Supramolecular Structure and Assembly of Neurofilaments Tracy Mac Donough Allan Hancock College Chemistry Major INSET Jayna Jones , Mentor Prof. Cyrus Safinya , Advisor Funded by: National Institute of Health National Science Foundation
Introduction � Neurofilaments (NFs) are cytoskeletal proteins located in the axon of neurons. � NF aggregation is a hallmark of several neurological disorders such as Parkinson’s disease and ALS. � We study the structure and assembly of purified NFs in vitro. Goal: 100 nm Mol. Bio.Cell, 3 rd ed, 73-74 Find the saturation ratio of NFs (1995). assembled from NF-L and NF-H.
NF Assembly 60kD NFL 100kD NFM 115kD NFH Mature NF Body Head Tail Alpha helix ~50 to 100 nm 45 nm • In vivo , mature NF have a Bundled Neurofilaments in vivo characteristic stoichiometric ratio of 7:3:2 (NFL:NFM:NFH) •Other ratios occur in developing, regenerating, and diseased neurons •We will look specifically at the ratio of NFs assembled from NF-L and NF-H 100 nm subunits *N. Hirokawa et al, J. Cell Biol. 98, 1523 (1984).
NF Purification from Bovine Spinal Cord 1. Homogenize spinal cord in blender 2. Centrifuge to get rid of cell waste 3. Incubate supernatant in glycerol and pellet neurofilaments 4. Clarify by centrifuging through sucrose gradient 5. Remove remaining impurities with ion exchange column Result: Pure NF subunits Delacourte et al, Biochem. J., 191, 543-546 (1980) and Scott et al, J. Bio. Chem., 260, 10736-10747 (1985).
NF-L H Assembly Monomers NFL NFH Coiled-coil Dimers Tetramers 10nm Overlapping Tetramers * Fuchs et al., Science, 279, 514 (2000)
Sample Preparation Samples taken from supernatant � Add increasing amount of 7:1 3:1 5:3 1:1 3:5 1:3 NF-H to NF-L � Centrifuge samples to NF-H form a pellet containing a network of NFs � Using gel electrophoresis, NF-L determine the ratio of NF-L:NF-H that formed the NF network versus what NFs remained in the supernatant Increasing amount of NF-H
Gel Analysis NF-L:NF-H (17:3) High Low P D NF-L:NF-H (3:1) P High Low D As higher concentrations of NF-H are added, more side arms are also added causing side arm interaction and assembling P: pixels D: distance down the gel
Saturation Point Slope ≈ 0 40 35 %NF-H in pellet (assembled NFs) Saturation 30 Point = 45% Slope ≈ 1 NF-H 25 NF-L:NF-H = 20 13:9 15 10 10 15 20 25 30 35 40 45 50 55 60 65 70 75 %NF-H in initial solution
Future Research � Reproduce the value for the NF-LH saturation point . � Use the saturation point ratio to calculate the distance between the sidearms on the NF core. ? ? ?
ACNOWLEDGMENTS Jayna Jones Professor Cyrus Safinya INSET Trevor Hirst Nick Arnold Liu-Yen Kramer Mike Northen (super mentor) MRL at UCSB National Science Foundation National Institute of Health
Saturation Point of Assembly for Neurofilaments NF-L Only NF-L:NF-H (17:3) NF-L:NF-H (3:1) NF-L:NF-H (7:13) 100 nm 40 nm 20 nm Saturation point is the limiting factor in how many NF’s can become coiled • around one another at any given time NF-L has no side arms. NF-M and NF-H have interacting side arms. • Theory: NF-H and M are attracted to each other causing a cross linkage. •
• Ion Exchange Column separates sub-units •Polyacrylamide gel to confirm fractions •Dialysis with MES buffer to reassemble proteins Protein Reassembly * Molecular Cell Biology, 3rd edition,73-74 (1995) Urea buffer denatures MES Buffer assembles sub-units back together. NF and separates into Sub-units refold to form mature NF. sub-units
100 nm *N. Hirokawa et al, J. Cell Biol. 98, 1523 (1984). 100 nm * Courtesy of Biozentrum *Courtesy of Simone Karrasch
Saturation Point NFL:NFH NF-L:NF-H 17:3 3:1 17:3 3:1 Increasing Amounts of NF-H Constant Amount of NF-L 2X NF-H Jayna Jones 2004
Protein Sample - VOLTMETER 200 V GEL ν= Ez/ f ν: Velocity BUFFER E: Electrical field Z: Molecular weight f : Frictional force +
Results NF-H NF-M NF-L CONTAMINATION Jones,Jayna 2003
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