Transcript Slide 1

Fibronectin
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Fibronectin exists as a dimer, consisting of two nearly identical polypeptide chains linked by a pair of C-terminal disulfide 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 α4β1 integrins. 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 III 910correspond 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 αVβ3 integrins on the cell surface. The "synergy site"
is in III9 and has a role in modulating fibronectin's accociation with α5β1 integrins.[5] Fibronectin also contains domains for fibrin-binding (I1-5, I10-12), collagen-binding (I6-9), fibulin-1-binding (III13-14),heparinbinding and syndecan-binding (III12-14).[3]
[edit]Function
Fibronectin has numerous functions that ensure the normal functioning of vertebrate organisms.[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 secrete proteases, including matrix metalloproteinases, that digest the plasma fibronectin, and then the fibroblasts secrete cellularfibronectin and
assemble it into an insoluble matrix. 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 α4β1 integrin-binding. These fragments of fibronectin are believed to enhance α4β1 integrins-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 migration duringembryonic
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 cavityand pharynx by potentially pathogenic bacteria.[10]
[edit]Matrix assembly
Cellular fibronectin is assembled into an insoluble fibrillar matrix in a complex cell-mediated process.[11] Fibronectin matrix assembly begins when soluble, compact fibronectin dimers aresecreted from cells,
often fibroblasts. These soluble dimers bind to α5β1 integrin 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 α5β1 integrins.[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 vertebrate androgen 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
promotelung tumor growth/survival and resistance to therapy, and it could represent a novel target for the development of new anticancer drugs.
The curious case of Sage’s
unfortunate roommate:
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?
We must interrupt your regularly scheduled lecture for
emergency purposes.
Now Presenting:
The ‘Dark Side’ of
Fibronectin
Chris Blum
Rikke Brogaard
Sage Foley
Dev Patel
Benjamin Mintz
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) Create an illustration of the interactions of fibronectin and
related proteins and their contribution to tumorigenesis [Level
5: Synthesis]
Fibronectin Structure
Genetic Roots
• Formed from fibronectin (FN) gene
• 20 Isoforms
• Occur through exon skipping and exon subdivision
Structure
• Glycoprotein consisting
of monomers ranging
from 230-250kDa
Module
• Monomers consist of
multiple domains
• Each domain contains
a mixture of 3 different
types of modules
Random Loop
Cysteine
Important cysteine dimers
Important sequence
for cell binding
Dimers
Super important!! Your
cells love this sequence
Disulfide Bonds
Without these bonds there will be no fibers!
Why doesn’t fibronectin bind to
everything all the time?
• Cryptic sites
Type I module
Type III module
Beta sandwich motif
Cryptic sites
Activity 1 (10 min)
For each component of the fibronectin molecule, match the bonds and
their interactions:
Disulfide bond
Module I
Covalent bond
Module III
Hydrophobic Interactions
Module II
C-terminal domain
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.
ECM Assembly
In order to form a matrix, FN must be converted from:
Inactive compacted form → active expanded form.
The exact mechanism behind the conformation
change is currently unknown, but ex. could involve:
• Interaction with other molecules ex. Heparin or
collagen
• pH or ionic strength
The assembly of the Extracellular Matrix
Assembly of the matrix
RGD sequence and the synergy
sequence binds to intergrin
receptor.
Q: Explain synergy
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”
The assembly of the Extracellular
Matrix
Integrin binding to FN
dimer → conformation
change , which expose
more binding sites.
FN-FN interactions →
Tighter connection and
matrix formation.
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
ECM Assembly
How would you explain
the fibril elasticity of
FN?
ECM Assembly
How would you explain
the fibril elasticity of
FN?
• Breakage of hydrogen bonds
between β-sheets in type III
could lead to the unraveling.
• Straightening of FN subunits
could provide extension.
• http://www.ks.uiuc.edu/Re
search/fibronectin/movies/
stretching-fn1.mpg
Fibronectin Receptors –
Integrins
Expression of fibronectin and its
receptors regulates:
• Anchorage
• Polarity
• Ability to migrate
• State of differentiation
http://endometriosis-bible.com/?hop=amyhart
Integrins
http://www.scq.ubc.ca/the-role-ofintegrins-in-wound-healing/
• Trans-membrane protein
• An α and β subunit
associated noncovalently
• β1 subunit = surfaces of most
vertebrate cells, cell-ECM
interactions
• α5β1 binds fibronectin
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/
Integrin Connections
Focal adhesions:
• Migratory and non-epithelial cells attach to ECM
• Clustered integrins interact with bundles of actin
microfilaments via several linker proteins
Signal Transduction
Proteins involved:
• Paxillin
• Vinculin
• Talin
• FAK
• Src
Signal Transduction
Fibronectin binds to other ECM proteins
• Fibronectin has binding domains for:
• Collagen
• Heparin
• Fibrin
• 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.
• Binds to heparin (proteoglycan)
• Two major binding domains. One near
C-terminal and the other near the Nterminal.
• 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.
• 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.
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
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.
Monocytes and ECM remodeling
via Fibronectin and MMP’s
• Monocytes
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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
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
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
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.
Meanwhile…
Prepare yourselves for the following picture
Case Study: Continued
The tumor is progressing!
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:
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Fibronectin
Integrins
MMP’s
Collagen
Heparin
Fibrin
Monocytes
Dedifferentiated cells
• For each interaction, list potential contributions to tumorigenesis.
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?
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) Create an illustration of the interactions of fibronectin and
related proteins and their contribution to tumorigenesis [Level
5: Synthesis]