Prof. Michelle Knowles and her group are interested in understanding how membrane proteins function on a molecular level. They use single molecule and quantitative imaging techniques to form spatio-temporal maps of fluorescently labeled proteins and develop methods for measuring transient protein-protein interactions in live cells and biochemical systems. Her lab currently focuses on two different proteins systems: SNARE proteins and P-glycoprotein. The SNARE proteins provide the energy needed for membrane fusion and are essential to neurotransmission, membrane repair, and intracellular membrane transport. VAMP, a SNARE protein located on a vesicle, and the plasma membrane SNAREs, Syntaxin and SNAP25, come together to form a ternary protein complex. When the cell secretes the vesicle contents, the three SNAREs interact and fold to form a very stable ternary complex that is thought to cause the membrane of the vesicle to merge with the plasma membrane. P-glycoprotein (P-gp) is a large membrane protein that removes toxic chemicals from the cell by hydrolyzing ATP. The upregulation of P-gp has been correlated to multidrug resistance in cancer and had been show to decrease survival rates. P-gp removes chemotherapeutic drugs and renders the cell resistant to treatment. We are developing methods to address how P-gp functions on a molecular level and how efflux is activated and halted with drugs. Ultimately, armed with a molecular understanding of P-gp, drugs that are capable of eluding or overcoming this drug resistance mechanism can be developed. To study these proteins, the Knowles' lab uses total internal reflection fluorescence microscopy, an imaging technique that preferentially illuminates the surface of the cell. In doing so, the background is greatly reduced and allows for single molecule imaging. Single molecules have been used as a calibration for measuring the number of proteins in a complex and are also used to study the dynamics of proteins. Single molecule techniques are ideal for characterizing how a protein works and have been used to reveal molecular mechanisms of protein function. Spatio-temporal mapping allows us to connect protein function with clustering, mobility and location. Technique and algorithm development plays a key role in this research as the Knowles' group aims to identify transient protein-protein interactions from imaging data.
Our group investigates the aggregation of Tau protein and its biological ramifications. The accumulation of Tau fibrils is a characteristic hallmark of more than 20 neurodegenerative diseases, including Alzheimer disease, frontotemporal dementia, and progressive supranuclear palsy. The formation of these aggregates is a multi-step process that starts with unfolded Tau monomers, progresses through oligomeric intermediates, and ends in highly ordered fibrils. One of the most remarkable findings in the past few years was that this process may originate in a single cell, as short fibrils can transfer between synaptically connected neurons. The recruitment of Tau monomers onto the fibril ends and the fragmentation of larger fibrils into smaller seeding competent units could then drive amplification leading to the spreading of Tau pathology throughout the brain. Cellular factors will play a key role in these events. Their identities and specific functions, however, remain unknown. Also, the structural characterization of Tau fibrils has traditionally encountered great difficulties because of the size and complexity of the involved aggregates. One strategy pursued in our laboratory that has proved very successful is the labeling of Tau with small reporter molecules that allow us to interrogate the structure using a technique called electron paramagnetic resonance spectroscopy. This method faithfully delivers structural detail regardless of the size and shape of the investigated species. Using this approach in combination with other biophysical techniques, we have discovered distinct conformers of Tau fibrils that vary in their ability to recruit Tau monomers. The findings are significant as they suggest that fibril structure could have a marked influence on fibril propagation. We are extending these investigations in order to obtain complete three-dimensional models of Tau fibrils. Similarly, we are analyzing the molecular mechanisms of Tau aggregation and spreading, and we are assessing the effects individual species have on cell viability. A molecular understanding of the aggregation of Tau and its cellular interactions and structural transitions will be an important prerequisite for the development of new strategies towards disease intervention.
Our group is interested in understanding the structure -function relationship of radical-S-adenosylmethionine proteins that are used to modify small peptides. We use traditional enzymology techniques (e.g. kinetic analysis and site directed mutagenesis) paired with biophysical techniques (e.g. isothermal calorimetry, electrochemistry, and surface plasmon resonance spectroscopy) to understand the structural attributes that lead to chemical function.