KNOWLES RESEARCH LAB
University of Denver
RESEARCH
Knowles Lab Research
Our research is focused on the regulation of membrane fusion processes. The first two projects are currently active and we use a combination of cell biology, quantitative microscopy, computational modeling, and biochemistry to identify what proteins and lipids regulate how molecules and vesicles are secreted from cells.
1. Membrane Fusion and Exosome Secretion
The fusion of multivesicular endosomes (MVEs) with the plasma membrane gives rise to the secretion of small vesicles, called exosomes. Exosomes are well-known for transferring cargo from cell to cell and cargo, such as miRNA, proteins and lipids, are thought to affect the target cells. Through bulk collection of exosomes and direct imaging of MVE fusion, the lab works to identify regulators of this process.
Figure shows a PC12 cell expressing CD63-pHluorin.
The CD63 protein is enriched in exosomes and MVEs. The fluorescent probe, pHluorin, is quenched within an MVE because it is acidic, then brightens upon membrane fusion and neutralization with the cell's plasma membrane. This cell has many more fusion events than the average cell. The total duration of the movie is 300 frames, taken at 100 ms/frame.
2. Membrane Fusion and Curvature
The fusion of lipid vesicles with the plasma membrane allows cells to release small molecule neurotransmitters, insulin, intraluminal vesicles, and other molecules. In neuroendocrine cells, the influx of calcium leads to the folding of SNARE complexes and an accessory protein, Synaptotagmin, is known to curve the membrane to possibly facilitate fusion. Despite knowing the membrane curvature is fundamental to the process, little is known about the relationship between SNAREs, lipids and membrane shape. It is thought that certain lipids, like phosphatidic acid, help stabilize the fusion pore. Phosphatidic acid is made by Phospholipase D from phosphatidylcholine, a lipid that cannot easily support membrane curvature. With the design of new assays for membrane curvature and microscopy assays for phospholipase D1, membrane shape can be incorporated into a model of SNARE mediated membrane fusion.
Funding: This work has been supported by the National Science Foundation and the University of Denver.
Membrane curvature is thought to lead to defect sites in a membrane where curvature sensing molecules can bind more readily.
Taken from our work (Black, Cheney, Campbell and Knowles, Soft Matter 2014)