Principles of Cell Signalling Flashcards
What are the 5 types of cell signalling?
- Contact dependent (membrane bound)
- Paracrine (acts on nearby cells)
- Autocrine (on same cell)
- Synaptic
- Endocrine (hormones in bloodstream over long distances)
What are the 3 types of receptors?
- Ion gated
- G-protein coupled (GTP binding dependent)
- Enzymatic coupled
How is a signal transduced from an enzymatic coupled receptor?
- Downstream elements activate or inhibit eachother via phosphorylation (kinases)
- Secondary messengers released in response to primary signal activate downstream pathway (e.g cAMP, PIP3, DAG and Ca2+)
- Scaffolding proteins act as adaptors to increase binding specificity
What are the two types of positive feedback?
Moderate - steepen a response to receptor activation
Strong - ‘all-or-nothing’ response creates bistable system
What is the result of moderate negative feedback?
- Dynamic equilibrium which stabilises response
- Less subject to noise
How are the patterns of sea shell shapes explained?
Activator-inhibitor diffusion system can activate itself or activate inhibitor which can inhibit the activator (long range)
How was wnt discovered?
- In screen for cancer genotypes
- Closely related to wingless in Drosophila, therefore named wingless-related intergration site
What are the characteristics of wnt?
- 19 wnt genes in mammals, 7 in drosophila
- Secreted molecule which is modified with palmitoleic acid (PAM region essential) and 2 glycoslylations (glycan regions)
What is the function of wnt signalling?
- Patterning (cell fate)
- Cell proliferation, polarity and adhesion
- Neuronal migration, axon guidance and synaptic differentiation
- Adult tissue regeneration
What is porcupine (Porc)?
- Multipass transmembrane O-acyltransferase located in ER of wnt producing cells
- Produces plamitoylation (PAM region) and maturation
- Loss of function leads to no wnt signalling and retention in ER
What is wntless?
- Sorting receptor needed for wnt secretion in endosomes, golgi and plasma
- loss of function results in retention in the golgi
How is wnt transported?
Through exosomes (extracellular lipid membranes) to receptor cells
What is the receptor for wnt signalling?
- Frizzled (Fz) a 7 pass transmembrane protein (also binds other signals)
- Wnt binds to C terminus via cycteine rich domain
- Activtion of N terminus results in cascade via DVL
What are the 3 wnt signalling pathways
Canonical (majority)
1. Wnt/β-catenin pathway responsible for cell fate specification, proliferation ect.
Non-canonical
2.Wnt/ PCP pathway where PCP is Planar Cell Polarity
3. Wnt/Ca2+ pathway responsible for cell migration and adhesion
What happens to β-catenin without the presence of wnt?
- Phosphorylation by ‘destruction complex’ leading to ubiquitination by β-TrCP for degredation at the proteosome (no gene trancription)
- Secondary role in cadherin junctions
What forms the β-catenin destruction complex?
- Axin (scaffold)
- ACP
- CK1 & GSK03 (kinases)
What happens to β-catenin in the presence of wnt?
- Fz binds to single-pass protein LRP5/6 (lipoprotein receptor related protein) which may then bind to destruction complex via dishevelled (Dvl/Dsh)
- β-catenin not degraded and can transolacte to the nucleus and activate gene trancription with TCF/LCF
- Can upregulate receptors in positive feedback response
What extra step can upregulate wnt/β-catenin signalling?
- Destruction complex endocytosed and recruited into multi-vesicular bodies which fuse with lysosome
- physical separation from β-catenin allows it to be stable
How can wnt signalling be inhibited?
- Dickobf proteins antagonise signalling by binding to LRP
- Wnt inhibitory factor (WIF) also binds to wnt
- Norrin (NDP) and R-spondins (Rspo) antagonise signalling by binding to Fz
How can inducing mutations determine signalling pathways?
- If overides other mutant suggests that it must be downstream
- If produces additive effect ay be a parallel pathway
How does the Wnt?PCP pathway work?
- Acts through receptor Fz and Dsh which activate trimetric G protein which through second messengers dishevelled and Rac produces regulation of actin cytoskeleton remodelling (however still unclear)
- Polarity believed to be established through cells comparing activity of Fz
How does Wnt/Ca2+ signalling work?
- release of Ca2+ from intracellular stores
- G protein actvation of PLC and Inositol 3-P
- Activates protein kinase C (PKC) and CamKII
- Role in gastrulation movements
What is the function of hedgehog signalling?
Morphogen - role in developmental patterning
Mitogen - survival factor and homeostasis of adult tissues
What is the pathology of hedgehog?
