Decriptions of BCMB REU Projects in “Sensing and Signaling”
Motile bacteria are capable of navigating in gradients of various physicochemical cues by constantly monitoring their surroundings using dedicated chemoreceptors. The ability to sense the environment using these chemoreceptors is essential as it allows these motile bacteria to modulate their swimming behavior to reach niches that are optimal for growth and survival. Our laboratory has recently uncovered a complex sensing and signaling between chemoreceptors and chemotaxis-like pathways, that ultimately modulate the swimming motility response (chemotaxis) as well as other cellular behaviors. We are currently analyzing the dynamic subcellular localization of chemoreceptors and putative chemotaxis-like protein targets using a combination of fluorescent tagging and imaging of chemoreceptors and chemotaxis-like proteins, biochemistry and molecular biology techniques. Several projects focusing on a subset of these proteins are available and suitable for undergraduate summer research experience.
Chloroplasts are the major site of primary productivity and biosynthesis in the biosphere. The vast majority of chloroplast proteome is encoded by the nucleus and must be post-translationally imported from the cytosol to their functional location. Precursor proteins targeted to the chloroplast contain a short, N-terminal extension called the transit peptide, which is required for proper targeting and translocation across the chloroplast membrane. The Toc complex (translocon of the outer chloroplast membrane) is thought to specifically interact with precursor proteins. We are particularly interested in the Toc34 component, which is widely hypothesized to act as a “gatekeeper” for recognizing precursor proteins and allowing them to enter the organelle. However, the current understanding of the Toc-transit peptide interaction is unclear. To aid in the elucidation of this complex process, we have several projects available in the lab, including:
- Mutagenesis of the Toc34 receptor component, followed by analysis of higher-order structure and GTPase kinetic activity. This project incorporates techniques including PCR mutagenesis, bacterial transformation, and recombinant protein expression.
- Mutagenesis of the transit peptide, followed by in vivo and in vitro analyses. We can track the localization of the transit peptide in vivo using a fluorescently tagged chimeric construct. Furthermore, we can also use purified chloroplasts to track the import of the transit peptide in vitro. This project involves techniques such as biolistic transformation, fluorescence microscopy, SDS-PAGE, and quantification/statistical methods.
- Investigation of the Toc34-transit peptide interactions using Fluorescence Resonance Energy Transfer (FRET). We have developed a project using thiol modifications of the conserved cysteine of Toc34 (C215) and several cysteine mutations selectively placed along the length of the transit peptide. These residues will be modified with a sulfhydryl-reactive fluorochromes.
Overall, we expect that this project will allow us to investigate the dynamics of the Toc translocon and interactions of the Toc components with transit peptides.
Thislab studies ethylene signal transduction with a focus on understanding ethylene receptor function. Ethylene is a simple, unsaturated hydrocarbon that is a plant hormone that affects diverse processes throughout the lifetime of a plant including seed germination, growth, senescence, fruit ripening, abscission, gravitropism, and responses to various stresses. While this work is aimed primarily at understanding ethylene signaling at the molecular level, the lab correlates observations at the biochemical level with time-lapse imaging of growing seedlings to provide links between events at the molecular level with those at the organ level. This use of multiple scales of investigation is critical to provide a broader framework to understand how organisms grow and develop. Recently, research in the lab has expanded to include cyanobacteria that contain putative ethylene receptors. The function of these proteins in cyanobacteria remains unexplored but preliminary results suggest that ethylene affects phototaxis through these proteins. More information can be found at the lab website: http://www.bio.utk.edu/binderlab/
One of the most important processes in multicellular organisms is communication between cells. In animals, this is accomplished by direct contact between adjacent cells, the movement of cells or the secretion of small molecules through the circulatory system. However, plant cells are bound by their cellulose cell walls, precluding the direct contact of cell membranes and preventing cell migration. Plant cells have therefore developed plasma membrane-lined, cytoplasmic channels that allow the direct transport of molecules between neighboring cells. These channels, called plasmodesmata, transport not only water and small solutes like sugars but also macromolecules including proteins, small RNAs and viruses. The Burch-Smith lab aims to understand how cells regulate the movement of these molecules through plasmodesmata. We also seek to understand how plasmodesmata form and how their structures change as plant tissues mature. We use cell and molecular biology techniques, microscopy and reverse genetics to address these questions, and there are several projects available to undergraduate researchers in the laboratory.
