تدريب Shadowing: Helge Ewers Seminar - تعلم التحدث بالإنجليزية عبر الفيديو

So my name is Helge Ebers.

I'm professor for membrane biochemistry at the Free University in Berlin.

And we are very much interested in membrane compartmentalization, something that we share with our host.

And we have been selected for today's journal club for a recent paper from a finishing grad student from the lab,

Jia Ahui Li, that was published earlier this year

and what she did there was she used tiny magnetic fluorescent particles

and pulled them using magnets over the cell surface

and i would like to share with you now why we did this

and what we think we may be able to do with it

and maybe this would be interesting for you or your work

so uh do we just jump right in yeah

so i mean we are very excited about this paper

so just start whenever you want all right

so uh here obviously this is a fluorescence micrograph of a cell here you can see trajectories of this particle

that we can pull all the way to the edge of the cell

and then it sort of travels around the edge of the cell here

and gets gets stuck and this is already one feature

that i really want to drag your attention to is

that we have very high accuracy right

so we can use the extremely high pointing accuracy of source

and particles together with pulling the molecule into a specific direction thereby we want to learn

something about the places where these molecules will get stuck

or where they stop moving where we come from is we

want to understand how membranes are compartmentalized all of you were

obviously familiar with the singer nicholson model from 1972 where singer

nicholson synthesized a number of recent observations on what plasma membranes may be made of

and consist of and how they may be assembled from lipids

and proteins into these fluent fluids and continuous bilates right

so a membrane protein would be embedded in a mosaic of lipids

and proteins in which these molecules are free to move right

and as an illustration i brought to you here a quantum dot

that is bound to a lipid in a supported membrane bilayer

and you can see that this molecule here is of course absolutely free to move

and just wiggles around in a random motion

through this membrane bilayer okay

so it's diffusing already pretty early after the 70s a number of investigators such as mike sheets

and naki kusumi and ken jacobson and and others

used modern video microscopy to investigate what is happening in membranes

and immediately they found

that in plasma membranes proteins are actually not entirely free to

move indeed the diffusion coefficient is like an order of magnitude

or more slower than would be expected in free lipid bilayers

and they started investigating this

and the most elaborate model here stems from decades of work

in the laboratory of akakusumi who has correlated movies with extremely high

frame rate imaging of of lipids or proteins bound to gold beads

or fluorescent particles and what he and others would observe is

that they would sort of seem to jump between domains in

which they would reside for a certain amount of time and when they did platinum replica EN microscopy of the plasma membrane,

they found that the cortical actin at the membrane would form these little corrals.

And that led to a so -called fence model

that Aki Kusumi has been putting forward on how these corrals

of actin filaments at the membrane basically compartmentalize the membrane

and proteins have a hard time jumping over these fences when they diffuse around in the plasma membrane.

So we now try to understand how this works, if that is really indeed the case, and what forms of membrane compartmentalization exist.

So diffusion barriers, like obstacles to the motion of membrane lipids, are ubiquitous in cell biology.

So the sperm head is divided from the tail by a barrier

that separates membrane proteins from diffusing back and forth there is the tight junction

and epithelia separates the apical from the basolateral membrane the cilial

plasma membrane is indeed a different compartment from the apical membrane so it's a different

uh complement of membrane proteins in here than in the remainder

of the membrane neurons have a diffusion barrier in the initial segment

that separates the axonal membrane proteins from the somato dendritic membrane proteins

and dendritic spines as well compartmentalized allowing only selective access to their head in

which the excitatory synapses sit finally dividing cells also have a diffusion barrier around the cleavage furrow

and that seems to be at least at least in yeast cells even be present in the endoplasmetic reticulum

and the nuclear envelope so the the specific regulation

and physical hindrance of membrane protein motion is a ubiquitous phenomenon in cell biology

and we we try to understand how this works

so in the initial segment here this diffusion barrier is one of the first ones

that has been described

and investigated in great detail first by a paper from the EMBL from Toshiri Kobayashi

and Carlos Dorsey's lab who found that

if they would use a pipette filled with fluorescent lipids

and touch the axon with it they would move through the axon

but they would not move out to the um side to the to the cell body okay

a couple of years later bettina winkler and numing poos lab found that this was likely due

to the soma just being so much larger than the axon

that you just couldn't detect this worsen lipids

and she could not define find a diffusion barrier here only

to a couple of years later uh doing some work with iran melman

and paul forscher a great expert on the normal cytoskeleton

to investigate using optical tweezers pulling membrane proteins through the initial segment

that they would get stuck in the initial segment

and they could not move there okay this was then further

investigated by aki kasumi who could correlate the motion of membrane

proteins to the accumulation of an adapter molecule called anchor

and g and that was published in nature biology in 2003

so uh yeah

so we are interested now in this um diffusion barrier here at the axon initial segment

that separates the somato dendritic domain from the axonal domain

and here what we find is a specific complement of adapter molecules cytoskeletal molecules such as spectrins ion channels

and adhesion molecules that make a specific array

that is about 60 to 90 microns alone

and this separates the external membrane proteins from the dendritic membrane proteins

which makes a lot of sense because of course the somatodendritic part integrates

and receives signals and the external part sends out signals so you don't want to mix that up

now it's long known that we have a diffusion barrier here so we got really excited when in In 2013,

the Xiaoyi Zhuang lab published the fantastic observation that along the initial segment,

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there would be an array of rings of actin circumfering the axon.

