherry and we benefited from data uh coming from the crystallography field where we know that the um that Aurora a has at least two confirmations an open inactive one here here and a close activated one here um this change in confirmation is induced by an autop
Phosphorilation of the kindness so the kindness can Auto phosphorate auto modify itself on this um residue um and this actually lets leads to a um a closure um of the kinetic pocket which also brings the two terminate together and allowing there for the threat effect so whenever you see a confirmational
Change so whatever you see threet uh you can infer on the confirmational change uh related to the activation of Aurora a um this sensor so this is the first version of the sensor was particularly instrumental to reveal um novel functions of Aurora a so uh we expressed
Our sensors uh our sensor at endogenous like levels and we started to monitor whether aora a could be active only at mitosis or in other cell cycle phases and this was the per first piece of evidence that confirmed an activation of Aurora a at my mitosis you see here um
The um poles of a mitotic spindle Aurora a is located there this is a fluorescent image of our sensor and here you see a lifetime cartography uh where you go from High um lifetime values to low lifetime values and uh so you already see in this map here in this pseudocolor
Scale that Aurora the lifetime of the Aurora sensor at mitosis is pretty low it’s towards the um yellow pseudocolor scale um levels um and you can see it here showcased in the graph uh so if you compare the behavior of the sensor uh the behavior of the biosensor here in
White at mitosis you will see that it is statistically different from um a daor only construct so a gfp orora a that Carri is no acceptor and therefore is uncapable of Performing threat but to our great surprise actually uh the expression of the sensor in other cell
Cycle phases um led us to observe that there is a strong activation of the sensor at centrosomes during G1 you can see it here and this activation was completely undiscovered before was completely unknown and uh it’s actually uh as strong as the activation of the sensor um in
Mitosis um very interestingly also if you look at centrosomes so uh fret measurements were done on these very bright spots which are the centrosomes um if you look at cells in S phase you will see that there is no activity no activation sorry of Aurora a at this location um which results in
Non-significant changes in the lifetime uh between the biosensor here in white and uh the donor only okay so we said if there is a uh an activation of the kinus there should be or there could be a potential role of the kinus at this compartment and um
Actually a role a new role for rora a um at this um during the G1 phase uh was detected um concerning um micral elongation because if you treat the cell with um a thermic shock and a depolymerizing agent called nocol to uh take out um the microtubules from the
Cell and you then you wash out the um the insult and you look at microtubules regrowing progressively over time you will see that in control cells that Express Alpha tubulin gfp uh microtubules will nicely regrow and uh go towards the EDG edges of the cells over time but uh in Aurora a depleted
Cells this regrowth process cannot occur and mro and microtubes are actually uh very fragmented and actually sto there um okay uh so this was a first piece of evidence for the usefulness of the sensor uh in concerning Aurora a but we also um identified few limitations of
Our tool first of all uh the Delta lifetime so the net difference in lifetime between the donor only construct and the biosensor uh in our hands was was maximum 120 PCS which is okay but it’s not um it’s not huge um and the gfp M Cherry configuration actually is uh preventing us from
Testing uh the Aurora a sensor with another sensor so a process called Multiplex uh because orora is a kindness so you want to see its activation but maybe you also want to see its activity towards the substrate because it’s phosphor relating substrates so if you want to use Multiplex threet uh this
Configuration might be um not super useful due to um the spectral properties of the sensor itself and on the biological uh side there is also another problem is that uh Fred By Flame experiments actually uh require adequate protein expression levels because you need a certain Photon budget to do uh to
Perform the experiments and this is particularly not convenient for Aurora a why because actually its levels are different according to the cell cycle phase uh so in g2m we have a lot of R so it’s easy to perform the measurement but uh in G1 they’re rather low um and this
Is also particularly uh problematic if you look at specific subcellular compartments that’s why basically so far I showed you only fret on centrosomes or on the mitotic spindle because it’s actually a very bright spot and you can detect it regardless of the cell cycle phase but if you want to perform threat
In the cytool for instance in G1 the kyes is so um we have such a poor abundance such a low abundance of the protein that is actually hard to perform thread by flim um that’s why we started to implement um or upgrade um the the biosensor by producing novel
