Transcript Collagen-Research

Discoidin Domain
Receptor 2 Inhibits
Fibrillogenesis 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
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
http://www.uic.edu/classes/bios/bios100/summer2003/lect06.htm
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
http://medical-dictionary.thefreedictionary.com/_/viewer.aspx?path=dorland&name=fibril_collagen.jpg
• Fibril assembly is spontaneous
• Energetically favorable due to electrostatic,
hydrophobic, and cross-linking interactions
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
microscopy
Se-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/C
oolStuff/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.olympusfluoview.com/theory/LSCMIntro.pdf
http://www.microscopyu.com/tutorials/java/virtual/confocal/
Figure 7 (B and C)
•
•
•
•
LSM images of mouse osteoblasts
Incubated for 6 hours without collagen
Nuclei stained blue
Collagen fluoresces green
DDR2+ FITC Collagen
WT + FITC Collagen
Figure 7 (D-I)
1h
3h
6h
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 upregulation 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 results
2. 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?