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So every 200 nanometer, you would have this actin ring that goes around the axonal membrane.

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Now, for us, of course, this was a fantastic way of testing whether these actin rings

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we find here in the excellent initial segment could be used

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as a paradigm to finally test the kosumi model right

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so they could never really look at the actin at the same time as the proteins

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because the actin cortex obviously is very plastic so it constantly moves

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and there's so much actin in the cell

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that it's very very hard even with modern super resolution methods to get a reasonable resolution of these compartments

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and in the time scale that is comparable to membrane protein motion

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so here we now have these rings that are persistent over longer time

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and we were hopeful to correlate that motion to the

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that structure to the motion of membrane proteins

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and we could indeed do this

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and here you could see you can see a single particle

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tracking data of lipid anchored gfp gpi gfp coupled to quantum dots

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and we could localize indeed that these stripes are right between these actin rings okay

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so the membrane molten protein diffuses between these actin rings

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and has a hard time in this wicked model of jumping over

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that actin ring now we are very happy to to publish this

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and we're really excited to prevent this to kusumi

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and others as a way of investigating membrane protein motion to find direct evidence for the Kusumi model.

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But of course, uh we always have to be skeptical right so one idea

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that is prevalent especially in the initial segment field stems from the observation that where we have these actin rings

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these actor rings are interspaced by spectrum tetramers okay

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so these elongated spectrum tetramers are about 190 nanometer long

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and they have binding sites for a large adapter molecule called anchoring g

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and this anchoring g recruits a lot of huge ion channels

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like five nanometer diameter eight nanometer they have a huge ion channels

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and also adhesion molecules and plenty of them

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so what we actually indeed have in between these actin rings

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is kind of like a forest of all these ion channels okay

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so when you talk to initial segment people they say well

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i'm not so sure that your membrane protein actually bumps into the actin rings maybe it's rather

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that it sort of gets stuck between all these ion channels and it's just not moving out

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so um we like uh complicated experiments i have to confess we like we just like really

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things that are complicated but are to the point so what we thought we should do

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is we should try and image a single membrane protein

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and then pull it across this area okay

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so now we would either just pull it through the membrane

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and it would kind of get stuck in between all these ion channels

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and then we could pull it again or

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when we pull it it will bump into the actin

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and then we can pull it through to the next ring

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and it will bump into and we can pull it on okay

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so and by combining a means to pull the membrane protein with

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very high resolution single particle tracking we were hopeful that we could achieve this

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so optical tweezers are you know expensive to set up

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and we didn't have them so we looked into much simpler means than

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that and we decided to go for a magnetic nanoparticles okay

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so we went through quite a few i have to say

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not all magnetic fluorescent particles you can buy are really very

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useful and nice so it was really um several months of work just to figure out

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which ones work well and which ones are mono dispersed

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and bind without background and you can still move them

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and they are bright enough and so on

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but we found some where we have an iron core

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that is surrounded by a starch shell and

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that is then coupled to striptavidin and

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so we can then bind it to biotimulated nanobodies

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or biotimulated lipids on supported membrane bilays okay

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so we can throw these nanoparticles into um light scattering uh device

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and we can see they're nicely in mono dispersed okay

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so we have a single peak around 200 nanometer in size

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um this is pretty much in agreement with the electron microscopy images we get of these iron cores

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so they the real particles will be a little bigger

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because there's a starch shell around it

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which we can't see i like this one it looks like a gummy bear a little bit

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if you like um so hopefully uh this is going to be a little more round

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when it then finally binds to the cell okay

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so we then made supported membrane bilers in these uh cover glass bottom dishes

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and we were thankful to get from friends a micro manipulator we could use to deliver a tiny magnet

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and attached to the magnet we have a metal wire

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that we pulled to be very very um uh uh thin at the tip okay

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so basically we have very strong neodyne magnet and a little wire

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that you can't see here it's so fine

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and then we dip it into the um solution where our cell sits

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and then we try to do microscopy okay so this is basically the setup you have your objective

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you you have you can bring it into less than 800 micrometer distance and then your magnet

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is parallel to the cover glass in a couple of microns above the cover so we can't really tell

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we just kind of try to almost touch it and then move the tip here you can see the tip

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towards the cell this is the tip of the wire right so the wire is here attached to the magnet

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this is what it then looks like here you can see this is a supported membrane bilayer

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we can now pull a couple of these particles you can see some of them are clearly stuck

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some of them don't really care so much about the magnet

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but a couple of them really clearly move towards the needle

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that is to the right here okay we can localize these

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particles very accurately you can see a couple of hundred subsequent

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localizations of a single particle as you can see it's just

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a few nanometers error we can achieve using these particles okay

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what we of course did was we looked at hundreds of trajectories of particles on supported membrane bilayers

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and then quantified the force they experienced according to the distance

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so the closer you go with the magnet the higher the

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force gets now the force is not extremely high we're talking femton newton here

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but this is of course good enough to pull it through water

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and also through the membrane bilayer

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but you would have a hard time competing against strong cellular forces

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and this is exactly what we want we don't want to

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disrupt anything in the cell we just basically want to move it

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and see if it experiences some kind of drag in some parts of the membrane.

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So if we go closer we can really see that according to the distance it really rises up to about one femtonut.

