 Thank you very much. I'd like to thank the organizers, Mikhail, Anik, Nadia and Nava for the opportunity to be here at this very, very interesting, enjoyable conference. Today I will tell you about our studies on the role of actin dynamics in the endocytic trafficking, and we haven't heard enough about the cytoskeleton here yet, so I thought I'd start off with a picture of the actin cytoskeleton, which is one of the main subjects of study in my lab. This is a scheme made by Dyke Mullins and Tom Pollard, which shows the force-generating machine that's responsible for the motility of cells and for other types of mechanical force generation in cells that use the actin cytoskeleton. So here the plasma membrane is depicted up here, the outside of the cell is up here and this is the inside of the cell, and what happens is there's sort of a short signaling cascade that activates a protein complex called the ARP2-3 complex depicted here in green, which then binds to the side of a pre-existing actin filament and then makes a branch on that actin filament and actin monomers polymerize and through a Brownian ratchet motion this actin actually polymerizes at this so-called barbed end of the actin filament or plus end and pushes, can generate a pushing force on the plasma membrane. And this whole process, it involves a cycle of assembly and disassembly and it's very interesting because actin assembles as an ATP bound protein, but then a short time after assembly a couple of things happen, a protein called capping protein caps the filaments, so filaments only grow for a couple of seconds before they're capped and that's very important because the filaments must remain short if they're going to be able to resist the tension of the plasma membrane as they push on it. And then also as the a short time after the actin assembles in these filaments the ATP gets hydrolyzed to ADP and that actually makes the filament susceptible to severing by a protein called cofillin and cofillin severs the filaments and then other proteins regenerate a pool of monomers which get recharged with ATP and then the cycle continues and so you get this constant assembly and pushing against the plasma membrane by this actin network. And this has been worked out in great detail where we know rate constants, concentrations, binding constants for all of the key factors involved in this process. This is through many labs, largely Tom Pollard's lab as well as Mary France Carlier's just down the road in GIF. And so for our work this has been a really important framework for our studies because what attracted us to endocytosis because we're really a cytoskeleton lab and many of the projects in our lab actually have nothing to do with membranes just have to do with actin assembly is that it turned out in budding yeast where we began most of our studies during endocytosis the assembly of actin here shown in red is absolutely essential to in to to invaginate the membrane and to pull off endocytic vesicles and to for the vesicles to undergo scission and so we study this system as a way to study in a biological context how the forces generated by the assembling actin are harnessed to to do work for biological processes and it's proved to be a very nice model and I should say my lab is sort of split half in yeast and half in mammalian cells. So how did we get involved in all this? Well we were working in budding yeast some time ago and we started identifying proteins that regulate actin. The first one that I found as a postdoctoral fellow I called ABP1 for acting binding protein 1 because it was the first actin binding protein found in yeast. Through genetics and biochemistry we and others found many other proteins that interacted with ABP1 and with each other using a synthetic lethal screen of the type Charlie Boone mentioned we found a gene called SLA1 for synthetic lethal with ABP1. What was curious and as we started to work out this network is that a number of proteins that sort of were entangled in this interaction network of physical and functional interactions were proteins that had been implicated in endocytosis and at that time in you know the 1990s those two processes weren't there really wasn't any good reason to think that they were directly involved with each other and that was something that we found curious. We kept running into genes for example called end genes that someone named Harold Rhysman in Geneva was studying because he was studying endocytosis in yeast cells but we didn't really know exactly what to make of it. So then this was around the time that after GFP had been found and different spectral variants of GFP had been found it was possible to start doing live cell two-color imaging and I had a new postdoc in my lab named Marco Kexonen who was very interested in this problem of how these proteins were working with each other and he decided to set to work by tagging pairwise combinations of proteins in this network with green fluorescent protein and red fluorescent protein and looking at them looking at live cells expressing both proteins at the same time so doing two-color imaging and so one of the I think the first pair he looked at he tagged ABP1 and SLA1 this SLA1 protein turns out to be interesting because it's an endocytic adapter it binds directly to endocytic cargo to the pheromone receptor in budding yeast and so this is what Marco saw. Now first of all what so other people had started to look at these proteins and they did this by looking at static images of cells and there was a paper published for example that looked at actin here the red is surrogate for actin and an endocytic protein here in green and concluded that for the most part they were present in different structures in a yeast cell you know occasionally you would see some yellow which meant that the two proteins were together but it's hard to know when it's just a low level of coincidence what to make of that but the real significance of this interaction and the explanation for how these proteins are functioning together in a network comes when you do two-color live cell imaging and Marco did a couple things differently from what other people did one one is he looked at two colors in real time another is he used a medial focal plane so yeast cells are spherical and if you use a medial focal plane