<TITLE: Cell Biology Club: Submicrometre Vertical Movements of Organelles Near Plasma Membrane Revealed by TIRF Microscopy
ACADEMIC DOMAIN: medicine
DISCIPLINE: cell biology
EVENT TYPE: lecture
FILE ID: ULEC23A
NOTES: continuation of and continued in ULECD160, event also includes ULEC23B

RECORDING DURATION: 42 min 50 sec

RECORDING DATE: 19.4.2007

NUMBER OF PARTICIPANTS: 10

NUMBER OF SPEAKERS: 4

S3: NATIVE-SPEAKER STATUS: Russian; ACADEMIC ROLE: senior staff; GENDER: male; AGE: 31-50

S4: NATIVE-SPEAKER STATUS: Russian; ACADEMIC ROLE: masters student; GENDER: male; AGE: 24-30

S5: NATIVE-SPEAKER STATUS: unknown; ACADEMIC ROLE: unknown; GENDER: male; AGE: unknown

S6: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: senior staff; GENDER: male; AGE: 31-50

SU: unidentified speaker>


<S3> thank you <NAME S1> very much thank you for inviting me , i have impossible a mission impossible today in 30 minutes i'm going through quite a few er things if i manage i'd like to introduce a new technique to you or maybe er some of you are already familiar with the TIRF microscopy can i see the hands of those who okay those who came to (the course) yes and also er to the (xx) er facility er in viikki the other task is to er present briefly what the interests of our group is that er we are in in the neuroscience centre and we are interested in the tripartite synapse this would be a foreign term for you right has every anyone ever heard about tripartite synapse <A FEW PEOPLE PUT UP THEIR HANDS> okay good erm not too @many@ and er i'll i'll show briefly three applications of TIRF microscopy and how we are er using it to advance our knowledge about sy- synaptic physiology , TIRF microscopy works great er but it only works in cultured cells and on the plasma membrane but luckily plasma membrane of cultured cells is a very interesting place and er er i don't have to convince you that er this is the site where communication between the cell and the external world is appearing and this is a a compartment in itself so if we roughly talk about the intracellular er compartments and the extracellular compartment then this is the third compartment which separates one from the other and this is where the communication of this cell with the neighbours is is taking place of course there are important transmembrane and membrane-associated proteins er receptors ion channels voltage-sensitive channels the transporters er of course the extracellular ma- matrix is not to be forgotten and important things such as neurotransmission er occurs at the plasma membrane er exo- and endocytosis is er er a very important event in communication between cells so if you are in- into cell biology or neurobiology er er mem- the plasma membrane of the cell is a a very very important er thing to look at , but with optical methods how do you look at plasma membrane it's not always an easy task because the resolution of optical microscopy of s- light microscopy er is is pretty low especially the vertical resolution er even in confocal microscopy so if you're interested in this these guys on the surface and you're not too much er interested in the the guys under the surface you want to have a technique which separates one from the other and er i'm going submit to submit to you today that TIRF is exactly the tool to do it in the cultured cells how does it work , first of all what does it stand for TIRF stands for total internal reflection fluorescence microscopy , and it's based on a very well-known phenomenon of total internal reflection of the light specifically we're talking about laser light which is a coherent beam of er the same wavelength and er er the phase er then when when you direct this beam onto an interface between a high refractory index medium and the low index medium for example if we're talking about glass-water interface the er refractory index of er glass is about 1.5 and er water is about 1.3 so we have now a difference between the er the optical density of of these two media then er there is always a bending er of the beam and at a certain angle which is called the critical angle none of the light is actually er trespassing the er the interface between the two media but all of the light is reflected back into the high dens- optical density beam , this feature is used in er er in telephone industry all the time the the light guides er and fibre optics they're all using this er this principle , what i didn't know of course i from the elementary physics course i knew that the er about the phenomenon of total internal reflection but what i did not know is that when light is completely reflected back from the er interface some of the energy is actually transferred on the other side but it is transferred there in a non-propagating wave light of course is a propagating wave of energy er and the er the the field or the wave which is created on the other side in the in the water er er on top of the glass coverslip er is actually er not propagating very far into the direction perpendicular to the surface it goes there er to a very short distance and the intensity drops off dramatically in about 200 nanometres or even 100 nanometres er then w- we'll see immediately what the