単一分子超解像顕微鏡を用いた細胞表面受容体の二量体化の調査
[Music] Hello everyone and welcome to today’s webinar investigating cell surface receptor drizzation using single molecule superresolution microscopy. I am Sydney Panila of Lab Roots and I will be your moderator for today’s event. Today’s web seminar is presented by Labroots and brought to you by Thermoffisher Scientific. To learn more, please visit thermoffisher.com. Now, we encourage you to participate today by submitting any questions you may have during the presentation. To do so, simply type them into the ask a question box and click submit. We will answer as many of your questions as we have time for at the end of the presentation. You may also use the ask a question box to let us know if you are having any trouble seeing or hearing the presentation. Okay. I would now like to welcome our speaker for today’s webinar, Katie Sherik. Katie is a post-doctoral researcher at the Francis Crick Institute in London. Katie, you may now begin your presentation. Thank you for the introduction and to the Fisher for inviting me to do a webinar about some of my work. Um, and today I’m going to be talking to you about some of my single molecule superresolution microscopy work looking at cell surface receptor drizzation. So this is just a disclaimer slide and I want to start off by giving a little bit of an introduction about me and my background. So I did my undergraduate degree at the University of Sheffield and I graduated in 2020 with a integrated masters in biochemistry and genetics. And in 2024 I finished my PhD which was at Imperial College London and I was investigating dopamine receptor drizzation which is what I’m going to be mainly focusing my talk on today. And at the moment I’m a posttock researcher at the Francis Crick Institute and I’m still working on membrane receptors but in a project in collaboration with Astroenica and I won’t be talking about this work today but just to highlight that um my interests really lie in membrane protein receptor signaling and drizzation and my work consists of a lot of cell culturing and microscopy which is what I will be discussing today. Membrane protein receptors are a fundamental part of signal transduction pathways and this means that they play a really key role in almost all physiological processes and they crucially allow signals to pass pass across the cell membrane and some of the key examples of this are the G-proin coupled receptors and the receptor tyrrosine kynases and because they they have a key role in many physiological processes. They are often altered in disease and because of this they have historically been highly tractable and successful drug targets with GPCRs alone making up over a third of FDA approved drug targets. So a little bit more about G-rotein coupled receptors. So these are a class of proteins that consist of over 800 members in mammals and GPCRs are grouped into different groups according to their sequence homology. And the largest and most diverse group of receptors is the class A or redopsson light receptors. And these are the targets of many different types of drugs and are also the focus of a lot of different research. So they’re the most wellstied group. So receptors can form dimer or olygras and they can do this either with themselves or with other GPCRs to form homodimer or hetrodimer and some types of receptors depend on drizzation more than others. So for example, the class C G- protein coupled receptor family has to form DS in order to function effectively. On the other hand, other receptors such as this class AGP PCR here, which is the beta 1 andurgic receptor, um doesn’t have to drize in order to function, but has been shown to drizz to in order to diversify their signaling. And a number of different GPCRs have been shown to form das in physiologically relevant pathways and some of these have a role in disease. Something that is important to note is that drizzation of g- protein coupled receptors and in particular class A gpcrs is often very transient. So these can be difficult to capture these short-lived complexes through high resolution structural techniques. So historically many people have used um biohysical assays such as fret or brea assays to study receptor drizzation and olymerization. And these are both proximitybased live cell assays and they can determine whether a protein is within 10 nanometers of another protein. But what’s important to note here is that um these techniques cannot determine whether a receptor is located at the cell membrane or whether it is located in intracellularly within the cell. And microscopy approaches ranging from fixed cells to live cell imaging have been used in recent years to more precisely measure receptor interactions within cells, including by only monitoring cells that are present on the plasma membrane. And these techniques include but are not limited to single particle tracking, fluesence correlation spectroscopy and single molecule localization microscopy. So today I’m going to be focusing on single molecule localization microscopy. And this is a group of techniques that is able to break the diffraction limit of light and achieve resolutions less than 200 nanometers and even down to only a couple of nanometers resolution. So in microscopy terms, we think of resolution as um the smallest distance between two points where they can still be seen as separate entities. And this is a limit of the microscope. So the lower the resolution, the greater your ability to see the precise detail of the molecules you are looking at. And this is particularly important when we’re looking at things like membrane receptors, as these tend to be less than 10 nanometers in size. So in a defraction limited wide field microscopy approach such as conventional confocal confocal microscopy the resolution of these techniques is low and you’re unable to determine the precise localization of the floror. You have this diffused area where it could be as I’ve shown in this figure on the left. um there is a