Answer Key: The Extracellular Matrix

BIOL230W Week 11 – The Extracellular Matrix The extracellular matrix (ECM) is easy to overlook but is a very important structure. It external is to cells and functions in tissue development, tissue repair and motility in multicellular organism; some single-celled organisms also form ECM as a community. This week will explore the structure and function of the ECM. We will also learn that in cancerous tumors the ECM is present in higher amounts compared to normal tissue and can have aberrant characteristics. Day 1 Learning Objectives 1. Analyze ECM composition with pie charts. 2. Describe the general concept of an extracellular matrix: • What are the two main functions of the ECM? • What are the components of the ECM? • Where are the components of the ECM produced? 3. Describe the structure of integrin proteins. 4. Define the role of the RGD motif. Small Group Activity: ECM Components Across Tissues This image shows the relative proportions of different categories of ECM and ECM-associated proteins in different tissues. We are focusing on proteoglycans, collagens, and fibronectin (glycoprotein). ECM regulators and secreted factors are typically enzymes or signaling molecules, respectively. SI = small intestine. If you are using a paper copy, use the color image on the screen to answer the questions. 1. What trends do you notice in the compostion of the ECM across different tissue types? Large portions of collagen in most tissue except liver and brain. Small portions of secreted facotrs 2. About what % of collagen makes up the brain? About 30% 3. Thinking about structure, why does the brain have less collagen than bone or cartilage? The brain is protected by the skull so it doesn’t need the protection from collagen From Beachley, Vince Z., et al. "Tissue matrix arrays for high-throughput screening and systems analysis of cell function." Nature methods 12.12 (2015): 1197-1204. BIOL230W Week 11 – The Extracellular Matrix Mini-Lecture: General Characteristics of the ECM (we will be using animal ECM for class) 4. What are the two main functions of the ECM? Adhesion and cell signaling 5. Add a description of each of the representative proteins of the ECM below. A. Proteoglycans: proteins with chains of sugar attached; negatively charged due to presence of sulfate or carboxyl group sugars. Functions to attract water. B. Collagen (example fibrous protein): Most common protein in body. Made in cell with 3 interacting proteins (tertiary structure – triple helix) that connect with covalent bond. Gives strength to connective tissue. C. Fibronectin (adhesive protein): Interact to form fibrils. Aids in migration and wound repair. Loss of function mutations are embryonic lethal. 6. Describe the activation of integrin. Alpha and beta subunit come together as a dimer. When they are inactive, they are not bound to a component of the ECM, they have bent conformation. Integrin is activated when a component is bound to it which changes conformation and makes it go from bent to straight. 7. The (integrin or ligand) contains a RGD peptide motif. Day 2 Learning Objectives 1. Outline the steps of collagen synthesis. 2. Analyze the steps involved in fibronectin activation in the ECM. 3. Predict what may happen if there are problems with collagen synthesis or fibronectin activation. 4. Compare and contrast collagen production vs. fibronectin production. Knowledge Recap: ECM Components Across Tissues 8. Collagen is a protein transported via exocytosis to the extracellular matrix. Outline (or draw) the steps of exocytosis below from mRNA in the cytoplasm to final collagen release to the ECM. Make sure to include all organelles involved. PremRNA Collagen mRNA Small Group Activity: Collagen Synthesis and Transport Some of the final protein components of the ECM (eg., collagen and fibronectin) are quite large and form complex networks. For example, ECM collagen fibers are upmm to 10 compared to intracellular BIOL230W Week 11 – The Extracellular Matrix microtubules at 25nM. These protein components are secreted by the surrounding cells and then must be assembled to their final structure in the extracellular space. 9. Below is a scrambled list of the steps associated with collagen gene expression (i.e., synthesis), transport and final assembly in the ECM to form collagen fibrils. Label the steps of the image on the next page using the steps below, as well as the nucleus, ER and extracellular matrix. A - Post-translation modification (adding hydroxyl groups to amino acids) to assist interactions between individual proteins. B - Crosslinking of single collagen helices to form thicker, more dense collagen fibrils. C - Formation of collagen molecule quaternary structure (triple helix structure). D - Function of V-SNARE and T-SNARE protein complex E - Completed by RNA polymerase F - Splicing of introns and addition of 5’ G-cap and 3’ poly-A tail G - Modification of collagen molecule (triple