<TITLE: Cell Biology Club: Biological Roles of Twinfilin Isoforms in Mammalian Cells
ACADEMIC DOMAIN: medicine
DISCIPLINE: cell biology
EVENT TYPE: lecture
FILE ID: ULEC23B
NOTES: continuation of and continued in ULECD160, event also includes ULEC23A

RECORDING DURATION: 18 min 4 sec

RECORDING DATE: 19.4.2007

NUMBER OF PARTICIPANTS: 10

NUMBER OF SPEAKERS: 1

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

SU: unidentified speaker>


<S8> so as mentioned i'm <NAME S8> from from viikki and i'm going to tell you about twinfilin isoforms but first er a little bit about actin er erm actin is this 43 kilodalton ubiquitous very conserved cytosolic protein and it erm has this monomeric form and these monomers er polymerise into filaments which are the functional form and each monomer has bound ATP or ADP molecule in them , erm this actin filament turnover is called treadmilling and erm (in this simplified) picture we have er actin monomer that has ATP-bound they come er into the plus-end the ATP is hydralised and the ADP-bound form comes off the nucleotype is changed and then the circle goes on . er well most people know actin from from muscles where it forms these (xx) structures with myosin and these structures are very stable but in non-muscle cells actin cytoskeleton is very dynamic and it can form these different erm forms like lamellipodia and stress fibres and filopodia and stuff which is not shown here it's involved in several important er cellular functions like movement cytokinesis polarity and endo- and exocytosis . and also it is known that some intracellular pathogens like listeria and vaccinia use this er er actin machinery er as a way to move in the cell during the infection process and this erm the way they erm use this actin machinery er can be exploited on on certain assays which one of them i'll explain later er there are really a huge number of proteins involved in regulating this actin cytoskeleton the proteins that er depolymerise and severe the old actin filaments the ones that start new filaments er and start branching er the ones that catch the unneeded filaments er and some that er control the monomer pool in the cells so er some of these proteins can be divided in families and one family is this ADF-cofilin family which has five members and all of they have this ADF er homology domain in them and i'm studying this er this one called twinfilin , which is about 40 kilodalton also very er ubiquitous and conserved protein it has two of these ADF homology domains the linker region and the tail region er it localises to the cortical actin cytoskeleton in yeast and mammalian cells and in yeast and drosophila and such there's only one twinfilin isoform but erm it has been discovered that in mammals there are two which have been named twinfilin-1 and twinfilin-2 and these are biochemically very similar to each other they both er bind actin monomers with one-to-one er stoichiometry and they both prefer ADP-bound form or ATP-bound form and they inhibit this er change of the nucleotype and that also inhibits the actin filament assembly they are known to interact with PRD2 which er inhibits their er actin interaction they also interact with capping protein erm this doesn't have any effect on the actin interactions but it's needed for the correct localisation of twinfilins they are believed to erm localise actin monomers to correct sites in cells , so here's the localisation erm actin staining twinfilin-1 twinfilin-2 you see that they have this er uptake staining and then also in these actin-rich filopodia areas er so they localise pretty much the same way but the er regulation of the localisation is different er for example when you transfect with active CDC42 you can see that twinfilin-1 goes to these cell-cell adhesions whereas twinfilin-2 doesn't and with active TRK1 twinfilin-1 goes to these membrane ruffles twinfilin-2 doesn't but that's pretty much all we know about this regulation so far , er they also have different expression patterns erm this er from er er embryonic mouse tissues twinfilin-1 twinfilin-2 you can see that twinfilin-1 is er the predominant one during the de- development and it's also more abundant in these adult tissues and it's erm expressed pretty much everywhere except in the skeletal muscle twinfilin-2 is also in almost all tissues and it's the only one in skeletal muscle and especially abundant in heart and er PRDC2 you can also see that they're quite widely expressed erm here you can see the muscle but no twinfilin-1 but twinfilin-2 is expressed there and also in the scheme there is a big difference , okay so here is erm stuff that has been published recently about this discovered new function for twinfilin-1 so er as i mentioned earlier it has been known that twinfilin sequesters these actin monomers and inhibits their er er polymerisation into these filament ends and the other function is binding capping protein and er erm but as i mentioned there's two of these domains and both of them have an actin-binding er region and none of these functions explain why there's two domains we just thought that these er actin monomer sequestering and capping protein binding er er reside in the C-terminal where the stronger monomer-binding (xx) . so er , i'm going to show you a couple of experiments from this paper and for you to better understand the next one i'll er er er i'll explain this a little bit so if actin is only sequestered it can still er er there's not enough twinfilin to sequester all of the actin from the cells so there's gonna be three monomers to attach to the filament ends so it just er inhibits the filament assembly slightly but if if it has some other function for example if it caps the ends and sequesters it can inhibit the polymerisation very efficiently . so erm from this experiment you see that er , this dotted line represents what would happen <MOBILE PHONE RINGING> if if if actin <SU> sorry </SU> er twinfilin just sequesters er like the maximum sequestering and when you look at the filament growth from the pointed-end you can see that it's above this line which means that is just sequesters but from the barbed-end you can see that it inhibits the growth much more strongly so there's something else