I am Yujie Ma, a rising senior undergraduate student studying biological engineering at Cornell University. Since the beginning of my sophomore year, I have been a dedicated member of Professor Mingming Wu’s laboratory working on research pertaining to how the physical conditions of the local tissues affect breast tumor cell migration. Specifically, my focus is on the influence of the flow of the surrounding bodily fluids and stiffness of the encompassing tissue on the cells’ ability to leave the primary tumor to invade into other parts of the body, as well as their behavior during invasion. In the human body, the flow of bodily fluids and the stiffness of the tissue would be controlled by habits, lifestyle, and predominantly age and would heavily influence the speed and reach of the spread of cancer. For one of my more recent projects, I made cancer spheroids, round balls of live tumor cells ranging from tens to a couple hundred microns in size that can act as three-dimensional models of actual tumors, and placed them inside some microfluidic devices. These devices are tiny scaffolds created to mimic the environment within the human body and allow for easy imaging. Collagen, the most abundant protein in the human body which can be found in every tissue, is mixed with cell media to form a gel and injected into the microfluidics device to complete the imitation. Inducing fluid flow and changing the stiffness of the collagen gel surrounding the spheroids replicates the tissue conditions of young and old individuals with active and sedentary lifestyles. After the spheroids within the microfluidics devices were imaged underneath a microscope for a couple of days, I analyzed the videos for the number of cells which broke free from each spheroid and the subsequent changes in area the spheroids occupied. Both are measures of the spheroids’ potential to “metastasize” using either single- or multiple-cell methods of movement. This project revealed that aging does indeed increase cancer’s ability to spread, as well as the efficiency of spreading. Fortunately, this project also found that regular and frequent exercise appears to help mitigate this trend.
I have learned much in my time in Professor Wu’s research lab. However, my expertise has been limited to this single field so far. Currently, I am still searching for the field that most suits me, but my aim to combat diseases of aging has never changed. I am extremely interested in the research opportunities offered by the SRF Summer Scholars program not only because of their emphasis on age-related issues, similar to what I am familiar with, but also because this program gives me the chance to branch out and expand my horizons.
Understanding protein homeostasis in Drosophila intestinal stem cells
This summer, I will be in Heinrich Jasper’s lab at the Buck Institute for Research on Aging, working under the mentorship of postdoctoral fellow Imilce Rodriguez-Fernandez. My two projects will use Drosophila melanogaster, better known as fruit flies, as a model system. For my first project, I will investigate the role of a member of the sirtuin family in regulating protein homeostasis (proteostasis) in intestinal stem cells (ISCs). My second project will assess whether altering proteostasis in ISCs influences the proteostasis of cell types found in different tissues.
Protein homeostasis, refers to all the biological pathways used by eukaryotic cells to maintain a balance between protein synthesis, folding, refolding and degradation (Figure 1).1 For the cell to remain healthy there must be a fine balance between synthesis/degradation and refolding of misfolded or elimination of damaged proteins. The most common mechanisms a cell can employ to degrade damaged proteins are via proteasome or autophagosome (Figure 1).2 Proteasomes are protein complexes that are specifically designed to degrade misfolded proteins by proteolysis, and autophagy is the process by which misfolded proteins and aggregates are enclosed in a double membrane and fused to the lysosome to form an autophagosome, where they are then destroyed by the strongly degradative enzymes within this organelle. Every cell in the body relies on protein homeostasis to remain healthy and properly operational.
Figure 1. The proteostasis network: protein synthesis is controlled by the rate of translation.
During or after protein maturation, any number of factors can cause damage, misfolding, and aggregation. All aggregates and misfolded and damaged proteins must then be degraded by the proteasome or autophagosome.1
Unfortunately, aging causes a decline in proteostasis; protein aggregates are more likely to form in certain cells of older organisms. For instance, the brain cells, known as neurons, accumulate protein aggregates which eventually leads to cell death in diseases associated with protein misfolding, such as Huntingtin’s, Parkinson’s, and Alzheimer’s. Interestingly, these diseases are usually observed in older patients, which suggests that the age-related decrease in proteostasis may have a negative influence in disease progession.2
I am interested in understating how adult somatic stem cells (SCs) maintain proteostasis. Adult somatic SCs are needed to regenerate our tissues throughout our lifespan. Although it has been shown that different stem cell types have different capacities to maintain proteostasis and this capacity may be affected during aging, the molecular mechanisms are not completely understood1.
For my projects, I will use Drosophila Intestinal SCs (ISCs) as a model system to study proteostasis in adult somatic SCs. ISCs generate all the differentiated cells in the intestine and are needed throughout the life of the organism to replace those cells that die by normal cell attrition or during stress. They are the only cells to undergo mitosis in the intestines. ISCs divide asymmetrically: instead of producing two identical cells, they yield another stem cell identical to the original and one differentiated intestinal epithelial cell with a specialized function ready to replace a dead cell (Figure 2).3
Figure 2. Diagram of Drosophila gut epithelium and ISC division and differentiation.
ISCs divide into another ISC and an enteroblast (EB), which then matures into either an enterocyte (EC) or an enteroendocrine cell (EE).3
Why use fruit flies for these projects? On top of being a well-researched model organism, Drosophila reproduce quickly, are easy to maintain in a lab, have a well-understood genome, and most importantly share approximately 44% of genes for tissue growth and structure with humans.4 Most discoveries made in Drosophila can eventually be translated to humans, making them a practical first step towards investigating our health needs (Figure 3).
