2007 Summer
UCSB Materials Research Laboratory Research Experience for Teacher Program
Binding Experiment of Microtubule and Tau
Proteins
Humphrey Kariuki
Objective
We will answer to the question “What % of tau protein binds to
MTs?”
The answer will give a key information for understanding the
structure and interaction of MTs and tau using x-ray diffraction.
Understanding Microtubule and Tau protein
assembly/ Neural Fibril Tangles (NFT)
Neurofibrillary tau tangles are one of the primary occurrences of
Alzheimer’s disease. (http://www.alzheimersupport.com/library/showarticle.cfm/ID/1690/e/1/T/Alzheimers)
Back ground information Alzheimer’s disease, the most common dementia
(NIH progress report on Alzheimer’s disease 2004-2005)
Alzheimer’s disease is an age-related and
irreversible brain disorder that develops
over a period of years.
People experience memory loss and
confusions. These symptoms gradually lead
to behavior and personality changes, a
decline in other cognitive abilities (such as
thinking, decision making, and language
skills), and ultimately to a severe loss of
mental function.
These losses are related to the breakdown of
the connections between certain nerve cells
in the brain and the eventual death of many
of these cells.
Problem statement “axon-specific distribution, tau has been implicated as
having a role in the unique organization of axonal microtubules but direct
evidence is still lacking.” [1]
Fig. a. Microtubules are abundant in the neuron and extend far into the axonal
growth cone. b. Distribution of tau (NFT) in 3 day culture of hippocampal
neurons. Note the presence of tau in the cell body, the axon, and the giant
growth cone (arrowhead). Scale bar, 20 µm.
[1] THE TAU PROTEINS IN NEURONAL GROWTH AND DEVELOPMENT, Roland Brabdt,
Institute of Neurobiology, University of Heidelberg, INF 364, 69120 Heidelberg, Germany.)
Peripheral problem statement
It should be noted that, to this point, none of the components with which tau interacts have
been identified and that the features of binding partners of tau (i.e. integral or peripheral
membrane proteins) are purely speculative.
www.nia.nih.gov/.../Part1/Hallmarks.htm
Healthy neurons are internally
supported by Microtubules
Outside
50Å
160Å
40Å
Inside
tubulin dimer


protofilament
4.9nm
www.nia.nih.gov/.../Part1/Hallmarks.htm
A special kind of protein, tau, binds to
microtubules and stabilize them.
PVPMPDLKN-VKSKIGSTENLKHQPGGG–KVQIVYKPVDLSK–
VTSKCGSLGNIHHKPGGG–QVEVKSEKLDFKDR–VQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVD
S-PQLATLADEVSASLAKQGL
AEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK(ESPLQTPTEDGSEEPGSETS
DAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVS
KSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDR
SGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTA
Projection domain
Binding domain
3RL
C
3RM
3RS
C
C
4RL
4RM
4RS
C
C
C
4 3
1
N
N
N
4 3 2 1
N
N
N
_Tau is a natively unstructured protein with no secondary structure. Tau is hydrophilic and charged at
physiological pH.
Purifying Microtubule/microtubule associated
proteins (MAPs) (biology department)
b. Gel electrophoretic separation of microtubule protein and purified tubulin.
Experiment 1 Microtubule polymerization
To polymerize Microtubule. Mw (tubulin) = 110k
on ice
at 36oC bath
procedure
1 Start with x-g of y-mg/ml tubulin
2 Defreeze tubulin on ice (repeat -touching gently and -put it on ice).
3 Add buffer for y'-mg/ml tubulin
4
5
6
7
8
9
Spin down at 16,500 rpm, 15min, at 4 oC.
with Sorval centrifuger (ss-3h rotor)
Take 90% of the liquid to avoid the bottom, and put it on ice.
1mM GTP (out of 50mM GTP)
5 wt% Glycerol in PEM 50 (put of 50% Glycerol in PEM 50)
Put at 36 oC for 20min.
Taxol (1:1 mole ratio taxol to tubulin) out of 2mM in DMSO.
Finally MT
x-g
0.0765
y-mg/mL
8.73
uL
add (uL) Tot vol
76.5
5
57.069
133.6
140.2
2.86
15.58
3.66
4.32
162.31
Viewing Polymerized microtubule using a
Differential Interference Contrast (DIC)
Microscope
CCD camera
Sample stage
Sealing glass slide and cover slip
DIC microscope @Safinya group
Polymerized Microtubule
Thread-like textures
Fig. DIC image of thread-like textures of MTs (arrow). scale bar
10m
Experiment # 2/3 UV/VIS analysis
procedure final Complex sample
•Mix tau/MTs
•Wait 10 min to settle
•Spin down for 30min/ @13.2k rev/min
•Collect supernatant 90% ~ 3.6ul( uv vis) repeat
•Boil for 45min /cool down to RT
•Spin down 30 min max rev
•Collect supernatant 90% ~ 30ul
•Uv/ vis analysis
Water bath @ 100oC
Denatured Microtubules
Denatured MTs
10m
UV/ VIS experiment
H
Bradford
Making calibration samples with bovine serum albumin
(BSA) and Bradford
bouman.chem.georgetown.edu
UV/VIS spectrometer
UV /VIS Calibration curve
0.4
Absorbance (a.u.)
0.3
0.2
0.1
y=0.04217 x - 0.02807
0.0
Supernatant
-0.1
0
2
4
6
BSA (g)
Fig. Calibration graph:
0.4mg/mL of BSA and 1mL of Bradford.
8
10
As a result, >90% of taus bind to MTs at 1:5 ratio of 3RS tau: tubulin
TEM Procedures
Negative Staining
• Load 10 l sample on to
grid
• Wait 2 min
• Wick off excess sample
• Add 10 l of cytochrome-C
• Wait 15 sec
• Wash with 3 drops of water
• Wick off excess water
• Add 1 drop of uranyl acetate
• Wait 20 sec
• Wick off excess Uranyl
acetate (UA)
Transmission Electron Microscopy (TEM) of
MTs/tau
Sample holder
loading TEM sample
Inserting sample holder in the vacuum
chamber
Transmission Electron Micrograph of MTs
Single protofilament line
Electron micrograph Microtubule-tau complex
Further studies
The binding experiment @ different tau to tubulin ratios.
@ six different tau isoforms.
@ different salt concentrations.
Acknowledgment
•M.C. Choi, Jayna Jones and professor Safinya
•Michelle Massie and professor Feinstein in Biology : providing tau
protein
•Herb Miller and professor Wilson : Tubulin protein
•Martina Michenfelder
…Thank you Martina