Cell Adhesion Flashcards
Why is adhesion to the ECM important?
Anchorage dependent growth Suppresses apoptosis Regulates gene. Expression Organisation of tissues Angiogenesis Wound healing and clotting Migration
What are focal adhesions
Connect cell to ECM
Integrins- link ECM to the actin cytoskeleton
4 major families of cell adhesion
Homophillic
Cadherins (e-cadherins)
Igs (NCAM)
Heterophillic
Integrins (avb3)
Selectins (p-selectin)o
Function of Integrins in epithelial cells
Connect the basal laminate to intermediate filaments, hemidesmosomes
Integrins a6B4, the B4 subunit is larger allowing it to connect to intermediate filaments
Function of Integrins in non epithelial cells
Integrins adhesome
Focal adhesions, focal complexes and podosomes
Connects the actin cytoskeleton to the ECM
Binds to RGD in fibronectin
190 adapter proteins,
Ways to visualise focal adhesions
GFP stained proteins e.g. Actin and paxillin
Integrin inside out signalling
Ab heterodimers
A has 4-5 extra cellular domains, B has 7
Movements near BA domain and propeller increase affinity
Separation of the cytoplasmic and transmembrane
A domains usually have ca or mg in one cation site, the other two sits are occupied by the acidic residues in the RGD sequence of the ligand
Structural adaptors
Talin, filamin and tension
Scaffolding adaptors
Paxillin kindlin Integrin-linked kinase, src
Used to propagate the signal transduction
Nanodiscs
Model membranes that resemble HDL particles
Small lipid bilayer patch with a membrane scaffold protein to shield from water
Has shown that talon binding is insufficient to activate
May also require kindlin activation
ECM and Integrins binding regions
How do pathogens use this?
Ligands such as fibronectin have an RGD loop that Integrins bind within the third repeat
Some viruses have an RGD on the surface to trick the cells into latching onto them
Activation of Integrins by glycoproteins
Glycoproteins Ia/IIa bind to exposed collagen
Outside in signal that leads to Integrins activation
The B3 Integrins
Where found
Disease
aIIbB3 Integrins found on platelets
Fibrinogen binds one Integrins receptor at each end which allows it to link platelets together
Glanzmanns disease- genetically deficient in B3 bleed excessively
RGD containing peptides can be used as antithromibics during surgery
Cdc42 pathway
Par6 -> polarity
WASP -> Arp2/3 -> filo podia
Rac pathway
WAVE
Arp2/3
Lamellipodia
Rho pathway
Activated by LPA
Rho kinase -> myosin LC pi -> myosin activity -> stress fibre
Formin -> actin polymerisation
PI3K AKT pathway
Phoshorylates lipids -> PIP3
PIP3 recruits the Ser/thr kinase AKT to the membrane
AKT inhibits transcription factors for cell death and can phosphorylase remodelling proteins
Rad Rho cdc42 and Integrins
Adaptors proteins of Integrins e.g. Paxillin and focal adhesions kinase can phosphorylase cdc42 etc to give focal adhesions turnover
Arp2/3 allows actin branching
ERK + c-Jun
FAK, Src and Ras activate MAPK
Increases transcription of c-Jun to promote cell cycle
ERK and Jnk
Jnk regulates c-Jun (cell cycle promoter)
CAS and crk regulate jnk
ECM stiffness and Integrins
Integrins clustering causes FAK (p) to act on B-catenin
B-catenin promote transcription of MYC
MYC signals miR-18a to stop BRCA1 and PTEN
PTEN stops migration and invasion
ECM stiffness promotes cancer
Integrins and calcium
Ca binds to the MIDAS and LIMBS sites and reinforces the bent active state
Activated in the presence of Mn
Integrins activation by talin
Talin binds to the B3 subunit
F727 of B3 embeds in the talin S1-S2 loop
Inter membrane salt bridge clasp loosens
K320 energetically compensates
Talin recruits vinculin to stabilise complex
Inter membrane clasps
Inter and outer membrane clasps hold the 2integrin tail segments together
Knockout mice to study Integrins isoforms
18a and 8b Integrins -> 24 Integrins
B1- inner cell mass day 5
A4- heart defect day 12
A5- abnormal mesoderm day 9
Signalling hubs
Groups of the Integrins adhesome
1-20 groups according to biological activity
Connectivity makes it robust to failures
When proteins with 16+ interactions removed it is still intact
30+ proteins such as Integrins, paxillin, FAK = embryonic lethal
EXCEPTION- Src is most connected but only osteopetrosis, support by other family members
Adhesion and disease
Of 232 adhesome genes, 22% caused specific disease
In the total genome, 11% are linked
Muscle and heart diseases because of the stress in these tissues
Haematological- cytoskeleton in RBCs
Dermatological- need for adhesion for barrier
Antibodies to measure Integrins activation
B subunit has 4 EGF like repeats
Unfolding exposes I-EGF-1
Exposes the epitope for antibody KIM127
Myotendinous junction
Primary site of force
Transmission from muscle proteins to tendon
Integrins link muscle cells via ECM
Also link tendon cells to ECM
Testing talin mutants
Contains 2541 amino acids
Replaced wild type talin with rescue transgenes
E1770A needed for autoinhibition, stops R9 and F3 folding together
Rod domain at 1655-1822 is amphipathic 5 helix
Tyr377 docks into bundle, basic loop interacts with acidic residues in helix 4
R3 has destabilising Thr residues- removal stabilises the domain and lowers vinculin binding
Integrins activation in blood clotting
