In this talk, postdoctoral researcher Claudia Vásquez (Alex Dunn Lab, Stanford) outlines her interdisciplinary investigation into how cells construct organs, focusing on the formation and shape stability of lumens. She begins by highlighting the clinical importance of understanding kidney development, noting the prevalence of chronic kidney disease and the need for engineered kidneys. Drawing on her PhD work in Drosophila, she explains two mechanisms of tissue folding: myosin-driven invagination and buckling due to tension differences. Transitioning to her postdoctoral research, she introduces the MDCK 3D culture system, where polarized epithelial cells spontaneously generate lumens. To dissect the contributions of luminal pressure, cortical tension, and apical‑basal polarity, she performs three key perturbations: (1) knockout of five claudins to compromise tight‑junction barrier function, revealing that lumens persist with normal shape despite a ten‑fold loss of pressure containment; (2) a cocktail of actin, myosin, and microtubule inhibitors to weaken the cell cortex, showing minimal impact on lumen morphology; (3) overexpression of RAB11A to enlarge the apical domain, which dramatically reshapes lumens toward irregular forms. These experiments indicate that, in MDCK cells, polarity networks dominate lumen shape stability, whereas pressure and cortical tension play lesser roles. Collaborating with graduate student Vipul, she presents a computational model that unifies pressure‑dependent and polarity‑dependent mechanisms, reproducing the regular blastocyst lumen (high pressure, low apical length) and the irregular MDCK lumen (low pressure, high apical length). Concluding, she outlines her future independent research agenda: integrating 3D cell‑culture and Drosophila embryo approaches to uncover how cells generate higher‑order 3D folding patterns, likening the process to shaping a balloon animal with varying internal pressures and material properties. The talk ends with an invitation to the next speaker.
Chapters
Thank you so much for this opportunity to share my work. I'm really excited to talk about the work that I've been doing in my postdoc in Alex Dunn's lab at Stanford. And so Giuliani really did a great job of introducing kind of the amazing things that humans can do, even just in sweating and all the kind of biosense, the different biomarkers that we can produce. And the question that I've been really fascinated about is how do we build ourselves? How do cells build us? And so today I'm going to tell you a little bit about my contribution to this big question. And so one thing that's fascinating to me is that we have all these organs that are packed into our body, and even in this cartoon depiction of these organs, they have these wildly different shapes and structures that are essential for their function. So if we zoom in on the kidney, even this human kidney that's at 15.5 weeks, it has this elaborate structure and these different folds, these different tubes that are all essential for its function. And kidneys are one of the organs that often fail. And so over 2 million people need dialysis or kidney transplants to remain alive. And about 10% of the human population has chronic kidney disease. So there's a need to be able to understand, to build new kidneys or to be able to fix kidneys when they're misfunctioning. And to do that, we really need to understand how do cells build organs. And so we actually have a pretty complete toolkit of these molecular components that are required to build different different organs. And so, you know, here I'm highlighting some of my favorite ones, different adhesion proteins and proteins involved in contractility. And we know a bit about how these molecular components fit into cells to generate different cell shapes, how they would change to make a columnar shape and how that would change to generate a different wedge-shaped cell. And we know a bit about how these cells kind of fit into an epithelial sheet to generate different, what I call epithelial motifs. These tissue folds or tubes. And these motifs stack together to form these higher-order 3D structures, these complex structures where it's complexes of these motifs stacked together. So tubes that fold, for example, to build our organs. And so if we could understand how all of this fits together, then we could really get to this idea of how to build an organ, and that would be truly transformative. And so my PhD, I contributed to this problem by working on understanding how tissues form folds.. And so I use the fruit fly embryo. And you can see here in this system, active contraction of the cells by myosin drives the invagination of the tissue. And so that this forms the mesoderm of the animal. Tissues can form folds in other ways simply by buckling, having a difference in tension. And so there's two, at least two ways to form a tissue fold. And what I'm going to tell you about today is actually work I've done in my postdoc, some of the work I've done in my postdoc contributing to understanding how do hollow openings or lumens form. How do you find, how do you make a tube? And so these lumens, these hollow openings, are prevalent throughout biology. They're prevalent at the beginning of human embryogenesis, the formation of a blastocoel. They're present in our kidneys, and they're present throughout many of our organs, including our livers. And what you can see in just these 3 examples of different lumens is that they have wildly different shapes. You have a kind of more regular shape over here, a very squished shape here, and then one that has this kind of bulging in. And despite these different shapes of these, these lumens here that I've pulled out, they all have kind of the same design parameters to make a lumen. So there's a need to be able to fill the lumen with fluid, the ability to hold cells together and maintain cell shape, and the ability to form an inside and an outside, or an apical and a basal side of the cell.. And what I'm interested in is how do different lumens depend on these different parameters? Are there lumens that depend more on pressure versus some that don't need pressure as much, for example, and can depend more on these other parameters? And to study this, I need a system that I can image and genetically manipulate, and I can do molecular— add