you're really focused on the surface of the cell just around the edges and you can see that all of these dot structures we called patches are present on the surface of the cell okay now if you watch in real time you see something really interesting and that is that every single patch has a very similar undergoes a very similar dynamic process when it first appears on the surface it's green and then invariably that green patch turns yellow in other words it first first an endocytic adapter appears on the surface of the cell and then actin filaments start to assemble at that site a short time later it happens in a very predictable order first the endocytic adapter and then the actin and then if you really study it's amazing a simple movie like this a question came up the other day in the discussion you know what can you learn from live cell imaging from a simple movie like this it's amazing what you can learn and one of the things if you really study this movie that you start to notice is that just when these patches start to turn yellow they move off of the surface into the cytoplasm okay as though perhaps the forces from actin polymerization are driving some sort of structure from the surface of the cell into the cytoplasm you can depict that nicely making something called a chimograph where you draw a line through one of these patches and sample that line in every frame of a movie and then you can see that this endocytic protein here is present over time on the surface of the cell then there's this burst of actin polymerization at exactly that moment the endocytic protein starts to curve off of the surface of the cell into the cells interior okay showing there's a very tight correlation between the assembly of actin and the movement of this structure into the interior of the cell so we've done this experiment over and over again with a lot of pairwise permutations it turns out there's about 50 or 60 proteins that are in this network and what we ended up with is this which is summarizes quite a few years of work from my lab and other labs in the field and what it shows is a cartoon of what we think is happening on the surface of the cell there we think that some endocytic proteins start to accumulate cargo starts to get captured and then there's a burst of actin polymerization the forces from the actin polymerization are harnessed to invaginate the membrane and pull off a vesicle and then along that timeline and color-coded with the cartoon above are about 50 proteins that we have ordered in this pathway all by doing different pairwise permutations of labeled protein so for example that SLA1 protein that I told you is an endocytic adapter is here and ABP1 is here that is showed you in the pair and so SLA1 arrives and then predictably ABP1 also arrives with actually a very predictable and minimally variable amount of time between the appearance of these proteins okay so we've so we've then what we've done is built this temporal map for the recruitment of many different proteins to these sites of endocytosis and when you do this for 50 proteins it gives you kind of a holistic view of this very complex pathway and series of events and so one thing you can do is you know it's really hard to think about the functions of 50 or 60 proteins at least for me is you start to see that you can cluster groups of proteins together within the pathway and for example early proteins were things like coat proteins and you can do that by by looking at genetic interactions physical interactions the dynamics of the proteins the lifetimes of proteins phenotypes when you knock out proteins and you know what we realize is that you could cluster these 50 proteins into maybe four or five groups of proteins and and then we we developed them the concept that these were modules of proteins and each module was carrying out a function and so the first module proteins shown in green and light blue or sort of a coat that you know would create the coat of the vesicle but also capture cargo this blue module are proteins that would link the coat to machinery that nucleates act in assembly then machinery that nucleates act in assembly shown in purple here and that generates forces on the actin would get recruited so a wasp protein that activates this R2-3 complex to nucleate actin a myosin that generates forces on actin these proteins start to accumulate interestingly there are a lot of of multivalent proteins the sh3 and proline rich proteins here we think that there might be a phase transition involved in and we published a paper last year on this in in linking the actin assembly to this acidic coat because there seems to be a threshold effect where having these multivalent interactions concentrates the the activators of the R2-3 complex because then what happens is we reach a threshold effect of a couple of key proteins and then there's this transient burst of actin assembly and the reason that it was so hard for people to detect an association of actin with the endoscopic machinery earlier is because this interaction is so transient okay that transient um transiently there's a burst of actin assembly knowing when things get recruited in the pathway can generate ideas about what things might be doing so the actin is recruited very late when the vesicle internalizes suggesting it's generating a force these uh bar proteins come really late in the pathway suggesting they might be involved in scission which was again verified by genetics now key to a lot of this work uh and our ability to make such a precise pathway was the fact that in yeast we could precisely integrate gfp and rfp into the genome because homologous recombination is very robust in yeast and so we could look at the um dynamics of each of these proteins expressed at their native levels because we didn't have to do what was commonly done in mammalian cells which is to make a cdna of a gene you're interested in uh clone gfp or rfp behind it and then reintroduce it into cells in other words and then overexpress that protein on top of the endogenous proteins what we decided to do so we started our eyes started looking towards mammalian cells because a number of proteins in that network that we found in budding yeast had homologs almost all of them did in mammalian cells and a great number of them were unstudied and so