Week 7: ECM Flashcards
extracellular matrix (ECM)
network of macromolecules (proteins/polysaccharides) that are secreted and assembled into an organized meshwork
how is the ECM regulated?
the ECM is dynamically regulated because cells keep making new components or degrading older components ⇒ helps to regulate cell behavior
- Cells control the ECM but the ECM also controls cells ⇒ mutual relationship
how do cells survive?
cells won’t survive unless they bind to a tissue culture dish floor/walls or the ECM ⇒ they divide and grow but stop when the cells are conformed to one another (stop)
anchorage dependent proliferaiton
Most cells want to attach to something but Fully cancerous cells will just grow however and aren’t specific
- Half cancerous cells still want to bind to a substrate ⇒ tissue culture plates are coated specially
are tissue cultures in vitro or in vivo?
The tissue culture mimics the ECM in the body for normal cell behavior => this is in vivo
functions of the ECM (3)
- Scaffold to support and stabilize tissues
- Substrates for cell adhesion and migration
- Regulation of cell survival, proliferation, differentiation, and functions
what are the 3 major macromolecule classes in the ECM?
- Glycosaminoglycans (GAGs) ⇒ sugars
- Fibrous proteins (insoluble) ⇒ collagen family
- Non Collagen glycoproteins
what do we call glycoproteins with unique sugars in the ECM specifically
proteoglycans
proteoglycans
core proteins with glycosaminoglycans
what is special about hylauronan?
it is just a glycosaminoglycan itself because is has no protein core
glycosaminoglycans
unbranched polysaccharide chains composed of repeating disaccharide units ⇒ an amino sugar usually sulfated and an uronic acid
what makes glycosaminoglycans unique? (2)
- Depending on the disaccharide species we classify them different
- Due to sulfate and carboxyl groups, GAGs carry a very high negative charge
4 main glycosaminoglycan groups?
- hyaluronan
- Chondroitin sulfate (aging supplements) and dermatan sulfate
- Heparan sulfate and heparin
- Keratan sulfate
→ 6 different types of GAGs but classified into 4 because some share the same core sugar backbone structure which make it the same repeated disaccharide with modifications
what do many GAGs have?
complex modifications on them where many sugar chains are highly sulfated ⇒ sulfate groups have neg charge
Hyaluronan (hyaluronic acid)
extremely large GAG with no sulfating and no core protein
- Forms a hydrogel in your body for resistance to compressive forces in tissues, joints, and soft parts of the body
what does Hyaluronan bind to?
water molecules (many)
how do GAGs link to core proteins? and what kind of bond is this?
covalently linked to a specific serine residue on the core protein (usually GAGs are 80 residues long)
T/F GAGs immediately start repeating their disulfide after linking to serine?
False; has a tetrasaccharide prior to the repeating
linkage tetrasaccharide
linkage of 4 sugars in proteoglycans that has to happen and be specific before the repeating disaccharide
- xylose - galactose x2 - glucoronic acid
decorin
(CS or DS) binds to collagen fibrils and regulates their assembly
- small with oney 1 GAG
aggrecan
major proteoglycan in cartilage that makes tissue flexible
- has many GAGS including chondroitin sulfate and keratan sulfate
what is the most abundant protein in animals?
collagen
fibrous proteins
major components of skin/bone providing tensile strength to tissues⇒ type 1 is the most common
- 25% of total protein mass and highly regulated for fiber formation
what are collagen molecules made of?
alpha chains that have a triple helix
alpha chains
series of triplet Gly-X-Y in collagen
- X is commonly proline
- Y is commonly hydroxyproline
triple helix
3 alpha chains that form a ropelike structure of collagen
- 40 different collagen molecules formed form different combinations of 3 alpha chains
what type of collagen is a triple helix?
type 1 or 2
- can also be type 4 but that won’t form fibrils
which type of collagen is a fibril association collagen?
type 9 => link and organize fibrils (2) and help with collagen fibrils interactions with other ECM components
which type of collagen is network forming?
type 4 => in the basal lamina sheets
how does collagen formation occur? (9 steps)
- Synthesis of pro alpha chain ⇒ polypeptides at the edges (translation)
- Precursor to collages still
- N terminal shown in green outside of 3 helix
- Red portion is the triplet Gly-X-Y structure - Hydroxylation of Pro/Lys
- Glycosylation
- Self assembly of 3 pro alpha chains
- Monomer finds a partner and automatically forms the triplets - Procollagen triple helix formation ⇒ in golgi end structure
- Secretion ⇒ via a secretory vesicle (still have green portion outside of triple helix structure )
- Cleavage of propeptides by extracellular proteases (collagen) ⇒ green is removed because it prevented the self assembly of larger collagen fibrils and then fibers
- Self assembly into collagen fibril
- Aggregation to form collagen fiber
why is fiber formation not triggered until outside of the cell?
because these are much larger than the actual cells so this cannot be triggered while in the cell or it is catastrophic
where do proteases exist?
only exist outside of the cell which helps the process stay tightly regulated
- cuts N terminus which form fibrils and then fibers of collagen
procollagen vs fibril vs fiber
- pro collagen includes a propeptide sequence on each of its 3 alpha chains => gets cleaves by proteases
- fibrils are made of cleaved collagen molecules self assembling together to form one round band like structure
- fiber is a bunch of fibrils that come together to form a bigger circular structure as the final product
what kind of links form within alpha chains and between collagen molecules of alpha chains?
