The existence of 'checkpoints' in the mitotic cell cycle is well characterized; however, much less is known about the possible existence of checkpoints controlling re-entry of terminally differentiated cells into mitosis. We are exploring this possibility in Chlamydomonas reinhardtii, a unicellular eukaryotic green alga that, in the presence of visible light, differentiates back and forth from an actively dividing 'vegetative' state to a non-dividing 'gametic' state depending on the presence or absence of nitrogen in the culture medium. The work of previous students in our group suggests that gametes of wildtype C. reinhardtii show a growth delay of about 12 hours after nitrogen is added to nitrogen-deficient media; UV exposure immediately prior to nitrogen addition significantly increases the length of this delay. This is consistent with the working hypothesis of a dedifferentiation checkpoint in this organism. However, the visible light requirement for dedifferentiation confounds this result, because C. reinhardtii also uses visible light for photoreactivation subsequent to UV exposure. To control for this, we are taking a genetic approach. We expect to find an even longer post-UV dedifferentiation lag for a photorepair-deficient mutant of C. reinhardtii compared to wildtype, which would strengthen the case for a dedifferentiation checkpoint in this organism. Of particular interest in the present study is our use of two parallel statistical approaches to analyze our experimental data: (1) a 'traditional' timepoint-by-timepoint statistical analysis, and (2) statistical comparison of logistic curves fitted to the same data. We have written programs that facilitate execution and comparison of the outcomes of both methods of analysis, and expect both methods to agree in confirming our hypothesis. This insight into the inner workings of cell cycle dedifferentiation, as well as the validation of the second, novel statistical approach could be beneficial to a broad variety of ecological and biomedical experimental contexts.

Self-assembling protein nanocages are hollow macromolecular structures that can be loaded with a desired payload. With proper functionalization they are readily endocytosed by specific cells and employable as targeted drug delivery vehicles. The delivery of a given therapeutic into a cell cytoplasm requires simultaneous disassembly of the nanocage and endosomal lysis. To this end, scientists at the Baker lab have engineered icosahedral nanocages using pH sensitive components that disassemble at low pH and disrupt endosomal membranes. However, current designs appear to be forming hyper-stable, partially assembled nanocage intermediates. We seek to improve the assembly kinetics of our 2 component nanocage by using the Rosetta macromolecular modelling suit to redesign current nanocage components. We hypothesize that weaker interactions between nanocage subunits will enhance assembly cooperativity and avoid formation of hyperstable intermediates. To test this hypothesis, we decreased the affinity between components by truncating portions of cage subunits which directly interact. We also modified charge distribution, and altered connections (protein loops) within subunits to simplify assembly. Designs are screened in varying expression conditions by transforming plasmids into Escherichia coli (E. coli) and purifying through immobilized metal affinity chromatography (IMAC) and Size Exclusion Chromatography (SEC). Finally, in vitro assembly of nanocage components is conducted to test potentially ideal stoichiometric equivalents for assembly. We expect that upon decreasing the affinity between individual cage components, we will observe efficient assembly of the complete nanocage. Successful design of a self-assembling nanocage with pH sensitive components will provide a secure intracellular delivery mechanism for targeted delivery of a wide range of therapeutics. Additionally, it will increase our understanding of the kinetics and thermodynamics of multiprotein complex assembly.

Ras is classified as a group of proteins that is involved in many signaling processes in the cell. Ras plays key roles in homeostatic cell proliferation, differentiation, and movement. However, the oncogenic point mutation from glycine to valine in the 12th position of H-Ras protein (denoted by RasV12) induces tumors and gives rise to metastatic cell behaviors. Cancer metastasis is characterized in collection of several steps, but the initial step – dissemination of cancer cells from the tumor of origin into the bloodstream – is not well studied. In this project, we use Drosophila Melanogaster as a model organism to screen for genes involved in the process of dissemination. Overexpression of RasV12 in intestinal epithelial cells caused them to disseminate from the intestine. Knockdown of genes known to be critical for the process suppressed the cell dissemination phenotype. Our image analysis in observing relative quantities of GFP-expressing RasV12 cells in the intestine allowed us to discern the importance of a gene on the cell dissemination process. In result, we identified several kinases and phosphatases involved in different steps of the cell dissemination process.