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So this is before there's no apparent force they just wiggle

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around in x as well as y direction it doesn't really matter

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but when we add the magnet we have a clear the femtonewton force in X direction, in this direction.

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So to test how accurately we can measure the motion of the particle, we looked at some defects in our membrane.

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So you can see a fluorescent supported membrane bilayer.

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This dark area is a patch on the cover glass where bilayer could not be formed.

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And we could see when we now pull these particles, they move

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towards the edge of the bilia and then they move along

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that bilia and you can see already here

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that from the trajectory you can see these very well defined little areas of membrane

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and if we now zoom into this little trajectory here you can see

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that we have very fine detail and a pretty straight line okay

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so this gives you already a little bit of a hint

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and we're going to further look into this one here this

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gives you a little bit of a hint as to how

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uh how accurately we can localize our particles here this is two microns

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and you can see this is where the fluorescence

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goes down where the edge of the membrane is and you can see that there's a very well defined edge

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where the particle approaches and approaches and approaches but can't come across okay

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so This is basically the error of the measurement of the membrane edge you can see from this particle trajectory.

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And this made us hopeful that by later correlating the motion of the particle to fixed cellular structures,

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we would be able to see where the particle then finally would get stuck.

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The next challenge was, of course, to go for cellular particles so we used our magnetic nanoparticles,

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coupled GFP nanobodies to them via biotin -striptavidin linkage,

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and then coupled this to a lipid -anchored GFP molecule, okay?

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So this is a CV1 cell that expresses GPI -GFP, and we then add the particles to the top of the cell, and then they move around the membrane,

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and we can pull them clearly in the direction of the magnet.

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You've seen this went actually pretty fast, right?

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Now, we did this for a number, so you can see the movie again.

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So we did this for a number of particles and a number of membrane proteins.

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So the simplest one would be the lipid -anchored GPI -GFP with the fluorescent magnetic nanoparticle attached to it.

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But we also had a standard LYFP -GT46.

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This is a standard transmembrane probe which has the transmembrane domain of the LDL receptor.

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coupled to a GFP on the outside and the transferrin receptor, which is also an often used probe for single particle tracking,

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where we also have a GFP on the outside here, the YFP.

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And no matter what protein we used, all of them, GFP, GPI, YFP, GD46, or transferrin receptor GFP,

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they would exhibit random motion before.

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We tracked them when we approached the sample with a magnet, they would move into the direction of the magnet.

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And then when we removed the magnet, they would resume random motion in the plane of the membrane.

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And we could show that this was specific binding.

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Here you can see this is in transfected cell.

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This is a neighboring untransfected cell.

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You can see that these particles, they only bind to the transfected cell.

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Using confocal microscopy, we can see that they are nicely on top of the cell

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and don't bind to the cover slip or anything.

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And when we then quantified the difference in diffusion coefficient of many, many particles and many, many cells before and after,

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we could see that globally speaking, there's no difference in particle diffusion before and after they were pulled.

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So it doesn't seem that we introduce any greater effects just by pulling them a little bit through the membrane.

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So this is a freely diffusing, normally behaving membrane molecule before you pull it.

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Then you pull it through the membrane and it can just resume its lateral motion again.

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Our aim, of course, is now to correlate the motion of these particles to obstacles inside the cell membrane

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and to do so we perform storm imaging of the actin cortex so we have a 3d capable

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storm microscope so we can distinguish the dorsal sides of the back of the cell from the ventral side

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here you can see ventral actin cables

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and then the dorsal um filaments you can see this is dorsal this is ventral actin

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and of course when we look at molecules

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that bind to the top surface of the cell we want to correlate motion to these dorsal actin fibers right

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so here you can see again gpi gft expressed in a

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cd1 cell we can then see as we pull this molecule

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through the membrane it kind of hits a barrier slides along

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that barrier and then so it hits the barrier it slides along the barrier

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and then it seems to get stuck okay um and when

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so of course this is where the magnet is right

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so it tries to move towards the magnet

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and then it somehow hits a barrier and it finally gets stuck

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so now if you look at the same area in wide field actin imaging we can see

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that there's some prominent actin structures there this is the dorsal actin

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and the 3d storm

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and here's the correlation here's the correlation of the trajectory with the actin filaments

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and you can see that it kind of moves between a couple of actin bits

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and then it gets stuck on this longer filament tries to find a way moving along it

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but finally gets caught in between the actin cortex

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so i hope i could convince you that using this method

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that we can detect obstacles to the motion of membrane proteins in the plasma membrane.

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So this is how far we got in this story.

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And we hope that in the future, that we will be able to use this to further investigate diffusion barriers in the plane of the membrane of cells.

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So our first aim is of course now to go back to the diffusion barrier in the neurons

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and then see if we can see any compartmentalization using this method.

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in neurons that correlate with these 200 nanometer distance actin rings.

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But of course there's other essays one can imagine that this could be useful for.

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Of course, one of the biggest problems that we face in membrane biology is quantifying molecular events such as receptor dimerization, right?

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So a lot of people try to do this by blinking

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or by co -localization studies or by clustering or by all kinds of statistics.

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However, if you imagine you would have two subunits of a dimerizing receptor,

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one of them would be fluorescent and the other one would carry a magnetic tag, and you would now add a magnet to it,

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you could see that the true dimers, so molecules that are really interacting, they would move.