Versions and so there were two major uh update routes that we wanted to take um the first one was to uh Implement Multiplex threet um approaches so uh you will see uh data concerning fluoresence Han isotropy that we uh use to uh potentially detect homofret and and dark
Acceptors again that allow us to accumulate uh our first sensor with the second one and secondly we also wanted to have a better dynamic range from our from our sensors so we also tested Novo a donor acceptor pair to improve the Delta lifetime differences um so first thing first um
We started with fluoresence anisotropy so we had a gfpm cherry version of the sensor we said okay what if we replace the acceptor with um gfp so we have a gfp Aurora a gfp sensor um this is particularly convenient because we know that um homo fret can be detected with
Anisotropy and that anisotropy decreases uh in the presence of fret and so basically you illuminate your sample with two uh laser uh excitations a parallel and a perpendicular one and you calculate an anisotropy index and as you can see here uh in this uh in this
Picture so we compared uh G gfp Aurora a so the donor alone again the biosensor gfp Aurora a gfp and from now on you will always see one mutant that we constantly use use in our analysis is the lysin 162 methionin which is an inactive um mutant of a keeping the kise
Open like this so for us it’s very important biological control and as you can see here in the micrograph so if you’ll focus on these anisotropy pseudocolored images or also if you want to make a look take a look at the graph well we were very disappointed because
We couldn’t see any threat occurring so any uh loss in a inotropy corresponding to threat in any of the conditions we were testing uh so we were very sorry but we couldn’t detect any threat using this tool so this is why actually we felt obliged to take a secondary route an
Alternative route and we focused on dark acceptors so this was an exciting time where U FL um dark acceptors so non florescent proteins such as Shadow G for instance were starting to be engineered and they were showed uh to be promising because not only they were non- fluorescent but they were also behaving
Um as excellent acceptors for um donor proteins so Shadow G was the first one that came out historically and it was optimized for San donors such as them tqu to and so here um we basically took cells in mitosis so where we knew that Aurora a was abundant and you will see
Here uh mitotic spindles from now on so here for instance and here and you can compare the donor only so this is always our standard control so in Aurora am2 close2 uh with or without Shadow G and as you can see here we start seeing decreases um so sorry we start to see
Differences in the um Delta lifetime profiles uh which are also showcased here in the graph so uh the Delta lifetime difference so the threet occurring between a donor only condition and the biosensor is significant here and with an inactive mutant we lose at least uh partially um the Fret effect uh
So we were very pleased with that because actually now we have the possibility of cumulating two sensors uh at the same time and this will come up later in my presentation also um but we also noticed that we had a difference in net difference between the Doner only
And the biosensor condition which was roughly 150 PCS so slightly better than our gfpm Cherry one which was 120 um but nothing Superior still um so this is why we at the same time we were focusing on improving our sensor and the dynamic range of it by replacing the
Donor and the acceptor pair and so um we were very uh grateful um to Maria and her team that provided us uh with a new bright uh yellow Flor for which is uh called nowadays tdan yfp back then we were calling it super yfp so in the original publication you will see it
Quoted as super yfp um but not we we put it in um in a donor receptor pair with m 2 so the very same um floro for as before uh being a donor of energy and again the principle is always the same you compare a donor only condition where
Rora a is expressed ad mitosis on the mitotic spindle and then uh you compare it with the um with the biosensor so carrying super yfp and as you can see here not only in these pseudocolored images but also on the graph here the difference between a Donner only condition and the biosensor dramatically
Increased so uh we still have a partial loss of the lifetime with um an inactive mutant uh but also we saw that the difference between these two conditions here is Al it can go up to 250 P seconds so we nearly doubled uh the uh Delta lifetime difference compared to the
First version of the sensor and what you might ask why are you interested in doing so well because maybe we reason that with this type of tool we could finally go in the cytool of G1 cells and measure fret um so we uh since the expression levels of rora a are so low
In the cell cycle phase and at this compartment we implemented more sensitive methods to sense fret uh in the cytool and these methods rely on two color fccs um and again you will compare uh we compared the profile I mean the um two Auto the autocorrelation curves from the
Wild type sensor here and the crosscorrelation curve here for uh this construct compared to an inactive um version of the of the sensor and as you can see there is a dramatic change in the ratio between the crosscorrelation and the cyan autocorrelation uh which is lower thereby indicating fret with the