Main binds to par1 Creates IP3 + DAG IP3 binds to ca channels PKC and CALDEG-GEFI acts as GEF for Rap1 Rap binds RIAM recruits to membrane RIAM binds talin PIPK creates acidic surface for basic talin to stick to membrane Talin switched off when phosphorylated
Talin phosphorylation
Talin recognises NPxY in Integrins tail
T144/T150 phosphorylation in the F1 loop can disrupt membrane association
Reduces + charge, stops loop from forming helix
Cross talk blood clotting and GPCRs
Many GPCRs activate PLCB
IP3 -> InsP3R
DAG + Ca -> CALDAG-GEFI
Many GPCRs could give outside in signalling
GTPase signalling cycle
GEF- changes GDP for GTP
GAP- hydrolysis GTP
Cross talk- GAPs and GEFs can be created by other signalling pathways
RIAM and talin activation
PIPK convert PI -> PIP -> PIP2 PLC converts PIP2 -> DAG and IP3 RIAM only binds folded talin PIP2 in membrane stops autoinhibition Out competes the R9 domain for the F3 domain
RIAM and vinculin
Talin R2-R3 binds RIAM in unfolded
Vinculin binds R3 in unfolded
Unfolding disrupts RIAM and link to rap1
Allows vinculin to bind to actin and talin
When talin is stretched by actin, vinculin allows stabilisation
Mechanobiology
Mechanosensing
Mechanotransduction
Forces contribute to cells and tissues
Sensing- protein conformation altered
Transduction- elicit cellular response proportional to stimuli
Forces that act on cells
Affect of stress on a cell
Stiffer ECM will incur more force -> B catenin
Nucleus changes genes in response to force,
B catenin -> miRNA -> PTEN repressor -> tumour
Shear stress of a cell moving
Stretching a talin molecule
Link from R1 to plate surface
Magnetic bead attached to R3
Jumps as each domain unfolds
Allows experiments in the presence of force
When unfolded to a random coil state, vinculin dissociates
Vinculin locks talin in the active state
Vinculin is biphasic
At 5pN R3 unfolds, switches RIAM for vinculin
Changes nascent adhesions to focal adhesions
Inhibited by - less than 5pN when RIAM dominated
>25pN as this is random coil
Mechanise siting transcription factors
YAP/TAZ recruited
TEAD transcription factors
Transcription of proliferation genes
Explains why a stiffer matrix causes cancer
Measuring forces in live cells
Pillar arrays- measure displacement of pillar
Magnetic tweezers- movement of magnet/bead
Optical trap- measures movement of point by diffraction
What are cadherins
Structure
Several PTMs - glycosylation, phosphorylation, cleavage
720-750 AAs
3-5 extra cellular repeats
Repeats 1-3 have a Ca binding site
N-terminus has HAV sequence for ligand binding
Cytoplasmic c terminal has LSSL that binds beta catenin
Cadherins subclasses
Cadherins binding
E, P, N only bind to same type
Tryptophan docking between cadherins
Needs calcium to strengthen flexible hinge region
Cadherins experiment to show in adhesive junctions
GFP tagged cadherins
Or transferred cadherins into leukocytes
Upon calcium addition, epithelial layer started to form
Embryo compaction- 8 cell stage compaction is seen
The types of cadherins in development
E cadherins- preimplantation, epithelial tissue
P cadherins- trophoblast
N-cadherins - nervous system, cardiac and skeletal
Cells also segregate into different levels of cadherins
Cells may express different subclasses
Block of EP cadherins then the blastula becomes disorganised
Cadherins at adherens junctions
Tail of cadherins link to B catenin
B catenin links to a catenin to F actin and vinculin
Alpha actin in cross links actin filaments
Vinculin VASP complexes can create focal adhesion bundles
PIP2 activates vinculin
Cadherins experiments under force
Cadherins catenin complex only under force
Need force to activate actin binding
Stretching of alpha actinin exposes vinculin binding sites which stabilises the focal adhesion
B catenin moonlighting in Wnt signalling
Wnt -> frizzled, associates with LRP cofactor
GSK3 phosphorylated LRP to bind axin
Means that CK1 and GSK3 can’t phosphorylated b catenin
Translocation to nucleus
B catenin binds to TCF and displaces Gro repressor
Recruits pygo, LGS
Activation of target genes
How does contact inhibition work
B catenin is sequestered at junctions
Cannot act in Wnt signalling
Suppresses growth and differentiation
Cadherins activity in mesenchymal epithelial transition
Part of embryogenesis
E cadherins switched off, allows cells to dissociate from epithelial layer
Seen in gastrulation
Cancer cells over express snail and slug with reduced cadherins
Loss of cadherins increases available B catenin to increase Wnt
A catenin as a mechanosensor
Force unfolds the D3a, D3b and D4 domains
Exposes vinculin and alpha actinin sites in D3a
Stabilises junctions under force
Unfolding at 5pN
Desmosomes
Link to keratin filaments
Specific cadherins- desmoglein and desmocollin
Plakoglobin and plakophillins intact with cadherins
Bind the cadherins to desmoplakin that binds to keratin
Gastrulation
Vegetal pore of the blastula flattens
Movement of cells upwards forming a blastopore and archenteron.
Other cells break free to become a primary mesechyme
More cells break free above the archenteron forming filopodia connections between the ectoderm and the archenteron
The filopodia contract, extending the archenteron
This causes the archenteron to have the endoderm cells that surround the archenteron and the blastopore forms the anus