advantage of of this er so-called evanescent field is if you put a living cell if you culture cells as we all do on top of a a glass coverslip then er with this totally internally reflected light beam you will be able to excite fluorophores which are in the membrane or very close to the plasma membrane but not all the other fluorophores that are inside the cell away from the plasma membrane and this is a huge advantage because now we are unlike the the epifluorescence when the light goes through the preparation and all of the fluorescent molecules are excited at the same time in the TIRF microscopy we're selectively producing fluorescence in the membrane-associated fluorophores these could be organelles which happen to be close to the membrane or in the membrane these could be er membrane-associated proteins fluorescently tagged these could be fluorescent indicators for calcium for er , chloride or anything you're interested in in the vicinity of the plasma membrane the background the signal-to-noise ratio is extremely good here because you're you don't have to deal with out-of-focus fluorescence at all all that you're interested in is this fluorescence and you can collect it with a CCD camera which is of course now much faster than the confocal scanning laser scanning microscope a little bit of er formula here this is just to draw your attention to the fact that the intensity this would be the intensity of this evanescent field becomes very low very quickly it's very high at the surface of the glass and then in an exponential fashion er it drops off so it's high here and it's low there and the intensity of the field would would depend on the distance from the surface and also on the penetration depth constant the penetration depth is proportional to the wavelength of the light and to the angle the er incidence angle and to er er two of the refractory indices er of of the of the media so for a normal er let's say 450 or 488 er nanometre er light which you use to excite your GFP or FITC er the er penetration depth er the constant of this exponential decay of the intensity er would be around 150 nanometres so immediately you get a vertical resolution er which is way higher than what you can get with confocal microscope i'm a great fan of confocal microscope myself but TIRF really beats it in vertical resolution have a look this is the point-spread function of the confocal microscope the lateral resolution of the confocal is approximately half of the wavelength that you're using so we're talking about 250 or 300 nanometres the vertical resolution er is much worse it's only one micrometre but with TIRF microscopy there is a little bit of an improvement in the lateral resolution and this is because you are not dealing with any background fluorescence you don't have to eliminate it you don't have it at all but the vertical resolution is better than 100 nanometres so </S3>
<S4> isn't the vertical resolution of confocal much improved by the convolution </S4>
<S3> er apparently <S4> [mhm-hm] </S4> [it is] with er er you can really resolve the smaller details especially we can improve your picture but yes , so there's a theoretical limit to all of the optical microscopy methods except the TIRF because here we are not dealing with er with refraction limit anymore we're in a sense using a trick of evanescent fields the the feature of de- decaying intensity to surpass the refractory er er limitation , how does it look like in practice well this is a hippocampal neuron er primary culture er which is er transfected with in this case it's TRKB receptor for BDNF tagged with GFP er and you can see the clusters the transport vesicles taking TRK around the cell and of course you're all familiar with the transfected neurons the soma is very bright because that's where most of your protein with the fluorescent protein tag is accumulated and then it's very hard to see the erm the dendroids and specifically especially the dendritic spines or er terminals or filopodia the small details if you look at the same neuron here in TIRF microscopy you can immediately see the difference now we are looking only at the footprint of this cell we're not looking at all the er elements er for example er these dendroids are not even seen in TIRF because they are sitting on top of glial cells neighbouring this neuron but those parts of the membrane which are attached to glass they appear at a very high er signal-to-noise ratio you can see the the details the filopodia and the spines very well and the same in here and epifluorescence doesn't show you this this well so lack of out-of-focus excitation and therefore lack of background fluorescence improves the resolution greatly you can also do er you you can combine epifluorescence and the TIRF and colour-code it as we did here so the green light is the er is encoding the epifluorescence image of this cell and the red is encoding er the TIRF image and then the colour actually now represents in a sense the distance from the coverslip so the er the closer it is the greener it is the further away it the redder it is and i'll show you how we used it to track mitochondrial movements near the plasma membrane in a moment what our group