Congenital malformations and cancer
Define a morphogen
A secreted molecule which forms a concentration gradient which then determines a specific response (e.g developmental patterning)
Give an example of Hh deterining pattering in development
Transplant of Hh expressing area in chicken wing results in mirror image or wing and additional tissue
What are the 3 vertebrate varieties of Hh?
Sonic hedgehog (Shh), Indian Hedgehog (IHh) and desert hedgehog (Dhh)
What are the characteristics of Hh?
- 45kDa precursor and 19KDa active protein, undergoes autoproteolytic cleavage in the ER
- Palmitate on N-terminal
- Cholesterol on C-terminal essential for spreading
How is Hh produced?
- Skinny hedgehog (Ski) membrane bound O-acyltransferase gives palmitate
- Autoproteolytic cleavage dependent on cholesterol and ubiquitination of HH-C
- Active protein reaches membrane is very hydrophobic
What are the 3 ways in which Hh is excreted?
- Dispatched and secreted to SCUBE2 (transmembrane molecule) which attaches to the cholesterol of Hh allowing it to be released
- Spontaenous formation of lipid structure
- Heparan sulphate proteoglycan (Dally) recruits lipophorin alipoproteins to form lipoprotein particles. Secreted phospholipase C-like protein Notum releases particles from membrane allowing them to travel in exovesicle
What is the receptor complex for Hh?
- Ptc (patched) main receptor
- Ihog
- Boi
- Dally-like protein
What happens to the receptor complex without the presence of Hh?
- Ptc stops smoothened (Smo) from reaching membrane
- Signalosome stays in the cytoplasm and Smo is degraded
- Phosphorylation of Cubitis (Ci) in signalling complex targets for phosphorylation by proteosome
What happens to the receptor complex with the presence of Hh?
- Hh binding to Ptc relieves Smo repression
- Smo and signalosome relocate to membrane
- Phosphorylation of Cos2 (Costal2 main signalling complex) and SuFu
- Releases unphosphorylated Ci in active CiA form can then enter nucleus and activate gene expression
What is interesting about Costal 2?
Has a microtubule motor domain suggesting that it can travel along them
Briefly describe vertebrate segmentation
- After the elongation of the axis formation of somites (paired blocks of paraxial mesoderm)
- Addition of one somite at a time in occilating genetic network termed ‘segmentation clock’
What are the 3 proposed methods by which a morphogen gradient is established?
- Diffusion through extracellular matrix
- Relay where signal is transduced at neighbour
- Protrusions
How can the distribution of a morphogen gradient be studied?
- Create genetically modified group of cells without the receptor, rest wild type, can signal pass these cells?
- In fly tissue yes, therefore diffusion is ruled out
What are cytonemes?
Filopedia-like structures which orient towards the source of a morphogen, causing recieving cells to grow towards the signal
At first thought tobe an artifact when visualising cells deprived of oxygen
How was the link between cytonemes and morphogen gradients established?
- Use of Hh Gal4 to visualise core Hh receptors Igog with RFP
- Filapodia in wing imaginal discs has long protrusions going from sending cells to recieving cells
- Actin-based structures
What are the main cytoskeletal strucutres of the cell?
- Stress fibres on the basal side attach to substratum and maintain shape
- Lamelopodia are protruding structures which propel cell movement at the front and contract at the back
- Filopodia are finger like structures
How is the cytoskeleton regulated?
RhoGTPases - small molecular swtiches which can be GTP bound (activate cytoskeleton effectors to activate remodelling or actin polymerisation) or GDP (inactive)
What regulates the activation and inactivation of RhoGTPases?
GEF - guanine exchange factor, activation
GAP - GTPase activating protein, inactivation
What RhoGTPases do filopodia use?
Cdc42
Describe actin polymerisation
- G actin polymerises in the nuclei to form F actin
- Capping protein can binf to F actin to prevent in from polymerising further
- Profinin can inhibit nucleasation
- R23 can lead to branching event to create complex networks
- Formin enables efficient polymerisation
How are filopodia formed?
- Cdc42 recruits to site of protrusion
- Formation of parrallel actin bundles
- Interaction of actin with the membrane
Where can cytonemes be observed in vivo?
- Formation of abdominal epidermis
- Fromed by histoblasts which are dormant in larvae and divide in pupae formation in dorsal and ventral direction
- Hh signalling in posterior department, Ptch in anterior department
What is observed with regards to cytonemes in the development of drosophila abdominal epidermis
- Cytonemes observed and are pointing towatrds to anterior compartment
- Form projections around 9 calls long
- Found everyhere where there was a Hh activity gradient
- Cytoneme length correlated with gradient length
What evidence is there that Hh is transported along protrusions?-
- When using Ihog marker, dots that move along protrusions
- Evidence that Hh is transported within exovesicles across protrusions
How are exovesicles formed?