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whereas other dimers apparent dimers

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that only appear to co -localize within a certain distance would tear apart the magnetic part of the dimer

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and we could move so we could calculate a fraction of molecules

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that would be dimers of course like all these dimer this would still be dependent on quantitative labeling,

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but this would be something we would have to figure out like in all assays.

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So with that, I am already finished with the work from this paper.

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This work has been done by Gio Hui Li, who is pictured here when she's just about to win the poster prize at the biophysical society meeting this year,

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so that was very exciting for us.

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She is soon to be starting, hopefully, her postdoc, and we look forward for a very exciting PhD defense.

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She has done this work together with the Paula Santos -Otte, an undergrad student who's been spearheading the initial experiments in the lab.

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Brady and Au has been computing software to overlay trajectories and super resolution data.

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Stefan Block helped us with calculation of forces in supported membrane bi -layers.

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And Jakob helped as well with super resolution imaging.

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And with that, I want to thank our funding agency, the Deutsche Forschungsgemeinschaft, who has been very generous.

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And I'm open for your questions.

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Thanks so much, Helge.

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Such a fantastic story.

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Thank you really really lovely and congratulations for gia

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and you as well um so i'd like to start with my own questions by tradition

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so you said you are not really changing the diffusion coefficient

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of the molecule by pulling it you're just directing it right

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yeah can you actually change the diffusion coefficient of the molecule can you apply more force let's say

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um magnetic field so we could get closer so there are people

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so when i looked around into this

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if it would make sense to go in

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that direction i found a guy who actually has a needle

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that hasn't that is like an electromagnet right

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that would of course be much superior to what we do

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and that would be really exciting to do

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because you could tune the the strength of the field and thereby more accurately influence membrane protein motion.

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But this was like a defense project.

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So this is outside of our budget.

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So I think that would be, of course, very exciting, but it's beyond our technical capabilities.

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So of course you detect, right?

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As soon as you pull, you detect an apparent difference in diffusion coefficient, but that is because you have two components.

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You have the diffusion and the pull.

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And related to this, now you're pulling things towards the side of the membrane.

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Can you pull the things towards the center of the membrane so that you actually collect some molecules towards the center to,

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let's say, induce some curvature or induce some crowding in the cell?

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I guess one could attack something from the top.

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Yeah, I think that should be possible.

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We never tried really to go like this with the needle.

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I guess it could be possible because the forces are weak enough, right?

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So this is based on, of course, indebted to the late Maxime Dahan and Jacob Pila and others,

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Fred Etock, who have been using intracellular magnetic particles, and they have been covering them with activated CDC42,

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so they could pull them around in the cell, this clump of magnetic particles inside the cell, and wherever they would then let it be,

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it would have a burst of actin polymerization and stuff like

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that, right? so i think that could be possible especially if you compare it with like i don't know holding

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a guv in direction of the magnet

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or something this of course one could imagine yeah yeah i

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think i mean this will be this is super super cool

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technique you can do endless things you can induce whatever in the cell you can induce a domain

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and you see you know

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if it recruits some other molecules okay i still have more questions

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but i'll start with the odd questions by the audience

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so ed ed jenkins from oxford is asking this is really

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cool can you apply this setup to achieve repulsion instead of pulling

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and have you tried to apply it in a perpendicular manner

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yes i think perpendicular we just talked about so we have not done

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that repulsion so the thing is with these tiny particles they are not actually magnetic like a magnet right

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so it's just too little iron in there and say they are paramagnetic

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or super paramagnetic so basically they can become attracted by the magnet but they themselves

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cannot act like a magnet and be repulsed by the respective pole of the magnet

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so this unfortunately doesn't work in these very very tiny magnetic particles

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so you need more mass to to achieve that to really have a permanent magnet

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thank you okay so the next question is by xin jiao very interesting story

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and fantastic tool can it be applied for intracellular membrane protein study for example er membrane proteins

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yeah i mean all you have to do is to get the magnetic particle inside

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um so you know it's possible um it may not be all

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that physiological i i was first i thought about this very in the beginning the first time

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when i heard about this uh thing called magnetogenetics right

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so there are people used ferritin which is a iron harvesting molecule

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and basically couple that to an ion channel and then they claimed

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that they could pull on the ferritin

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which basically has a tiny iron cluster to open the ion channel now that um has been very very controversial

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because some physicists that know a lot about magnetism could quickly demonstrate mathematically

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that this is entirely impossible because there's not enough iron in there.

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So you really need like 50, 100 nanometer iron core to even be possible to be able to move it at all.

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So it's unfortunately so far, I haven't found a way to genetically encode a magnetic particle.

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That would of course be wonderful.

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The closest that gets to this may be these bacterial

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um iron capsules so some bacteria make capsules that can enrich iron inside

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and these can be big enough for sensing of magnetic fields

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and so on so these are like 100 nanometer like like a virus kind of capsules

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and they can have iron inside so this would be something

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but it's not very much explored thanks elga

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so next question is by ej she's asking can these nanoparticles

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be studied in receptor signaling could these nanoparticles for instance show how they activate the catalytic domain in receptor dimerization

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which you touched upon a bit yeah i mean

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so i think uh what what would be an idea that

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that we would have is that basically you you pull on a protein

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and then you would think that

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if the protein is dimerized to another receptor you would pull it with right

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so so this could then

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if the other molecule would also be fluorescent you would then see all right i'd pull on the red protein

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and when i pull my 50 red proteins to the side 20 green proteins come with

