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Cell signalling > Cell Adhesion > Flashcards

Cell Adhesion Flashcards

1
Q

Why is adhesion to the ECM important?

A 
Anchorage dependent growth
Suppresses apoptosis
Regulates gene. Expression
Organisation of tissues
Angiogenesis
Wound healing and clotting
Migration
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2
Q

What are focal adhesions

A

Connect cell to ECM

Integrins- link ECM to the actin cytoskeleton

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3
Q

4 major families of cell adhesion

A

Homophillic
Cadherins (e-cadherins)
Igs (NCAM)

Heterophillic
Integrins (avb3)
Selectins (p-selectin)o

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4
Q

Function of Integrins in epithelial cells

A

Connect the basal laminate to intermediate filaments, hemidesmosomes
Integrins a6B4, the B4 subunit is larger allowing it to connect to intermediate filaments

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5
Q

Function of Integrins in non epithelial cells

A

Integrins adhesome
Focal adhesions, focal complexes and podosomes
Connects the actin cytoskeleton to the ECM
Binds to RGD in fibronectin
190 adapter proteins,

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6
Q

Ways to visualise focal adhesions

A

GFP stained proteins e.g. Actin and paxillin

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7
Q

Integrin inside out signalling

A

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

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8
Q

Structural adaptors

A

Talin, filamin and tension

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9
Q

Scaffolding adaptors

A

Paxillin kindlin Integrin-linked kinase, src

Used to propagate the signal transduction

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10
Q

Nanodiscs

A

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

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11
Q

ECM and Integrins binding regions

How do pathogens use this?

A

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

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12
Q

Activation of Integrins by glycoproteins

A

Glycoproteins Ia/IIa bind to exposed collagen

Outside in signal that leads to Integrins activation

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13
Q

The B3 Integrins
Where found
Disease

A

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

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14
Q

Cdc42 pathway

A

Par6 -> polarity

WASP -> Arp2/3 -> filo podia

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15
Q

Rac pathway

A

WAVE
Arp2/3
Lamellipodia

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16
Q

Rho pathway

A

Activated by LPA
Rho kinase -> myosin LC pi -> myosin activity -> stress fibre
Formin -> actin polymerisation

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17
Q

PI3K AKT pathway

A

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

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18
Q

Rad Rho cdc42 and Integrins

A

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

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19
Q

ERK + c-Jun

A

FAK, Src and Ras activate MAPK

Increases transcription of c-Jun to promote cell cycle

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20
Q

ERK and Jnk

A

Jnk regulates c-Jun (cell cycle promoter)

CAS and crk regulate jnk

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21
Q

ECM stiffness and Integrins

A

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

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22
Q

Integrins and calcium

A

Ca binds to the MIDAS and LIMBS sites and reinforces the bent active state
Activated in the presence of Mn

23
Q

Integrins activation by talin

A

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

24
Q

Inter membrane clasps

A

Inter and outer membrane clasps hold the 2integrin tail segments together

25
Q

Knockout mice to study Integrins isoforms

A

18a and 8b Integrins -> 24 Integrins
B1- inner cell mass day 5
A4- heart defect day 12
A5- abnormal mesoderm day 9

26
Q

Signalling hubs

A

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

27
Q

Adhesion and disease

A

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

28
Q

Antibodies to measure Integrins activation

A

B subunit has 4 EGF like repeats
Unfolding exposes I-EGF-1
Exposes the epitope for antibody KIM127

29
Q

Myotendinous junction

A

Primary site of force
Transmission from muscle proteins to tendon
Integrins link muscle cells via ECM
Also link tendon cells to ECM

30
Q

Testing talin mutants

A

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

31
Q

Integrins activation in blood clotting

A 
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
32
Q

Talin phosphorylation

A

Talin recognises NPxY in Integrins tail
T144/T150 phosphorylation in the F1 loop can disrupt membrane association
Reduces + charge, stops loop from forming helix

33
Q

Cross talk blood clotting and GPCRs

A

Many GPCRs activate PLCB
IP3 -> InsP3R
DAG + Ca -> CALDAG-GEFI
Many GPCRs could give outside in signalling

34
Q

GTPase signalling cycle

A

GEF- changes GDP for GTP
GAP- hydrolysis GTP
Cross talk- GAPs and GEFs can be created by other signalling pathways

35
Q

RIAM and talin activation

A 
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
36
Q

RIAM and vinculin

A

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

37
Q

Mechanobiology
Mechanosensing
Mechanotransduction

A

Forces contribute to cells and tissues
Sensing- protein conformation altered
Transduction- elicit cellular response proportional to stimuli

38
Q

Forces that act on cells

Affect of stress on a cell

A

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

39
Q

Stretching a talin molecule

A

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

40
Q

Vinculin locks talin in the active state

Vinculin is biphasic

A

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

41
Q

Mechanise siting transcription factors

A

YAP/TAZ recruited
TEAD transcription factors
Transcription of proliferation genes
Explains why a stiffer matrix causes cancer

42
Q

Measuring forces in live cells

A

Pillar arrays- measure displacement of pillar
Magnetic tweezers- movement of magnet/bead
Optical trap- measures movement of point by diffraction

43
Q

What are cadherins

Structure

A

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

44
Q

Cadherins subclasses

Cadherins binding

A

E, P, N only bind to same type
Tryptophan docking between cadherins
Needs calcium to strengthen flexible hinge region

45
Q

Cadherins experiment to show in adhesive junctions

A

GFP tagged cadherins
Or transferred cadherins into leukocytes
Upon calcium addition, epithelial layer started to form
Embryo compaction- 8 cell stage compaction is seen

46
Q

The types of cadherins in development

A

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

47
Q

Cadherins at adherens junctions

A

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

48
Q

Cadherins experiments under force

A

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

49
Q

B catenin moonlighting in Wnt signalling

A

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

50
Q

How does contact inhibition work

A

B catenin is sequestered at junctions
Cannot act in Wnt signalling
Suppresses growth and differentiation

51
Q

Cadherins activity in mesenchymal epithelial transition

A

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

52
Q

A catenin as a mechanosensor

A

Force unfolds the D3a, D3b and D4 domains
Exposes vinculin and alpha actinin sites in D3a
Stabilises junctions under force
Unfolding at 5pN

53
Q

Desmosomes

A

Link to keratin filaments
Specific cadherins- desmoglein and desmocollin
Plakoglobin and plakophillins intact with cadherins
Bind the cadherins to desmoplakin that binds to keratin

54
Q

Gastrulation

A

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

Cell signalling (5 decks)

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