- Via endocytic pathway
- Escort complex attaches to endosome to produce late endosome and multi vesicular bodies (needed to transport lipophilic Hh)
- Rab GTPases are important regulators of protein trafficking and are used for markers of different compartments
What is an example of the use of cytonemes in notch signalling?
- Spacing of bristles in the epidermis
- Long protrusions allow lateral inhibition over many cell diameters
Give an example of cytonemes playing a role in wnt signalling
- Wnt8a transported along filopedia-like petrusions during the formation of the zebra fish neuronal plate
- However not in drosophila. Possible diffusion?
What is the role of the notch pathway?
- Cell specification: lateral inhibition, lineage and boundaries
- Segmentation in vertebrates
- Proliferation and growth
What are the receptors and ligands of the notch pathway?
Transmembrane ligands:
- Delta, serrate/jagged
Transmembrane receptor
- Notch
What does notch ligand binding result in?
Two proteolytic cleavages
- Extracellular cleavage by ADAM-family metalloprotease
- Within transmembrane domain- gamma secretase complex – this releases notch intracellular domain (Nicd) where it can then activate target gene expression by interacting with CSL and Mastermind (Mam)
How is the notch receptor synthesized?
- Glycosylation by O-fucosyl transferase which chaperones notch to the membrane
- Glycosylation by fringe is important to binding ligand interactions
- Notch is produced as a pro-protein, must be cleaved by covertase
- Receptor transported to membrane
Which two E3 ligases ubiquitinate notch? What function does this have?
Deltex - recycling of notch for the membrane
Itch - signals for degradation
What does the sending cell express in notch signalling?
- Delta but also the receptor (can interact on the same cell for inhibition)
- E3 ligases (neutralized and mindbomb) are also crucial for endocytosis and actibity of the ligand
What occurs once the notch signal is bound?
- Transendocytosis of the receptor bound with the ligand
- Sending cell exerts pulling force an then cleaves
Describe lateral inhibition
e. g neuronal and epithelial cells
- Both cells have delta and notch, however through random fluctuations one cell will recieve more receptor
- These differences are exaggerated through positive feedback e.g more delta produced inducing neuronal fate
- Contact-dependent method for differentiating cells
How is the spacing of bristle patterns carried out in drosophila?
By lateral inhibition (notch signalling) through cytonemes
What do cells directly interact with and how?
- Each other through tight junctions, usually apically
- The extracellular matric which acts as a scaffold which stabilises tissue and also acts as the substratum upon which cells move, usually basally
What are focal adhesions?
- Integrin mediated adhesions occurring on the basal side of the cell
- Integrin heterodimers bind to ECM creating focal contacts allowing the cell to move forwards
- Transmembrane complex which also interacts with intracellular complex and actin cytoskeleton
What are the 3 main components of the extracellular matrix?
- Proteoglycans and glycosaminoglycans (GAGs)
- Fibrous proteins (collagens)
- Glycoproteins
What is the basal lamina?
- Sheet like struture basally secreted by epithelial cells composed of laminin and type 4 collagen
- Acts as filter and is also structural
- Promotes cell survival and proliferation
- Route for cell migration
What is laminin?
- Primary organiser of the basal lamina
- Heterodimer (with alpha, beta and gamma chain)
- Interacts with glycoproteins, perlecans and itself (to form a network)
What are the properties of integrin?
- Made from single pass transmembrane heterodimer containing alpha and beta subunit
- Coupled to cytoskeleton and bound to ECM
- Different combinations of alpha and beta determine types of ligand bound (different between cell types)
- Folded when inactive, unfolded when active
What structures exist downstream of integrin and what is their role?
- Talin is an important activator of integrin, can bind directly to actin
- ILK (intergrin-linked kinase) phosphorylates a number of targets
- FAK (focal adhesion kinase) has a role in cell adhesion and spreading, allows release of focal contacts
- Alpha actinin bundles actin filaments
- Paxillin functions as scaffold
What is inside-out integrin signalling?
Strength of adhesions and events on cytoplasm modulate effects of integrins on the outside of the cell
Describe the mechanism of inside out integrin signalling
- Signal causes cascade of second messengers, small GTPase RAP1 can then recruit talin to the membrane
- Activation of talin activates integrins
- Clustering of integrins can lead to changes in cell polarity, survival and proliferation
What is outside in integrin signalling
- Typical receptor
- Can mediate cell adhesion, migration and ECM assembly
Describe the mechanism of outside in integrin signalling
- Very complex
- ECM binding regulates cell proliferation
- integrin signalling can also activate Rac and have effects on cytoskeletal remodelling
- Mechanotransduction: integrins mechanically connect the cytoskeleton with the ECM and so forces on ECM may activate signalling – allowing cell to sense its environment and react to it