Figure 3. Parallels between the mammalian and Drosophila digestive track.5
The Jasper Lab previously observed that an accumulation of protein aggregates, clumps of misfolded proteins, around in the cytoplasm leads to a cell cycle halt until all the aggregates are destroyed. This was termed a ‘proteostatic checkpoint’ (unpublished). This step is very important to keep the stem cell and its differentiated offspring functioning correctly.
The goal of my first project will be to study the role of a member of the sirtuins family in regulating proteostasis in Drosophila ISCs. Sirtuins are a family of proteins known for their role in regulating metabolism and aging, but not much is understood about them.6 Members of the sirtuins family could potentially play a part in the proteostatic checkpoint, and a previous screen performed by the Jasper Lab supports this hypothesis. My focus will be on one particular sirtuin that has shown the most promise out of the other members tested during this screening.
For this first project, we will be using the TARGET system (Figure 4), which combines the UAS/GAL4 system with a temperature-sensitive (ts) version of the GAL4 inhibitor known as GAL80ts. Using this system, we will express fluorescently-labelled protein aggregates in ISCs and enteroblasts (EBs) using temperature control.7 GAL4 expression is driven by the escargot (esg) promoter in ISCs and EBs, and all genes downstream of the Upstream Activating Sequence (UAS) sequence will be expressed. GAL4 expression is inhibited by GAL80ts, a temperature sensitive molecule that is active when the flies are in a cool environment but degrades at higher heat, which permits us to control when the transgene is expressed using temperature.7
Figure 4. Schematic of the temporal and regional gene expression targeting (TARGET) system.
Aggregates and GFP will only be expressed in ISCs and EBs at the non-permissive temperature of 29°C, where the GAL80ts is degraded. At 18°C, GAL80ts is active and inhibits translation of both the GFP and the mRFP-HttQ138 markers. Modified from reference 7.
The transgene is specifically mRFP-HttQ138, a red fluorescently-labelled, pathogenic version of human Huntingtin (Htt) with 138 polyglutamine repeats.8 Upon expression, these mutant proteins will form aggregates that are easy to track with microscopy, making them excellent tools to track protein aggregate removal. We will induce proteostatic stress in ISCs by expressing mRFP-HttQ138 aggregates with and without decreasing the levels of a sirtuin to measure ISC proliferation using the following experimental paradigms: Paraquat treatment or bacterial infection. Both are well-known and refined methods to purposely cause realistic damage to the fly gut for experiments on ISCs. Paraquat treatment simulates ingestion of a toxin, while bacterial infection mimics disease. Further, we will conduct lineage-tracing experiments to track how much each ISC has divided and whether they activated the proteostatic checkpoint or not.
For my second project, I will be using the UAS/GAL4 system again alongside the QUAS/QF system (Figure 5), which has a similar overall function to the TARGET system, just with different genes and proteins.9 The advantage of having two such systems is that you can control the expression of 2 different proteins individually, without affecting the expression of the other. Using these systems I will study the relationship between ISCs proteostasis and other tissues.
Figure 4. Overview of the QUAS/QF system.
This method is very similar to the UAS/GAL4 system, but with QUAS and QF replacing UAS and GAL4, respectively. X refers to a gene of interest.9
Future Plans:
This fall, I plan to return to the Wu Lab and continue the projects where I left off. Upon graduation from Cornell University, I immediately intend to apply to a PhD program and eventually pursue a career in pharmaceutical research.
References:
[1] Vilchez, D., Simic, M. S., and Dillin, A. (2014). Proteostasis and aging of stem cells. Trends in Cell Biology, 24(3), 161-170. doi:10.1016/j.tcb.2013.09.002
[2] López-Otín, C., Blasco, M. A., Partridge, L., Serrano, M., & Kroemer, G. (2013). The hallmarks of aging. Cell, 153(6), 1194-1217. doi:10.1016/j.cell.2013.05.039
[3] Putting DNA to Work. (2004, March 02). Retrieved June 13, 2017, from https://www.koshland-science-museum.org/sites/all/exhibits/exhibitdna/intro03.jsp
[4] Apidianakis, Y., and Rahme, L. G. (2010). Drosophila melanogaster as a model for human intestinal infection and pathology. Disease Models & Mechanisms, 4(1), 21-30. doi:10.1242/dmm.003970
[5] Houtkooper, R. H., Pirinen, E., and Auwerx, J. (2012). Sirtuins as regulators of metabolism and healthspan. Nature Reviews Molecular Cell Biology, 13, 225-238. doi:10.1038/nrm3293
[6] Wang, Y. and Zhong, Y. (2004). TARGETing “when” and “where”. Science Signaling (220), 5.
[7] Weiss, K., Kimura, Y., Lee W.C., and Littleton, J. (2012). Huntingtin aggregation kinetics and their pathological role in a drosophila Huntington’s disease model. Genetics 190, 581–600.
[8] Potter, C. J., Tasic, B., Russler, E. V., Liang, L., and Luo, L. (2010). The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis. Cell, 141(3), 536-548. doi:10.1016/j.cell.2010.02.025