we decided why don't you know why don't we study them and then when we decided to look at the dynamics in mammalian cells we decided to use genome editing to make precise integrations so for example we tagged the clathrin coat with rfp and the dynamin protein that mediates the scission of the vesicle with gfp but we did this using first zinc fingers but nowadays crisper cast 9 to make a precise integration of these tags and so as tomi showed you yesterday you can now you can make look at these events in real time in mammalian cells and you know many other labs have done this including tomi's before us but i think you know one innovation we made was to do this at endogenous levels by genome editing so here you could see the red clathrin coat appearing in this turf this is a sort of early turf movie of ours and then each of these spots very predictably would turn sort of yellow and green when the dynamin would come to mediate the the fission event and so it's a fair amount of trouble to do the genome editing and you can ask is it is it worth that extra work and so one way we looked at this question was to compare cells in which we had overexpressed clathrin and dynamin as rfp and gfp and another to cells where they were endogenously tagged and so to do this we made 3d climographs so i showed you a 2d climograph before but in this case we took a four minute movie like this and showed the entire movie in one picture by putting time in the z dimension so these are basically a stack of frames from a movie and you can see each endocytic site when we overexpressed clathrin and dynamin and you can see that the two colors kind of blur together and so it's really hard to distinguish when one if one protein is arriving much before the other and that sort of thing when you when you overexpress the proteins but when you genome edit the cells you find that there's a nice period when the clathrin is assembling and you have primarily clathrin and then the end of the process is punctuated when dynamin is recruited and vesicle scission occurs so we think that having these genome edit cells and we now have probably i don't know 130 140 lines in the lab with various proteins engineered allows us more sensitivity to both look at the normal cells but also to look for effects of perturbations in the cells but we also coming from a yeast background we wanted to try to more closely replicate some of the features of yeast cells and oh sorry this is shows you sort of a profile of looking at the average you can look at the kinetics of clathrin recruitment and dynamin recruitment the clathrin largely comes first and there's a spike of dynamin and the two disappear together when a vesicle forms okay so so but what are the other sources of variation maybe from one lab to another one experiment to another different labs we're looking at cell lines in mammalian cells from different species different cell types fibroblasts versus whatever liver cells almost all the cells that were that are studied in tissue culture have chromosome abnormalities because they're cancer cells and those cancer cells are in a cancerous state they don't represent normal physiology so we wanted to establish a robust system where we could study cell proteins at their endogenous level in as close to the physiological normal physiological state as possible and what we chose to do is to start studying cancer cells and we use this cancer line from Bruce Conklin's lab at UCSF called WTC it's an induced pluripotent stem cell we've also used ES cells this is a karyotype of a hella cell and you can see the chromosome numbers are quite aberrant there you know are five copies of some chromosomes and three of others there are massive translocations and this I should note is a snapshot of the of a cell because these cancer cell lines are not stable when you have this kind of karyotype it's constantly changing this is a snapshot of a dynamic change for one thing's chromosome instability is a hallmark of cancer cells whereas you can get stem cells that actually have a normal karyotype and normal physiology and by many indications are are normal and so we decided a few years ago to start doing all of our genome editing in stem cells there are other advantages like you can take your stem cells and you can induce them into many different cell types so now you can compare a process a cellular process like endocytosis in cells that are genetically identical okay the only difference is the epigenetics you're differentiating them to different cell types and at first we've concentrated on comparing the stem cells to fibroblasts and neural progenitors so we've made a bank of genome edited stem cells that we then differentiate into these different cell types and it's really interesting because if you look my postdoc Daphne Demberne if you look in the stem cells by EM we in a collaboration with Justin Tarasca we see sort of large class encoded vesicles when we differentiate to fibroblasts we see these large structures that have been referred to as plaques that often seem to have vesicles emerging from their sides and then neuro progenitor cells have superfast endocytosis in extremely regular smaller vesicles and so and this the EM level ultra structure recapitulates very well what we see by the dynamics of looking in real time and we've begun to dissect you know what's happening as cells differentiate to differentiate this pathway and adapt it for these different cell types and we think because it's an isogenic model we now have a lot of control to do very you know well controlled experiments and to really get to the to the source of these different phenotypes the other thing that you can do with stem cells is to show is to make organoids okay so again if we want to get closer to physiology and we heard about this yesterday from Tommy and it was beautiful zebrafish studies another I think complementary way is to make organoids in this case we're looking at a intestinal organoid this is Daphne d'Ambrenet she's French from Paris and and a collaborator in Dirk Hockemire's lab at Berkeley Ryan Forster who's helped us make organoids and you know these epithelial cells have an apical surface which happens to be in the lumen this is a and then this basolateral surface and so you know they're in real cells in tissues there's stuff like