Cell adhesion and communication Flashcards
1
Q
Tell me the common features of junction complexes
A
Common features of junction complexes
- Transmembrane adhesion proteins
- Intracellular link to cytoskeleton
- Extracellular link to outside structures
- Cadherin and Integrin superfamilies
- Cadherins mediate cell-cell attachments
- Integrins mediate cell-matrix attachments
- Some cadherins link to actin forming adherens junctions
- other cadherins link to intermediate filaments to form desmosome junctions
2
Q
Name 4 things that help make a tight junction
A
Claudin
Junctional adhesion molecule (JAMs)
Occludin
ZO
3
Q
What is a claudin?
A
A four-pass transmembrane protein (goes through the membrane 4 times) that constitutes TJ strands
4
Q
Whats a junctional adhesion molecule (JAMs)?
A
A class of cell-cell adhesion molecules with two Ig repeats that localise to TJs
5
Q
Whats an occludin?
A
A four pass transmembrane proteins localised at TJs
6
Q
Whats a ZO?
A
A ZO is a Zonula-occluding family proteins: TJ- undercoating scaffolding proteins
7
Q
What type of binding is present in tight junctions?
A
Homophilic binding is present in tight junctions
e.g. occludins to occludins e.g.
8
Q
What are cadherins and where are they found?
What are they important for?
How can they exist?
A
Cadherins (cell-cell attachment)
- Proteins
- Found in all multicellular animals and Choanoflagellates
- Not present in plants, fungi, bacteria or archaea
- Important component of being an animal
- Can exist as free-living individual organisms or as a colony
9
Q
Label the structure of a Choanoflagellates
A
10
Q
What are cadherins thought to be part of?
What may the presence of them be important for?
A
They are thought to be part of the group of protists from which animals evolved
Presence of cadherin may have been important in this evolutionary process allowing multicellularity
11
Q
What is the name of cadherins derived from?
A
Name derived from the face that they require Ca2+ to mediate cell-cell adhesion
This can be demonstrated in the image…
12
Q
Tell me about the role of Ca2+ with cadherins
A
- Important during embryogenesis to stick cells together
- Loosely held together until the 8-cell stage – then compaction occurs, and cells become tightly adhered
- with cell junctions forming
- Add a chelating agent (EDTA) to remove Ca++ and cells can separate
- Reassembly when Ca++ is added back
13
Q
What were the first cadherins identified and how were they named?
A
First cadherins identified were named based on the cell type they were discovered in
- N-cadherin – nerve cells
- E-cadherin – epithelia cells
- P-cadherin – placental cells
However not restricted to single types of cell e.g. N-cadherin also found in fibroblasts
Within a particular tissue there is diversity in the different cadherins present
14
Q
Tell me the non-classical types of cadherins…
A
15
Q
Phenotypes of cadherin defects
A
16
Q
Tell be about binding between cadherins?
A
- Binding between individual cadherins is relatively weak and the bonds are parallel
- Strength comes from many such links close together (think of Velcro)
17
Q
Why is Ca2+ important for cadherin function?
A
- Flexible hinge regions between the cadherin repeats
- Ca2+ binding to the hinge prevents it flexing
- Removal of Ca2+ also reduces binding affinity at N-terminus
- Destabilisation leads to proteolytic degradation
18
Q
Tell me about the role that cadherins play in tissue organisation?
A
- Classic experiment from the 1950’s (Townes & Holtfreter J. Exp. Zool. 128: 53-120)
- Early amphibian embryo – mesoderm, neural plate and epidermal cells have been disaggregated and mixed
- The cells are able to arrange themselves according to cell type and assemble into structure
- Homophilic attachments between cadherins likely to be key in this reassembly
19
Q
What do changes in cadherin expression help to regulate?
A
Neural tube development
20
Q
What do catenins form a link between?
A
form a link between the intracellular cadherin domain and actin filament
21
Q
What do catenins play a key role in?
A
Key role played by β catenin and/or γ catenin (plakoglobin)
22
Q
What additional things to adheren junctions have?
A
- Adherens junctions have an additional related protein p120-catenin
- If the intracellular domain of cadherin is absent then cell adhesion is weakened
- The link to actin is important
23
Q
Label this adherens junctions
A
24
Q
What does Beta- catenin have an important role in ?
A
wingless/Wnt signalling
25
Tell me the dual role of beta- catenin
1) Intracellular anchor protein at adherens junctions
2) Transcriptional regulator in Wnt signalling
26
What does the breakdown of the adherens junction release?
Breakdown of adherens junction releases β-catenin to move from cytoplasm into the nucleus - there it can affect transcription
27
What does Wnt signalling regulate?
Wnt signalling regulates phosphorylation and degradation of β-catenin controlling its availability to form adherens junctions
28
Wnt signalling and cell adhesion is linked...
What happens in the absence of Wnt signalling?
What happens in the presence of Wnt?
29
What is vascular endothelial cadherin (VE Cadherin) required for?
* Vascular endothelial cadherin (VE Cadherin) is required for endothelial cell survival
* Required for the response to Vascular Endothelial Growth Factor (VEGF)
30
When VEGF binds to a receptor tyrosine kinase, what does it require?