intramolecular cross linkage between alpha chain parts and intermolecular modified lysine side chains in the formed fibril
Fibril associated collagens
types IX (9) and XII (12) do not aggregate into fibrils and are smaller than type 1/2 because they Mediate interactions of collagen fibrils with other ECM molecules and link the type 2 collagen together
Network forming collagens
type IV (4) that does not form fibrils and instead is composed of 3 alpha chains that interact via terminal domains to assemble into a flexible network that gives tensile strength to the basal lamina
- key component of the basal lamina
where do type 4 collagen molecules connect to one another?
at 7S boxes to make tetramers
multi domain glycoproteins functions (3)
functions to organize the matrix, cell ECM attachment, and guide cell movements
what do multi domain glycoproteins bind to?
have multiple binding sites and many bind specifically to other matrix molecules (collagen, proteoglycans) or receptors on cell surfaces (integrins)
fibronectin
large glycoprotein composed of 2 (heterodimer) similar but not identical subunits held together by disulfide bonds at their C termini
- functionally distinct domains separated by flexible hinge regions and serves as an organizer and connecter of ECM
what kinds of domains do fibronectin have?
collagen binding, heparin (heparin sulfate GAGs), and integrin binding
- Both collagen and heparin binding is associated with different ECM components and orchestrates and stabilizes the structure of the ECM
Also binds integrins which makes it a cell binding site
what is the cell binding domain?
tripeptide RGD (Arg-Gly-Asp) binds to integrins on the surface of cells
- Organizer in the ECM as a connector
- This is captured by alpha beta subunits of integrins
basal lamina
thin, tough, flexible sheet of specialized extracellular matrix
- under epithelial tissues directly surrounding muscle
components of the basal lamina? (4)
- Laminin
- Type IV collages ⇒ network forming
- Nidogen ⇒ small glycoprotein
- Perlecan ⇒ heparan sulfate proteoglycan
laminin
composed of 3 polypeptide chains (alpha, beta, and gamma) arranged as an asymmetric cross
- Binding sites for integrins and other ECM components
- Heterotrimers self assemble into a network
integrin
major cell surface receptors that bind the ECM
Transmembrane heterodimers composed of alpha and beta subunits ⇒ similar to cadherins
difference between integrin and cadherin
integrins are a heterodimer and cadherins don’t form those but bind to one another homophilically
what adaptor proteins connect integrin with actin filaments?
- talin binds to the beta subunit of integrin when active
- kindlin also binds to the beta subunit of integrin but doesn’t touch talin and is on the tail end
- vinculin binds talin to the actin filaments it isn’t already connected to
what allows hemidesmosomes to bind to both collagen on the extracellular side and keratin on the intracellular side?
type 4 collagen binds to laminin which binds to type 17 collagen and integrin on the outside of the cell
- on the inside pectin binds to integrin, BP230 binds to pectin and keratin binds to BP230
what alpha and beta integrin type receptors are responsible for collagen, fibronectin, and laminin?
a1B1 is a collagen receptor
a5B1 is a fibronectin receptor
a6B1 is a laminin receptor
- the binding is determined by the alpha/beta pair
–> different combinations provide specificity of binding particles
Glanzmanns disease?
B3 subunit deficiency; failure of platelets to bind fibrinogen and results in defective clotting and excessive bleeding
Leukocyte adhesion deficiency
B2 subunit deficiency; failure of white blood cells to migrate from blood at sites of infection, resulting in repeated bacterial infections
inactive integrin state
both external and internal segments are unable to bind ligands ⇒ means no binding for either end
active integrin state
both external and internal segments are able to bind ligands ⇒ means binding occurs when even 1 side is activated to activate the other binding site as well simultaneously
outside in activation of integrin
binding of an external ligand to extracellular domain leads to activation of the intracellular domain
inside out activation of integrin
binding of the adaptor protein to intracellular domain leads to activation of the extracellular domain
what is unique about integrin?
they are only active/inactive on both their intracellular and extracellular segments
inside out activation example with platelets
- extracellular signals activate intracellular regulatory signals ⇒ monomeric GTPase Rap1 which activates Talin
- Rab1 unfolds talin so it binds to integrin intracellular domain as an adaptor protein
- Talin also interacts with adaptor proteins (vinculin) to form an actin linkage
→ now has a high affinity for the ligand
Rab1
once activated by GEF, it activates Talin
- GDP to GTP state and binds to talin via RIAM
Talin
when active it interacts with the beta subunit of the integrin to trigger integrin activation
outside in activation example
tissue culture cells in the lab are spread on the plate and cells want to attach to the surface which allows them to grow
- The extracellular binding is integrin mediated for proliferation
- FAK is recruited to the site where integrin makes contact with the ECM and FAK molecules undergo cross phosphorylation which signals Src to bind
- when these are activated the FAK will binds to paxillin and talin on the cytosolic membrane side to activate integrin binding with the ECM
Focal adhesion kinase (FAK)
recruited to the site where integrins are at the site of contact with ECM
Ribonuclease
Short and branched oligosaccharides chains (Proteoglycan)