Wab sensor and Which is higher indicating a loss of threat um with the kind is dead sensor so again accumulation of of approaches and of new um donor acceptor pair to urate our Fred readout and to potentially to detect pools of the proteins which were undetectable
Before and right now we clearly have a pallette of aorai sensor for uh for biology so relying not only on standard flat by fret By Flame but that can be implemented for Multiplex fret and that can be more sensitive uh for instance allowing us to use um two color fccs and
Now we can finally go back to mitochondria right uh because these are great tools for us to go and sense uh Aurora a pools in um discrete subcellular locations um nevertheless the first experiments that we performed were done with the version 1.0 of the sensors so gfpm cherry and as you can
See here again the experiment is uh standard in our hands we compare the donor only condition here and uh by expressing endogenous like levels of the cise uh we see that it we can detect a discrete pattern uh which um corresponds to the mitochondrial pattern here with which with its own
Lifetime and this lifetime decreases uh when we have a biosensor construct so gfp or am Cherry no news Under the Sun you’ve been seeing this before in my previous slides it’s just that this time it is at mitochondria and very interestingly this pool is sensitive to Inhibitors so if we take Inhibitors that
Actually are used in the clinic to counteract Aurora a specific cancers we can actually revert this activation of the sensor uh so meaning that there is no more difference between a DA or only condition and the biosensory condition uh these compounds derive from mln type of compounds and this uh compound is the
Most promising one that we have uh although it has a poor efficiency in Vivo but it’s called mln 8237 so um we retrieved an activation of the sensor um at mitochondria what for and of course if you’re in the mitochondri field you’re going to be interested in looking at the
Mitochondrial uh morphology and so so we performed these experiments in cells and um we benefited from the collaboration with Tomar Ral in uh in marsill uh that actually provided us with a very nice model of uh drosophila uh expressing mitenda 2 so mendra 2 I mean it’s a
Photo convertible protein you heard this before during the week so far but just a brief reminder so it is expressed only at mitochondria and when you photoc convert a subset of mitochondria in the roar you can have two outputs so either they are uh not connected so upon photoc
Conversion mitenda will become uh red but over time mitochondria will remain in the region of Interest or in the close vicinity or your mitochondria might be connected and you’re going to be experiencing the effect of water in a pipe basically it will diffuse through uh The Matrix and you will reach um
Mitochondria or pieces or portions of the network which are more far away and so this is exactly what we did in drosophila uh in in dropil uh we have a very nice model to keep uh the fly alive uh and to image in a non-invasive manner uh mitochondrial functions in the notum
So which is the back of the fly itself and here you see an epidermal cell of the notum of the fly expressing mendria 2 this is before photoc conversion so everything is green and no red and you see your tiny little mitochondria some of them are uh behaving as threads some
Of them they are dots and after photoc conversion so we photocon converted in this Roy here our mitochondria become red but over time they are mainly staying in the Roy or in close proximity of it indicating that the connectivity in this case is limited but if we overexpress Aurora a here again it’s
Before the photo conversion so the this is a cancer-like paradigm and if we photoc convert in this Roy you will see that over time almost the entire mitochondrial Network becomes red so meaning that Aurora a uh sorry mitoch Andra is diffusing throughout the network and actually this is happening
Not only at 2 minutes this is happening very very rapidly meaning that the pipe is really working and water is like really flowing and if we um quantify mitochondrial connectivity by normalizing mitenda red objects at different time points uh we can actually um see that we have higher connectivity
Here in flies that overexpress orora a compared to Wild Ty flies so long story short you express overe Express orora a like cancer uh and you induce mitochondrial hyperfusion is this just a cosmetic change of your mitochondrial shape well the answer to this question is no because again if you’re in the
Mitochondrial field you um might be wanting to know how much ATP your uh mitochondria are producing so one way of testing of assessing ATP levels is through mitochondrial respirometry assays and so uh we provided evidence that this confirmation of mitochondria actually is increasing mitochondrial respiration so mitochondria produce more
ATP when Aurora a is overexpressed um compared to cells for instance that Express only a gfp so we have a selective Advantage for the cells they change the mitochondrial shape in an aurora adependent Manner and this induces more ATP in the cell uh so to cut a long story short so
Far I showed you that Aurora a is a mital protein is getting in uh inside the organel it’s reaching the Matrix and uh the fact that you have a cancer-like situation where you have an an overe expression of aora a leads you to have a hyperfusion of organel so you have this