Post-translational modification with the small protein Ubiquitin (Ub) controls virtually every process in the cell and ubiquitylation malfunction is associated with various disease pathologies. The purpose of this research is to understand crucial functional interactions between Ub and its E2 conjugating enzymes. During the modification process, E2s form thioester conjugates through their catalytic cysteine with the C-terminal tail of Ub; E2~Ub conjugates represent central ubiquitylation intermediates. Ub usually shows great flexibility in these conjugates and this flexibility is associated with low discharge activity. Upon stimulation, Ub’s flexibility is restricted towards so-called closed conformations which are associated with high discharge activity and involve non-covalent interactions between helix2 of the E2 and Ub. Unpublished work from the Klevit Lab identified the yeast E2 Ubc6 as an enzyme with an intrinsic predisposition towards closed conformations and high base-line activity. Comparing E2 structures, I found that the distance which Ub needs to stretch from the catalytic site to helix2 is potentially shorter for Ubc6 than for other E2s. I hypothesize (1) that this shorter distance allows Ub to more efficiently engage in closed conformations in the Ubc6 conjugate, and (2) that there is an opposing strain force operating against closed conformation contacts for other E2 conjugates. My hypotheses predict that decreasing/increasing the length of Ub’s C-terminal tail will decrease/increase E2 activity and closed conformation interaction. These predictions are assessed via functional in vitro assays and 2D NMR, respectively. Apart from Ubc6, I am testing the well-studied E2 Ubc13 whose conjugate experiences no intrinsic bias towards closed conformations. In addition, this project extends to the ubiquitin-like (Ubl) protein SUMO1, which is structurally but not functionally related to Ub, to verify the universality of my structural model. Thus, results from this project can give broad insight into the mechanism of E2 activity regulation.

With the aim of better understanding developmental disorders, researchers have expended great energy to elucidate the mechanisms that regulate the development of the peripheral nervous system (PNS). Larval Drosophila melanogaster presents a highly useful model organism for studying PNS development due to the larva’s rapid development and neurological parallels to mammals. Thus, to investigate the development of pain-sensing neurons in the PNS, we studied nociceptive class IV dendrite arborization (C4da) neurons in larval D. melanogaster. Randomly inducing mutations with the mutagen ethyl methanesulfonate ultimately identified a miRNA, miR-14, that when absent perturbs C4da neuron development. Confocal microscopy revealed that miR-14 knockout results in greater detachment of neurons from the extracellular matrix (ECM) than those in wildtype specimens. Furthermore, imaging showed that miR-14 knockout leads to far greater ensheathment of c4da dendrites in the epithelial cell-cell junctions than observed in wildtype specimens. These observations suggest that miR-14 plays a critical role in directing proper C4da neuron development. Additionally, relative to wildtype specimens, miR-14 mutants demonstrated significantly increased responsiveness to numerous stimuli including noxious touch, noxious chemicals, and blue light but not noxious heat. These findings indicate that miR-14 influences pain sensitivity in a modality-specific manner. Finally, analysis of miR-14 mutant transcriptomes by RNA-seq demonstrated decreased expression of innexin and integrin—proteins associated with epithelial cell-cell junction formation and ECM adhesion, respectively. These results suggest that miR-14 regulates proteins that enable proper attachment of C4da neurons to the ECM and prevent abnormal C4da ensheathment in epithelial cell-cell junctions. Taken together, these data support the hypothesis that miR-14 regulates sensitivity to pain by controlling C4da neuron-epithelial interactions.

