Cell adhesion and motility are important cell processes that are necessary for embryo morphogenesis. Prevous studies have identified p120-catenin as a key player in coordinating cell movements by either stabilizing E-cadherin complexes to strengthen adhesion or activating cytoplasmic CDC42 and Rac1 GTPases to initiate motility. Most of these studies were done in-vitro using cells grown in a 2-dimensional plane. Comparatively, few studies have attempted to describe p120-catenin's function in a 3-D space. In an effort to investigate p120-catenin's function in embryos, we are co-immunoprecipitating p120-catenin from developing zebrafish embryos. We are specifically investigating how the tyrosine 228 (Y228) residue of p120-catenin mediates binding to E-cadherin. The hypothesis is that phosphorylation of Y228 causes p120-catenin to bind to E-cadherin. To test this, we are injecting zebrafish embryos with p120-catenin containing either an Y228F of Y228E mutation. If our hypothesis is correct, the Y228F mutation would prevent phosphorylation of the 228 residue and decrease affinity between E-cadherin. The Y228E mutation mimics the negative charge of phosphorylation and would instead cause p120-catenin to bind to E-cadherin. 

Gastrulation is an early phase in the embryonic development of vertebrates during which the three germ layers differentiate. The movement of the cells during this phase, is mediated by actin structures stimulated by integrins, extracellular matrix, and ligands. Migration of the cell is activated by Rho GTPases and focal interactions with cadherin-p120 catenin interaction stabilizing cell-to-cell adhesion. To be more specific, the protein, p120 catenin, is required for the extension of the dorsal axis and normal migration of the presomitic mesoderm. This lab studies the molecules that define the pathways of cell migration and tissue migration in zebrafish embryos. Specifically, we are studying how various mutations affecting the phosphorylation state of p120 catenin family genes were produced by undergraduate students and their respective effects on the dorsal axis extension were visually observed. In this experiment, zebrafish embryos were injected with mutated mRNA and analyzed using our in situ hybridization of two probes. Somites were stained using the myoD probe while the presomitic mesoderm was stained using the papC probe. Staining the mutated mRNA, allowed the team to witness phenotypic abnormalities of the dorsal axis much more clearly. Quantifications of the stained somites and presomitic mesoderm will allow a statistical analysis on various p120 catenin mutants. Overall, this experiment provides a deeper understanding of cell migration during developmental processes.

In cells, phosphorylation of p120 catenin causes it to dissociate from cadherin transmembrane proteins, which leads to the activation of downstream pathways and cell motility. We have mutated amino acids in p120 catenin in order to locate the functions of specific sites of phosphorylation in zebrafish embryos. The phosphorylated tyrosine amino acid, Y335, in p120 catenin is a target of the phosphatase "PTP-PEST” and controls epithelial cell motility and Rho GTPase activity in colon carcinoma cells. We designed a knock-out of normal activity by mutating the tyrosine to a phenylalanine, which cannot to be phosphorylated. We also mutated Y335 in p120 catenin to a glutamic acid (E), which mimics the negatively charged phosphate. These mutants were transformed into bacterial cells and the isolated DNAs were transcribed into mRNA. We have also injected zebrafish eggs with the mRNAs to analyze the effect of these mutants on development. We have also mutated several other serine and threonine phosphorylation sites on p120 catenin including, S252, T310, and S312 to determine if the mutation to an alanine or to a glutamine will result in altered downstream activity. We have injected mRNAs with the S252A and S252E mutants into zebrafish eggs and determined that the S252E mutation rescues a knockdown of the endogenous p120 catenin, while the S252A mutation does not. The results suggest that phosphorylation of S252E is required for the migration of presomitic cells to the dorsal axis. In fibroblast cells, phosphorylation of p120 at T310 promotes adhesion junction disassociation, allowing cells to mobilize in a “treadmill” fashion. We used p120 catenin mRNAs with the mutations T310A and T310E to measure their impact on cell motility in zebrafish. These individual mutations may give insight on the downstream effects p120 catenin in cell motility, including morphological bone abnormalities and metastasizing cancer cells.