VE cadherin as a cofactor
31
What do integrins function as and where are they embedded?
They function as a dimeric unit and are embedded in the plasma membrane
32
What domains do integrins have?
Intra and extracellular domain
33
What are integrains comprised of?
2 non-covalently associated glycoprotein subunits
34
Tell me the transmembrane proteins of the integrin?
* short intracellular C-terminal
* large extracellular N-terminal domain
35
What do the extracellular domain of the integrin bind?
Extracellular matrix proteins or cell surface ligands of other cells
36
Label this integrin...
37
What does the intracellular domain of the integrin link?
(usually via Talin) to the actin cytoskeleton
38
The role of integrins in Hemidesmosomes
39
Cell adhesions via integrins needs to be what?
dynamic
40
Tell me about cell adhesions via integrains in animals...
* Cells in animals can migrate and so cell adhesions need to be broken and reformed
* Allosteric regulation allows switching between active and inactive states
* As an integrin binds or detaches from a ligand, it affects conformation of both intra- and extracellular domains
41
Tell me about outside-in activation of integrins...
* Binding of an extracellular ligand to an integrin results in binding to the cytoskeleton
* Transmission of a force via the cytoskeleton
42
Tell me about inside-out activation of integrins
* Intracellular regulatory molecules such as phosphoinositide (PIP2) activate Talin
* Causes strong binding of Talin to β integrin chain
* In turn this activates the extracellular domain of integrin to bind extracellular ligands
* PIP2 can be produced in response to extracellular signals.
* Complex crosstalk between different signalling processes
43
Tell me the consequences of defects in integrins
* Humans have 24 different Integrins – combinations of 8 different β chain and 18 different α chain genes
* Various combinations eg α5β1 is a fibronectin receptor, α6β1 is a laminin receptor
* Defects in either the α or β subunits can lead to genetic disorders
44
What are **selectins?**
Subgroup of lectin proteins
45
What type of binding do selectins have?
* Heterophilic binding- bind to molecules of a different type
* Cell surface carbohydrate binding proteins – lectins
46
What do selectins mediate?
Mediate transient cell-cell adhesion in the bloodstream
47
Provide and explain an example of how selectins mediate transient cell-cell adhesion in the bloodstream
* Control binding of white blood cells to the endothelial cells lining the blood vessels
* White blood cells move between the bloodstream and tissues – requires changes in cell adhesion
* Controlling where and when selectins and integrins are expressed regulates movement of white blood cells
48
Label this P-selectin...
49
Name at least 3 types of selectins and where they are
**L-selectin** on white blood cells
**P-selectin** on platelets and endothelial cells activated by an inflammatory response
**E-selectin** on activated endothelial cells
50
In lymph organs, what do endothelial cells express and what are these recognised by?
In lymph organs endothelial cells express oligosaccharides recognised by L-selectin on lymphocytes causes lymphocytes to bind and become trapped
51
At inflammation sites whats expressed and what recognises these?
At inflammation sites endothelial cells express selectins that recognise oligosaccharides on white blood cells and platelets
52
Tell me about ICAMs, VCAMs and NCAMs. What do the names stand for and what type of binding do they do?
* **ICAMs –** Intercellular Cell Adhesion Molecules
* **VCAMS –** Vascular Cell Adhesion Molecules
* **NCAMs –** Neural Cell Adhesion Molecules
* **Members of the immunoglobulin (Ig) superfamily**
* **ICAMs** have an extracellular domain characteristic of antibodies
* **ICAMs and VCAMs –** heterophilic binding to integrins
* **NCAMs –** homophilic binding
53
Label these structures of the NCAM and ICAM...
54
How can multiple forms of NCAM be generated?
By alternative splicing
55
What can NCAMs have a high level of?
How does this effect the charge of the molecule?
Sialic acid chains making them negatively charged
56
If the NCAM has a negative charge, what does this mean?
The negative charge can inhibit cell adhesion
57
Tell me about Cadherins and the Ig superfamily
* Cadherins and Ig superfamily proteins present in the same cell types
* Cadherins have stronger adhesion properties – important for tissue integrity
* NCAM likely to function in fine tuning of structures
* Cadherin mutations usually lethal but NCAM mutations have more subtle effects
58
What does Axon outgrowth rely on?
Axon outgrowth relies on regulation of adhesion, chemotaxis and signal factors
59
Do the Ig superfamily have a role in the creation of a synapse?
yes
60
Tell me about mutations in *Drosophila Fascicilin 2 **?***
* Mutations in Drosophila Fascicilin2 (related to NCAM) have abnormal direction of axon growth
* Fascicilin3 functions in recognition of the target tissue by neuronal growth cones
* Fascicilin3 forms homophilic adhesions.
* Transient expression of Fascicilin3 in motor neurones allows synapse formation with muscle expressing Fascicilin3
* Abnormal synapses can be made by ectopic Fascicilin3 expression
61
Synapse formation is complex with many components
Cadherins, Ig superfamily members, neuroligins and neurexins hold the pre- and postsynaptic membranes together
62
Junctions
63
How do plant cells attach and detach?