cell cell interactions and polarity where different activities are happening at different surfaces and so we think ideally what we want to do is to be able to watch these things in organoids and and Tommy already showed you that we've we also collaborated with the Betsy lab to start imaging these things and I just wanted to mention if you look at a volume like this Tommy alluded to this too one of the problems that is becoming really acute in with these advanced microscopes like the lattice light sheet with adaptive optics is the amount of data that you can generate and so in this first frame here we just did some simple segmentation just looking at the nuclear volumes and starting to look at the membranes and commercial software you know a file like this gets to be about two gigabytes which is the point at which you start overwhelming commercial software one of our movies from our study with Eric Betzig so Daphne spent just eight days at Janelia Farm she generated 30 terabytes of data and a typical movie like this one is 72 gigabytes and so it's hard to manipulate these kinds of images and it's hard to do particle tracking and so we have a team now of people who are trying to catch up with Tommy who who can develop software for analyzing these things so these three folks are all a student postdoc an undergraduate computer science major are all have all been improving our particle tracking software for 2d and then Joe Schoenberg who's a new data science fellow in the lab has now got things working well in 3d so we can start to look at these things and you know this is a movie that compares different modes and of you know with the lattice light sheet and you can see with the adaptive optics you can hopefully start to see you can see the individual in the civic events and start to quantify things in real time and this was this is part of a paper that Tommy and I are both authors on that will be out in science sometime soon with Eric Betzig's lab so this is work all done in Betzig's lab and Tommy actually showed this movie yesterday so that's now with the adaptive optics and this is with particle tracking from Tommy's lab okay so um so with this project you know we're we're now um making we started with intestinal organoids but there are a lot of people on my campus who are interested in making other things and so Joe uh just this data science fellow is now making brain organoids with another lab and I think um the organoids have are complementary to things like zebrafish because you can make many different tissue types you can create huge banks of stem cells and there are now resources one of the things that people didn't say the yeast or drosophila field enjoy are shared resources of knockout collections uh tagged gene collections and so on so forth the allen institute of cell science which which i'm also involved with is making a big library now using the same parent cell line as we are of with virtually every cell organelle cytoskeletal structure signaling protein so on so forth tagged with gfp or rfp and so all of these things can now be imaged using um the lattice light sheet for example to look at whatever your process is and then you can engineer in your favorite disease mutation like we heard from dr. shen before me uh I don't know what how you say that disease but the awful skin disease you could start to um take these cells and differentiate them into keratocytes and uh and look at these things so I think this has a lot of promise back to actin okay so this is a an old experiment that um marco and uh edson did in my lab some years ago again this is a budding yeast cell and these are these cortical patches and when you look just in a wild type untreated budding yeast cell in a chimograph you see these hook-like structures where the endosyct protein is present on the surface and then at the end of its lifetime it curves into the cell when the membrane invaginates in a yeast if you add an actin inhibitor latrumpulin A to the cells you completely block this internalization okay actin is absolutely essential for generating that force and so we want to use this as a system to study force generation but we're now starting to look at this in mammalian cells and so with our genome edited cells one question was whether actin is really integral to the um endosyct machinery in mammalian cells like it is in yeast cells and so when we genome edited the cells for say clathrin and actin we found that essentially every endosyct event in a mammalian cell involves a burst of actin assembly again it's it was alluded people for many years because it's transient and um is really christian merafield uh who found this originally that the actin there's a burst of actin assembly late in the endosyct pathway but i think our work with these genome edited cells added to that by showing that it's really something that happens at essentially every endosyct event and so and no i'm sorry so what this experiment is it's looking at actin at endosyct sites i'm sorry so i've we've labeled actin with rfp and we've labeled uh dynamin with gf gfp and showed that it essentially every site there's a burst of actin assembly sorry yeah thank you for slowing me down okay so then using our genome edit cells now where we think the events are more regular and it's easier to detect perturbations so we've done a lot of sort of drug screens and RNA eyes and here again whether actin was involved in endocytosis in mammalian cells and how important it was had been a question for many years and a lot of inconsistent data in the field as we titrated latrunculin this actin inhibitor and looked these are chemographs looking at the lifetimes of dynamin we found you know in wild type cells it's it's very regular it's in our cell lines generally around 18 seconds of lifetime but as you titrated more and more actin inhibitor the lifetime of the dynamin got longer and longer showing that that final step of vesicle formation is getting delayed uh and impaired in the absence of actin okay so now we we want to think about how actin might be working to help make endocytic vesicles and so for quite some time I've been collaborating I started collaboration on mathematical modeling with Georgia Oster in my department lately Georgia's retired and it was my collaboration has been handed off to Padmini