is interested in is plasticity of er the tripartite synapses first of all what is the tripartite synapse we firmly believe that thinking of the synapse as a simple connection between two neurons is incorrect because this connection is tightly enwrapped by glial cells specifically in the s- in the brain these are astrocytes er which a- are like foam around in the neural field they're foam around the synapses and each synapse is to some extent and this is a variable parameter er enwrapped or ensheathed by the astrocytic fine processes so we think of the synapse not as a dipartite or or dual structure but rather as a tripartite structure three parts presynaptic neuron postsynaptic neuron and the perisynaptic astrocyte , and what we specifically are interested in is the link between the functional and morphological plasticity of the synapses we think this approach is is particularly er important for finding the link between morphology and function because astrocytes er are very motile the fine processes that are close to synapses they move all the time er and we see it with the multiphoton or confocal imaging in brain slices er and the the movements of the astrocytes towards the synapse or away from the synapse have a dramatic effect on the uptake of the neurotransmitter clearance of potassium or the spillover of neurotransmitter all these things that have been studied for for years but not many people are looking at the role of astrocytes in in this so we sort of decided that this would be our niche to to look at er the astrosynaptic communication and er the the function of the synapse at the same time so mostly we're using combination of electrophysiology with imaging but today i'm only talking about er imaging techniques the TIRF er was established in our lab about three years ago and er it was simply er introduced to me by er mikko nokkonen who works for olympus here in finland he said there's this thing called TIRF have you heard of it i said no what is it then he explained me briefly and i started to read about it and got really excited so my mission today is to get you excited about it how is TIRF useful in our research well one of the things we're looking at is the activity-dependent release of substances such as ATP from the astrocytes ATP inside the cell of course is the form of er energy storage and accumulation er produced by mitochondria mostly but it can also be released in a vesicular form exocytotically from the cells and then it becomes a powerful neurotransmitter er there are specific receptors for ATP on the astrocytes and also on neurons on the terminals and er on postsynaptic neurons and we are interested in the mechanism of release of ATP and also we're interested in trafficking of ATP receptors h- how they get to the membrane what are the mechanisms to maintain them there and remove them et cetera so TIRF is really good to look at vesicles because you can really track each vesicle coming to the membrane and then releasing its content into extracellular space the second project is a m- er membrane trafficking of proteins such as receptors or channels er and there TIRF is also very very useful and the third one is motility of different organelles we happen to focus on mitochondria because er we have discovered that er they are incredibly motile not only er in the horizontal direction but also in the vertical direction they come er to plasma membrane and move away and i'll show you the data on that and we think it's very interesting and very important for cellular function so how does the vesicular release look in TIRF first of all how does the trafficking of vesicles whether it's transport vesicles or secretory vesicles dense-core or synaptic vesicles how does it look in TIRF as any organelle as any fluorescent object er which is too far from the plasma membrane to be excited by er evanescent field the distant er vesicles which sit in GOAG or in endoplasmic reticulum they are not visible in TIRF but when they come closer to the membrane they become visible and this is shown here th- first they appear in the red colour only epifluorescence then the TIRF signal increases and finally when they they fuse to the plasma membrane if they have some fluorescent content then it looks like an explos- explosion i'll show you this little movie and pay attention to this particular vesicle can you see it <SU> mhm-hm </SU> er it it will be a little er blowing in end now and the vesicle disappears i'll play it again . so now it's the exocytotic release . er what we do we load er cultured astrocytes with a a dye er which is called quinacrine or guinacrine er which accumulates in the vesicles containing high concentrations of ATP in a peptide-bound form and then we use two imaging modes TIRF and epifluorescence to visualise those vesicles and here you can see that each vesicle has its certain its own distance from the plasma membrane some of them are seen only in red and these are the vesicles that are far from they are not docked on the plasma membrane whereas the other here here in the green colour they are very close to the plasma membrane and then we stimulate the cells with er increased potassium or with specific agonists like ATP or