also going on and capping function would fit this very well and this is to show the same experiment with the isolated er N-terminal and the C-terminal domain and the C-terminal domain with the linker and all of these merely sequester they don't have this capping activity so we conclude that this capping activity requires both of the domains in the twinfilin , er another experiment exploits this er this pathogen er movement er which i showed before if you have erm lis- listeria which has this er ACTA protein that activates ARP2/3 complex which nucleates new actin filaments and this er growing of the filaments pushes the bacteria forward so you also need er capping proteins to stop growth of unwanted er filaments or the ones that are left behind and then you need some for example cofilin that depolymerises these old filaments and so that you don't <SIC> ran </SIC> out of er actin monomers that you can recycle them to the growing end so these are the minimum requirements needed for listeria to move and er it has been deve- er developed in assay that uses these er proteins and instead of bacteria there is these beads that are coated with NWASP which is also an ARP2/3-activating protein , so with this erm it's called minimum motility medium when you er er here's the bead and here's the actin tail and the beads move and when you add twinfilin nothing happens but when you take out the capping protein from this er from the these components erm the beads don't move but the actin erm tail just grow in every direction and form these weird shapes but then when you add twinfilin it is actually the tails start to look normal and the beads start to move again so twinfilin can replace the capper in this in this minimum medium and here's to show that er most twinfilin does this but yeast and drosophila twinfilin don't rescue this movement , so we have found the function of twinfilin which needs these both both of these domains here's to conclude er twinfilin-1 caps both domains are needed yeast and drosophila twinfilin don't and this is involved in motility and endocytosis , so another part of my talk will i'll tell you about twinfilin knockouts mhm er previously there has been er made er (xx) in yeast which erm results in enlargement of co- cortical actin patches and lethality with er some cofilin and profilin mutants and er this hypomorphic mutant in drosophila er results in this er (xx) bristle morphology so these erm phenotypes we thought that er twinfilin may play some role in development of multicellular orgasi- organisms and decided to create knockouts or isolated isoforms and if they survive hopefully a double-knockout and hope this will tell us more about the importance of twinfilin and how the different isoforms er what are their roles and this is done in collaboration with doctor reinhard fässler's lab in in max planck institute so here's just er to show you briefly the knockout constructs in both of these the knockout cassette has been inserted under the first exon , and so far we have the twinfilin-2 knockout mice but unfortunately they don't have any obvious phenotype twinfilin-1 mice are still under process , but we found something interesting (xx) while we were erm making sure that the twinfilin-2 knockouts are really knockouts so from the western blot you can see that twinfilin-1 expression is fine it was low in heart and and in muscle and actin expression is fine there was some strange (xx) in the heart and muscle and er and then on checking the northern blot you can see that really in the heart and muscle there is some RNA present while the other tissues seem like correct knockouts , and and we did some searching and discovered actually that there is a second possible slice variant for twinfilin-2 so for example here is the actual twinfilin-2 gene it has nine exons and then there's this other one that has eight exons and the , well this one's second exon which is here the first er s- it is slightly longer . and here would be the sequence for this one and they only differ from the very N-terminus but the er knockout cassette was inserted into this first exon here so it prevents from this twinfilin so we named these twinfilin-2A and 2B so it erm prevents this from er this expression but not this one , and we confirmed this with RT-PCR so it really shows that twinfilin-2B is expressed only in the heart and muscle , of these mice , so what erm are we doing doing is to clone and express this twinfilin-2B and the isolated domains of these these two and check if they have the same er biochemical functions actin-binding capping protein binding and capping and hopefully some day we can make a construct that will knock out all of this twinfilin-2 so here's some er data about these they er the full-length protein have a pretty much the same er affinity for G-actin erm the surprise came here that the twinfilin-2B N-terminum domain binds G-actin much more tightly than twinfilin-1 N-domain or the 2A N-domain so it seems to bind actin more bi- er i believe than these two and this is to check for the capping er this is er this doesn't have the dotted line that was on the er earlier picture but you can imagine it here but it seems that all of these forms also cap , and they all seem to bind capping proteins here's the native gel from capping protein (xx) there's twinfilin-1 er twinfilin-1 with capping protein here's erm twinfilin-1 where the capping protein binding domain er or the domain pathosequence has been removed so there's no binding and then 2A and 2B they can really bind . so so far we haven't found a lot of differences for these three isoforms and it seems that twinfilin have er mammalians have three twinfilins that are biochemically similar but the expression patterns differ slightly and all of them have these three func- functions sequestering binding capping protein and capping actin filament pathogens , so hopefully we'll find some difference between these (areas) , so , er then thanks to pekka lappalainen and all our group and past member maria vartiainen who's now in cancer research UK erm these knockout things were done in collaboration with reinhard fässler's er lab and and the capping things with marie-france carlier and her people , thanks </S8>
<APPLAUSE>
