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?