Discoidin Domain Receptor 2 InhibitsFibrillogenesis of Collagen Type 1

Lucy Greetham

Mark Nowey

Britney Tappen

Lauren Canova

Casey Pham

Learning Objectives

• Identify the objectives of the paper (level 1- Remember)

• Distinguish the advantages and disadvantages of in-vivo and in-vitro experimentation to study collagen. (level 2 – Understand)

• Demonstrate ability to understand SPR techniques and interpret new data. (level 3 – Apply)

• Design an experiment to test your hypothesis for the mechanism of cellular response to a change in DDR expression (level 6 – create)

Objectives

• What are the main objectives of this paper?– Show interaction between DDR2 and collagen type

1– Study the binding kinetics – Determine how DDR2 effects fibrilogenesis

Discoidin Domain Receptors (DDR1 and DDR2)

• Widely expressed cell surface receptors, which bind and are activated by collagen

• Activation results in downstream signaling which is known to regulates the ECM

• Both bond the native triple helix, ut only DDR1 binds nonfibrilar Type 4 collagen

DDR2

http://www.uic.edu/classes/bios/bios100/summer2003/lect06.htm

Discoidin Domain Receptor 2 is a Receptor Tyrosine Kinase (RTK)

Ligand binding leads to: oligomerization trans-autophosphorylation phosphoryation of cytosolic molecules association of other cellular components

Signal Transduction from the exterior to the interior of the cell

Production and Characterization of DDR2

• The extracellular domain (EDC) of mouse DDR2 was fused to human IGg1 and overexpressed in COS cells

• Secreted DDR2-Fc fusion protein purified and used for in vitro experiments

• Oligomers made by incubating fusion protein with anti-Fc antibodies

• Oligomers were characterized using SDS-PAGE, Western Blot, and Size-Exclusion Chromatography

Fibrillogenesis

Fibrillogenesis

• Fibril assembly is spontaneous• Energetically favorable due to electrostatic,

hydrophobic, and cross-linking interactions

http://medical-dictionary.thefreedictionary.com/_/viewer.aspx?path=dorland&name=fibril_collagen.jpg

In vitro vs. In vivo

In vitro – performing an experiment in a controlled environment. Ex) test tube or a petri dish

In vivo – observing an experiment inside a living organism

Ex) Clinical trials

VS

Activity 1 (10 Minutes)

It has been determined that protein X binds to collagen at a specific binding site.

Can you list the advantages and disadvantages of studying this interaction in vivo vs. in vitro?

Surface Plasmon Resonance

• Label free detection of molecular interactions

• SPR measures the formation of surface plasmon polaritons (SPPs) at the interface of a metal and a dielectric substance (liquid or air)

• As SPPs travel along the interface, the electric fields are generated

• The wave vector of SPPs at this interface depends upon the local refractive index,

• Any change to the local index of refraction results in a change in the SPP wave vector, which is the basis for measuring binding interactions using SPR.

Example Data

Figure 2

Interaction of DDR2 with immobilized collagen type 1

a. DDR2-Fc dimers and oligomers (22 nM)

b. Disassociation constant was determined by washing DDR2-Fc oligomer Top to bottom: 21, 16, 11, 7 nM

Activity 2 (10 minutes)

• SPR data was taken for a monomer, dimer, and oligomer of cell surface receptor X.

• The monomer has the highest affinity for collagen, while the oligomer has the lowest affinity for collagen.

• The intern that took the data lost the excel file.

• Draw what the data should look like for each form of the receptor.

Figures 3 and 4

• Purpose–How DDR2 affects the fibrillogenesis

process• Methods–Turbidity measurements–Hydroxyproline (HP) assay

Turbidity Measurements

● What does turbidity measure?

● The cloudiness of a solution caused by individual particles.● Collagen fibrillogenesis when collagen solution warmed from 4°C to 37°C

● UV-spectrophotometer

● Multiple cuvette assembly● Temperature control chamber

● Diluted in PBS

● Absorbance (313 nm) vs. time

● Ran for 8 to 10 hours. Why so long?

● To ensure maximum amount of collagen had been formed for each sample.

TrkB

• What is TrkB?– A catalytic receptor for growth factors– Mediates neuronal differentiation and survival

• Why is it used in this paper?– It’s used as a negative control– Confirms outside things aren’t affecting the

experiment

Figure 3

Presence of DDR2 Affects Fibrillogenesis• DDR2 concentration 1/5 of collagen

concentration• PBS only concentration of fibers

rapidly increased then plateaued• TrkB has a short lag time (2 hr)

– Why?• Steric hinderance by TrkB

• DDR2 have a long lag time (4 hr) and rate of increase is gradual.

• Increased DDR2 = increased lag time

• What does this say about DDR2?