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so i guess two out of five of my receptors at this time are engaged as a dimer right

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so this is a conclusion that that i could imagine would be accessible experimentally um

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but of course one would have to have a very well suited system

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and as quantitative as possible labeling

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which i think are the the as always the challenges right i mean we know that

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thank you i guess our next question is by tara sitch

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from here excellent talk thank you very much i have two

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questions the first one is how such barrier enforces face control is it mainly size based

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or are there any specific mechanism what passes and what is not

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yeah that's a really a good question

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so we don't really know akikosumi has two models for how his diffusion barrier works right

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so the the i guess the question you're referring to is

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how does a molecule in the right

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so the membrane has two leaflets how does the membrane molecule

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in the outer leaflet like a lipid how does it even sense something

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that is below the bottom leaflet how does that work

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and the idea from akikosumi is that you basically have your actin

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and bound to your actin are a number of membrane proteins

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that stick through the membrane and they are kind of like a pillar so

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if you have several of them your molecule whenever it wants

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to cross the actin even though it doesn't interact directly with the actin it can encounter one of these pillars

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and you can then do mathematical modeling and you can calculate

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that if you have 20 30 percent coverage of your actin filament by pillars this is already leads to measurable

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confinement in these these zones one could also imagine other means

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so for example there could be interaction with the cytoskeleton via um phosphatidylserine

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so there's like charge interaction of the cytoskeleton with the bottom leaflet

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and then lipids in the bottom leaflet interact with lipids in the top leaflet

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and this could be some sort of lipid interdigitation

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or lipid phase phase dependent local confinement so there's some evidence suggesting

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that this could be possible as well thank you

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so second part of the question is does actin create an obstacle

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or a trap in other words if you pull particle back

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when it's at the obstacle will it be trapped

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or will it be trapped in the obstacle

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or it will go back yeah so um

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if it's just a straight line like this i would expect it to just go back right

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so um if you imagine having these little pickets then of course

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if you pull it into an area where there's a lot

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of pickets until it can't move anymore it may be confined in an area

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and they kind of have a hard time getting out of there right

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so um this i guess will be dependent on the circumstances

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and that's why we are

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so excited about this axon model where we have just these actin rings every 200 nanometer

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because it's it's like it's designed for this experiment right

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so it's very uh clearly defined we know where there's actin

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where there's no actin um we can fairly straightforward image

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that after the experiment so hopefully um this will allow us to ask questions exactly like

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that in the future

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so can you actually turn off the magnetic field in the middle of the trajectory to see

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if it starts free diffusion again yeah i think this is what uh gia did

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so he has this micromanipulator and i mean she's not microsecond you know

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so so basically you just have to turn the screw

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and then it moves out um so so this works

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and we we actually did this

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so you can see i think there's even this time tags

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no not here um i think we have a time tag in the paper let me see if i can open it

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what do i share do i share my screen

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or do i share that i don't share anything

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so let me just share this share

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so in the paper we have a time scale so you can see this is seconds

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can you can you see that yeah so this is seconds up here

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so for 24 seconds we observe it wiggling around

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and then moves in the magnet and it moves for 24 seconds in

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that direction and then she removes the magnet

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and then she needs to find the particle again

448

and then she lost some time

449

so it's i you know it's a couple of seconds

450

or a second that you need to remove it

451

but whenever we did this you know we almost all of

452

the time we were able to look at the trajectory before then move it

453

and then have it wiggle around this was kind of our control to make sure that

454

you can remove it and the particle resumes diffusion right

455

because what we wanted to evade was

456

that we just sort of introduce a big artifact in the protein and just destroy something inside the cell

457

this is very good so i have two questions by dig allah from here as well

458

so taking asking how do the different membrane anchors perform in

459

terms of pulling force do longer transmembrane anchors get stuck more

460

often that's the first part of the question about the tm second part is the intracellular part

461

and does the length of the intracellular part have an influence on

462

which kind of structures they get stuck

463

and i myself expand the question a bit let's say you are looking at a cd44

464

which according to sergio greenstein that it interacts with actin cytoskeleton

465

but but according to aki most of the membranes actually interact with actin cytoskeleton directly

466

or indirectly forming the barrier if you look at different proteins

467

that would in the intracellular part directly in track with actin

468

or not would it make a difference

469

or would it be an interesting question yeah

470

so this is uh this would be very very interesting

471

so this is definitely something where we we want to pursue um it is hard work

472

so uh you have to you know this is like a single particle methods.

473

So We are at it, but we can't really tell you anything about it yet.

474

So now we are focusing first on the, in the remaining time we have GIA.

475

We're focusing on the neuron story, but of course we would be very excited to see, you know, what is with the huge ion channel.

476

You know, we would expect it more likely to get stuck.

477

What is, if you have a long transmembrane domain, is it more likely to interact with the actin? um this is of course questions

478

that we would like um to investigate further using this

479

one extension of this question is about the tm i mean it would be probably also interesting

480

if you check a um let's say the raft preferring ordered domain preferring tm versus disordered preferring tm yeah um

481

and maybe one thing also i don't know probably you have done this

482

because this would be obvious thing

483

that would come to a person who is doing all this phase separation

484

and if you pull one thing from ordered membrane to this ordered phase to disordered phase

485

or disordered phase to ordered phase to to calculate the force

486

that is needed absolutely i think i think one one thing

487

that would also be very interesting just on the very basic

488

side on this whole membrane lipid domain field would be to pull a lot of

489

raftophile molecules on one edge of the cell

490

and then do some lardin staining and see if indeed you have a change in, yeah, this is something.