Gigantic mitochondria there are not just gigantic but also that produce a lot of ATP and um actually if you’re in if you’re a cell biologist like me you want to know how this is happening right so you want to start looking for the partners in crime
For these roles of Aurora a so we started looking for partners and we benef we were benefiting first of all from from our own proteum of aora a which was making actually life harder because we were detecting approximately 250 partners of aora a at mitochondria which yeah it’s a little bit complex to
Dissect uh but actually uh we were helped by the literature because ainal paper came out um depicting um one key partner uh of Aurora a which was prohibiting to that in this study was found to be uh the receptor of lc3 on the inner mitochondrial membrane and
This if this looks like Chinese to you don’t worry I’ll guide you through why is this important to us well uh again if you’re in the cell biology field you might have heard of a pathway to clear the damaged material from the cell which is called autophagy and lc3 so this
Autophagy pathway actually consists in building double membran vesicles called autophagosomes that actually grow and grow and grow and encapsulate the intracellular material including mitochondria and lc3 is a key partner in this pathway so without lc3 is really really hard to do autophagy and lc3 is also very peculiar because it’s there
From the beginning to the end of the pathway so at the end of the pathway uh the autophagosome here will fuse with the lome and thanks to the acidic pH of the lome you will degrade the cargo in the vesicle itself and so we went back to our Fred
By flim analysis we were very intrigued by all these potential partners of Aurora a at mitochondria and first of all we validated our uh hit that we obtained from the pro from the interatomic analysis um by performing Fred By Flame in a standard protein protein interaction mode um and as you
May invisage here you we have Aurora hfp which is a donor and again here you have the donor itself and we have this time two acceptors because prohibiting 2 is found in a complex with it’s cognate protein prohibiting and we tested they are mitochondrial and so we tested
Threet at the mitochondrial on the mitochondrial surface here which is dashed in Black concerning prohibiting and concerning prohibiting to and you can already foresee that we have um significant changes in the lifetime only in this mitochondrial area and for for your convenience this is Quantified here and we detected um a significant threat
Between auror and prohibiting but uh threat was even more significant between Aurora a and prohibiting 2 and at this point if you remember what I just told you meaning that there is a third protein in this game um we started wondering whether it takes only two to
Tango or maybe sometimes it takes also three to tango um so we started to probe protein protein interactions uh 2 by two using Fred by flim this is what I just showed you before so Aurora a it interacts with prohibiting to uh but also we found out that it interacts with
Lc3 itself so the second protein is still um effectively in the game and this um this is dynamic if we remove prohibiting we lose this interaction you see it here compared to compared to here positive inter interaction no interaction but also in cells that overexpress aora a we have um as we were
Expecting an interaction between prohibiting 2 and lc3 itself so uh if these look uh if this data looks look too complex um you find you will find a cartoon here indicating you that Aurora a is forming a tripartite complex with prohibiting to NLC 3 and this complex is taking place only at
Mitochondria and again this is not just cosmetic what’s the purpose of this complex um well actually we started to call um these mitochondria super mitochondria why because actually we provided evidence that if you uh buil this tripartite complex uh this is a way for the cell driven by aora a to degrade
A portion of the mitochondrial Network and select only the mitochondria that produce High ATP levels so um it’s a way an active way that Aurora a has to degrade the dysfunctional mitochondria and keep only only the ones that are super duper good and why do we say so
Because uh not only if we have a a formation of the tripartite complex we have high ATP levels but if we block it using for instance pharmacological approaches or uh mutants if we block it not only we restore the mass but we also restore normal ATP levels uh so these
Super mitochondria are indeed important in Aurora AP pathogenesis in cancer or they seem to be relevant at least but we’re still o microscopy people so we’re not only cell biologists we are also microscopy people and I was really frustrated because actually okay we depicted a role for the tripartite
Complex but what we could see where the entry point so here uh mitochondria was still there in the cell and I could only we could only detect the end point here so mitochondria at one point no mitochondria um at later time point and we couldn’t follow the degradation of a
Mitoch of mitochondria in real time and with a robust tool the tools that we have what he had in the field were actually not that U were not performing very well at least not in this um not in this paradig and so if you’re in the microscopy field you might uh appreciate
