Velia M. Fowler was kind enough to give us some in-depth answers to our questions about her research for the article “Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens,” which she co-authored with Roberta B. Nowak in J Histochem Cytochem June 2012 60: 414-427, doi:10.1369/0022155412440881.
Tell us about your work?
I want to begin with some background. This study is about the hexagonal packing geometry of cells in the eye lens. We got interested in this problem because we were studying the actin cytoskeleton, and how it links to the plasma membrane. We made a knockout mouse, to delete one of the actin binding proteins of this membrane- associated actin cytoskeleton (tropomodulin) and found that there was a disruption in the hexagonal packing geometry of the fiber cells of the eye lens. Our first observation1. was published in the Journal of Cell Biology, where we reported the disordered packing of the lens fiber cells, and by doing some biochemistry, we also characterized the disruptions in the actin cytoskeleton in the mutant lenses.
Getting back to the hexagonal geometry of lens cells. The lens cells are very long and thin and stretch from the front to the back of the eye lens. If you cut the lens at the equator so that you can see how the cells are packed geometrically together, you see that they are packed in this beautiful hexagonal packing pattern. This is the most perfect hexagonal packing geometry of any tissue that we know of; certainly the most perfect in a vertebrate, although in invertebrates, the Drosophila eye is exquisitely regular as well.
Since we had discovered this defect in the hexagonal packing, I got interested in that problem and started doing lots of reading about it — the field that studies this mostly is the developmental biology field of epithelial morphogenesis, for example, in flies. In certain stages of embryonic development, there are movements of epithelial sheets during dorsal closure or germ band elongation, where the cells change their shape and their packing geometry and rearrange. There is a huge amount of work in this area showing that cell adhesion and mechanical forces influence the packing geometry.
I think, that’s the bigger picture area that our work is related to, the broader area of cell biology. Also, these movements of epithelia sheets happen in vertebrates during gastrulation, it’s not just something that happens in invertebrate embryos. In our study that’s published in JHC, we wanted to see what the extent of the hexagonal packing disruption really was, we wanted to quantify it. Basically, we had observed these disordered cells in the lens sections but we didn’t know whether all the cells were disordered, what percent of the cells were disordered, and just how disordered were they.
Now the field of epithelial morphogenesis that I mentioned earlier has developed computational image analysis software in order to actually count the number of nearest neighbors each cell has in order to define their nearest neighbors. We had a summer student come to the lab, he was an external Masters student from Strasbourg, France, and his project for the summer was to apply this software that was developed for a completely different problem to our images of the lens. That was one of the things that we worked on and we found that the disordered packing of the cells was only in patches and not in all the cells. That was a major finding that the disorder was in patches and the other major finding was that these patches arise as the cells mature and get older. So, now I need to tell you that the lens cells form on the outside of the lens and then the cells elongate and are laid down like layers of an onion, and they are laid down in this perfect register with this hexagonal packing pattern. We looked in these sections at the youngest cells, which are on the outside and then a little further in older, older, and older cells and we could see that these patches of disorder were very infrequent in the outer layers of cells and they became more abundant as the cells moved inward and matured, and also the patches became larger.
The summer student from France, Martin Dressler, although not an author on the paper, made a very important contribution to the study in working out the computational analyses. He was thinking along the lines of mechanical forces and how they affect hexagonal packing. Now here is where we get into a whole other area that is another basis for the study and contains the conceptual underpinnings of our ideas.
There are physicists that study hexagonal packing in nature and it turns out that soap bubbles, the lowest energy form of packing is hexagonal, a hexagonal geometry, which is true for many objects. If you have a lot of spheres and you put them together, they are going to fit together best in a hexagonal packing geometry. This area of physics talks a lot about minimizing forces, minimizing energies of interactions and so taking from that field, we’re thinking about forces and the people studying the fly epithelial sheets are also thinking about these forces.
From these ideas, we conceptualized that the changes in the packing from no disordered patches and very small patches to bigger patches as being due to the mechanical stresses on the cells in the lens as the lens is growing and getting bigger. The idea is that there would be more physical forces pushing on them due to the size of the lens as they get packed into the center, as well pulling forces on the lens from the extracellular fibers which attach to the outside of the lens capsule and hold the lens in the eye. There are also osmotic pressure forces in the lens, due to water fluid flowpumps and channels, which can create mechanical forces. Thus, maybe in a normal lenses, a wild-type lens, the cells have a cytoskeleton that can exert tension against these forces and keep them balanced and keep the cells packed properly in a symmetric hexagonal pattern, but in the knock out lens where the cytoskeleton is disrupted, the cells cannot resist the forces, leading to asymmetric distributions of forces, and this leads to disordered cell slippage and disordered packing. That’s kind of a long way to describe the main points of the study!
My lab (http://www.scripps.edu/fowler/) is basically a cell and developmental biology lab, and we’re also interested in physiology, but we like and are drawn to the study of order in biological systems with a precise geometric organization. We’ve trying to understand how molecules and cells that are normally not like this become constrained to assume these beautiful arrangements. So the lens is one of the systems that we study. We also study muscle which has a precisely organized arrangement of sarcomeres and myofibrils that’s very important for contraction, just as a side point. But in all the systems I study, we are interested in how molecules can impose order on chaos, basically. And it’s interesting, at least in this field of hexagonal packing, the physicists think that it uses basic principles of minimizing energy, in order to achieve ordered geometric packing.
How will this paper contribute to relevant fields?