64
What do plant cells walls provide ?
* Structural and mechanical support.
* Maintain and determine cell shape.
* Resist internal turgor pressure of cell.
* Control rate and direction of growth.
* Regulate diffusion through the apoplast.
* Carbohydrate storage.
* Defence.
* Source of signalling molecules.
* Cell-cell interactions.
65
What are the components of the plant cell wall?
66
Tell me about cell adhesion at the middle lamella- Pectin rich domain in the plant cell wall
* Role in cell adhesion
* Form a hydrated gel in the cell wall
* Breakdown products, oligogalacturonides (OGAs) function as signalling molecules.
67
What are the different domains of pectin?
* Homogalacturonan (HGA)
* Rhamnogalacturonan II (RGII)
* Rhamnogalacturonan I (RGI)
* Xylogalacturonan (XGA)
Not much known about the enzymes needed for pectin synthesis
68
Cell adhesion via cell wall components?
69
defect in plant cell adhesion
70
Why do we use model systems in cell biology
* A simplified/convenient version of a complex entity.
* The model captures key characteristics of the entity needed to address the task at hand.
* It is not possible/ethical to study what we want directly (e.g., human disease)
71
Model organisms are non-human species used to help understand biological processes
72
Tell me the advantages and disadvantages of single cell organisms- E. Coli (Escherichia Coli)
**Advantages**
* Fast growth in chemically defined media
* Relatively cheap culture media
* Several molecular tools for manipulation
* Extensive knowledge of its genetics and genomics
* Extensive knowledge on its transcriptome, proteome, and metabolome
* Several strains are considered biosafety level 1 (K12)
**Disadvantage**
* doesn’t have a nucleus/chromatin, not all protein modification mechanisms e.g. glycosylation
73
What are the two types of single cell organisms of yeast?
**Simple eukaryotic organism**
* many essential cellular processes are the same in yeast and humans.
* S. pombe resembles animal cells in terms of (division pattern; centromeres; introns; heterochromatin)
* S. cerevisiae is more similar to animal cells in terms of (relative lengths of G1 and G2; synaptonemal complexes).
74
Tell me the advantages of yeast?
* Rapid growth (1.5 or 2.5 hours)
* Non-pathogenic
* Efficient transformation by exogenous DNA
* Efficient homologous recombination
* Simple genetic screens
75
Why are the multinuclear organisms- Nematode worm (Caenorhabditis elegans) Less complex than humans?
* No bones, heart or circulatory system.
* But does share many genes. and molecular pathways with us.
76
What is each nematode worm made up of and what can this be used to study?
* Each worm is made up of about 1,000 somatic cells, a third of which are nerve cells.
* Can be used to study reproduction and development.
77
Why is the mouse Mus Musculus) closely related to humans?
The mouse is closely related to humans with a striking similarity to us in terms of anatomy, physiology and genetics.
78
Tell me about time taken for a mouse being born and giving birth
* The time between a mouse being born and giving birth (generation time) is short, usually around 10 weeks. This means several generations can be observed at once.
* Amenable for genetic manipulation
- Many knock out, knock in and conditional expression lines exist
79
Tell me about mammalian systems cell culture?
* Cells isolated from a system and grown in vitro
* Cheaper to study cells in culture than a whole organism
* Can do more perturbation and measurements
* Most cell lines are transformed
* loose some properties compared to normal cells in vivo – can lead to artefacts
80
**Organoids** are a recent advance, Tell me about their growth?
* in vitro 3D cellular cluster (retain more structure and architecture than other in vitro cells)
* derived from stem cells
* ESCs, iPSCs or tissue resident stem cells
* self-renewal
* self-organization
81
How do you grow and organoid?
82
Modulating cell signalling is key...
What is Matrigel comonly used as?
83
When and what was the first organoid system to be described?
**2009- mouse intestinal organoids** (first organoid system to be described)
Distinct crypt-like and villus-like domains border a central lumen containing dead cells extruded from the constantly renewing epithelial layer.
84
Organoid development through the years
85
What do organoids represent an important bridge between?
Why is this the case?
Organoids represent an important bridge between traditional 2D cultures and in vivo models.
* more physiologically relevant than monolayer culture models.
* far more amenable to manipulation of niche components, signalling
86
Tell me some advantages of organoids?
* Drug efficacy testing / Drug safety testing
* Disease modelling
* Regenerative medicine
* As model systems for basic research: In particular for human biology
87
A simplified view of cell biology
88
At a transcriptional level, how can mRNA be measured?
* in situ hybridisation
* qPCR
89
At the transcriptional level, how can proteins be detected?
* SDS PAGE + Coomassie (A)/ Western blot B
* Immunochemistry
* ELISA
90
Summary of lecture 18 and 19
* Plants and animals stick cells together in different ways
* **Animals –** predominantly via protein interactions
Cadherins
Integrins
Selectins
Immunoglobulin superfamily members
* **Plants –** predominantly via polysaccharides
* There are different types of model’s systems each with their specific advantages & limitations
* Recent advances have generated new types of models to study cell biology
organoids
* When designing an experiment, it is important to consider what you need to measure and what techniques you may use
