Fibronectin

1. Fibronectin • Fibronectin exists as a dimer, consisting of two nearly identical polypeptide chains linked by a pair of C-terminaldisulfide bonds.[3] Each fibronectin monomer has a molecular weight of 230-250 kDa and contains three types ofmodules: type I, II, and III. All three modules are composed of two anti-parallel β-sheets; however, type I and type II are stabilized by intra-chaindisulfide bonds, while type III modules do not contain any disulfide bridges. The absence of disulfide bonds in type III modules allows them to partially unfold under applied force.[4] • Three regions of variable splicing occur along the length of the fibronectin monomer.[3] One or both of the "extra" type III modules (EIIIA and EIIIB) may be present in cellularfibronectin, but they are never present in plasmafibronectin. A "variable" V-region exists between III14-15 (the 14th and 15th type III module). The V-region structure is different from the type I, II, and III modules, and its presence and length may vary. The V-region contains the binding site for α4β1integrins. It is present in most cellular fibronectin, but only one of the two subunits in a plasma fibronectin dimer contains a V-region sequence. • The modules are arranged into several functional and protein-binding domains along the length of a fibronectin monomer. There are four fibronectin-binding domains, allowing fibronectin to associate with other fibronectin molecules.[3] One of these fibronectin-binding domains, I1-5, is referred to as the "assembly domain", and it is required for the initiation of fibronectin matrix assembly. Modules III9-10correspond to the "cell-binding domain" of fibronectin. The RGD sequence (Arg–Gly–Asp) is located in III10 and is the site of cell attachment via α5β1 and αVβ3integrins on the cell surface. The "synergy site" is in III9 and has a role in modulating fibronectin'saccociation with α5β1integrins.[5] Fibronectin also contains domains for fibrin-binding (I1-5, I10-12), collagen-binding (I6-9), fibulin-1-binding (III13-14),heparin-binding and syndecan-binding (III12-14).[3] • [edit]Function • Fibronectin has numerous functions that ensure the normal functioning of vertebrateorganisms.[1] It is involved in cell adhesion, growth, migration and differentiation. Cellular fibronectin is assembled into the extracellular matrix, an insoluble network that separates and supports the organs and tissues of an organism. • Fibronectin plays a crucial role in wound healing.[6][7] Along with fibrin, plasma fibronectin is deposited at the site of injury, forming a blood clot that stops bleeding and protects the underlying tissue. As repair of the injured tissue continues, fibroblasts and macrophages begin to remodel the area, degrading the proteins that form the provisional blood clot matrix and replacing them with a matrix that more resembles the normal, surrounding tissue. Fibroblasts secreteproteases, including matrix metalloproteinases, that digest the plasma fibronectin, and then the fibroblasts secrete cellularfibronectin and assemble it into an insolublematrix. Fragmentation of fibronectin by proteases has been suggested to promote wound contraction, a critical step in wound healing. Fragmenting fibronectin further exposes its V-region, which contains the site for α4β1integrin-binding. These fragments of fibronectin are believed to enhance α4β1integrins-expressing cell binding, allowing them to adhere to and forcefully contract the surrounding matrix. • Fibronectin is necessary for embryogenesis, and inactivating the gene for fibronectin results in early embryonic lethality.[8] Fibronectin is important for guiding cell attachment and migrationduringembryonic development. In mammalian development, the absence of fibronectin leads to defects inmesodermal, neural tube, and vascular development. Similarly, the absence of a normal fibronectin matrix in developing amphibians causes defects in mesodermal patterning and inhibits gastrulation.[9] • Fibronectin is also found in normal human saliva, which helps prevent colonization of the oral cavityandpharynx by potentially pathogenic bacteria.[10] • [edit]Matrix assembly • Cellular fibronectin is assembled into an insolublefibrillarmatrix in a complex cell-mediated process.[11] Fibronectin matrix assembly begins when soluble, compact fibronectin dimersaresecreted from cells, often fibroblasts. These soluble dimers bind to α5β1integrin receptors on the cell surface and aide in clustering the integrins. The local concentration of integrin-bound fibronectin increases, allowing bound fibronectin molecules to more readily interact with one another. Short fibronectin fibrils then begin to form between adjacent cells. As matrix assembly proceeds, the soluble fibrils are converted into larger insoluble fibrils that comprise the extracellular matrix. • Fibronectin’s shift from soluble to insoluble fibrils proceeds when cryptic fibronectin-binding sites are exposed along the length of a bound fibronectin molecules. Cells are believed to stretch fibronectin by pulling on their fibronectin-bound integrin receptors. This force partially unfolds the fibronectin ligand, unmasking cryptic fibronectin-binding sites and allowing nearby fibronectin molecules to associate. This fibronectin-fibronectin interaction enables the soluble, cell-associated fibrils to branch and stabilize into an insoluble fibronectin matrix. • [edit]Role in cancer • Several of the morphological changes observed in tumors and tumor-derived cell lines have been attributed to decreased fibronectin expression, increased fibronectin degradation, and/or decreasedexpression of fibronectin-binding receptors, such as α5β1integrins.[12] • Fibronectin has been implicated in carcinoma development.[13] In lung carcinoma, fibronectinexpression is increased, especially in non-small cell lung carcinoma. The adhesion of lung carcinoma cells to fibronectin enhances tumorigenicity and confers resistance to apoptosis-inducingchemotherapeutic agents. Fibronectin has been shown to stimulate the gonadal steroids that interact with vertebrateandrogen receptors, which are capable of controlling the expression of cyclin D and related genes involved in cell cycle control. These observations suggest that fibronectin may promotelungtumor growth/survival and resistance to therapy, and it could represent a novel target for the development of new anticancer drugs.

