Download - The AFM detection of ligand-receptor interaction on a surface of living cells Małgorzata Lekka
The AFM detection of ligand-receptor interaction on a surface of living cells
Małgorzata Lekka
Sample
Scanner
LaserPosition Sensitive Photodiode
Tip
Cantilever
AFM working in IFJ
atomic force microscopy
FN
FL
scanner linearization
spring constant
A–B signal
geometry: = 10º
tip – shape
Hoh et al.
force spectroscopy
scanner linearization
scanner
photodiodelaser
sin2max
ns
without protein (conA)
nominal value
= 7 kHz, k = 0.01 N/m
measured value
= 5.8 kHz, k = 0.007 N/m
resonant frequency of a thermally excited cantilever
with protein (conA)
0.3 mg/ml
= 5.8 kHz, k = 0.007 N/m
1 mg/ml
= 5.4 kHz, k = 0.006 N/m
Sader et al.
!
TkkF B min
k = 0.03 N/mRT
Fmin 11 pN
probe
Standard TGT01
Si3N4 + APTES (4%) + glutaraldehyde (GL; 2.5%)
Si3N4 + APTES (4%) + GL (2.5%)
+ conA (0.3 g/ml)
R = 54 +/- 7 nm R = 275 +/- 10 nm
probe size protein concentration immobilization procedure
Moy et al.
Grandbois et al.
number of molecules on probe
Single molecular pair
bond type energy [kJ/mol]
bond length [nm]
force [nN]
covalent 250 (for S–S)
0.20 4.42
noncovalent ionic 20 0.25 0.27
Van der Waals 2 0.35 0.02
hydrogen 740
0.300.30
0.080.45
rough estimation of the bond strength
2
2xkE
+ hydrophobic forces
Kim et al.
Distribution of vitronectin receptors on a living MC3T3-E1 cell (murine osteoblastic cell)
Grandbois et al.
AFM tip functionalized with Helix pomatia
N-acetylgalactosamine in membrane of group A of RBC
mixed red blood cells
No
of e
vent
s
Force [nN]
Tees et al.
Lee et al.
Eb 10 -20 [J] koff[s-1] xb [Å]
AGD – αIIbβ3
RGD – αIIbβ3
- 2.67
- 2.64
47.58
1.53
1.09
1.03
No
of e
vent
s
Force [nN]
5 s
0.3 sretraction velocity 3.5 μm/s
ConA-CaY
How to check what it is measured ? not functionalized AFM probe
measurements of known ligand-receptor pair
HCV 29 cellssilicon nitride tip
ConA–PC-3 ConA–ASA ConA–CaY
Force [pN] 116 17 790 32 940 39
CaY – Con A+ 1 mg/ml Con A
non-specific interaction
interaction between ligand – receptor pair
blocking of the binding sitesCaY– Con A free amount of ligand in solution
all or certain number of binding sites can be blocked
different types of interaction characteristic for cancerous cells
HCV 29 non–malignant transitional epithelial cells of ureter
T24 transitional cell cancer of urine bladderAFM, contact mode
SNAPHA-L
ConA
lectinslectins
sialic acidsialic acidNN-acetylglucosamine-acetylglucosamine
mannose, glucosemannose, glucosecarbohydratescarbohydrates
binding force Cell lineCell line LectinLectin Binding force [pN]Binding force [pN]
HCV29HCV29 ConAConA 43.343.3 3.43.4PHA-LPHA-L 59.959.9 77..11SNASNA 167.2 167.2 5.5 5.5
T24T24 ConAConA 123123.6 .6 1818..11PHA-LPHA-L 152152..66 88..22SNASNA 76.276.2 1010.9.9
verification
50 µg/ml ConA
conclusions AFM allows detecting molecular interaction on a surface of living cell
The spatial arrangement of functional carbohydrate groups on cell surface was attributed to the density of all types of the carbohydrate structures (mannose, N-acetylglucosamine, sialic acids).
The maximum range of force distribution (presented in histograms up to 1.2 nN), the size of the adhesion spot (i.e. one single point on the distribution map ~ 0.95 μm2), the number of bonds (2–3 for cancerous cells) suggested that ligands present on a surface of T24 cells formed groups composed of several single carbohydrate chains involved in adhesion process in the lectin recognition.
Institute of Medical Biochemistry Medical CollegeJagiellonian University
Piotr LaidlerJoanna DulińskaMaryla Łabędź
The Henryk NiewodniczańskiInstitute of Nuclear PhysicsPolish Academy of Sciences
Zbigniew StachuraMałgorzata LekkaJanusz LekkiJan Styczeń
PhD studentsJoanna GrybośKateryna LebedGrażyna Pyka
Atomic Force Microscopy
Histogram
max
min
)()()(x
xdxrxfxfrC
Autocorrelation function
bin size
large number of data
force between single pair: CaY-ConA
F = 960 +/- 110 pN
more complex interactionsF const
single interactionF const
FF
FFF2
22
22 2
m = m2
F 2
= m · F + F0
F0 – non-specific force
F · F0
f1(F, F2, F0 )·
f2(F, F2, F0 )