How is planar cell polarity established?
- when an epithelial sheet is being established, there is already a very subtle asymmetric distribution of some of these protein complexes
- It’s not until you get a signal that binds the Fz receptor that you get amplification of polarity - Dsh, Pk, Dgo
- during development epithelia tend to not be so polarised, but as they develop they gain this polarisation - a signalling event
- intercellular signalling event: involves Frizzled protein, affects proteins expressed by both cells (e.g. Flamingo) and other proteins e.g. Strabismus –> form complementary complexes, Fml is expressed on all sides while Fz and Stbm are planar polarised on opposite sides of the cell
How are primary cilia in PCP organised?
Primary cilia:
- important signalling centres for various growth factors (Wnt, Shh)
- Sense the environment
- Some are motile and can move secreted factors (early embryo) (in the node): important because by an unusual mechanism they can actually distribute growth factors from one side of the embryo to the other and are therefore very important for determining left to right asymmetry in the early embryo
- in most epithelial cells they are not motile and what happens during development of PCP is that they often start off in the centre of the cell and as planar cell polarity becomes established, the cilium become localised to one pole - you also find that a lot of the proteins associated with establishing Planar cell polarity are localised where the cilium is (Fz6, Vangl2, Abi2)
- Become localised to one pole of the cell in development
- Regulated by Fz/Dvl pathway
What is the Wnt/PCP signalling pathway?
- Wnt Canonical signalling vs non-canonical signalling
- Frizzled activated Dishevelled to regulate actin cytoskeletal polarity not gene transcription
- Fz looks like a G couple protein receptor but is not
- The Canonical signalling pathway uses two key domains of Dishevelled - DIX and PDZ which inhibit APC, thus allowing Beta-catenin to enter the nucleus and alter gene transcription
- PDZ domain particularly tethers proteins to the cell membrane
- DIX domain - interacts with axin therefore inhibiting complex
non-canonical
- DEP domain together with PDZ interacts with other proteins which ultimately leads to the activation of Rho, the JNK cascade, actin cytoskeleton which regulate planar cell polarity
- different ligands will trigger different pathways from FZ
How do integrins act as cell matrix receptors?
- transmembrane receptors for ECM proteins
- composed of two polypeptide chains (alpha and Beta subunits)
- cytoplasmic attachment proteins (talin, vinculin, kindlin) link Beta-integrins to the actin cytoskeleton
- integrins are by-directional signalling molecules
What are the multiple integrin types?
- a variety of alpha and beta subunits can combine to form integrins with different binding properties
- 24 different integrins are present in humans
- alpha subunit is critical for engaging the specific extracellular ligand, while beta subunit interacts with intracellular ligand
- different alpha subunits give integrins different binding specificities - the combination of beta and alpha subunits determines to what they bind
How do integrins bind ligand?
- alpha and beta subunits combine to generate specific ligand binding sites
- mainly determined by alpha subunit head domain (‘I’ domain)
- can exist in active or inactive states
- active state requires divalent cations (Mg2+, Ca2+)
How is integrin activity managed by the cell?
- activity state is a conformational change
- mediated by:
- internal signal from the cell (in vivo)
- ligand binding (eg RGD peptide) in vitro
What is the difference between integrin avidity vs affinity?
Clustering of integrins can allow functional adhesion by increasing avidity (increase probability that it will engage ligand) but high affinity requires opening of the binding site
What is inside-out activation of integrin activity?
Integrin activation state (hence affinity for ligands) changes in response to: cytoplasmic signals initiated via activation of other pathways
cell receives a signal which activates talin –> engages with beta subunit as a dimer thus activating the beta subunit (and from this the alpha subunit)
Inside-out signalling involves propagation of conformational changes from integrin cytoplasmic domains to extracellular binding site in response to intracellular signalling events (e.g. growth factor signals, actin cytoskeleton changes, integrin phosphorylation, binding/release of proteins to beta-subunit tail)
there are multiple inside out activators (including Rac/Cdc42 –> link between integrins and apico-basal polarity)
inside-out signalling affects cell adhesion, migration and ECM assembly
How does outside-in signalling contrast with inside out?
