Supramolecular Structure and Assembly of Neurofilaments Tracy Mac - - PowerPoint PPT Presentation

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

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: Find the saturation ratio of NFs assembled from NF-L and NF-H.

Introduction

• Mol. Bio.Cell, 3rd ed, 73-74

(1995).

100 nm

Head Body Alpha helix 45 nm Tail ~50 to 100 nm NFL NFH NFM Bundled Neurofilaments in vivo

*N. Hirokawa et al, J. Cell Biol. 98, 1523 (1984).

100 nm

NF Assembly

60kD 100kD 115kD Mature NF

• In vivo, mature NF have a

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 subunits

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

NFL NFH Coiled-coil Dimers Tetramers Overlapping Tetramers Monomers 10nm

* Fuchs et al., Science, 279, 514 (2000)

Sample Preparation

Add increasing amount of

NF-H to NF-L

Centrifuge samples to

form a pellet containing a network of NFs

Using gel electrophoresis,

determine the ratio of NF-L:NF-H that formed the NF network versus what NFs remained in the supernatant

NF-H NF-L Increasing amount of NF-H 7:1 3:1 5:3 1:1 3:5 1:3 Samples taken from supernatant

Gel Analysis

P: pixels D: distance down the gel NF-L:NF-H (17:3) NF-L:NF-H (3:1)

High Low High Low P P D D As higher concentrations of NF-H are added, more side arms are also added causing side arm interaction and assembling

Saturation Point

10 15 20 25 30 35 40 45 50 55 60 65 70 75 10 15 20 25 30 35 40

%NF-H in pellet (assembled NFs) %NF-H in initial solution

Slope≈1 Slope≈0

Saturation Point = 45% NF-H NF-L:NF-H = 13:9

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

• 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.

NF-L:NF-H (17:3) NF-L:NF-H (3:1) NF-L:NF-H (7:13) NF-L Only

40 nm 100 nm 20 nm

* Molecular Cell Biology, 3rd edition,73-74 (1995)

Urea buffer denatures NF and separates into sub-units Protein Reassembly MES Buffer assembles sub-units back together. Sub-units refold to form mature NF.

• Ion Exchange Column separates sub-units
• Polyacrylamide gel to confirm fractions
• Dialysis with MES buffer to reassemble proteins

*Courtesy of Biozentrum

100 nm *Courtesy of Simone Karrasch

100 nm

*N. Hirokawa et al, J. Cell Biol. 98, 1523 (1984).

Saturation Point

NFL:NFH 17:3 17:3 3:1

Jayna Jones 2004

Increasing Amounts of NF-H Constant Amount of NF-L

NF-L:NF-H 3:1 2X NF-H

+ BUFFER GEL

• VOLTMETER

200 V

Protein Sample ν=Ez/f

ν: Velocity E: Electrical field Z: Molecular weight f : Frictional force

Results

NF-H NF-M NF-L CONTAMINATION

Jones,Jayna 2003