helix) by enzymes previously secreted via exocytosis H - Ribosome docking with Sec61 10. Collagen hydroxylation requires an enzyme that uses vitamin C as a cofactor. Scurvy is a disease that presents when a person Eis vitamin C deficient. Nucleus Hippocrates describes: “the F mouth feels bad; the gums are detached from the teeth, blood runs from the nostrils… ulcerations on the legs; some of these heal… skin is thin.” H A What is the molecular outcome of a lack of vitamin C as related to collagen? Thin blood vessel walls and less effective collagen, basement membrane of epithelium isn’t strong, bruising easy. C D 11. What is the relationship between the molecular outcome and the disease presentation? BIOL230W Week 11 – The Extracellular Matrix Due to thinner blood vessel walls, they rupture more easily and therefore bruising easily is common as well as an inability to keep teeth in sockets. G B Small Group Activity: Fibronectin Activation in the ECM Fibronectin is another ECM protein that is synthesized in the cell, secreted to the ECM and then activated to create a protein network. Use the image to answer the questions about the signaling pathway that activates fibronectin. Fibronectin (FN) matrix assembly model. (A) Binding of inactive FN to integrins induces receptor clustering and colocalization of protein kinase (FAK) and talin. FAK autophosphorylates and allows SRC to bind to the complex. SRC is critical for integrin interaction with actin (a cytoskeleton organelle) when active (an interaction that aids with cell migration – see question 15). (B) Clustered integrin complexes co-localize with syndecan and organize the actin cytoskeleton (green lines). Signaling molecules Ras/MAP kinase (orange), Rho GTPase (violet) and protein kinase C (PKC, blue) further regulate actin. Contractile forces between active:integrin:FN aid in converting inactive FN into active FN. (C) Increased active FN at the integrin cluster promote further FN:FN interactions (black lines between FN) and fibril assembly. 12. Highlight the RGD motif of fibronectin found in Image “A”. 13. Based on this signaling pathway, is the RGD domain accessible when fibronectin is in an inactive form? Are FN:FN binding motifs accessible in the inactive form? Why is this important? Yes, it is accessible when fibronectin is inactive which is important because it cannot interact with other fibronectins, but it can interact with integrin when it is outside the cell. BIOL230W Week 11 – The Extracellular Matrix 14. The binding of integrin to fibronectin causes the integrins to become clustered in the plasma membrane. This, in turn, concentrates fibronectin to promote fibril interactions. Is this an example of a positive or negative feedback mechanism? Positive feedback mechanism 15. In some cancers, the src protein has a gain-of-function mutation. Overactive src causes the above mechanism to occur unregulated. How do you think this affects cancer development? Promoting cell migration when it shouldn’t, which causes metastasis. 16. In what ways is collagen synthesis similar to fibronectin synthesis? Both are translated and transcribed in the cell, specifically translated in the ER, and exported. Both interact between molecules outside of the cell (FN:FN, CL:CL). 17. In what ways is collagen synthesis different from fibronectin synthesis? Fibronectin requires cell signaling in the cell, fibronectin also doesn’t undergo modifications by enzymes (collagen is enzymatically assembled). Day 3 Learning Objectives (Sci Lit – Week 11) 1. Describe the general anatomy of breast tissue as related to breast cancers. 2. Use micrographs to quantify ECM characteristics. 3. Compare and contrast normal breast tissue ECM with cancerous breast tissue ECM. 4. Outline the progression of cancer as related to changes in the ECM. Small Group Activity: Data Analysis Summarize the results from each of these graphs based on the stains discussed. Make sure to include the significance of the result to changes in the ECM. 18. Fig. B Top Summary: Increase in trichrome stain as we move from normal, to DCIS, to IDC. This indicates an increase in collagen, which indicates an increase in stiffness in the tumor. 19. Fig. B Bottom Summary: Increase in staining, as with the top image. This means alignment of collagen is increasing. This equates to a lump being formed due to increased stiffness. 20. What is the significance of elevated integrin and FAK activity in cancerous cells? BIOL230W Week 11 – The Extracellular Matrix Promoted motility which creates a moving cancer cell = metastasis (moves out into surrounding tissue) 21. Use the image below to summarize the relationship between normal vs. breast cancer ECM and the progression of the tumor. You only need to focus on the terms discussed in class. Breast cancer ECM will have increased collagen, and increased collagen alignment and therefore ECM will be stiffer. Additionally, cancerous tissue will have more matricellular proteins.