491

Don't tell me if you don't see any difference, okay?

492

Okay, so the next question is by Abhi from Berlin.

493

asking I wonder that have you also tried this in combination with expansion microscopy?

494

Hmm.

495

No. Can you do expansion microscopy with live cells where there's still diffusion?

496

Not really.

497

I guess that would be an instant kill like electron microscopy that's still we're waiting for movies.

498

um we haven't done it i guess as a postdoc imaging

499

method why not right it might work as well as storm does

500

so we might as well do

501

that i don't see a reason why we should not um thanks again

502

so next question is by betul from turkey the three -dimensional

503

structure of the proteins may change due to the magnetic field would

504

that would that lead to any breaking or reorganization of the bonds

505

and interactions would it change actually the force that yeah yeah i think

506

so that's a very good question i think it would not

507

so one should not underestimate um the forces that keep a protein together

508

so hydrophobic effect is an extremely strong thing

509

so it would need really many many piconewtons to pull a

510

single lipid out of a membrane for example right this is something

511

that is many orders of magnitude beyond this i think we are here in the femton obviously,

512

all membrane proteins, they are never really the same, right?

513

So they all kind of like wiggle around and fold and unfold a little the whole time.

514

So I guess you could sort of say you may favor one confirmation a little bit.

515

I can imagine that.

516

But in terms of structural changes that are accompanied by strong binding,

517

I would think that we are below the energies

518

that this can be right so

519

if you imagine like a virus binding to its specific receptor these are significant amounts of energy

520

that get released in such a process so i would think we are below

521

that kind of level thanks again related to this next question is by arta

522

and he is asking with this magnetic manipulation would it be possible to modulate an ion channel

523

yeah i mean so this people try to do this using uh what they called magnetogenetics um

524

but this i as i said was highly doubted i think

525

people have in animals used magnetic beads to do meccano uh

526

like this piezo channels the the meccano sensitive channels this i think this has been done.

527

but it has not been combined with tracking.

528

So I think magnetic beads are used in a lot of different applications already in humans,

529

or you can also use it for molecular imaging and so on.

530

And I think it has been used to pull open these mechanosensitive channels.

531

So earlier there was one question asking if you can do intracellular imaging

532

and as you said the challenge would be getting the magnetic nanoparticle inside the cell

533

now we're talking about cellular level but if you think about an organismal level

534

do you think this would be possible to somehow label with magnetic nanoparticles the extracellular

535

proteins and somehow move them in a larger scale so

536

that you can create let's say a signaling morphogen gradient

537

or something um do you think this would be possible in a larger scale

538

i guess yeah why not um yeah so you think

539

that maybe in an embryo you would sort of inject them in an embryo

540

and then try to artificially enrich in one part of the embryo your magnetic particle coupled i guess

541

that should be possible especially because the forces are not

542

that strong so depending on the distance right so the

543

so you'd have to adjust probably very carefully

544

because with magnets the distance dependence is extremely high right so

545

so force goes crazy so one would have to be be careful about

546

that but it basically compares to i mean

547

if you tried these magnetic beads to pull down proteins right if you put the magnet on the side

548

So imagine that in a little weaker, and I guess it should be possible, yeah, why not?

549

Thanks, so next, usually we have a lot of questions.

550

No, it's wonderful, I think it's really, congratulations to you.

551

I mean, this is an amazing format that everybody is so open to discuss, that you really create a great atmosphere here,

552

and I'm very happy that it's appreciated so much by the students and the audience.

553

I mean, this is a compliment for you and your setup.

554

Well, I told you earlier, I'm going to join in the future.

555

Yeah, this is great.

556

Yeah, actually, I'm not doing anything.

557

I'm just sending you a link and reading the questions.

558

That's my only job.

559

Thanks to you.

560

You are doing all the job, Helgen.

561

It's the style and the atmosphere that makes this happen.

562

That's really nice.

563

So next question is by Christina, and she's asking, are structural changes observed between the Acton ring

564

and the encode protein during stimulation between axon and dendrite

565

and may these changes be observed with magnetic nanoparticles i wish

566

um yeah i mean this is this is very very exciting

567

so there's myosin motors that basically change the the these rings um uh there's membrane molecules

568

that dynamically become recruited to these rings and then execute some signaling.

569

Right now, I have to say the biggest problems we face is actually cleaning up the particles,

570

getting really nice particles that bind well, that behave well.

571

So this is something that just isn't fantastic at the time, right?

572

So this is still not really out of the box particles.

573

You can buy them from many companies

574

but it has been a very very frustrating journey to get some particles

575

that really work well so so they have not achieved a level

576

that maybe quantum dots have right so i think 20 years ago

577

when the quantum dots were first around there was only one of them

578

that you could use only the 605 ones were any good

579

and and now the 655 ones are still the best

580

so I think there's still some way to go for these

581

to be really really nice hopefully I've been now approached by some companies

582

so hopefully we can work together with them to make this a more homogenous

583

and more useful and more tractable sample because this is I have to say right now our biggest problem

584

to get them onto cells

585

in a way

586

so we have to get rid of a lot of them to have the good ones left over

587

and then put them on the cell.