Tree lines play a vital role in plant community composition in montane ecosystems. Over the past several decades, increasing annual temperatures have led to shifts in tree lines in montane environments. However, little is known about how local topography may have an effect on tree line advance. My research incorporates historical and repeat, ground based and aerial photography to assess the change of alpine tree line in a range of topographical conditions in Mount Rainier National Park over the past century. My study quantifies changes in tree cover and tree-line elevation in ridge and cove environments of the park. To track treeline change, historical and recent photographs were aligned, and areas of the photograph were broken up into polygons based on their small-scale topography. Random points were overlaid in these polygons to assess the rate of change of tree line shift. By looking at the change in tree cover in each random point relative to the polygon type, we are able to determine the amount of tree thickening that has occurred. We predicted that tree lines on sun exposed ridges will shift more quickly than those in cove environments. Snow is prevalent for longer periods of time in concave valleys limiting the amount by which the trees can shift up. Our results thus far indicate that small-scale ridge regions have the highest rate of change for tree line shift followed by cove. Subalpine meadows often occur in cove topography. Tree thickening in these regions over time may impact the wildflower populations and decrease species richness in subalpine meadows. In all, our research adds to the growing body of evidence that tree lines are moving up in elevation as well as provides the National Park Service with information about tree line influences on the plant communities of the park.

Masting refers to annually variable seed production; whereby plant species release large quantities of seeds one year, followed by one or more years of significantly lower seed production. Seed masting behavior is thought to be evolutionarily favorable, a technique for survival. Two main theories exist to explain mast seeding, predator satiation and increased pollination. The purpose of this research study was to determine A) if a group of six conifer species within Mt. Rainier National Park exhibit mast seeding behavior (i.e. synchronized seed production over large areas, annual variation in seed production exceeds annual climate variation); B) Whether seed viability is greater in 'boom' years than 'bust' years (which would support wind pollination efficiency as an evolutionary driver of masting); and C) whether these patterns vary by species and/or elevation. We examined six species of conifers: Abies amabilis, Callitropsis nootkatensis, pseudotsuga menziesii, Thuja plicata, Tsuga heterophylla, and Tsuga mertensiana. Data was collected from 108 seed traps located in 18 stands at multiple elevation ranges within Mt. Rainier National Park. Seeds were sorted based on tree species, and classified as filled (viable) vs. unfilled (not viable). We then calculated mean total seed production for each stand and used correlations to assess how correlated annual seed production is across 18 stands. Next, we compared annual variation in climate within each stand, to assess whether species showed higher variation in seed production in comparison to climatic variation. Finally, we compared results for our six focal species to assess species specific similarities and differences. Understanding the mechanisms behind masting is important, as masting can control tree population demography, and influences ecosystem dynamics. Moreover, our study may provide greater understanding of the implications of climate change on masting dynamics for forests at Mt. Rainier National Park.

We know that climate sensitivity can vary within species, but the factors that influence which climatic variables influence population dynamics remain unclear. In this study, I am assessing whether macroclimate influences the climate sensitivity of three prominent conifer species: Pacific silver fir (Abies amabilis), mountain hemlock (Tsuga mertensiana), and Alaska yellow cedar (Callitropsis nootkatensis) on Mt. Rainier. I am analyzing tree core data from three high elevation sites. Each site is located in a distinctly different climatic area on the mountain. I am comparing standardized tree growth to climate variables (mean growing-season temperature, total growing-season precipitation, snow duration, etc.) using linear mixed effect models. This will determine how influential climate variables differ by location and by species. Gaining more insight into which climate variables have the greatest influence on growth in different areas will allow us to better predict how specific plant populations will react to ongoing climate change. Moreover, studying tree communities at high elevations is particularly important, since behavior at treeline will determine whether a species’ range expands, contracts or shifts. Range expansion of one species could lead to the crowding or elimination of another. For example, conifer range expansion on Mt. Rainier could negatively affect the alpine wildflower meadows, a major attraction for tourists. Widespread range contractions could also decrease forest density and potentially reduce the ability of those forests to sequester carbon from the atmosphere. Thus, with better predictions of tree shifts, we can make more informed management decisions for ecotourism and conservation.