different molecules that I can easily kind of test each of these components.. And so I chose to study the MDCK cells. So these are Madin-Darby canine kidney cells embedded in the extracellular matrix. And what's amazing about these cells is that within 48 hours they polarize, and you can see the lumen surrounded by this orange cage right here. And the same mechanisms that these cells use for polarization are the ones used in different organogenesis, including the kidney.. And so, when I first started looking at these cells forming lumens, you can see that some of them form these very irregular shapes, and some of them form these very irregular shapes. So, here's another example where you can see the faces of these cells pushing into the lumen. And when I looked at a whole bunch of these, I saw that cells can form a wide diversity of lumen shapes just by putting them in this extracellular matrix and allowing them to polarize.. And so this suggested that maybe pressure alone wasn't contributing to forming these lumens. But I wanted to test each one of these experimentally. And so what I'm going to show you first is how I tested luminal pressure. And so I did this by effectively kind of poking holes in the lumen and seeing how stable was the lumen shape. And you can do this by— I did this by knocking out 5 of the claudins, which are components of these tight junctions, and these tight junctions are essential for epithelial barrier function. And when you knock out these 5 claudins, the ability of the epithelia to maintain its barrier decreases tenfold. But surprisingly, even though these cells are extremely leaky, they not only still polarize, but they still form a lumen, and these lumen shapes are not significantly different from their wild-type counterparts. And so pressure alone is not a driver for determining lumen shape stability, in the system. So, now that I've tested pressure, I wanted to look at cell cortical tension, and I did this by weakening the cell cortex. And so, what I'm going to show you here is an experiment that I did out of frustration. I first started by trying to affect each one of these components alone, and I found very little effect. And instead, I just added them all at once, and to my surprise, I found that even though I had just ablated the actin, myosin, and microtubule cytoskeleton, there was still a lumen at the end. And you can see this here in these 3D contour plots of the lumen shape, where the blue contours are 2 minutes before and the green contours are 18 minutes after. And so even though the lumen is quite wiggly, you can see that when adding the cytoskeletal drug cocktail, there's not a very dramatic change in lumen shape. So just that the cell cortex is not an important contributor to lumen shape. So that left me with testing this last component. And I did this by increasing the size of the apical area. And I did this a couple of different ways by manipulating different components of the crumbs complex and the PAR complex. But what I'm going to show you today is about RAB11A. And so RAB11A delivers these apical components to the apical side of the cell, both the crumbs and the PAR complex.. And if you overexpress RAB11A, you get an increase in this apical domain size. And when you look at the shapes of the lumens in these cells that are overexpressing RAB11A, you can see this marked change in lumen shape that's for this more irregular shape. And so, what I've shown you today is that in this MDCK system, in fact, they're more dependent on this apical-basal polarity networks to make a lumen and to have its lumen shape stability and less so on these other two parameters. But I was curious if that was the case for many lumens. And in fact, we know that for the mouse blastocyst, there's a high dependence on this pressure parameter. And so Vipul, a talented graduate student in the Dunn lab, built a model that could try to unify these two different ideas. And so what I'm showing you here is the output of various simulations. Where each one of these is a mean of the simulation at these conditions, showing the solidity of these lumens. And so a lumen that has a very high pressure and a lower preferred apical length has a very con— a very normal-looking regular shape. In contrast, a lumen with a very low pressure can still have a lumen, but it has a very irregular shape. And this is very similar to what we see in the shapes of the blastocyst and compared to the shapes of our MDCK cells, which actually when we fit these parameters, they fall around here with. And so, lumen shape can be stabilized by both pressure-dependent and pressure-independent mechanisms. And so, what I've talked to you a little bit about today is some of my PhD work showing how cells can make folds, some of my postdoc work showing how cells can make lumens, and I haven't had a chance to talk about how these molecular components fit into different cells. And what I'm really excited to tackle in my independent group is this next step: how do cells make these higher-order 3D folding patterns? And I think I'm really well positioned to do this because of my background in studying these different scales of how cells build different shapes. And to do this, I want to leverage my experience not only in 3D cell culture, but also go back to the Drosophila embryo and really be able to couple a top-down and bottom-up approach to understand how do cells make different 3D folding patterns. And I think a way to kind of like— mechanism to take away is if you think about how you can make a balloon animal, you can fill it up and this can have— and if you— the different amounts of pressure required and forces required to make a balloon animal of this nature are different than if you didn't fill the balloon at all. In fact, if this balloon were an active material, it might be able to hold its shape without any external forces. Or if you fill it up differently, you might get a different balloon animal shape. And finally, if you use a different material, the forces involved in creating this balloon animal is very different than the forces involved creating either of these two balloon animals. And these are the sorts of questions that I want to attack, but instead of with balloons, I want to understand them for cells. Thank you all for your attention, and I'd like to introduce the next speaker, Ivan Huapi.
