<TITLE: Cell Biology Club
ACADEMIC DOMAIN: medicine
DISCIPLINE: cell biology
EVENT TYPE: lecture discussion
FILE ID: ULECD160
NOTES: continues in and continuation of ULEC23A and ULEC23B

RECORDING DURATION: 18 min 30 sec

RECORDING DATE: 19.4.2007

NUMBER OF PARTICIPANTS: 10

NUMBER OF SPEAKERS: 10

S1: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: senior staff; GENDER: male; AGE: 31-50

S2: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: unknown; GENDER: female; AGE: 31-50

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

S7: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: senior staff; GENDER: female; AGE: 31-50

S8: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: research student; GENDER: female; AGE: 24-30

S9: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: unknown; GENDER: female; AGE: unknown

S10: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: research student; GENDER: female; AGE: 24-30

SS: several simultaneous speakers>


<S1> okay let's get started , now obviously some people's s- stuck in a traffic jam somewhere but they come as they g- as soon as they get here so i'm really happy to see all of you he- all of you here you're highly welcome to this er cell biology club meeting and it's very nice that so many of you made it here despite the late , beginning er time point i er frankly i don't know the reason why this er s- the seminar is later today than than usual [nor-] </S1>
<S2> [i know] </S2>
<S1> you know </S1>
<S2> yeah there wasn't available room [before this @kind of space@] </S2>
<S1> [uh-huh okay okay] okay </S1>
<S5> and your english is doing fine you're still </S5>
<SS> [@@] </SS>
<S1> [okay okay great so er] </S1>
<S2> @keep going@ </S2>
<S1> we have two er presentations today the first one is by by <NAME S3> from the viikki neuroscience centre , <NAME S3> er erm , did his er undergraduate studies at the university of kazan in biophysics and er he finished his PhD in 1997 in trieste in italy thereafter he did two post-docs in the united states one at the duke university and one at N-I-H . and er he's been in helsinki since 2003 and he's presently group leader at the neuroscience centre so <NAME S3> is interested in syna- er er primarily interested in synaptic physiology and he's today talking about movements of organelles near plasma membrane as revealed by TIRF microscopy please <NAME S3> </S1>
<LECTURE ULEC23A by S3>
<S1> thank you very much er for the for for a fascinating talk now there's some time to take some questions <NAME S2> </S1>
<S2> now <COUGH> this er move- movement of mitochondria was very interesting er the the images you showed sort of er showed like mitochondria having a fixed size but i- i think that at least in in many cell types it's it's like a network and there is constant branching and and and frag- fragmentation and and fusion <S3> yes </S3> so can can you detect that kind of er changes with the TIRF <S3> [erm] </S3> [not] just sort of er moving up and down [or or] </S2>
<S3> [yeah] th- this kind of changes you can detect even with the normal epifluorescence <S2> [mhm-hm yeah but] </S2> [microscopy w- we do] see them er attach or detach from each other er , the interesting thing is that segments of mitochondria exhibit different distances from plasma membrane so it looks like this part is attached to one @microtubule@ perhaps and and the motor protein a separate motor protein is driving it at this moment towards the plasma membrane whereas the other part of this train is attached to a different motor protein and it's going up so this seems to be really stochastic and er er independent if you look at this movie again and er you know they're here for instance this part behaves completely independently from that part and you know and and there and you can notice also some fission and fu- er fusion events here erm , i don't think TIRF is er specific er specifically useful for detecting fission and fusion because there you you will have two processes going on at the same time erm but otherwise yes you could s- study vertical motility and fission and fusion at the same time </S3>
<S1> <NAME S6> </S1>
<S6> so related to the last part of your talk so are there virgin means to detect these er ATP synthesis or the ROS generations so that you could <S3> [yes] </S3> [m-] monitor them at the [same time] </S6>
<S3> [yeah] i removed the slide unfortunately but er there are some ROS-sensitive er indicators <S6> [mhm] </S6> [for] instance dichloral er fluorescein or diha- dihydro-dichlor-fluorescein which when it's oxidised it becomes fluorescent and actually changes the excitation probability er and we did look at it and we do see an increase in er reactive oxygen species in TIRF which means near the membrane and outside as well er during the activation of astrocytes however there are other sources of er ROS as well er like er NAPPH er in the plasma membrane itself er which can also contribute to that and the contribution is pretty large from other people's work er in order to dissociate that we would need more pharmacology a little bit but we're working on this er actually a- another word to promote TIRF it's very difficult to work with er ROS-sensitive dyes because they are er they can er spontaneously oxidise and change the fluorescence er they are very sensitive to ambient light er et cetera and the re- reactions for all of them are irreversible so the same molecule can go- cannot er go er back from the oxidised state however here when when you use TIRF or for that matter multiphoton microscopy to produce excitation only in a very thin plate then after you have er oxidised one molecule of the dye then it can very quickly exchange with a different molecule er which can now detect er reactive oxygen species again so in a sense you make them you make the reaction reversible but this is only possible either in multiphoton microscopy or here in TIRF because we are specifically exciting the perimembrane volume and there is a large reservoir for the new er fresh molecules for coming there so it's that's a good question </S3>