Rangamani who's now at the University of California San Diego and these two folks in my lab a graduate student Julian Hassinger and post-doc Matt Akamatsu have been doing mathematical modeling Julian has been doing continuum modeling and mat agent-based modeling and there's been sort of a nice synergy between the two of them for Julian's work he views the membrane as an elastic sheet and then starts to vary parameters to see how uh how they affect the vesicle formation so for example he can vary the spontaneous curvature of these proteins that generate the that are formed the coat or the surface area of the coat and he can show that he can form vesicles but then over sort of a physiological range of membrane tensions he finds that he can stall these the endocytic process and that depending on how much tension there is it will stall either at this sort of u shape before the u to omega transition or it can stall at a very early stage and Julian went on to show in his paper that um you can if you stall these events at higher memory tension but still within a physiological range and we've measured the memory tensions with our colleague Dan Fletcher that you can add forces that that might be provided by Acton to push the pathway towards completion now it was very satisfying to us that these structures accumulated in the stalled cells because it fit well with work from mainly I think from Tommy's lab and from Sandy Schmidt's lab and Tommy had this very nice study where he both varied memory and tension and looked in cells where there were natural differences in polarized epithelial cells the apical surface has high memory tension compared to the basolateral surface and found that you could with Acton inhibitors inhibit endocytosis at the high tension apical surface but that the basolateral surface was much less sensitive to Acton inhibition and the basically the modeling work that Julian had done had fit very well with some of the experimental work from from Tommy's lab and Sandy's lab suggesting that Acton was really required when you have high memory tension okay so then in Julian's model he varied you know where the Acton forces might be acting and in one scheme he had the Acton sort of working like we think it works from our yeast studies by pulling the vesicle in and he also had the Acton generating a pinching force and actually both of these could help drive the process towards towards completion so Matt then started to use his agent based modeling where he's you know looking at every single acton filament and individual molecules in this process but in doing so we had a whole wealth of information from studies like those from Tom Pollard's about the you know all the relevant physical properties and physical constants and levels for Acton cytoskeleton but there were a few things we didn't have at end of six sites and so one of things Matt did was built a really robust system for translating a fluorescent signal into a number of proteins and you know I think this is something that I want to become part of our regular workflow in the lab is every time we get a fluorescent signal is to be able to immediately read out a number of proteins and so what Matt did is he adopted a system built developed by David Baker where he builds these synthetic nano cages well he designed he engineers them proteins that make nano cages of different numbers 12 24 60 120 and then puts GFP on them and then expresses these in cells and so these individual punta each represent a certain number a known number of GFP molecules and it makes a really nice standard curve over a range of numbers that's relevant for most of the numbers involved in endocytosis and so we can use that for example to look at here dynamin and the arp 2 3 complex which nucleates act in the diamonds and purple and the arp 2 3 is in green and we can count the number of arp 2 3's and so now for Matt's modeling we know that there are about 150 arp 2 3 complexes and so on and so forth so more numbers to add to this model so so Matt has built this model using Francois Nelex cytosim program and he models the vesicle as this object hanging from a spring which is the plasma membrane and then he puts in arp 2 3 complexes he caps filaments he does a sweep of parameter space so that we can you know explore sort of within a bio all biologically reasonable range these various parameters that we that are all no known or that we've determined for his mathematical model and the question is can he generate a system that self-organizes itself around an endocytic site and that generates sufficient force to overcome the highest membrane tension that we think occurs in a physiological context where endoscopic vesicles are forming and so here he's plugging in numbers that he's either determined or that come from the literature and he's done these simulations and now he can generate a network in fact that self-organizes itself around this vesicle and generates sufficient force to create to pull pull and then stick vesicle against this spring okay so then from from Matt's work we can generate this sort of self-organizing actin network sort of the one we have now is more in this mode and then some of the conclusions I already mentioned he can generate about 15 piconewtons of force which we think is sufficient to overcome even very strong membrane tension okay okay I'm sorry I'm going fast I'm not explaining everything there's two classes of proteins the nucleators are blue and they are generally at the base on the membrane in the bud in the in the in the these purple proteins are coat associated actin filament binding proteins sorry and that's an important it's not a nucleator so for example there's a protein called hip1r that binds to clathrin it binds to pip2 and it binds to actin filaments and it's a part of the coat my former student osa inkvis goldstein showed that so the purple protein thank you for asking that the purple protein is a filament binding protein that captures filaments nucleated at the base okay so all right so okay so in this model then I said there are two modes in which julian found actin could help to overcome the you know high membrane tension and so we want to know what actin actually looks like at endocytic sites and actin is really hard to see in the EM compared to say microtubules but Tatjana