With concern about climate change increasing, assessing how plant reproductive success is impacted by increases in temperature is crucial in order to fully understand how climate change influences ecosystem structure and dynamics. This study measures how population variation and the climate of different elevations affect the capsule size and seed mass of the high-elevation plant mountain monkeyflower, Mimulus tilingii, at Mt. Rainier National Park. If climate is the primary limiting factor determining the range of M. tilingii, plants within the species’ climate range will produce the most abundant capsules and seeds whereas plants grown outside the species’ distributional range will produce smaller amounts. Sample seedlings were planted at two elevation sites: 1)  M. tilingii’s range center and 2) below M. tilingii’s range to mimic the increase in temperature due to climate change. Populations were categorized as either within-population (for crosses within the same elevation site) or between-population (for crosses between the two elevation sites) with equal amounts at both sites. This study quantifies M. tilingii’s reproductive success both in the context of the site as well as the population type, allowing for comparisons on these two levels. Results show that parent plants from the lower site produced more flowers and grew faster, indicating that plants in the lower elevation site might be more successful. These unexpected results suggest that M. tilingii may be better adapted to a warmer environment and that climate change might even be beneficial for this reason. Determining the exact impact of temperature on reproductive success is crucial not only for measuring the species’ fitness but also for understanding how these new adaptations will play an even bigger role in competition for resources among different plant species and how those new interactions will alter the ecosystem as a whole as the climate warms.

Protein glutathionylation is a post-translational modification that can take place under mild oxidative stress. It occurs when the antioxidant glutathione (GSH) forms mixed disulfide bridges with low pKa cysteine residues on proteins. This may play a role in reversibly regulating the activity of proteins that have key cysteine residues or protecting those cysteines from further irreversible oxidation if oxidative stress worsens. GSH itself is synthesized in a two-step process, and the rate limiting step is catalyzed by glutamate-cysteine ligase (GCL), a heterodimer composed of the catalytic subunit (GCLC) and modifier subunit (GCLM). GCLM acts to increase the activity of GCLC. Thus, in the absence of GCLM, GCLC is less efficient in making GSH. We have generated Gclm null mice and these mice have liver GSH levels that are about 10% of normal, resulting in an oxidized thiol redox environment. An important question that has not been answered is whether Gclm null mice have different levels protein glutathionylation than Gclm wild-type mice. The oxidized thiol redox status of Gclm null mice is predicted to increase the level of protein glutathionylation. On the other hand, there may in fact be less protein glutathionylation in Gclm null mice because they have less GSH to begin with and there is a compensatory increase in sulfiredoxin and thioredoxin reductase expression, both of which are known to reverse glutathionylation. Using assays of protein glutathionylation, this project seeks to provide confirmation of the hypothesis that Gclm deficiency results in adaptive regulation of glutathionylation under different degrees of oxidative stress.

Developmental phenotypic plasticity can be a vital means to buffer the sudden and persistent effects of anthropogenic climate change, through facilitation of species persistence and possible facilitation of species range shifts. Integrating our understanding of plastic physiological growth mechanisms with functional traits may prove useful for interpretation of the ecological impacts of climate change. We investigated the influence of water-stress and light limitation on leaf mass per area (LMA), leaf anatomy, and leaf gas exchange of juvenile Populus tremuloides trees. Leaves that developed while water-stressed had significantly elevated LMA, due to increased leaf density from significantly higher spongy mesophyll surface area per leaf area (A_smes/A) and reduced stomatal conductance (g_s) but only marginally reduced photosynthesis (Pmax). In shade, leaves had significantly reduced LMA, due to reduced thickness, density, and combined A_mes/A, but maintained the same A_smes/A, and reduced palisade mesophyll surface area per leaf area (A_pmes/A), which was coupled with a reduction of g_s and P_max. Furthermore, water-stressed leaves had elevated intrinsic water use efficiency (WUE_i: P_max/g_s) while the shaded leaves, despite reduced g_s, maintained similar WUE_i. Our results suggest that, under water-stress and full sunlight, greater A_smes/A conferred maintenance of photosynthetic rate possibly through reduction of mesophyll resistance (r_m), at the cost of reducing expansive leaf area (and increasing density) as leaves were more dense but were 20 % the area of control leaves. In shade, suppressing A_pmes/A while maintaining A_smes/A may be a plastic strategy to increase laminar light capture, as leaves had reduced density and thickness but were still 50 % the area of control leaves . Together, in P. tremuloides saplings, these stresses induced developmental plasticity in LMA with differing plastic underlying anatomy and function; we discuss future implications specifically in the context of developmental plasticity, growth trade-offs, and the ecological impacts of climate change.