<S6> er if i may just briefly another [er] <S3> [yeah] yeah </S3> ju- just i mean this is an impossible question to answer but er but er regarding your model so this tripartite er er <S3> [synapses] </S3> [s- synapse] synapse so er this is unfortunately i mean here it's er restricted to this er glass er interface er water interface so just could you just sort of comment on that how well actually these sort of things that you now model in this interface do then actually spatially represent are represented in the <S3> yeah </S3> er your real object of study and </S6>
<S3> er very badly </S3>
<S6> [yeah so that er] </S6>
<S3> [@@ we can] only look at the bipartite synapses <S6> [yeah] </S6> [actually] if there is an astrocyte under it we will only see the astrocyte we will not see the terminal and the spine simply because it will be out of the reach of the evanescent field so here we are limited to the non-physiological synapses and that's why we're only looking at the astrocytic signalling separately actually or neuronal signalling separately er what the preparation that we use for the real tripartite synapse studies is of course brain slice or with some ambition in vivo @@ and er we use multiphoton microscopy combined with electrophysiology there but that's the topic of the second talk @@ which </S3>
<S1> i'd like to ask about the astrocyte er systems er in the tripartite er synapse the astrocyte is obviously polarised and forms close contacts with the neuronal part of the synapse a- are the ATP-containing vesicles also localised to active zone-like things or or or ho- how are they organised in the in the in the [tripartite synapse] </S1>
<S3> [yeah] er we are working on it at the moment we're trying to use the quinacrine approach in slice it's a little more difficult there because you have to electroporate the cells with the dye and it's not working yet for another neuroactive substance glutamate er which is also released by exocytosis from astrocytes it is known that the vesicles containing glutamate are er enriched at the perisynaptic sites er and @more@ more specifically they're actually er at the presynaptic site where er er the specific subtype of NMDA receptor extra- extrasynaptic NMDA receptor NRDP containing is localised so there's a beautiful study by andrea volterra and the group er in this year's er science check it out they they follow the whole sequence from you know molecules to er in situ recordings and show that synaptic plasticity specific form in hippocampus is mediated actually by astrocytes @@ <S1> okay </S1> so it's it's really really nice </S3>
<S1> yeah , <NAME S4> <NAME S4> </S1>
<S4> when you use GFP fluorescence GFP-tagged proteins you get very nice er spatial clustering formation er so two questions come er did anybody try to compare these with the immunofluorescence for non-tagged er proteins for natural proteins to figure out possible artificial effects of GFP fusion and the other question if in many cases obviously immunofluorescence will not be specific enough so can you use any logical tricks any parameters of GFP fluorescence which would indicate how natural this behaviour versus er what could be artificial er result of abnormal mass or concrete mass and abnormal shape [of the (neurons)] </S4>
<S3> [yeah yeah] well that's er er a nasty question from any referee who who wants to sink er a paper with the GFP-tagged protein er the usual ways that people use is either immunofluorescence as you say but immunofluorescence is tricky in nonpermeable astrocytes you have to use the extracellular epitopes you have to you know be be careful with that we have done some attempts but not successfully the other approach is to use overexpression of the proteins with a small tag , er but most of the small tags are not fluorescent so you cannot follow them in living cells in time-lapse there's a trick a very interesting one er which i found very useful er you can insert the sequence of of the bungaritoxin binding domain of let's say er of of er nicotine receptor alpha-seven nicotine receptor into your protein of interest it's only like seven millimasses or 27 i can't remember it's really really short and then you use er fluorescently tagged bungaritoxin which is also a very small molecule and then you can follow the trafficking of of this receptor er and we've done that that works well and the trafficking doesn't seem to be different er for specifically alpha receptors which we are looking at </S3>
<S1> there was o- one short question from the back </S1>
<S7> so okay i think so related to this same thing can you show one more time this PSD95 <S3> sure </S3> GFP i was just working on that this day and i was wondering how it should look like [@@] </S7>
<SS> [@@] </SS>