glutamine and what we see is the selective release of those vesicles which are docked on the plasma membrane which makes sense and of course when we er we can track the movement of these vesicles and you can appreciate that from that movie for instance this vesicle is not docked and it's very far from the plasma membrane and it appears in the red colour only whereas the the other molecules or vesicles that are close they are not moving very much , er this here just shows an example of the specific behaviour of different vesicles and i'll play you some more movies here we have the spontaneous release of the vesicle that used to be here and the others stay around i play it again , this vesicle disappears the others stay and this is a an in- er induced exocytosis which would be associated with a massive release of vesicles and you can see that before they are released before they disappear they come closer to the plasma membrane they change their colour from red to green and then after that they disperse , we can track each vesicle individually because we are doing time-lapse imaging and here is an example when we apply ionomycin to increase calcium inside the cells in low concentration then this vesicle becomes docked so its TIRF signal actually increases which means it becomes more excitable by the evanescent field because it's closer to the interface to the coverslip right so the behaviour of this vesicle was to come closer to the plasma membrane to get docked this vesicle first got docked and then it released its content and this one was already docked and it just released and then we can calculate what's the er distribution of the times of release or the kinetics of release it's a very er useful and and interesting information because there are of course other methos- methods to study exocytosis for instance you can measure capacitance of the cell but then you don't have any spatial information of where the release is taking place whereas with the optical imaging with the TIRF you can really say which vesicle and where and what's the history of this vesicle et cetera , briefly the second er project and i i'll just show a few examples you can fluorescently tag the protein of interest overexpress it in the cell and then look at its trafficking not just along the dendrites er and axons but actually towards and away from the plasma membrane here is an example er we're looking at some MAGUK proteins which are synaptically associated SAP97 PSD95 , again this is an epifluorescence image and you can see some clusters there but the resolution is is not as great is it er it appears more or less like a big mess @@ but when you look at the same neuron with the TIRF microscopy you can immediately see all the clusters er both of the soma and also er the dendrites and these are (xx) the dendritic spines where SAP97 went the same here you can see some clusters er and follow them but it's not very easy er but with TIRF microscopy the resolution is increased and er you are looking selectively here at the clusters that are already in the membrane and you can study their dynamics and we also used a an even more er complicated technique which is a combination of TIRF with FRAP the TIRF-FRAP er you know what FRAP is of course right er it's fluorescence recovery after photobleaching but instead of photobleaching a region in the X-Y er plane er what we do here we photobleach specifically the membrane-inserted or membrane-associated proteins we just expose er the cell for a l- extended period of time to the TIRF excitation and then we we we bleach all of the proteins that are that are sitting in the membrane and watch them come back they don't come back the bleaching is irreversible but they are replaced by the non-bleached er er molecules here we're looking at the specific subtype of a potassium channel tagged with EGFP and TIRF is shown in green er epifluorescence in red , watch this movie you will see the green dots disappearing and then one by one some of them will come back so now we bleach them and here you can see this one coming back there this actually came back and then moved away so er this way you can you can really estimate the kinetics of the insertion you can estimate the er just as in other applications of FRAP er you can estimate the er mobile pool the immobile pool et cetera er it's a really really nice way of studying that er it's just an example of how to how you can trace the the kinetics , finally i would like to spend a little more time on telling you about the mitochondria mitochondrial motility because this is a novel er we like to claim that it's a novel er mode of signalling of way of signalling of mitochondria , er this image just shows you er an astrocyte we are interested in astrocytes as members of the synaptic er er tripartite structure which was er er loaded with mitotracker green this dye accumulates specifically in mitochondria and then we stain the membrane with a a styryl dye with FM143 with a very low dose we keep it in the bath so just s- so to see where the the cell appears and when you look more closely at the mitochondria in a time-lapse mode just normal epifluorescence you notice that they move a lot , so these are not