Most people that will be interested in this paper will be people studying the lens. Probably not people studying the fly morphogenesis because we drew from them rather than they drawing from us. In the lens field, outside of the concepts that I discussed above, one of the main advances of this paper is applying quantitative methods to measure the disorder and irregularity in these lens cells. Other people studying lens fiber cell organization have sometimes gotten a mouse knockout where they’ve seen the disordered packing but nobody has quantified anything. We have developed some relatively simple methods to quantify the percent of disorder in the lens, which actually is already being used by some other lens researchers. First that’s really nice to see, but then hopefully, when more people apply these approaches to their mouse lens mutants we will be able to understand, looking at a bigger picture, what controls the regular geometry of lens cell packing.
Now why do we care whether fiber cells are disordered? There is a general hypothesis in the lens field that the ordered packing of the cells is essential for the clarity of the lens. So the idea is that if you have disorganized cells with altered spaces between them and irregularity, then you are going to get more light scattering and that could be leading you to having a cataract. It could underlie some of the human cataracts. But, if we can’t quantify the percent of disorder, we’re never going to be able to figure out how much disorder can the lens tolerate before it starts to scatter light and result in opacities that lead to cataracts. I think in the long view, that by applying these quantitative methods, we’re going to be able to quantitatively correlate cellular organization to the degree of light scattering to understand how the cellular organization contributes to lens transparency. Looking forward, that is one of the main applications of what we have done.
Are you going to continue working in this particular field?
One unsolved questions is that the mouse knockout we have been studying, was created to delete the actin binding protein tropomodulin that we’ve been studying for years, but this deletion turned out to be on a background of a mutation in an intermediate filament protein. So there’s always been the question of to what extent does the absence of the special lens-specific intermediate filament protein contribute to the defects in the actin cytoskeleton due to the absence of the tropomodulin? A study we are working on now and have nearly completed compares the four different genotypes: wild type lenses, lens missing only the tropomodulin that have a disruption of the actin cytoskeleton, then lenses missing only the intermediate filament protein, and lenses missing both the tropomodulin and the intermediate filament protein. Thus, we are comparing two single knockouts and a double knockout with the wild-type lenses. This study uses the same methods that we developed here in the JHC study to quantify the amount of disorder, and also uses some additional approaches to quantify the degree of transparency. Basically, we’re trying to get a better handle on what’s causing what.
Then additionally, we have added on another set of assays to look at lens resilience. The lens transmits light so you can see and you can focus on objects and in order to focus, it has to change shape, as opposed to a microscope lens. A microscope lens does not change shape, it’s a fixed shape and it moves up and down in order to focus on near or distant objects. But in the human eye, we don’t move our lens back and forth, our lens actually changes shape to focus on a close object or a faraway object. This change in length and shape requires a certain degree of flexibility and resilience in the lens. Now the mouse lens doesn’t actually change it’s shape but it likely does have resiliency properties that are similar to primate’s lenses. So we looked at the mechanics of the lens from our single and double knockouts, and basically we ended up finding that the transparency, the disorder and the mechanics are determined synergistically by the two cytoskeletons, and that none of these biological properties are really independent of the other ones. So the work just got really complicated.
Did that make sense to you as a scientist that they would be synergistic or did that surprise you?
As a scientist it makes sense, since there are a lot of proteins that have been discovered in the last ten years that link between actin filaments and intermediate filaments. Although the tropomodulin protein we are investigating is different in that it is a small protein and the other linkers are very large, it could still be linking actin filaments to intermediate filaments. As a scientist, this totally makes sense and what you are really talking about is structural cross-talk between two different cytoskeletal systems, similar to signaling. That all makes sense to me but I was really hoping there wouldn’t be because it is very expensive to maintain all these lines of mice and now I find that I have to study a lot more mice than I thought I would study. and where I thought I could actually narrow my focus, instead I have to broaden my focus. So it is more expensive and a bigger field of literature, reagents etc. However, it is very interesting!
This goes back to fields of research tending to be very narrow because we want to simplify things and because we have this principle of reductionism, scientific reductionism, where we would like to find the root cause and we would like it to be one gene or one protein or one interaction and then we can study that in great detail. In reality however, for any cell or tissue in your body, there are many interacting systems both in signaling pathways with cross-talk, and in structural systems where things are connected to one another in order to accomplish the purpose of the tissue. In general, you are seeing all fields moving past reductionism to try and understand what are the connections between systems and what is the meaning of it. So again, while we don’t study the signaling pathways where cross-talk is well-established, we study the structural linkages and I think my work is moving in that direction.
I would like to end by noting that future studies in the lens and other tissues will involve looking at the cross talk and interactions between structural networks that achieve different functions in the tissues. For example, a contracting muscle uses actin and myosin filaments in sarcomeres to generate forces, but the contracting sarcomeres are attached to the edge of the cell with intermediate filaments– otherwise the muscle can’t actually move anything and you’re not going to go anywhere. Similarly, in the lens, you need to link the systems in order to accomplish the goals of fiber cell organization, for transparency and for mechanical resiliency.
If you think of other areas such as genomics, a good comparison for readers is the field of bioinformatics. Bioinformatics is a huge growing area of systems biology; these are ways to look at connections between gene expression, and between different mutations. That’s another type of investigation, but again it’s focusing on the systems and the connections and we’re trying to do a similar thing on a structural level.
Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens by Roberta B. Nowak, Rebecca K. Zoltoski, Jerome R. Kuszak, and Velia M. Fowler
J Cell Biol 2009 186:915-928. Published September 14, 2009, doi:10.1083/jcb.200905065