<S3> oh it shouldn't look like this @@ i would say that it it it should rather look like that with er a lot of s- spine insertion this was done in very young cultures the thing another disadvantage of TIRF is that , er you have to use rather young cultures and low-density cultures so that each cell is attached to the glass covered with laminin which is unnatural right it is not good for them er lots of studies have shown that if you don't have an astrocytic sublayer in cultures the synapses are not formed normally so these are abnormal cells they are attached to glass they are unhappy and probably that's why we don't see many synaptically associated clusters and we don't we can't wait two or three weeks for the s- synapses to be formed completely so you should get a better picture if you're working with mature cultures with lots of healthy astrocytes there to feed your synapses to ma- to help them er be formed and maintained erm but other than that you know , they wouldn't be sitting in the dendritic spines and some of them are still in the dendrites </S3>
<S7> yeah so also this [SAP97] </S7>
<S3> [SAP97] yeah the same the same thing this just happens to be er larger in the expression of protein there but otherwise they they look similar sorry for taking so much @time@ </S3>
<S1> no problem thanks a lot <NAME S3> <S3> okay </S3> then we move on to the second presentation by <NAME S8> from the institute of biotechnology who is er telling us about twinfilin isoforms she works with with pekka right </S1>
<LECTURE ULEC23B by S8>
<S1> okay thank you very much <NAME S8> for a really nice talk , questions <NAME S6> please </S1>
<S6> so i mean there was this really small @unfortunate@ N-terminus you had bad luck with the not bad luck but er sort of er with the twinfilin-2B that there was such a so it was very interesting but so is there a structure i mean how what's the role of the very aminoterminus of er twinfilin is it known because </S6>
<S8> well in [twinfilin] </S8>
<S6> [how] much they differ o- otherwise they were identical </S6>
<S8> yeah well it's known that for twinfilin-1 at least the very N-terminus is really important for the actin monomer binding <S6> mhm </S6> so we thought that there might be some some difference there and it seems that it binds actin monomers <S6> [yeah] </S6> [more tightly] but we don't know why </S8>
<S6> but there is no structure </S6>
<S8> er not really i think ville is working on the structure <S6> mhm </S6> i think er at least for twinfilin-1 i don't know about twinfilin-2 we haven't (xx) </S8>
<S1> okay any other questions <NAME S9> </S1>
<S9> if you are working on the protein from er any any actin-binding protein from mice and then you don't see a phenotype do you then is it customary that you challenge it somehow that you make them extra excited or something </S9>
<S8> erm we're thinking that probably because there's twinfilin-1 and there's twinfilin-2A they're both in almost all the tissues so probably they can compensate for each other and we're trying to make this twinfilin-1 knockout to see if it if that one has er a phenotype and then if they survive er cross them and see when you get all of these out and hope hopefully then there's something er something erm after that probably some behavioural and this kind of testing , yeah </S8>
<S1> <NAME S10> </S1>
<S10> so there are other actin monomer-binding proteins as well or <S8> yeah [yeah] </S8> [yeah] are there knockout mice for any of those </S10>
<S8> erm yeah maybe some </S8>
<S10> do you know what the phenotypes are like </S10>
<S8> er . i think erm . i know that a- at least for profilin there are some mice because bu- but i think that was lethal so now <S10> [yeah] </S10> [th- they] just have like er conditional <S10> yeah </S10> knockout erm , so . i'm sure there are others but right now i can't [think of any] </S8>
<S10> [yeah] i was just wondering if you you know have any idea what to expect if you get the knockout </S10>
<S8> yeah well we did but we didn't find @anything yet@ so </S8>
<S1> i guess the problem you encountered with the knockout model is a pretty universal one people er the classic er approach is to target the first exon but there's so many genes there are slice variants which are skipping the first exons er <S8>  yeah </S8> i guess e- e- every every single person who is planning to make a to make a knockout is is struggling with the same problem <S8> yeah </S8> how to how to how to create a knockout construct </S1>
<S8> yeah i think it it was because this project started like even before i came to the lab and it was designed years ago so i think then there was no indication of this second slice variant it was just discovered and put on the database </S8>
<S1> i guess the result anyway indicates that that er that the muscle that that the muscles they somehow need another slice variant of the twinfilin er to strengthen to to sort of sort of er , put more emphasis on on the on these specific functions in the tissues where mo- where where more efficient actin function is needed or something like that so <S8> [yeah] </S8> [i- it] the result in itself is is really interesting </S1>
<S8> yeah we are thinking maybe er the muscle where you have these very stable structures you can't have any actin filaments going like <S1> yeah </S1> in any direction you need something very strong twinfilin <S1> [yeah yeah yeah] </S1> [to keep them] in in check </S8>
<S1> if there are no further questions we s- we thank the speakers once again please give them a hand <APPLAUSE> thank you all of you for your attention </S1>