just er er fuel tanks if you wish or you know the gas stations but these are rather the gas stations on the wheels that move wherever you need them my computer is acting up . er and this shows you that there's a lot of lateral motility but when we combined here the TIRF microscopy with epifluorescence we noticed that they don't only move left and right but they also move up and down . er but before i go into that up and down movement i would like to er tell you a little bit about why mitochondria may wants to move to certain places mitochondria in addition to being ATP er power stations er they also are very strong er th- they have a very strong calcium buffering capacity they can take up a lot calcium actually much more than endoplasmic reticulum can take up however this calcium buffering capacity is only activated when mitochondria are exposed to a very high calcium concentration about one millimole . or was i supposed to yeah i think it is about one millimole in any case in the cell the the high calcium concentration occurs only at the site of either calcium release from endo- endoplasmic reticulum stores or the influx from the outside world so the overall calcium doesn't go up that much because it's very carefully buffered including by mitochondria in the cells so in order to take up calcium mitochondria have to be very close to the calcium source otherwise they will never take up calcium and this is illustrated er here in this concept of microdomains of ca- high calcium is illustrated by the spatial interaction between endoplasmic reticulum and mitochondria when calcium is released from er er endoplasmic reticulum stores due to activation of IP3 receptors this calcium can diffuse to a neighbouring er IP3 receptor and increase the probability of calcium release from from there but and this will give rise to the calcium wave we all know about the calcium wave generation right this i don't have to explain very carefully calcium-induced calcium release does it ring the bells CICR okay good so CICR will be very strong if calcium can diffuse from one store to the other with no inhibition and this happens when mitochondria don't er collocalise with endoplasmic reticulum but if they do and if they have the membrane potential then they can take up all o- all this calcium or most of it so instead of going to the neighbouring IP3 receptors it it will go into mitochondria and then no wave propagation the propagation is blocked so mitochondria wherever they are they buffer calcium around them very efficiently so this was known about collocalisation between mitochondria and endoplasmic reticulum but what about the other important source of calcium in the cell the plasma membrane where calcium can come from the outside well we checked that using the TIRF technique so we s- we stained the mitochondria or we actually tagged them with er YFP mitochondrial er er YFP and then we exposed the cells to the evanescent field to the er the TIRF in the TIRF mode this approximately shows you the distances there if the evanescent field permeation depth er penetration depth is about 150 nanomolar and the plasma membrane is within let's say 100 nanomolar then it doesn't leave much space for mitochondria to be excited these are s- large organelles and the closer they come to the membrane the more they get excited if they move away they're not excited anymore okay that makes sense so the vertical mobility or m- vertical movement of each mitochondrion towards the plasma membrane will be er revealed as an increase a sharp increase in the intensity of the TIRF signal and the movement away is a decrease of the signal , let's see how mitochondria look in the in a cultured cell this is the normal epifluorescence er way of looking at it and then we switch to TIRF we get a quite a different picture er the mitochondria which are close to the plasma membrane they are greatly revealed whereas some of the mitochondria that are er not very close they are not even visible so if we colour-code it here we'll we will see that some , i switched the colour here right the the red now is close to the membrane green is far from the membrane just to confuse you to keep you alert rather erm no this comes from two post-docs' projects and they just like different colours so this , er what we also see here is that each mitochondrion can can er have some parts of it that are close to the plasma membrane and some that are far away and if we now look at its endodynamics if we do the time-lapse imaging we'll see that this distance also changes in time we see that mitochondria not only move around but they also change colour for instance here it becomes red and then yellow and then green and so tha- this is these sort wiggling movements up and down but th- this is er er er non-stimulated the resting condition what happens if we now stimulate the cell well two things will happen first of all they will stop moving and second of all they will all or or mostly they will drop on the plasma membrane er like just like leeches and and get stuck there er let's see how this will look w- we focus on this specific region and then this is the control condition unstimulated and then we stimulate with glutamate