2. The curious case of Sage’s unfortunate roommate:

3. Diagnosis • What are some characteristics of a malignant tumor? • What tests are run diagnose a cancerous tissue? • What would you screen for in a biopsy?

4. WARNING We must interrupt your regularly scheduled lecture for emergency purposes. Now Presenting:

5. The ‘Dark Side’ of Fibronectin Chris Blum RikkeBrogaard Sage Foley Dev Patel Benjamin Mintz

6. Objectives 1) Learn the basic structures of fibronectin [Level 1: Knowledge] 2) Compare the structure and strength of fibronectin and collagen [Level 4: Analysis] 3) Createan illustration of the interactions of fibronectin and related proteins and their contribution to tumorigenesis [Level 5: Synthesis]

7. Fibronectin Structure

8. Genetic Roots • Formed from fibronectin (FN) gene • 20 Isoforms • Occur through exon skipping and exon subdivision

9. Structure • Glycoprotein consisting of monomers ranging from 230-250kDa • Monomers consist of multiple domains • Each domain contains a mixture of 3 different types of modules Module Random Loop

10. Cysteine Important cysteine dimers

11. Important sequence for cell binding Dimers Super important!! Your cells love this sequence Disulfide Bonds Without these bonds there will be no fibers!

12. Why doesn’t fibronectin bind to everything all the time? • Cryptic sites Type I module Type III module Beta sandwich motif Cryptic sites

13. Activity 1 (10 min) For each component of the fibronectin molecule, match the bonds and their interactions: Disulfide bond Covalent bond Hydrophobic Interactions Module I Module III Module II C-terminal domain

14. ECM Assembly Two types of FN are present in vertebrates • Plasma FN - a major protein in blood plasma, produced by liver cells. • Cellular FN – which is secreted by various cells, primarily fibroblasts. Cellular FN is a soluble dimer that can self assemble into the insoluble matrix → This matrix separates and supports the organs and tissue of an organism.

15. ECM Assembly • In order to form a matrix, FN must beconverted from: • Inactivecompacted form →activeexpandedform. The exact mechanism behind the conformation change is currentlyunknown, but ex. couldinvolve: • Interactionwithothermolecules ex. Heparin or collagen • pH or ionic strength

16. The assembly of the Extracellular Matrix Assembly of the matrix RGD sequence and the synergy sequence binds to intergrin receptor. Q: Explain synergy

17. The assembly of the Extracellular Matrix Assembly of the matrix RGD sequence and the synergy sequence binds to integrin receptor. Q: Explain synergy A:The effect of two or more agonists together is greater than the sum of their individual effects. ” 2+2 = 6”

18. The assembly of the Extracellular Matrix • Integrin binding to FN • dimer → conformation • change , whichexpose • more binding sites. • FN-FN interactions → • Tighter connectionand • matrix formation.

19. Activity 2: 10 min.Fibronectin vs. collagen Predict how collagen and fibronectin matrices will respond to applied tension. How may differences in mechanical properties affect cells? Explain

20. ECM Assembly Howwouldyouexplain the fibrilelasticity of FN?

21. ECM Assembly Howwouldyouexplain the fibrilelasticity of FN? • Breakage of hydrogen bonds between β-sheets in type III could lead to theunraveling. • Straightening of FN subunits could provide extension. • http://www.ks.uiuc.edu/Research/fibronectin/movies/stretching-fn1.mpg

22. Fibronectin Receptors – Integrins Expression of fibronectin and its receptors regulates: • Anchorage • Polarity • Ability to migrate • State of differentiation http://endometriosis-bible.com/?hop=amyhart