The fact that okay you don’t have a tool you want a better tool you better build it but of course it also means that you’re back to back to square number one and you have to start again by validating a new tool and so this was um
A project carried out by beum a PhD student in the team and now a postu uh who actually uh started to dissect the molecular uh signaling of the autophagy pathway so I told you the lc3 is a key player in it but uh unfortunately for bigum that had uh to
Deal with this uh back then it also has a very complicated signal in Cascade everything starts right after the um the production of lc3 uh and as soon as is um translated actually it’s taken care of by atg4 which acts as a molecular scissor and which recognize this this
Residues and cut this residue here uh this Glycine and cuts a portion of um of lc3 and then this is called priming so it’s a real oophagy initiation step but then lc3 is undergoing a wide series of intermediate processing steps and this is the market list I don’t expect you to
Um to remember any of this unless you are in the autophagy field and you’re interested in this but uh you may be interested in kn that uh lc3 is literally taken care of by intermediate atg proteins that actually take it and carry it physically um at the interface
With the fofor vesicle so this vesicle that degrades the material and at this point other atg proteins will conjugate so will attach a molecule of lipid to lc3 that will allow for the integration of lc3 itself on the Figo for membrane so you’re sticking lc3 on your membrane
And then the vesicle grows and grows and grows until until it closes is up and this is a go sign autophagy at this point is turned on and of course whenever you say that you can turn something on it means also that you can turn something off uh so it’s sufficient
To remove the lipid here to detach lc3 which is freed in the cytosol and then the pathway can begin until the source of stress is solved and degradation is no longer uh needed um why am I telling you this story well because in the field and this
Is might is part of the reason why the tools we had back then were um not sufficiently robust or uh informative is that autophagy initiation was back then currently uh was undetectable meaning that the uh great majority of tools were reporting only on this step and people in the autophagy field use this
Conjugation step to in to infer whether autophagy is on or off but actually everything that comes before is non-detectable um so if you want to have a more sensitive tool we reason that we um should have designed a sensor to um detect autophagy initiation um and again
We went back to fret and we built uh so we basically flanked uh lc3 itself with a donor acceptor pair here uh and the Fret readout allows us to say okay autophagy is turned off and wherever autophagy is initiated remember this atg4 uh partner that remember the scissors actually it’s cleaving off
Apart of lc3 so we uh the the donor and acceptor Moyes are actually um teared apart and no threat uh is longer detected and then autophagy can still go on and on but we still have uh a way to sense its progression because our lc3 fused with the donor is a regular lc3
Based construct that uh sticks to the fofor membrane and so we can quantify with a regular confocal microscopy readout the abundance for instance of lc3 positive vesicle uh vesicles and infair on the status of autophagy and for this sensor and you see me coming it relies on uh the
Improvements and the knowledge that we uh obtained from the orai sensor because we benefited uh from uh aquamarine and tdan yfp um which are a particularly convenient uh pair of donor receptor pair and why are they convenient in this uh in this context is because they are
Totally pH resistant uh so they resist to acidic pH so in theory this allows us to follow the pathway really from the beginning until um the uh incorporation of the vesicle and its fusion into the lomes um so first of all this was the design right so um you need a proof of
Concept that what you design is actually happening and first uh we want you to um understand whether the sensor responds to an atg4 dependent cleavage so remember the scissors here so we compare the WAP sensor where these residue here is accessible to atg4 uh we compared it to a
G120a sensor where uh by replacing this glycin with an nanin you basically block the action of atg4 atg4 does not longer recognize lc3 and lc3 cannot be cleaved and this was a go noo step for us um and you can appreciate here in uh in the images here uh concerning the WAP and
The g20a sensor that we have a dichotomic Behavior Uh here with the wild type sensor we basically detect no fret here you have the life the fluorescence and the Delta lifetime cartography so basically these all these pixel correspond to no fret values they uh they they have a lifetime which is
Similar or almost identical to the donor uh whereas wherever you have uh a mutated biosensor here you see a very significant threet and here I would like to drive your attention on Delta lifetime differences we go from um of course no um no uh variations or very
Limited variations here with a wild type to 500 PCS of Delta LIF type difference for the mutant and here we were taking so this this difference is is is really um much more significant than the Aurora a for instance where at maximum we could have 250 PCS um but also here we were