and we'll see that the TIRF signal is greatly increased whereas epifluorescence is not changing i'll go back to control and now with glutamate the TIRF is increased and you can see the change in the colour here from green to mostly yellow and red if we now plot the average er TIRF signal we will see that it is gradually increasing during the one minute of glutamate exposition er and the epifluorescence signal doesn't change which means that we're not losing the dye from the mitochondria we're not changing the brightness of mitochondria in- individually or anything we are simply moving them with this stimulation towards the plasma membrane , the other er important thing to remember with TIRF if you are going to do these experiments is that , er , it is incorrect to say that we are monitoring distance from the plasma membrane because we don't know always where the plasma membrane is er what we're measuring is the distance from the coverslip from the interface er between glass and water so i- in each experiment where we want to claim that an an organelle comes closer to the plasma membrane we want to make sure that the plasma membrane is not moving down at the same time so that's what we did we er used er double TIRF two lasers at the same time the plasma membrane was stained in red here with er synapto-red dye and mitochondria in green , so now if the plasma membrane moves at the same time we will see that er that as well and what we found is that the plasma membrane doesn't react to glutamate application in the same way so we can really claim that there is an increased spatial association between mitochondria and plasma membrane </S3>
<S5> s- so what about the overall thickness of the cell [s- so does the cell wall] </S5>
<S3> [er that] w- we did some er control experiment to test for that there is er this assay er with er calcines we can load cells with calcine which is theoretically calcium-sensitive but it er its er sensitivity is so low that er it doesn't doesn't change er much in the cells er but what will ch- would change is er when the cell changes the morphology the density of the dye er will change and therefore the intensity of the signal in the optical frame also changes it has been used before to check with cell volume changes and we don't see that er so th- that indicates that it's it's not that the cell is sitting down altogether but it it's rather er the specific movement of mitochondria the other two things that are are happening there is an increase in calcium which is known to be there when you stimulate astrocytes with glutamate and also the drop in motility as i said they come closer er to the plasma membrane and they stop moving er we did this this assay er to check their mobility , if you add BAPTA-AM or incubate cells with BAPTA-AM to block er to chelate intracellular calcium none of this will happen we can stimulate the cells but there is no increase in calcium no change in the mobility and no association of mitochondria with plasma membrane if you use other agonists now ATP is a potent agonist for astrocytes er very similar picture a sharp increase in calcium a gradual increase er in the TIRF signal meaning they're coming closer to the plasma membrane no change in epifluorescence and er the change er er transient degrees in the motility you can also er increase intra- extracellular calcium to drive calcium inside the cells and now the er movement of mitochondria to the plasma membrane becomes even more efficient it it's very rapid so we think that it's actually the influx of s- er calcium inside the cell that triggers this behaviour in mitochondria , here's a the movie which shows you this effect if you look at the green and red colour you will see that at a certain point the red colour goes the intensity goes up whereas green doesn't change that's er during the stimulation with er with increased extracellular calcium the stimulation was now and we can see that and now we remove high calcium and it goes back to the base level , what we know is that mitochondria move inside cells er along microtubules and er er the filaments so we wanted to at least get an idea of what whether this vertical movement is also dependent on the cytoskeleton er microtubules and er other elements we used a very crude tool of course er n- nocodazole and nocodazole is a drug which er prevents polymerisation of actin i'm not expert in that so what would happen is th- the that the microtubules would become shorter and shorter right there is no polymerisation of the plus-end the minus-end there is depolymerisation continuously it's a very slow effect but what happens is that they stop moving which was shown previously in other types of cells and also surprisingly this is what we didn't know they actually come closer to the plasma membrane again due to this treatment with nocodazole and this is no- not associated with an increase in calcium anymore because nocodazole doesn't do anything to the calcium </S3>
<S6> sorry sorry did you here check the plasma membrane movement <S3> [yes] </S3> [with nocodazole] around some of the cells and </S6>