diffuse area where it could be and this is what is known as the point spread function of an emitting floror and in these defraction limited techniques such as confocal microscopy you’re unable to distinguish between two separate points meaning the resolution is low and this is because these point spread functions are overlapping. So single molecule localization microscopy techniques achieve this high resolution by breaking this defraction limit and ensuring that point spread functions do not overlap and this allows the precise localization of a flororahor to be determined and this is what I’ve indicated on these images as a cross so you’re able to determine the precise location rather than this diffuse area of a high a lower resolution image. So the most common types of single molecule localization microscopy are storm, palm and paint. And what I will be talking about today mainly is palm. So generally with these techniques we achieve non-over overlapping point spread functions by only activating a very small subset of flororahores in each ch in each of the frames and this ensures that we are temporally separating floror emissions so that the point spread functions are not overlapping and after imaging over many frames the localization of all emitting florohors can be precisely determined. And from this we can extract information such as drizzation um by doing algorithms such as neighborhood analysis. So in this example here um where I’ve noted die or olymer quantification you would see that within a defined search radius that we caught or are in this example the orange receptors here would be monomers the blue ones would be dimer and the the green ones would be tetras as they are within a defined search radius of each So to focus a bit more on PD palm which is the technique that our lab focuses on the most. So this stands for photoactivation localization microscopy with photoactivatable dyes. And what we do is we um conjugate an antibbody with a photoactivatable die that is activated and um excited in a stocastic manner. So in each frame we only activate a very small subset of these conjugated antibodies and this appears as blinking when we are imaging and this is where you get excitation and emission of the floror and this is followed by bleaching. And if we conjugate the antibbody and the die in a 1:1 ratio we can be sure that one blinking event and one localization is one receptor. So now that I’ve introduced single molecule localization microscopy as a technique to look at membrane protein receptor drizzation, I want to talk a bit more about my PhD work which was looking at G2 dopamine G- protein coupled receptor drizzation. So D2 dopamine receptors are class A GPCRs and they have been implicated in a range of different diseases in particular schizophrenia and Parkinson’s disease and they have been shown to form both transient homodimer with themselves and hetrodimer with other GPCRs and importantly post-mortem schizophrenic brains have been shown to see an increase in D2 homodimer. So what we really wanted to look at with this project was to uncover the precise molecular interface between the two proas or individual receptors within D2 homodimer. And the hope is that this information could be used to design better diamond specific drugs. So D2 dopamine receptor signaling is mediated by G-proins and varestessins. So this receptor usually couples to G alphaig G- proteins which have an inhibitory effect on adenel cycles reducing cyclic KMP production in the cell. However, it also couples to butressin 2 which is an adapter protein that is known to act as a scaffold to recruit pro proteins to turn off signaling from the receptor. But it is also known to signal itself through pathways including activating the map kynise pathway and mediating internalization of the receptor. And when we have um equal signaling from G-proin and breastin we call this balance signaling. So when we have a preference for one pathway over another, we have what we call bios signaling. So for example, preferring the betestin over the G-proin pathway will produce um more signaling effects downstream of betestin. And this has been shown to have effects in signaling and um physiological or path pathophys physiological responses as well. So with this project we wanted to first stabilize the receptor DR. So as I mentioned previously class A GP PCRs are often transient DR which makes them difficult to capture with high resolution structural analysis. So in order to stabilize the receptor, we used a mutagenesis based approach and we identified residues based on computational molecular models of the D2 homodimemer and these residues were predicted to increase the dimer stability. So after making all these mutations and validating them, I wanted to look at how these mutant G2 receptors affect diamond stability. So I used a number of different techniques to assess this including western blood analysis, um bioluminescence resonance energy transfer and PD palm. So from this a particular mutant really stood out and this was the double V96S V97 C mutant and this is shown in the turquoise color here. So you can see that relative to wild type D2 receptor you see an increase in relative DMER levels by western blood analysis an increase in bremax which indicates an increase in proma proximity within the dmer with this mutant and through beauty palm we showed that there was an increase in total olygras in this double mutant expressing cells compared to wild type expressing cells. So since the focus of this talk is single molecule imaging, I want to talk a little bit more about the PUD palm imaging in more detail. So we use photoactivatable KH500 dyes conjugated to an anti flag antibbody that recognized the flag tagged receptor that is transiently transfected into hex 293 cells. So we did this with the wild type receptor and um the mutant receptor to compare levels of dmerization in these transient transiently transfected cells. So pedopal imaging of fixed cells allowed a cell surface