Svitkina's lab has done I think the nicest study looking at actin around endocytic sites and what she does is she does platinum replica shadowing of unroofed cells and she sees something that looks much more like that first model where the actin is helping to pinch off the vesicle because the actin is concentrated around the base of the endocytic vesicle now this work is beautiful and I love it and I think it's showing you how some actin is organized but there is there are a couple problems with this kind of analysis one it's EM and you you can't look at a very large number of events you can't vary parameters like numbering tension very easily just because there's so much work needed to generate these images and then also because these cells are unroofed so they did in order to get these kind of images they did something very violent to these cells they ripped the top of the cell off and then they looked at what was left behind so if part of the machinery that's associated with these endocytic sites is more tightly associated with what's being ripped off than what's left behind it will be gone so we for us for our modeling and for understanding this process it's really important to understand how actin is organized and so we decided to strike up a collaborator with my colleague Cous in the chemistry department at Berkeley and my postdoc Charlotte collaborated with Cous postdoc Sam Kenny and generated by super resolution imaging storm imaging thousands and thousands of images of actin around clathrin sites and Joe has come in and helped us to do some quantitative analysis of these imaging but what's really interesting now is that we find basically two classes of structure so clathrin is shown in red and actin is shown in teal or whatever that color is and in many sites and in the majority of just normal growing cells what we see is that the clathrin is higher than the actin so the actin is around the base of the clathrin coated vesicle this is exactly like what was seen in that em study but from Tatiana Svitkina's lab however we also see this other kind of figure where the clathrin is completely engulfed in actin and i should tell you for their data set they use a surrogate timer to figure out where they are in the endocytic pathway which is they used labeled dynamin so dynamin appears very late in the pathway so they only analyzed endocytic sites that had dynamin associated with them and what they found is that normally the endocytic vesicle the clathrin is higher than the actin and that's shown here because the the blue is the height of the actin and the red and i can tell you one of the things that's really cool about the super resolution imaging is um you know it's done in x y but you if you put this spherical lens in the path you can actually generate z data so this is actually a z projection from data that was collected in x y which i i think is really cool um anyway that's an aside so so anyway this is the height of the actin and the red is the height of the clathrin now if you do a transient osmotic treatment to raise the membrane tension what you see is that you shift this distribution so now the majority of the endocytic sites the actin is engulfed the clathrin is engulfed in actin and so we think that the cell responds to high membrane tension by assembling more actin and actin with a different geometry and it's interesting to think about what might be the membrane the tension sensor that's sensing the higher tension and whether it's a different nucleator that is nucleating the actin around the top of the clathrin pit as opposed to the bottom of the pit and so this data is really really rich and we think we can do things now like look at class averages and and actually get some structural more structural information from this um and we can look at other perturbations to the system so and then in the last couple of minutes I just want to tell you about one short little vignette about a project we've had so a long time ago with George Oster we shared a postdoc named Jin Lu who collaborated with my longtime postdoc specialist Edie Sun and did a did a theory paper which we know one of the notions in that paper which which is you know I think very commonly discussed now but I think was a little less common than was that there's a crosstalk between the geometry of the membrane and the biochemical reactions that are occurring throughout this endocytic process and so you can think of endocytosis as a you know sort of cascade of events where the curvature the geometry is constantly changing for the membrane and that these geometries are being read back by the biochemical reactions so for example there are proteins that bind specifically to curved membranes bar proteins for example and so if one of those well proteins binds um if one of those protein binds that will make the adjacent membrane have the ideal curvature so more proteins can bind and then also enzymes that act on the bilayer if the bilayer is flat they may have a hard time accessing bonds but as it becomes curved they could act more quickly and so on so forth so with this notion that there's crosstalk between the curvature and the biochemical reaction rates um so we like that notion but it just kind of sat there for a while and um thinking here's some words from George George says that modeling can tell you how things might work they modeling can also tell you how things cannot work but modeling cannot tell you how things do work for that you need experiments and so we what we wanted was a way to um experimentally test whether curvature was affecting this process and we've seen some elegant studies here from of pulling out um tongue tongues from uh gubs um but we we came up with another way to look at curvature in live cells which was I met this woman named Ben Xiaosui who's a material scientist at Stanford University and they make nano arrays and we saw micro arrays in the previous talk these are arrays that she makes by etching on a nano scale on quartz glass and what happens is you can sit cells down on these nano arrays you can also put supported bilayers on them and the bottom of the cell actually tightly conforms to the curvature of the pillars and you can dial in all sorts of curvatures and then we have all these genome-edited cells so we could put the cells on these substrates