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Katelyn's Project Page - RISE Summer 2008 |
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Intern: Katelyn Cahill-Thompson, Biomedical Engineering, University of California, Davis
Mentor: Joanna Deek
Faculty Supervisor: Cyrus Safinya
Department: Materials |
CHARACTERIZING THE Ph AND SALT DEPENDANCE OF NEUROFILAMENT GRAFTING DENSITIES
Conditions leading to the accumulation of neurofilament networks are implicated in such neurodegenerative diseases
as Lou Gehrig’s disease, Parkinson’s and Alzheimer’s. This study characterizes the pH and salt dependence of NFnetwork
grafting densities. Neurofilaments (NF), structural proteins within the intermediate filament family, are
present predominately in neuronal cell axons and are composed of three subunits: high, medium and low (NF-H,
NF-M and NF-L). Once assembled into filaments, the C-terminus tails of these subunits radiate outward, interacting
with the sidearms of adjacent filaments to form extensive NF networks within the cell. The grafting density is the
percentage of a particular subunit incorporated in a neurofilament, which we compare to the percentage initially
present during assembly under various conditions. We have succeeded in isolating and indentifying the three
subunits through anion exchange chromatography, gel electrophoresis and Bradford assays. The tails of NF-M and
NF-H contain regions of dense phosphorylation; by altering the pH of the networks’ assembly conditions, the
charges of the tails will be altered. To similar affect, varying the pH changes the side-group charges of present
amino acids. By altering charge, changes in pH may affect the subunit interactions and thus grafting densities. Salt
concentration may shield the subunit tail from its own repulsive charges, allowing it to fold in onto itself rather than
radiate outward, thus blocking the filament from further extension and altering grafting density. This study tests
eleven subunit ratios at pH’s of 6.0 and 6.8 with salt concentrations of 40mM, 90mM, 150mM, 240mM and
500mM. The grafting densities are monitored using stained polyacrylamide gel analysis and quantified stain
intensity.
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