map of the D2 receptors on the surface of HEC 293 cells to be determined and neighborhood analysis allowed the olymoric state of the receptor and these transfected cells to be determined. So what we were able to see from this data is that we saw a increase in total olygras in hex93 cells that were expressing the double mutant compared to those that were expressing the wild type receptor. And what’s really good with PD palm is that you can look at the stochometry of the receptor. So you can see um whether these receptors are made up of um just dimer or trimers or higher order and this can be particularly important for some receptors that may have differential effects depending on the complex that they are in. But what we saw in this particular case is that when when you break down and look at what these olygic complexes are, there did not really seem to be any particular population that was enriched. So because these were transiently transfected cells, um there were variation in the expression levels of the receptors. So we did also do controls looking at um a range of different receptor densities as well. And we did show that these had an effect on um the extent of drizzation but didn’t really influence the trends that we saw um when comparing wild type receptor to mutant receptor. So I don’t have time to go into detail about the signaling profiles of these mutant receptors today but I want to highlight some of the key points here. So what we showed was that the signaling profile of D2 double mutant expressing HEC 293 cells was altered compared to D2 wild type expressing cells with a decrease in G-proin mediated signaling enhanced basil but restin recruitment and um enhanced both basil and agonist induced internalization and this all suggests a switch towards more beta rest in mediated pathways over GI. So we’re seeing this signaling bias. And what was interesting is that we saw in the mutants that showed increased drizzation compared to wild type, we also saw altered signaling with a bias towards beerestin. And to investigate this theory a bit further, our collaborator carried out molecular modeling with the predicted D2 homodimer in complex with either B2Sin 2 which is shown on the left hand side or with G- protein shown on the right hand side and this revealed that when you have a dimer of the receptor it can accommodate two betestin molecules. However, when it’s in a dimer, steric clashes mean that only one G-proin molecule combined per dmer. So, this provides some explanation for the observed effects of receptor mutants which showed an increased drizzation and also showed a bias towards betestin 2 associations. As within a dimer, you’ve got more space for more betestins to bind than gro protein. So it makes sense that you would therefore get more signaling of beta rest mediated pathways. So in conclusion, this study found that using molecular models of D2 DRS, we were able to identify stabilizing mutations which increase the stability of the G2 DS relative to wild type. And importantly, one of these receptors which I focused on today, the double mutant showed an increased propensity to form olygras through a range of different techniques including PD palm and overall this more generally suggests that D2 diamonds may favor associations with betestin 2. So on the left hand side of this diagram here I’ve shown what um the wild type receptor tends to do and then on the right hand side I’ve shown what this double mutant seems to do. So we she we see increased dmerization increased um associations of betaestin in the absence of any liant weaker g- protein binding betest independent downstream signaling activation and an increase internalization. And to summarize overall why you would use single molecule superresolution microscopy to study membrane protein receptor drizzation. So we’re able to quantify receptors at the plasma membrane specifically. And this can really be complemented with other techniques such as the resonance energy transfer and western block to ensure that you are looking at associations happening within the plasma membrane that you’re interested in. And this allows single molecule resolution of receptor di and olymers. And PDP palm specifically allows you to determine the receptor complex stoometry. And this could have really important effects whether you’re looking at das or bigger complexes. These might have distinct effects and you wouldn’t be able to determine this through bulk assays such as resonance energy transfer. So I would like to thank the co-authors of the paper of the study that I presented here today from Imperial College London and our collaborator Francesca Finelli who did the modeling that I presented here. And I’d also like to acknowledge my my current position even though I didn’t show any of the work today. Um I’m still working on PD palm to look at receptors but a different type of receptors looking at receptor tyrosine kynases this time. And here are my contact details in case you want to get in contact. um my email, my social media where I I really try to show what it’s like working in academia in a transparent way and also do a little bit of science communication. So feel free to add me on there and thank you everyone for joining this webinar. All right, thank you very much Katie for your very informative presentation. We will now move into the Q&A portion of the webinar. If you happen to have a question that you would like to ask, please do so now. Just type your question into the ask a question box and click submit. We will answer as many of your questions as we have time for. All right, let’s go ahead and get started here. The first question that we have says, how are you able to determine which is the best superresolution microscopy approach for your application? Yeah. So I think it really depends on what you want to look at and what your focus is on. So for example, what type of cells you’re using or what type of material you’re using for the microscopy. So things like fixed cells or live