this one this one happens to have bars sometimes they're pillars the bars are flat in the middle and they have a very high curvature at the ends um if you put our genome-edited cells on them here's two of the bars it turns out that the highly curved ends of the bars become hot spots and this is a now a chimograph and you see a clathrin dynamin clathrin dynamin clathrin dynamin so they're just streaming off vesicles on these highly curved sites and so this is very exciting to us you can see it actually happening in em here's a cross section of a pillar there's an endoscic vesicle budding from the edge of the pillar and the only problem with this system is that it was very hard to make these nano arrays fortunately at Berkeley we found that we have another way of making these we now etch molds and then we use a polymer and we can stamp out these nano arrays where now this one happens to have ridges and you can see looking at the diamond for example that endoscic vesicles are coming from the crowns of these ridges and so it's nice now because we can make lots of these and so now we can do things like RNAI screens and see if we've bypassed certain steps in the process you know do you you know our hypothesis is that there's some rate limiting step where we have to generate initial curvature and then you attract more curvature sensing proteins and so on so forth uh if you're interested in this I just some have some i-biology talks that were just posted online so these are the people who did the work I tried to mention everyone as we were going along and I don't see anyone that I didn't mention this is the lab there's me and I'm here it's a joint lab with my wife Georgian Barnes who's here in the audience at the meeting and I thank you very much for your attention what do you think determines the size of the vesicles is there much variability in the size of endoscopic vesicles you can make and and and what what determines so naturally what you mean yeah well it's interesting uh you know when we differentiate these so the stem cells actually have sort of a very variable and there's kind of a range I'm going to say 80 to maybe up to 200 nanometers is that too big or yeah too big a little smaller okay but you know in these neuro progenitor cells they're very very regular and very small on the small end of that range so you can vary them and in fact Tommy's done these beautiful studies with viral infection showing how adaptable clathrin is the coat can actually encase something quite large but that's given by what is being taken so naturally do you mean naturally and if you play with your your member intention right do you change the size yeah that's a good question yeah Tommy says no so I don't think I haven't yeah looked at the absolute numbers so what make sure that if they don't collapse the clathrin self-assembles is a rigid molecule it's rigid enough as it builds it puts hexagons and pentagons so the overall curvature is defined by the ratio of hexagons against the pentagons so what exactly does that make clear as David says the neurons inside the smaller guys they could vesicles that you have in the secretary pathway in all cells these are small but yeah yeah huge pressure inside the cell no no the no no no the vesicles that are forming from the from internal vessels inside the cell the end of the cells they're all small guys no but at the plasma at the plasma membrane it doesn't we have never seen changes on the overall shape just by changing tension right what does it is a little bit cargo so they adapt a little to the size of the cargo with an upper lip we go beyond 100 nanometers they stall it's flat we've also done mass spec to look at the differences in the clathrin associated proteins in the three cell types that we've compared the stem cells and neural progenitors and fiber lesson we found some interesting differences and at least one of them if we modulate it we can change the geometry of the vesicles so ap2 actually have you looked at how the combination changes in your different differential itself no that's it's very interesting no we have not done that and you know again some nice probes available now so the young guy this might be a basic question but do you know what the why are they why are those ridges hot spots for endocytosis yeah that's what we that's what we're trying to figure out now our idea is that there's some that the curvature is a signal that's attracting there's some limiting step where some initial curvature may help to recruit proteins and so like there there are proteins that bind specifically to curb memories like clathrin for example likes to make a a cage and so that by giving the cell curvature you're attracting those proteins and so we we have a strategy what we haven't we haven't been able to do many experiments because it it takes a it the the quartz substrates we have take a day they're made at stanford it takes a day to make each one at a cost of about thousand dollars we can make about 15 of these in two hours now so we're just in a place it's taken us two years but we're just in a place where we can stamp these things out and ask that kind of question maybe i can continue on this question so now it's not only what you expected the first protein which would be sensitive to that kind of curvature which is not that high would be the f bar right so if you flip at bar you change the localization that's we're doing we're setting up to do those experiments literally we've got these substrates you know we had to go through all these polymers we're finding things that weren't autofluorescent work we had to my student learned the CAD programs and how to go Lawrence Berkeley National Lab had to learn how to etch everything so now he's bob he's ready to go so those are good questions yeah i'm going to continue on this there's in epithelial cells you have all kinds of specializations infoldings and brush borders and all those and endocytosis is actually preferentially coming from the the pit from the curvature no i don't think so i i mean i know not in i think that there it's possible the cell might exploit some natural fluctuations and trap a state but um you know one of the things we're going to explore now as we're trying we want to set up some kind of systematic study i think you heard today on the micron