Sensing and Processing in the Six-protein “Brain” of Bacteria
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.
Developing New Approaches to Target Tumors
Cancer is the second leading cause of death in the U.S. This disease is caused by dozens of tumor types, with very large physiological and biochemical differences between them. This makes a painfully slow process their detection and treatment, since it requires tailoring to each specific tumor type. However, there exists a unifying principle shared by most solid tumors. This is that the microenvironment surrounding tumor cells is acidic. Our laboratory designs peptides that sense the acidic tumor microenvironment. Acidity triggers a conformational change in the peptide, resulting in the specific accumulation in the plasma membrane of tumor cells. These peptides have promising applications for diagnosis and treatment of a wide range of tumors. To evaluate these peptides we employ biophysical, biochemical and cell biology techniques.
Ethylene Receptors: From Prokaryotes to Plants
This lab 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/
How are Membrane-Cytoskeletal Interactions Established?
Cell growth and division involve important cellular events such as endocytosis (a process by which the cell engulfs molecules) and cytokinesis (a process by which the cell divides into two). Defects in these cellular events have been shown to be associated with cancer development and progression. Central to cytokinesis and endocytosis is actin cytoskeleton associated cell membrane remodeling. Membrane remodeling is a complex process involving several players the details of which are poorly defined. The lab is focused on understanding how membrane-actin cytoskeletal events are established. Recent results from this lab have shown that the major cell growth regulator Cdc42 promotes membrane-actin cytoskeletal events. Several projects are available for students to study how Cdc42 regulates membrane-cytoskeletal events. Students will be trained in fission yeast genetics and cell biology approaches, and particularly in fluorescent confocal microscopy.
Water and a Primitive Enzyme
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.
Dynamic Dance of the Enzymes
Cytochrome P450s are a class of heme-containing enzymes that catalyze chemical reactions in various lifeforms, such as biosynthesis of steroid hormones and hydroxylated fatty acids, metabolism of an extensive variety of xenobiotic compounds, including a high percentage of drugs produced by the pharmaceutical industry. They are also of high relevance to the chemical industry due to their potential in catalyzing difficult monooxygenation reactions that may be necessary in chemical synthesis, refinement of petroleum products and degradation of pollutants. The catalytic cycle of these enzymes entail many steps during which they bind specific substrate molecules, add an oxygen atom to them in a series of oxidation and reduction reactions, and form product with high regio- and stereo-selectivity. Structural studies on several P450s have thus far revealed that they have highly conserved structural features. Given this fact, it is likely that diversity of substrate binding and catalysis observed in various P450s is regulated by intrinsic characteristics of the protein that are not observable in static representations of their structures. One such factor is the dynamic flexibility inherent in the active site and the substrate binding regions for these P450s that allows them to sense multitude of substrates and monooxyenate them at a variety of positions. It is being increasingly observed that there is a varying degree of plasticity within these regions in all P450s and such dynamic motions have become a topic of intense research inquiry, both in drug metabolizing enzymes and bacterial enzymes of importance in chemical synthesis. Of the techniques currently available to observe dynamic effects in proteins, spectroscopic techniques in concert with molecular dynamic simulations are most useful in investigating dynamics on timescales spanning from picoseconds to seconds. Our lab has been instrumental in application of these techniques to characterize dynamic motions in individual amino acids and to assess the degree to which they are correlated to the functional dynamics of the molecule as a whole on all relevant timescales. Several projects are available within the lab in this regard to study the effect of dynamic movements on substrate binding and catalysis by various P450s. Characterizing these in-step movements or the dynamic dance of these enzymes can potentially be exploited in the creation of improved, more efficient pharmaceutical and chemical products.
Are Chromatin Insulators Epigenetic Landmarks of the Genome Architecture?
A major challenge in modern Biology is to understand how the organization of the DNA within the nucleus affects its function. Analysis of chromatin structure and function have revealed that DNA is capable of transmitting, through cell divisions, two major levels of information: The well know level of information residing within the DNA sequence, and the not so well understood information level that resides in the peculiar manner in which DNA is packed within the nucleus, which is known as “epigenetic” level of information. Epigenetic information is responsible, among other things, of the differences in expression profiles existing between different stages of development or between tissues. Most of epigenetic information is found as chromatin structures associated to local DNA sequences such as regulatory sequences and promoters, but there is a growing body of evidence suggesting that higher order chromatin structure, mediated by long-range interactions within the chromatin fiber also plays a major role in gene transcription regulation, and may as well be considered a critical component of the epigenetic identity of each of the cell lineages occurring in metazoans. Chromatin insulators are regulatory elements found in Drosophila and in vertebrates that are considered to have a major role in higher order chromatin organization based on their capacity to mediate long range interactions within the chromatin fiber. Chromatin insulators are DNA sequences that have the ability to block communication between enhancers and promoters when located between them and to prevent heterochromatin spreading. It is generally accepted that these properties result from cis-interactions between insulator proteins, which loop out the intervening DNA sequences to form functionally independent chromatin domains, and providing a model that supports their ability to mediate higher order chromatin organization. The long term goal of research in my laboratory is to elucidate the role of chromatin organization during cell differentiation and gene expression regulation and whether chromatin insulators play a major role mediating the long-range interactions necessary for the formation of the higher order chromatin structures that lie at the base of chromosome organization.
Nano-Motors, Micro-Movements, Macro-Effects
Myosin motor proteins are responsible for the rapid movement of organelles through plant cells, a process commonly referred to as cytoplasmic streaming. Loss of myosin motors leads to loss of cytoplasmic streaming, reduced cell expansion, and smaller plants, demonstrating the importance of these intracellular movements. Closer inspection of these movements reveals that different organelles move with different speeds, suggesting that their movements can be regulated independently. In addition, in some tissues cytoplasmic streaming can be triggered by external signals, e.g. light. We want to understand these regulatory processes better and are therefore investigating localization and activity of myosin motors in plant cells. In this project, we will investigate whether organelle movements can be manipulated by altering conditions or adding specific inhibitors of cellular signaling. This involves live-cell microscopy with fluorescently labeled myosin proteins or organelles and computational analysis of the observed movement patterns.
Development and Roles of Neuropeptide Producing Neurons
Neuropeptides, as produced by neurosecretory cells in the central nervous system, are major physiological signaling regulators in insects and mammals. One of the research goals in this lab is to elucidate biological functions of the neuropeptides using Drosophila melanogaster 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 and environmental signals. Another project studies the effects of ethanol or caffeine on the neuronal degeneration. Ethanol and caffeine consumption is a major factor that influences human behaviors. To understand the impact of these chemicals on the neuronal degeneration, studies will investigate how they influences development of neurons in fruit flies.
Cell to Cell Communications During Plant Development
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.