Once the integrins engage with the extracellular matrix and form a very strong attachment you get what’s called outside-in signalling. Further conformational changes to the integrin occur (not quite clear) but one the things you get is large clusters of integrins forming structures called focal adhesions.
Once you get focal adhesions you get other complexes coming in and then you get what is called a very large signalling complex involved in cell polarity, survival and proliferation, cytoskeletal structure and gene expression
Binding of activated integrins to ECM ligans results in “outside-in” signalling
The integrin alpha and beta chains move apart and expose the inding site for talin on the beta chain
Talin binding leads to formation of focal adhesion complexes
How are integrins involved in cell migration?
- interaction of integrins with their substrates plays an important role in cell motility via generation of cell traction forces (CTF) in combination with actin assembly and disassembly
- focal adhesions need to form for cell migration but at the back of the cell they also need to be broken
- very dynamic
Metastasis
- integrins play roles in cell migration and metastasis
- upregulation of a particular integrin subtype can lead to metastatic ability by allowing the cell to migrate over a variety of substrates
How can integrin activation reorganise the ECM?
Two pieces of tissue containing fibroblasts plated on a collagen gel
Tension on the collagen fibres aligns them - cells will migrate along the aligned fibres
Fibronectin and actin at the leading edge of a migrating fibroblast. The extracellular fibronectin accumulates at focal adhesions and becomes aligned in fibrils parallel to the actin filaments due to integrin binding
What are focal adhesions?
Focal adhesions are sites of anchorage for intracellular actin filaments and extracellular ECM molecules
They also concentrate other signalling molcules
formation of lipid raft containing receptor tyrosine kinases
What is FAK?
Focal Adhesion Kinase:
- ligand-bound integrins recruit intracellular proteins (talin, vinculin, paxillin) –> contraction of the actin cytoskeleton
- contraction promotes integrin clustering
- FAK is recruited by binding to paxillin/talin and Beta-integrin tail
- clustering of integrins results in FAK trans-phosphorylation
- FAK phosphorylation can transduce signals into the cell
- integrins themselves to do not have a kinase domain - need to form a complex, need to recruit other kinases for signalling to occur
- FAK regulates intracellular signalling cascades (eg Ras/MAPK, PI-3K (promotes cell survival), Rac) and formation of focal adhesions
FAK knockout cells have more focal adhesions than normal: this suggests that FAK normally plays a role in focal adhesion disassembly. Many cancer cells have elevated levels of FAK - does this assist their motility or altered survival
How does FAK regulate survival?
- when displaced from the extra-cellular matrix epithelial and endothelial cells undergo apoptosis (anoikis)
- Anoikis in vivo may prevent detached cells from reattaching to new matrices and growing dysplastically
What is the concept of anchorage-dependent cell proliferation?
- suspend cell in agar: probabilty of entering S phase: 8%
- perched on a small adhesive patch, very rarely proliferates, doesn’t spread out much etc: probability of entering S phase = 30%
- spread on big adhesive patch: approx 90% enter S phase, cell spreads out etc etc
How do integrins-FAK regulate proliferation?
- focal adhesions serve to cluster integrins and growth factor receptors
- adhesion dependent G1 progression rquires joint regulation of the ERK/MAPK pathway by integrins and growth factor receptors
- much more efficient because concentrated –> increased chances of binding ligand, phosphorylating each other, more efficient entry into cell cycle
How is cell proliferation dependent on cell shape?
- integrins may act as mechanoreceptors
- tension on the cytoskeleton may combine with growth factor signalling to promote G1 progression (clustering effect?)
- if you grow a cell on a defined amount of fibronectin in a single patch the cell will die via apoptosis
- if you grow it on the same amount of fribronectin distributed in small spots the cell spreads, survives and grows
What is the tensegrity theory?
- cell shape could also change the nuclear matrix via the cytoskeleton?
- integrins can act as mechanoreceptors and facilitate chromatin changes
- Don Ingber
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