588

So we need to do quite some preparation to get them going well.

589

And is it challenging to actually label

590

the proteins on the surface with biotin tag so

591

that you can label the proteins and pull the proteins in

592

and out no no that's that's that's very straightforward

593

so we have the the nanobody that is bound to a biotin

594

so the nanobody gfp interaction is in the nanomolar range and

595

by controlling the amount of biotin nanobody we add we can control

596

a little bit that you bind monovalently right so

597

if you add less nanobodies then you assume to be surface proteins then

598

or if you pre -coat your particles with nanobodies then you can

599

have the equilibrium shift towards having mostly only one molecule bound to your nanoparticle

600

because of course the cross -linking would also be a worry

601

if you have these 200 nanometer size bits but that is something

602

that has been done by many labs for many different particles it's a very common coupling chemistry.

603

Okay

604

so then a related question comes from Hussein Emre can neuronal

605

stimulation be modulated by moving NMDA channel let's say in neurons

606

out of the synaptic space using fluorescent magnetic nanoparticles um yeah uh

607

well you have to ask okay so uh um

608

so daniel okay in his lab he has been able to show

609

that um in the post -synaptic density of excitatory synapses there are glutamate receptors

610

and once these are opened they close and then they are desensitized for a while so they can't reopen and this is why

611

if you do two quick pulses one after the other the

612

first pulse is going to be higher than the second pulse it's called paired pulse depression

613

and what indeed happens is that these desensitized receptors they diffuse out of synapses and can become replaced by

614

fresh receptors so if you abolish diffusion inside the synapse the pelt balls depression is much stronger

615

so the difference between the first peak

616

and the second peak is much higher than if you have diffusion

617

so diffusion is actually important for the exchange of used receptors

618

for for fresh ones to achieve high cadence high frequency signaling

619

so i could imagine that indeed if you would forcefully remove molecules

620

or keep them out of the synapse you could have a similar effect i do not think

621

that if you have a receptor

622

so receptors are linked to the post -synaptic density via stargazin

623

and pst95 i do not believe that you can pull using the magnetic force

624

an anchored ion channel out of the postsynaptic density for

625

that there's just too many binding sites

626

and it's not gonna i don't think that's gonna work

627

so one one thing for me to understand is rings the

628

actin rings um are they like a like a spring

629

or they are rings next to each other so then they are not continuous i don't think they are continuous um

630

now that it has been shown that myosin actually crosslinks them

631

and seems to pull them together and they can widen

632

and become closer again um i think it's really even an exciting question

633

if they if they have actin always in one direction right

634

so if there's only plus directed act in one way

635

that would be weird because that would mean an axon would have a left

636

and a right right because there would be the plus side this way always this way

637

so it would always go up

638

with the plus end on one side of the axon

639

and down on the other side of the axon i can't imagine

640

that so i would assume that you have them go plus

641

and minus in both directions every single ring

642

so i would think it's rather ring structures than one continuous spiral also that would be a very long spiral.

643

Yeah, yeah.

644

So last question, Helge, by Jan Su.

645

Is it possible to temporarily block the ion channels between the actin rings with biochemicals

646

and reduce the electrical activity of the ion channels to reduce membrane protein stacking possibility?

647

Okay, I do not feel qualified to answer that.

648

That's, can you pose that question again?

649

It sounds very interesting.

650

Is it possible to temporarily block the ion channels between the actin ring with the biochemicals

651

and reduce the electrical activity of the ion channels to reduce the membrane protein stacking possibility?

652

Okay.

653

Well, there are pharmacological inhibitors of voltage -gated channels in the initial segments.

654

So that should be possible um i could imagine calcium having

655

or a calcium influx having an influence on membrane protein motion

656

because calcium of course for example um uh interferes with pip2 your charged lipid cytoskeleton interaction

657

so that's for example one important uh process in dendritic spines

658

when calcium flows in the the membrane relaxes from the cytoskeleton and you can sort of like

659

through new polarization you can make a bigger spine um but i haven't thought about that and

660

that would be a hard experiment to do i guess

661

but uh yeah yeah i could imagine it i'm not saying it's happening

662

but uh yeah okay thanks thanks

663

so much as always it is such a pleasure to yeah

664

likewise this is really great i'm going to join you

665

when is the next one next week yeah next week every

666

week wednesday that's really nice very nice format i i thoroughly enjoyed it thank you i'm sorry

667

that i'm the only person that you see whole talk

668

but um you know there are always downsides yeah no no

669

it's great i really enjoy it it's very uh nice atmosphere

670

here it doesn't feel as weird as it should um sitting

671

in front of your uh your computer the only thing i can't take is

672

that you have a talk sitting down right i think that's

673

so strange um yeah i've seen actually people giving talks standing

674

up in front of the computer as well maybe i should do that next time

675

all right yeah thank you much for having me i hope

676

you enjoyed the paper i everyone enjoyed the paper a lot i really get good feedback on this paper

677

and thanks very much for joining us having time for us today for a fantastic presentation

678

and congratulations again gia

679

and you for this great paper thank you gs in the audience please feel free to write us anytime

680

if you have questions we'd be happy to help thanks a lot helge

✨ فيديو موصى به

146 جمل

The Most Complex Language in the World

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72 جمل

المفردات وملاحظات التحدث لهذا الدرس

يحتوي هذا الفيديو على 680 جملة و8384 كلمة للتدرب على الترديد. مدة الكلام 57:31. يتكلم المتحدث بسرعة ثابتة، نحو 146 كلمة في الدقيقة، وهي سرعة مريحة للترديد. 78% فقط من الكلمات ضمن أكثر 3,000 كلمة شيوعًا في الإنجليزية، فالمفردات صعبة نسبيًا.