<S3> er yes er w- actually i i i can't remember i don't er i don't want to lie here er this would be a good good good thing to check er i have to ask julia whether she did that experiment or not erm then after th- this treatment with nocodazole you can stimulate cells now with high calcium and nothing happens they do- don't er stop th- they don't decrease their motility because their motility is already @very@ low er they don't come closer to the plasma membrane but they er normally increase in their intracellular calcium so here er we think that this motility is also mediated by microtubules this is roughly what we think happens there there are at at least two motor proteins that are associated with mitochondria dynein and kinesin and they are pulling mitochondria towards plus- and minus-ends of microtubules and under normal conditions they can move freely when calcium is low they can move freely along the microtubules but they're also the the microtubules are attached to the adhesion plaques so some mitochondria would come closer to the plasma membrane just spontaneously like testing going around when microtubules are disrupted then th- they will er mi- mitochondria will be gradually dragged towards the plasma membrane and this cartoon er tom and jerry style illustrates this just as i said normally there's a there is a s- spontaneous testing behaviour of mitochondria they move left and right up and down as long as calcium is low when calcium is increased specifically when it's increased in the microdomain here at the plasma membrane then the next time mitochondria happen to come close they get stuck , we don't know exactly what is the signal erm which is mediating that what is the calcium censor there but it could be associated with actually uptake of calcium into mitochondria and maybe their depolarisation . then when the microdomain is removed they can move freely again . why er we think this is important well the the first reason it is important for calcium buffering the mitochondria are made unable to come close to the plasma membrane they will not be able to prevent the calcium overload by er voltage-ga- gated calcium channels or er NMDA receptors or any calcium-permeable channels so the cell will actually be poisoned by calcium and er this mechanism we think may be involved in er ALS where mitochondrial motility is lower in an amyotrophic lateral sclerosis and er it could also be involved in other apoptotic mechanisms where mitochondria er are known to participate not only by releasing the er cytokines but also perhaps by this kind of mechanism we don't know yet but we can hypothesise a second very interesting thing is that mitochondria due to their respiration all the time are producing reactive oxygen species the free radicals , and these free radicals they don't move too far from mitochondria normally because there's lots of antioxidants and targets for oxidation inside the cell but once er reactive oxygen species get out of the cell they can easily permeate into the neighbouring cells because they they go through the the bilayers no problem they're gases basically very er cell-permeant and there er they can find different targets like the synaptically associated proteins et cetera we have recently shown that astrocytes when stimulated in the brain slices they can generate a lot of er reactive oxygen species and these reactive oxygen species would er change the probability of release of GABA at at the synapses in the young hippocampus so how do reactive oxygen species get out of the cell , one mechanism is that , they get out at this moment when mitochondria get close to the plasma membrane because now the distance is so short that reactive oxygen species can get into extracellular space where there are not too many targets for them not too many (xx) and then they get into neighbouring cells whereas under normal conditions when mitochondria are far away from the on average from the plasma membrane er there is not much of the intercellular signalling mediated by reactive oxygen species but this is er yet to be proved and yet to be tested , so in summary , i hope to have shown you that TIRF microscopy is a very useful tool for studying the membrane-associated signalling , er unfortunately it's limited to cultured cells you cannot do it in brain slices or in the in vivo experiments , er it is very useful to visual- for visualising docking and fusion of individual vesicles the spatial resolution is great , it also provides you unique ways of characterising the er the membrane trafficking membrane recycling of er fluorescently tagged proteins and er this is about the mitochondria we have shown that the er stimulation or stimuli which increase intracellular calcium they drive mitochondria close to the basal plasma membrane er and this may be important for calcium homeostasis and also for reactive oxygen species signalling and finally i would like to thank the people who did the work evgeni is looking at ATP release from astrocytes julia er studied mitochondrial motility and ramil er was looking at reactive oxygen species and this work is done in cooperation with our friends er in italy in the US and in slovenia and we're funded by the academy and er CIMO thank you very much </S3>
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