Hydroxyproline Assay

• What is HP and where is it located?–A postranslational modification of proline –Only in collagen

• Purpose of Assay– Estimate the amount of fibrillar collagen

formed–HP in hydrolysates (substance produced by

hydrolysis) direct measure of amount of collagen

How They Did It

• Samples of collagen centrifuged to separate collagen fibrills and resuspended

• Mixed with hydrolysate and chromatophore (pigment containing and light reflecting cells)

• Absorbance measured and compared to standards to determine concentration of collagen

Figure 4

Estimation of Collagen Content

• Standard curve acquired by measuring standards in 0-50 µg range

• Amount of collagen fibrills present at 1.5 hr and 18 hr

• DDR2 collagen at 1.5 hours less than half TrkB at 1.5 hours

• Very similar amounts of collagen fibrills at 18 hours

• 50-55% collagen used in fibrillar collagen

• What does this experiment support?– Confirms turbidity measurements – DDR2 delays fibrillar production

Figure 5

Purpose: To analyzed and “characterize” the change in collagen morphology with and without the pretense of DDR2

Methods: Atomic Force Microscopy and Transmission Electron Microscopy

Molecular imaging of membrane proteins and microfilaments using atomic force microscopySe-Hui Jung1, Donghyun Park1, Jae Hyo Park2,Young-Myeong Kim1 and Kwon-Soo Ha

EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 42, No. 9, 597-605, September 2010

http://www.nanowerk.com/spotlight/spotid=14045.php

How Does AFM Work?- Sample is loaded into the

machine- Flexible Cantilever Tip

connected to a piezoelectric scanner (allows for controlled x,y,z movement)

- Laser Reflections monitors the movement of the lever.

- Data is analyzed - Topographical image is

produced.- Involves complex physics

beyond scope of this class.

(Hooke’s Law, ect)

Different Modes of AFM

- Direct Contact

- AC or “Tapping” Mode

- Key: Cantilever Probe is flexible and forces between it and sample can be measured (Hooke’s Law)

- For this research and many other biological imaging , “Tapping” Mode is used. Why?

AFM Imaging is like Reading Brail.

http://loveyalyn.blogspot.com/2011/01/world-braille-day.html

Advantages of AFM:

- High Resolution, beyond light diffraction limit. <1nm

- Real time imaging- Easy preparation of

samples- Less risk of

damaging sample being imaged.

http://umanitoba.ca/chemistry/courses/chem222/Support/CoolStuff/MolecularStructure/AS-AFM.jpg

Collagen without DDR2 present

Collagen with DDR2 present

Data suggest that with DDR2 present,Collagen 1 is primarily in monomeric form,

Transmission Electron Microscopy

Use of electron beam to visualize a specimen

Higher resolution than light microscopy due to the short wavelength of electrons

- Collagen, Collagen/TrkB, Collagen/DDR2 observed at 7000x 30,000x and 50,000x

Fiber diameter was measured at the trunk of the fibers - Fiber consists of 5 triple helix monomers

Figure 6

DDR2 alters the d period of collagen fibrils

Figure 7

• Purpose– To determine how cellular expression of DDR2

effects collagen fibrillogenesis

• Methods– Viral Transfection– Western Blotting– Laser Scanning Confocal Microscopy

Confocal Microscopy

• Principles – Uses fluorescence to image live

specimens– Only emissions which are in

focus with the pinhole aperture are collected

• Benefits– Reduces background image

noise– Higher resolution– 3-D images

http://www.microscopyu.com/tutorials/java/virtual/confocal/http://www.olympusfluoview.com/theory/LSCMIntro.pdf

Figure 7 (B and C)

• LSM images of mouse osteoblasts• Incubated for 6 hours without collagen• Nuclei stained blue • Collagen fluoresces green

Figure 7 (D-I)W

T +

FITC

Col

lage

nD

DR2

+ FI

TC C

olla

gen

1h 6h3h

Figure 7 Take away

• In the wild type cells, collagen can be seen forming thin fibrils as soon as 1 hour

• Fibrils became longer and thicker over the course of the 6 hour incubation

• DDR2 mutants exhibited large aggregates of collagen

• Size of DDR2 aggregate remained similar during incubation, but intensity increased

Conclusions

• Oligomerization of DDR2 increases the ability to bind to collagen

• DDR2 inhibits collagen fibrillogenesis• Type 1 collagen in the presence of DDR2 forms

amorphous aggregates as opposed to fibrils

DDR2 and MMP

• DDR family of receptors has been linked to up-regulation of MMP

• Deletion of DDR2 has been shown to cause dwarfism, skeletal malformation, and delayed wound healing

• Bone morphogenesis and skin healing are dependent on the function of MMP’s

Activity 3 (20 Minutes)

• Intima: the innermost coat of an organ (as a blood vessel) consisting usually of an endothelial layer backed by connective tissue and elastic tissue.

• DDR1-null mice had decreased neointimal thickening after vascular injury

Activity 3 (20 minutes)

1. Hypothesize a reason and or mechanism for these results2. Design an experiment to test your hypothesis

Learning Objectives

• Identify the objectives of the paper (level 1- Remember)

• Distinguish the advantages and disadvantages of in-vivo and in-vitro experimentation to study collagen. (level 2 – Understand)

• Demonstrate ability to understand SPR techniques and interpret new data. (level 3 – Apply)

• Design an experiment to test your hypothesis for the mechanism of cellular response to a change in DDR expression (level 6 – create)

Questions?