Performing our our analysis by calculating the mean Delta lifetime meaning that we get one value uh over uh mean Delta lifetime throughout the entire cell area so the entire cell area is one value and with this type of conditions we I mean it was particularly sufficient actually I would say because
It allowed us to determine that the Fret first of all here depends on the presence of this glycine or not uh which is targeted by atg4 if you remove it you have uh you start to see uh a lot of threat um and of course we want you to
Take the uh question the other way around so not acting on lc3 but acting on the enzyme and of course we employed um atg4 SI rnas to answer this question um so here you will always see the mutated version uh in the cartography you see that this huge uh Delta lifetime
Differences are maintained you see them here in the pseudocolor scale where purple means um means fret and you can see it here showcased in the graph no matter what we do to the uh to the mutant we see um this huge um Delta lifetime difference occurring but we
Were actually very um very disappointed to see that if we look um at the wild typ sensor uh in uh a in uh atg4 uh sna uh condition actually we couldn’t see any significant difference uh with compared to the control ones we could see some but it was actually never or
Almost never significant and um actually our frustration when looking at this data led us to ask us a further question uh we started to reason that maybe mean Delta lifetime fails where pixels don’t meaning that maybe by using just a mean Delta lifetime throughout the entire cell area was limiting us um in
Extracting pertinent information still occurring in the cell and so uh we implemented a more sensitive uh type of analysis uh which consists first of all of what we call a histogram analysis uh five minutes no it’s over oh sorry uh okay I can finish okay thank you and uh
You will see here that uh at the end of um of the tail uh we have a shift in the two curves um from control to atg4 SNA and you have pixels in the SI atg4 sna um that actually fret as much as the uh as the mutants so apparently I’m running
Out of time uh so I’m not going to be telling you this entire story and how this actually is pertinent to to uncover new ways of regulation of autophagy during the cell cycle but I will be happy to discuss with you further should you be interested and so uh the O original
Question and I promise I’m done uh the original question in understanding uh whether this sensor is pertinent for R dependent mitophagy well we still don’t have an answer to this question and maybe uh next time um that we discuss together in Mobo we will finally have um
An answer to this question but the experiments will be conducted soon so in conclusion I hope I convinced you that Fred sensors are indeed uh a nice tool to detect um the roles of Aurora a and potentially autophagy uh within cells ated mitochondria that we can image um
Complex uh complexes made of different proteins at mitochondria and that we can Implement both Broad and sensitive analysis to extract more sub spatial and temporal information and so if you want to know more come and see our Workshop so there is a workshop animated by CLA
PhD student in the team um that is uh understanding protein protein colocalizations and mitochondria using storm so this is a new uh line of research for us and uh with Sav panson engineer on the um macroscopy facility in Ren we are trying to implement um methods to uh both extract the super
Resolution information and uh the threet information and this afternoon there is also a round table on uh biosensing for organal imagings that is animated uh by sandin P and myself uh on new developments and challenges in this topic and with this I thank you for your
Attention this is our team in Ren ouch um I’m particularly grateful to um collaborators that were instrumental in detecting the roles of Aurora a at mondria at mitochondria such as Beatrice durel sh kran Marie of course who is here in the audience that are provided us with tdan yfp and Arno goer who
Actually um engineered uh hybrid versions of the eurai sensor relying on the fast um on the fast tag today I presented you data from from bigum I’d like to thank the sponsors of course and you for your attention and I’m happy to take questions thanks Julia for nice
Beautiful and clear interesting talk uh two rapid question first have you tried to follow see something about P6 two on this process and uh the there exist a mutant where you have for example Aurora B Aurora a sorry mainly expressed in cytool cytosolic level that revers the
Level ex expression so there are two unrelated question yes okay so I’m starting with the second one so yes we tried mutants that only retain their cytosolic activities they’re really not very uh nice for the cell I mean the cell doesn’t like to have cytosolic or
Only or a so the mitocondrial pool is really important and concerning the p62 I guess it’s more for the uh for the um lc3 sensor uh part uh so we are wondering if these uh huge clusters of uh fret that we see in the cells are p62
Related we still don’t have an answer for that but this is indeed on our to-do list and then the rest of my question at your uh Round Table of course