أهم المفردات في هذا الفيديو

15 كلمة من الفيديو تستحق التعلّم، مع النطق والمعنى:

الكلمة	النطق	المعنى
membrane اسم	/ˈmɛm.bɹeɪn/	غِشَاء
particle اسم	/ˈpɑɹtək(ə)l/	جُسَيْم, ذَرَّة
molecule اسم	/ˈmɑ.lɪ.kjul/	جُزَيْء
magnetic صفة	/mæɡˈnɛtɪk/	مِغْنَاطِيسِيّ
magnet اسم	/ˈmæɡ.nɪt/	مَغْنَاطِيس
barrier اسم	/ˈbæɹi.ə/	حَاجِز
receptor اسم	/ɹɪˈsɛp.tə/	مُسْتَقْبِل
plasma اسم	/ˈplæzmə/	بِلَزْمَا
obstacle اسم	/ˈɑbstəkəl/	عَقَبَة, حَاجِز
trajectory اسم	/tɹəˈd͡ʒɛktəɹi/	مَسَار
label اسم	/ˈleɪ.bəl/	مُلْصَق, بِطَاقَة
experiment اسم	/ɪkˈspɛɹ.ə.mənt/	تَجْرِبَة, اِخْتِبَار
interaction اسم	/ˌɪn.təˈɹæk.ʃən/	تَآثُر, تَفَاعُل
wire اسم	/ˈwaɪɚ/	سِلْك
biology اسم	/baɪˈɑ.lə.d͡ʒi/	عِلْم اَلْأَحْيَاء, بِيُولُوجِيَا

الأفعال المركبة التي ستسمعها

الكلمة	المعنى
go down فعل	نَزَلَ
go up فعل	صَعِدَ, يَصْعَدُ
sit down فعل	جَلَسَ, قَعَدَ

القواعد في هذا الفيديو

أكثر التراكيب التي يستخدمها المتحدث، مع العبارات نفسها من الفيديو:

التركيب	في الفيديو
الجمل الشرطية if + جملة، will/would + فعل — شرط ونتيجته	if you imagine you would have · if the other molecule would also · if you have questions we'd be
المضارع التام المستمر have/has been + -ing — حدث بدأ سابقًا وما زال مستمرًا	has been putting · has been computing · have been using
المضارع التام have/has + اسم المفعول — حدث ماضٍ ما زال له أثر الآن	have been selected · has correlated · has been described
المبني للمجهول be + اسم المفعول — التركيز على ما يحدث لا على من يفعله	have been selected · was published · are compartmentalized

نطق يحتاج إلى انتباه

يستخدم المتحدث 65 من الصيغ المختصرة، مثل don't, I'm, can't. انطقها بصيغتها القصيرة كما تسمعها.

أصوات “th”: thereby /ðɛɹˈbaɪ/, thin /ˈθɪn/, thoroughly /ˈθʌɹ.ə.li/
أصوات “sh” و“zh”: interaction /ˌɪn.təˈɹæk.ʃən/, congratulations /kənˌɡɹæt͡ʃəˈleɪʃ(ə)nz/, shell /ʃɛl/, structural /ˈstɹʌk(t͡)ʃəɹəl/, observation /ˌɑbzɚˈveɪʃn̩/
الكلمات الطويلة — انتبه لموضع النبر: investigate /ɪnˈvɛs.tə.ɡeɪt/, trajectory /tɹəˈd͡ʒɛktəɹi/, experiment /ɪkˈspɛɹ.ə.mənt/, interaction /ˌɪn.təˈɹæk.ʃən/, biology /baɪˈɑ.lə.d͡ʒi/

كيف تتدرب بهذا الفيديو

استمع إلى الفيديو كاملًا مرة واحدة دون أن تتكلم، ودوّن الكلمات التي لا تعرفها.
ردّد جملة جملة بالسرعة العادية، وكرّر كل جملة حتى يتطابق إيقاعك مع المتحدث.
سجّل صوتك وقارنه بالأصل، مع الانتباه إلى كلمات مثل membrane, particle, molecule.

ما هي تقنية التظليل الصوتي؟

التظليل الصوتي (Shadowing) تقنية تعلم لغة مدعومة علمياً، طُورت أصلاً لتدريب المترجمين الفوريين المحترفين. الطريقة بسيطة لكنها قوية: تستمع لصوت إنجليزي أصلي وتكرره فوراً بصوت عالٍ — كظل يتبع المتحدث بتأخير 1-2 ثانية. تُظهر الأبحاث تحسناً كبيراً في دقة النطق والتنغيم والإيقاع وربط الأصوات والاستماع والطلاقة.

تقنية الشادوينغ: اقرأ الدليل الكامل خطوة بخطوة →

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المفردات وملاحظات التحدث لهذا الدرس

أهم المفردات في هذا الفيديو

الأفعال المركبة التي ستسمعها

القواعد في هذا الفيديو

نطق يحتاج إلى انتباه

كيف تتدرب بهذا الفيديو

ما هي تقنية التظليل الصوتي؟