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 )