scale about septans recognizing curvature and the clathrin pathway be being influenced by curvature we want we think there's probably a lot of other things that are happening this other effect by curvature so we want to use this system to look systematically at a lot of processes and see what else is responding but i don't know so in a natural setting um yeah i don't i don't but comment on that otherwise there's something funny about this at least in the fish we haven't seen that oh that there's a preference or where they come from that yeah now this must be an old question for you i was wondering whether you think or i'm thinking about whether force is the only way acting would act to make the poses or that might be other mechanisms i'm thinking of a paper by g2 mayor and gluggen johannes and you still propose that acting plays a role in phase separation oh yeah and that would be important for supervision or many other ways we had it so we had a paper um in e-life this year where we actually propose something like that that they're that this multivalent um interactions make a phase separation to nucleate the actin but there is your question also about whether acting is doing something else in budding yeast it's really clear that acting does something else which is it sends back a negative signal to take everything apart to turn off actin nucleation and to uncoat the vesicle if if you remember i showed a image from yeast where we use this latrunculin A and when you look at a coat protein like clathrin when you block actin it assembles on the surface and it just stays there and which normally it forms and turns over and we've actually found a couple of the proteins which may be yeast specific i don't know one is a protein kinase that phosphorylates an actor with the R2-3 complex and turns it off another is a synaptogenin which binds to it binds to indirectly to actin to recruit it to facilitate the encoding step so there the actin not only generates a force but it also negatively feeds back on the processes that set up the site so i was wondering about uh the type 1 myosins that are involved in yeast endocytosis do you know what they do there and are they also involved in mammalian cell yeah so there are type 1 myosins in mammalian endocytosis in yeast they are almost essential for the invagination step and so i have a very good student ross peterson working on that process and they're interesting proteins because they both nucleate the one in yeast nucleates actin assembly and it has a motor domain and we have a um collaboration with Michael Ostap at Penn uh who's a single molecule biophysicist and we're characterizing the type 1 myosin in budding yeast they're they're two kinds of myosin motor you can roughly classify them some are tension sensitive uh clasps that when you pull on them they just bind very tightly to actin and the others are force-generating motors and so with the single molecule experiments you can differentiate which type it is we were at least i was really betting that we had a tension sensitive clasp that that myosin was actually holding everything together but in fact from the kinetic profiles it looks much more more like a force generator and so um so budding yeast uh according to uh the theoretician at Fred Chang collaborates with in in fission yeast says that the the amount of pressure you need to make to make an endoscic vesicle in yeast which grown a tremendous turgor pressure would be equivalent to pushing your finger into the tire of your car so to put it in sort of real-world sense so so i think it may be more cute in um mammalian cells but we definitely we've cloned it and tagged it and it's the same person doing the super resolution work is studying the myosin in my lab we just got an inhibitor through the mail from someone in germany of the type one myosin and we're trying to figure out what it does in the mammalian cells if there's time for one more we want to take coffee there's this guy in the back who's been patient but David this is probably philosophical i mean cup one and cup two vesicles are roughly the same size they don't use actin they don't use bar domain proteins i mean is this acting the driven process entirely due to perhaps the surface properties because there is so much um of the cytoskeleton to begin with is it to do with the tension i mean although Tommy says tension has no role what no no he said the opposite he said the opposite Tommy actually did a nicest study i think to to clarify the role is that it because the membranes are so tense that you need extra force to imagine it i think that might be true but you know actin is also used for some intracellular trafficking events this it's it's a p1 yet with the the um and i mean basically the but i'm talking about cup two and cup one is that is that it is that it is not it is not you have a lot of excess area so that the plasma membrane is not tense but sometimes but sometimes there is some some events like probably you agree that the plasma membrane normal condition the liquid barrier is not tense right so in fact in fact if you follow up i mean one point i wanted right is you said that all the intercity classroom codes in european and are recruiting actin right the the events well 90 percent according to me so when we did with there we did the uh the structure illumination yeah so there we were looking right the acting only appears in about 60 percent of the end of some day the pits they don't don't have they are perfectly functional they're coming in how did you tag actin excuse me what i'm sorry how are you looking at acting what would be used for acting i don't remember but it was acting i can't remember it was uh go good i should google i should look at my own paper why is that why is that important i'm just kidding i'm just kidding we see 90 you saw 60 i don't know but you know maybe no but there was a difference the guys that had the acting were shorter lift and yeah they were fast okay there was no difference in the size of the pit but those were faster the kinetic was faster and the other ones were a bit slower that was that's interesting yeah okay well you know when they're latrunculin titration we saw it slowed events yes we tried to look acutely but now we've also we actually get exactly the same thing with our two three inhibitors