cells or adherent cells or tissue samples. Different microscopy techniques tend to favor these different sample types more or less. And one of the main things is you want to decide whether you want high spatial resolution or high temporal resolution. So do you want a really high resolution image or do you want to know about the kinetics and things like that. Um so in reality it might be a combination of different techniques. So things like looking at for example PD palm to get the high spatial resolution but then combining that with something like single particle tracking so you get more of the dynamics as well. All right fantastic thank you. The next question that we have here says what could be the therapeutic implications of the biased signaling of D2R dimer? Yeah. So we know that in schizophrenic postmortem schizophrenic brains there is a an increase in the D2 dimer. So if we’re what this study showed was that um there is a relationship between D2 DS and association with beta restin. So it might be that with these specific mutations we’re locking the receptor in a a more active like confirmation. And if we’re we want to target D2 dimer specifically, um we need to be aware of what signaling implications that might have. So for example, breaking apart the D2 dimer might um switch the signaling back to a more balanced signaling away from this bias. Um so I think really exploiting the dimer and using that to change the signaling would be where therapeutic approaches would go. Perfect. The next one that we have here says, “How do transiently transfected HEC 293 cells compare to what would be seen in the brain in terms of expression or receptor density? Yeah. So with transiently transfected cells um within different transfactions and also within the same transaction, we were getting different receptor density and different cells. So we did image across quite a range of different cells of different receptor density and within the analysis we’re able to determine the total receptor number. So we can say whether it’s a high density or a lower density and we did find that receptor density had an effect on the proportion of the receptors that were in a monomeic complex. But regardless of whether you had a low receptor density or a higher receptor density, the trends we saw were the same. So we still saw this difference between wild type and the mutant. So it seems to be at least the trends we’re seeing seem to be receptor independent. And sometimes people say, oh, if you’ve got high receptor density, you’re not just form forcing the receptors together. But we saw even at lower receptacy we still saw das and in the brain the density of the receptors tends to be quite high. So if anything with this technique we’re actually underestimating rather than overestimating the drizzationization. Okay great thank you. The next question that we have here says, “What are the main advantages or disadvantages of PD palm?” Yes. So, I think the main disadvantage, as I sort of talked about in a previous answer, is that with this technique, you’re only getting really a snapshot because they’re fixed cells. So, you you might miss some of the dynamics of the interactions between the receptors. Um but what’s really powerful particularly with um PD palm is that you’re able to determine the stochometry of the complex. So you can say whether it is in a dimer, a trimer or something else. And with certain receptors, we know that the type of complex can influence how it signals. So looking at one type of receptor complex might be really useful for this. All right, we have um a couple more that have come in here. We have a comment that says, “Thanks for the opportunity to watch this webinar and learn something new.” Um the next question that we have says, “How does the cell decide which pathway they have to follow to make homodimer or heterodimer in terms of cell surface receptors?” So that’s something we don’t actually understand that well. So it’s likely that this is dependent on the context. So dependent on the cell context. So things like um different lians. So ligan dependent formation of receptor complexes. So homodimer or hetrodimer. It can be influenced by the type of lian. So one lian you might favor homodimemer complex. another ligan might favor a heptodimer complex and it’s also likely um cell type dependent and dependent on the receptor the density of other receptors. So for example we know that in the brain different brain regions um you tend to form either more homodimer or more hetrodimer depending on what other receptors are there and how highly they’re expressed. All right, fantastic. We have another here. It looks like this is a two-part question. It says, “I was wondering in the context of drug interactions, how can this technique be applied to monitor drug induced receptor drizzation at the cell surface?” And then the second part says, “And what kind of insights can it provide into mechan mechanisms of action or resistance particularly in the case of targeted therapies?” Yeah. So what I’ve touched upon a little bit is how we can really exploit what we know about diamonds for drug interactions and designing specific drugs that might target one complex over another. So what we could do um and what I didn’t go into too much is that we can do this technique. we can treat the cells or whatever sample you’re looking at with specific drugs and then monitor um before drug um addition and then after and see how that changes the drizzation. So that would be one way you could look at that. Um and to answer the second part of your question to do with the mechanisms of action um really understanding the downstream implications of this would be really useful. So things like um if we using this microscopy if we saw that treating the cells with a specific drug causes changes in drizzation we can look at how treating the cells with this drug affect signaling. So things like the betestin um assays or the groin assays that I was talking about before. All right, wonderful. Um, it