Martian regolith is very different from Earth soil. In order to colonize, or have an extended stay on Mars, agriculture must be established. The purpose of this experiment was to investigate growing plants in Martian regolith in a manner that would be fuel-efficient, by using the existing soil of Mars with minimal interference and minimal materials brought from Earth. The regolith used was Mojave Mars Simulant-2 (MMS-2), developed by The Martian Garden. MMS-2 is more than a 90% match to the chemical composition of the regolith on Mars. Plant growth was compared between Earth soil (control), 50% Martian regolith MMS-2/50% Earth soil mixture (Mars Mix A), and 50% Mars regolith MMS-2/25% coffee grounds/12.5% Earth soil/12.5% vermiculite mixture (Mars Mix B). Plants were grown in all three mixtures and growth was measured during three month cycles. Although several plant species were planted, only kale produced any significant measurable data. Plant growth decreased with decreased percentages of Earth compost additive as measured by plant length and robustness. Efforts to reduce the mass of additives required to support plant growth include an exploration of acidifying Martian regolith MMS-2 prior to planting. Acids have been chosen for their ability to add critical nutrients of nitrogen and phosphorus. Nitric acid and phosphoric acid have both effectively lowered the pH to 6, similar to the optimal pH range for plant growth. The implications of this study indicate that Martian regolith and Earth soils on their own will not be sufficient to begin agriculture on Mars. Further research on chemical soil amendments will be needed for sustainable agricultural development on Mars.

Growing sustainable crops on Mars is an important aspect of developing a colony on the red planet. The goal of this research is to modify Martian regolith simulant to support plant growth. Results will be presented for the readjustment of the pH of Mars soil (pH 8) simulant to match that of typical fertile earth soil (pH 6) using nitric and phosphoric acid. The acids used were chosen based on their viability for transport to Mars and their ability to add crucial nutrients for plant growth in the unfertile soil. During the project, both acids effectively lowered the regolith pH, but in the hours and days following the pH increased significantly, which has motivated testing the buffer capacity of Mars soil simulant. The data collected was used to prepare three samples of Mars soil simulant; the first was modified with phosphoric acid, the second with nitric acid, and the third was also modified with nitric acid and had a buffer of dihydrogen phosphate added. The growth of kale was measured in the three modified soils, each mixed with equal parts potting soil. Kale growth was compared to trials performed without the acidification or buffering of Mars simulant soil.  Our research presents progress towards growing food in Mars regolith to sustain colonization efforts on the planet. This work can also be applied to the potential need to grow food in adverse conditions on Earth as the human population increases and the impacts of climate change advance.

Harnessing the power of enzymes to carry out synthetically relevant reactions is rapidly emerging as a powerful tool for sustainable chemistry. Iron-dependent enzymes have been of particular interest to the field because of their ability to facilitate complex reactions with broad substrate scope. Recently, we found that Fe(II) 2-oxoglutarate dependent hydroxylases (Fe(II)/2OGs) exhibit non-native activity towards either epoxidation or allylic hydroxylation. Oxyfunctionalizations are important in chemical synthesis as they allow for diversification to a range of more complex molecular scaffolds from simple olefinic precursors. Because of this, reactions that produce hydroxides or epoxides are of high interest, especially in an asymmetric fashion. However, current methods are not broadly applicable, as they are limited in substrate scope, selectivity, and tolerance towards other functional groups. Enzymes could rival traditional methods, offering greater selectivity and substrate scope while using inexpensive and Earth-abundant reagents. Here, we explore the ability for Fe(II)/2OGs to catalyze non-native asymmetric epoxidations or allylic hydroxylations. First, we will determine whether the reaction taking place is an epoxidation or allylic hydroxylation. Second, we will identify any additional Fe(II)/2OGs capable of oxyfunctionalization beyond our initial hit. Third, we will use directed enzyme evolution to improve oxyfunctionalization activity and enantioselectivity of a selected enzyme candidate. Fourth, we hope to expand substrate scope of an evolved Fe(II)/2OG to develop a general “epoxidase” or hydroxylase capable of reacting with a variety of functionalized olefinic small molecules in an asymmetric fashion. Generally, our lab seeks to use Fe(II)/2OGs mutagenesis libraries and a developed liquid chromatography-mass spectrometry screening platform to exploit the existing wide catalytic diversity of Fe/2OGs into a useful array of synthetically relevant transformations.