23. Integrins • Trans-membrane protein • An α and β subunit associated noncovalently • β1 subunit = surfaces of most vertebrate cells, cell-ECM interactions • α5β1 binds fibronectin http://www.scq.ubc.ca/the-role-of-integrins-in-wound-healing/

24. Fibronectin and Integrins • Recognition sequence: RGD (Arg-Gly-Asp) • RGD sequence is common cell recognition site • Fibronectin assembly activated • Integrin is also activated, conformational change http://www.ks.uiuc.edu/Research/fibronectin/

25. Integrin Connections Focal adhesions: • Migratory and non-epithelial cells attach to ECM • Clustered integrins interact with bundles of actin microfilaments via several linker proteins

28. Signal Transduction Proteins involved: • Paxillin • Vinculin • Talin • FAK • Src

29. Signal Transduction

30. Fibronectin binds to other ECM proteins • Fibronectin has binding domains for: • Collagen • Heparin • Fibrin

31. Binds to collagen • Binding domain includes type I and type II although FN binds more effectively to gelatin. • What does collagen do? Why might it be important for collagen to bind to fibronectin? • Function: Remove gelatin from blood and tissue. Also, collagen improves cell adhesion. Collagen does not accumulate without FN.

32. Binds to heparin (proteoglycan) • Two major binding domains. One near C-terminal and the other near the N-terminal. • What do proteoglycans do? Why might it be important for proteoglycans to bind to fibronectin? • Function: Fight infections in the area of tissue damage. Also, heparin enhances cell adhesion.

33. Binds to fibrin • Binding domains are Fibrin I and Fibrin II. • What does fibrin do? Why might it be important for fibrin to bind fibronectin? • Function: Augment cell migration and cell adhesion during wound healing.

34. Metastatic tumor – ECM interactions • Metastatic tumors invade basement membranes • Proteins (enzymes) that degrade ECM • Serine Proteases • MMP’s • What is the dominant collagen structure of basement membranes? • Collagen IV http://www.ndt-educational.org

36. Breaking down the ECM • Two MMP’s important for Col IV breakdown: • MMP 2 (Gelatinase A) • MMP 9 (Gelatinase B) • MMP2 and 9 both have 3 tandem repeats of fibronectin type II-like module. • Fibronectin type II-like modules bind collagen IV.

37. Monocytes and ECM remodeling via Fibronectin and MMP’s • Monocytes • Wound healing/tumorigenesis response • Secrete MMP-9 • MMP-9 degrades plasma fibronectin • Fragmented fibronectin greatly enhances cell migration and expression of other MMP’s and pro inflammatory agents. • Cellular fibronectin inhibits MMP-9 expression

38. Tumors and Dedifferentiation • A. Normal pathway. B. Daughter cell fails to proliferate causing a tumor. C. Stem cell fails to create a daughter cell and keeps dividing causing a tumor. www.wikipedia.com/tumor

39. Dedifferentiated Cells and Fibronectin • What do dedifferentiated cells need to proliferate? • What do dedifferentiated cells need to infiltrate a tumor? • Stem cell migration has been demonstrated to be influenced in the presence of fibronectin, Stem Cell Factor has a binding site on fibronectin and has been demonstrated in vitro to influence direction of stem cell migration and stem cell proliferation

40. Tumor vs. Normal Cells • Normal cells grow, migrate, and proliferate using the same processes and proteins as metastatic tumors. So what makes your normal cells different from cancer cells? • Cancer is not a “disease”. Cancer is normal functioning of your cells gone wrong. Normal cells can become cancerous through various ways resulting in out-of-control growth and proliferation.

41. Meanwhile… Prepare yourselves for the following picture

42. Case Study: Continued The tumor is progressing!

43. Final Activity (20 min) • After all standard methods failed, it is up to you to convince Sage’s poor friend’s doctors to choose fibronectin instead of another ECM protein to screen for metastasis! Create a persuasive diagram with material you learned from today’s lecture to sway them. • Diagram all interactions between the following: • Fibronectin • Integrins • MMP’s • Collagen • Heparin • Fibrin • Monocytes • Dedifferentiated cells • For each interaction, list potential contributions to tumorigenesis.

44. For Friday • Think about other situations in which undifferentiated or precursor cells may require signaling and rapid matrix development. • Ask yourself: Is fibronectin all bad?

45. Objectives 1) Learn the basic structures of fibronectin [Level 1: Knowledge] 2) Compare the structure and strength of fibronectin and collagen [Level 4: Analysis] 3) Createan illustration of the interactions of fibronectin and related proteins and their contribution to tumorigenesis [Level 5: Synthesis]