There are many mutualistic interactions that occur naturally in the world. Some of these mutualistic relationships evolve to be obligate, where the presence of one species is dependent on the other for its survival. We study the evolution of an obligate relationship with two model organisms, a sulfate reducer, Desulfovibrio vulgaris, and a methanogen, Methanococcus maripaludis. 22 populations of these organisms have evolved in an environment that has forced their cooperation. When we sequenced these populations, we discovered that at least 13 of the 22 populations of D. vulgaris have lost the ability to reduce sulfate To confirm if these mutations are beneficial or not, we measured the fitness of mutants with knockouts in the sulfate-reducing gene pathway relative to the wild-type strain with either an ancestral or evolved M. maripaludis partner. The final population size of each D. vulgaris mutant will be measured using FREQ-Seq; a technique that quantifies frequencies of various mutations in an amplicon. In previous iterations of this experiment, results presented a discrepancy between replicates (I and II) due to a failed cell lysis technique. We found that only a small fraction of the cells were lysed. With a better cell lysis protocol, we expect to see an increase in D. vulgaris mutant frequencies when paired with either an ancestor or evolved M. maripaludis compared to the wild-type. The data collected from these experiments will help us have a better understanding whether losing the ability to reduce sulfate is a beneficial mutation for D. vulgaris or not.
 

When isolated subpopulations adapt to the particular features of the predators, prey, or cooperative partners that are specific to their local environment, populations may become specialized to their local partner and less able to interact with other populations. To what extent might this hypothesis explain the diversification of microbial species, which cooperate or compete with each other in complex communities? We tested whether specialization was a common outcome of evolution between the bacterium Desulfovibrio vulgaris and the archaeon Methanococcus maripaludis after they evolved together in a beneficial interaction for 1000 generations. To test if D. vulgaris populations tended to become specialized for the M. maripaludis populations they evolved with (and vice versa), we revived 6 independently-evolved communities, separating the D. vulgaris and M. maripaludis populations from one another, and then paired each population back up with its sympatric partner (evolved in the same community) or with 5 allopatrically-evolved partners (evolved in different communities). We used microscopy to quantify the density of D. vulgaris and M. maripaludis to test whether the absolute fitness of either species was higher in sympatric versus allopatric pairings. We also competed allopatrically-evolved and sympatrically-evolved D. vulgaris against one another. We conclude that variation across cocultures in population yield of D. vulgaris or M. maripaludis is not necessarily correlated with whether they evolved in sympatry or allopatry, suggesting that the first 1000 generations of adaptation to a new mutualism may not be characterized by specialization. Our results show that some sympatric pairings were more efficient than allopatric pairings;however no distinct trend was identified. Broader implications of our research include roles in  byproduct inhibition which is an important factor in global carbon cycling within diverse anaerobic environments, including bio-digestion for fuels, wastewater treatment, and experimental evolution. 

Do species evolve in diverse communities, similar to the simple community of our research? We are looking at the evolution that we've seen in a species of sulfate reducers found in our simple laboratory environment and comparing this evolution to that of a large, more complex anaerobic digester of the wastewater treatment plant. In our 'simple community', a methanogen, Methanoccocus maripaludis, and a sulfate reducer, Desulfovibrio vulgaris, were paired. When two species are forced to live together over time we expect to see evolution take place. It was found that after 1000 generations D. vulgaris loses its ability to reduce sulfate in this paired relationship. Using this laboratory pairing as a model, we have looked at the evolution of sulfate reducers in  these two different anaerobic digesters from the University of Washington and Brightwater Municipal Waste Water Treatment Center. Both digesters have similar conditions; they are both anaerobic with low levels of sulfate present. To test our hypothesis we have currently been extracting DNA from weekly samples of both digesters. The dsrAB gene is first amplified, as it is a conserved region throughout sulfate reducers and their relatives. We are now doing Illumina sequencing in order to better determine the diversity of sulfate reducers present. Using this information, we will be able to design primers for the sat apsA and apsB genes. This will show us if any mutations are present among the population of sulfate reducers in both digesters. This will be means for the comparison between real world and experimental conditions. Future work will look for mutations in the sat, apsA and apsB genes. By identifying these loss-of-function mutations in both the digesters, we will be able to see the impact of evolution on sulfate reducers in complex communities.