looks like we have another question here that has two parts as well. This one says, “Thank you for this insightful presentation. I have a couple of questions. How do you distinguish between real DRS and random uh and random in your analysis? Are there technical limitations for studying DRS in live cells versus fixed samples?” Yeah. So to answer the first part of your question, generally this is controlled for by doing controls. So things like um doing negative controls so we can monitor what the background level is um when we have for example no antibbody on there or no cells in the dish, you can monitor the background that way. And then we have within the analysis pipeline we have um filtering parameters which we have optimized so that we are sure that what we’re counting is um real interactions. Um and then the second part of the question so to answer it basically yes there are technical limitations. So it’s a technical limitation based on the technique. So for PD palm because we are looking you’re imaging over a time course. So you will add your conjugated antibbody for the um for PD palm specifically you add your conjugated antibbody to your fixed cells and then you will image for for example 10 minutes and then because we’re stochcastically activating only a few receptors at the time we will build an image of that fixed sample. So, we’re not able to do this in live cells because then the the image that we’re looking at will change. So, we’re not able to build this detailed highresolution image. Hopefully, that answers your question. All right. Wonderful. So, we will go ahead and wrap up with this final question that we have here. It says, “Since your study focuses on palm, could you share what advantages it had over other superresolution techniques like storm or paint for your specific application?” Yeah. So, the main differences between palm and storm is that palm uses photoactivatable dyes whereas storm uses a oxidation reduction buffer system. So really it was um easier for our applications to use the PD palm technology as the storm protocol often requires you to optimize the buffer solution. Um and with PD um with DNA paint sorry um with that technique you were not able to determine the stochometry of the complexes like you are with PD palm and we really wanted to make sure that we were looking at specific populations so either dimemer or higher order and with DNA paint you’re not able to determine that it’s just bulk um receptor on its own or receptor in an olymeric complex. So that that was our rationale for choosing that particular technique. All right. Fantastic. Well, thank you again Katie for your time today and for your important research. We would also like to thank Labroo Roots and our sponsor Thermoffisher Scientific for underwriting today’s educational webinar. Before we go, I would like to thank the audience for joining us today and for their very interesting questions. Questions that we did not have time for today and those submitted during the on demand period will be addressed by the speaker via the contact information you provided at the time of registration. This webinar can be viewed on demand. Lab Roots will alert you via email when it’s available for replay. We encourage you to share that email with your colleagues who may have missed today’s live event. Until next time, thanks everybody. Goodbye.
Presented By:
Katie Sharrocks PhD
Speaker Biography:
Katie is a postdoctoral researcher at the Francis Crick Institute in London. She currently works on a project as part of a Crick-AstraZeneca alliance collaboration scheme, using single-molecule super-resolution microscopy to investigate Epidermal Growth Factor Receptor di/oligomerization in Non-Small Cell Lung Cancer cell lines. In 2024 she completed her PhD in Molecular Cell Biology at Imperial College London, investigating the impact of D2 dopamine G-protein coupled receptor mutants on dimerization and signalling. She has expertise in proximity based cell assays, signalling assays and microscopy imaging, including super-resolution microscopy. Prior to starting a PhD, she completed an integrated Master’s degree in Biochemistry and Genetics in 2020, graduating with first class honours. Her work is driven by a desire to learn and gain new knowledge which could ultimately help patients in the future. Katie is passionate about communicating science in an accessible way so that as many people as possible have access to science and understand its significance in daily life. As part of this, she participates in outreach activities with schools and the general public and runs social media pages which communicate science and share the realities of working in science.
Webinar: Investigating cell surface receptor dimerization using single molecule super resolution microscopy
Webinar Abstract:
Cell surface receptors, such as the G-protein coupled receptor (GPCR) and receptor tyrosine kinase (RTK), play central roles signalling pathways. Due to their central role in regulating a wide range of physiological processes, membrane protein receptors are often altered in disease, with ~34% of FDA approved drugs targeting GPCRs alone. Dimerization and oligomerization of multiple receptors plays an important role in their function. For some receptors, dimerization is essential for its function, while for others it is a way to increase signalling diversity. Super-resolution microscopy techniques allow a level of resolution beyond traditional diffraction-limited light microscopy techniques. This allows the locations of individual membrane receptors and the oligomeric state of the receptor on the cell surface to be accurately determined. In this talk I will outline the super-resolution microscopy techniques used to study receptor di/oligomerization. This will focus on work on the D2 dopamine GPCR an example, where super-resolution microscopy was used to investigate differences in receptor oligomerization between mutant receptors.
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