Research in the Howell lab has focused on characterization of R67 dihydrofolate reductase (DHFR) as a good model for a primitive enzyme. One surprising result associated with this enzyme is weaker binding of substrate (dihydrofolate, DHF) in the presence of small molecules called osmolytes. This result is unusual as binding of most ligands is tighter in solutions with low water activity. In other words, binding is usually accompanied by release of water from the interaction surfaces and when the water concentration is low, binding is enabled. Our model to understand the unusual result for DHF is that it is a sticky molecule and that it interacts with the osmolytes. These interactions must be stronger than those with water, so the osmolytes act as inhibitors to the binding process with the DHFR enzyme. To test this hypothesis, we are examining the binding of various redox states of folate to other folate utilizing enzymes, which should also show weaker binding. We are addressing this hypothesis using purified proteins as well as with genetic selections.
While water is ubiquitous & occurs at high concentration, it is often ignored. In vitro experiments typically use infinite dilution conditions, while in vivo, the concentration of water is decreased due to the presence of high concentrations of molecules in the cellular environment. Thus our hypothesis of osmolyte interaction with folate/DHF is novel. High osmolyte concentrations can exist in the mammalian kidney, some plants, cartilaginous fish & coelacanths, bacteria, etc), and our model predicts varying osmolyte concentration will impact function. We propose that weak interactions are unavoidable in the cell due to intracellular crowding. In other words, our studies address the basic question of whether the in vitro behavior of folate and its derivatives accurately reflects their behavior in vivo.
Neuropeptides, as produced by neurosecretory cells and inter-neurons in the central nervous system, are major physiological regulators in insects and mammals. One of the research goals in this lab is to elucidate biological functions of the neuropeptides using Drosophila as a premier genetic model system. Using available mutants lacking neuropeptides or their receptors, students are involved in the characterization of phenotypes associated with the responses to various stresses. Further molecular analysis will highlight the mechanisms underlying neuropeptide-regulated stress responses. Another project studies the effects of ethanol on the neuronal degeneration. Ethanol consumption is a major factor that influences human behaviors and ethanol-triggered behavioral and neurological changes are remarkably similar in invertebrates. To understand the impact of alcohol on the neuronal degeneration, studies will investigate how ethanol influences development of the peptidergic neurons in fruit flies.
TheSerpersu lab uses biophysical and biochemical techniques to study interactions of aminoglycoside antibiotics with several enzymes that modify these antibiotics and cause resistance to their action against bacteria. Projects involve studies of aminoglycoside-enzyme interactions by kinetic and spectroscopic methods including fluorescence, EPR and NMR spectroscopy to determine effects of aminoglycoside binding on structure and dynamics of these enzymes. In addition, these studies require the use of chromatographic methods to purify either enzymes or, in some cases, aminoglycosides and their analogs. Other approaches include culturing cells in isotopically enriched media, computational work to determine relaxation rates of nuclei, and analysis and interpretation of spectral data. Projects involve a wide assortment of techniques starting from molecular biology to highly sophisticated spectroscopic techniques within the framework of a biological problem.
This lab is interested in the mechanisms regulating coordinated growth and differentiation in plants. Currently the research is focused on a particular signal transduction pathway that determines size and shape of plant organs and regulates cell differentiation during development. This pathway facilitates communications between cells; one type of cell secretes small proteins that are recognized by plasma membrane receptors located on other cells. The receptors belong to the ERECTA family of Ser/Thr kinases and they activate a downstream signaling cascade by still an unknown mechanism. In the model organism Arabidopsis, loss of these receptors leads to severe dwarfism, infertility, and changes in epidermis development. The goal of the summer project is to identify and characterize novel components of this signaling pathway using forward genetics. The project will involve phenotypic analysis of plants by microscopy and use of molecular biology techniques.
Climatologists rely on satellites, neuroscientists have electrodes and magnetic resonance imaging, behavioral biologists peek through binoculars, but how about cell biologists? How does one observe the inner workings of living cells in real time? Biosensors are tools designed to visualize one specific cog of the cellular machinery in real time. One of these, the green fluorescent protein, has taken the community by storm; indeed, modern cell biology would be unthinkable without it. The von Arnim lab is developing a class of biosensors based on a fluorescent protein that is paired with a luciferase, a protein that converts chemical energy into light. Biologists love a pretty picture, yet luciferases are notoriously difficult to image. Until recently, simultaneous microscopy of two different luciferases was not for the faint-of-heart. An NSF funded project to develop a bioluminescence ratio-imaging microscope is beginning to change that. Undergraduate researchers have already been involved in optimization of this advanced imaging technique and future opportunities exist for undergraduate researchers to participate in this project.