External Factors Flashcards
1
Q
What is cell behaviour?
A
The term used to describe the ways in which cells interact with their external environment and their reaction to this, particularly proliferative and motile responses of cells
2
Q
What external influences are detected by cells?
A
- Chemical - hormones, growth factors, ion concentrations, ECM, molecules on other cells, nutrients and dissolved gas concentrations
- Physical - mechanical stresses, temperature, the topography or ‘layout’ of the ECM and other cells
3
Q
What external factors can influence cell division?
A
- All external factors can potentially influence cell division but the ones that are best understood are:
- Growth factor
- Cell-cell adhesion
- Cell-ECM adhesion
4
Q
What is the Basic behaviour of cells in culture?
A
- Initially you get an isolated cell on the culture medium, which will then settle down on the culture surface due to gravity
- It will then spread across the culture medium and it will usually obtain some polarity - it will have a front and a back (the cell has no obvious direction on it)
- The front is usually the motile part
- This is showing a vertical view of how the cell will initially settle on the surface and then it will spread across the surface and acquire polarity
- Then it will become motile
- This is NOT a passive process; you need energy to modulate cell adhesion and the cytoskeleton of the cell, it is an energy dependent process.
5
Q
What is the relationship of Cell-ECM Adhesion with Cell functions?
A
- Only patches of the culture medium are adhesive in this experiment
- The degree to which the cells proliferated on adhesive surfaces of different sizes was observed
- If they suspended the cells in agar (non-adhesive) few cells entered S phase
- If they were able to stick to a small adhesive patch that did NOT allow them to spread out fully, a small proportion of cells proliferated
- If the cells were allowed to stick to a larger adhesive patch, which allowed them to spread out fully - almost all the cells started proliferating
- Throughout this experiment, growth factors were available to the cells so this variable did not affect the proliferation of the cells
- It was therefore concluded that the ability of the cells to respond to the growth factors required them to be adhered and spread
- So natural cell-ECM adhesions are required for proliferation
6
Q
The Importance of Cell Spreading, how can this be demonstrated, how’s it called?
A
- Most cells will stick to fibronectin (matrix molecule that is also found in the blood)
- If you have a defined small patch of fibronectin and put a cell on it, the cell would stick but it cannot spread and it will die of apoptosis
- It is not about the amount of contact but about the structure of it
- If you take the same amount of fibronectin and distribute it over a number of small spots, the cell will be able to spread and it will survive and grow
- So it’s not just adhesion that is required for the cells to proliferate, the cells also need to be able to spread to enable the cells to respond to growth factors and proliferate
- Summary of Cell-ECM Adhesion:
- In suspension, cells do NOT significantly synthesise proteins or DNA
- Cells require to be attached to ECM (and a degree of spreading is required) to begin protein synthesis and proliferation
- Attachment to ECM may be required for survival (e.g. epithelia, endothelia)
The requirement of attachment to ECM for survival is called ANCHORAGE DEPENDENCE
7
Q
Cell-ECM Adhesion Molecules
A
- Cells have receptors on their cell surface, which binds specifically to ECM molecules
- These molecules are often linked, at their cytoplasmic domains, to the cytoskeleton
- This means that there is mechanical continuity between the ECM and the cell interior
8
Q
Integrins
A
- Integrins are the most important of the matrix receptors
- Integrins are heterodimer complexes consisting of alpha and beta subunits
- They bind to the ECM via their heads
- Each of their tail regions cross the plasma membrane and project into the cell
- There are about 10 alpha and 8 beta subunits that form more than 20 known combinations
- Each combination specifically binds a short, specific peptide sequence
- Such peptide sequences are often found in more than one ECM molecule
- e.g. RGD is found in fibronectin, vitronectin, fibrinogen and others
- Intracellularly, the integrins are linked, via actin-binding proteins, to the actin cytoskeleton (most integrins do this)
- Exception: a6b4 integrin complex found in epithelial hemidesmosomes - these are linked the cytokeratin(intermediate filament) cytoskeleton
- Integrin complexes cluster to form local adhesions (most) or hemidesmosomes (alpha-6 beta-4)
- These clusters are often involved in SIGNAL TRANSDUCTION - the integrins are not just an adhesive patch for adhesion of cells, it is also a platform for signalling
- This allows the cells to interpret the matrix composition of the environment
- Many integrins are also designed to bind to specific adhesion molecules on other cells
- This is particularly important in the immune system and blood clotting
Which are the varying degrees of integrins?
- Some integrins bind to more than one ECM molecule
- Some only bind to a single ECM molecule
9
Q
What are the different signals to and from ECM receptors?
A
ECM receptors (e.g. integrins) can act to transduce signals in both directions
- E.g. ECM binding to an integrin complex can stimulate the complex to produce a signal inside the cell
- The signals can be from ‘outside-in’
- The signals can be from the ‘inside-out’
Inside-out integrin signalling:
A signal generated inside the cell (e.g. as a result of the hormone binding to the receptor) can act on an integrin complex to alter the affinity of an integrin (i.e. alter its affinity for ECM binding)
- Integrin complexes could be folded over, in which case they have a low affinity for matrix molecules, so they don’t stick particularly well
- There are signals generated within the cell that makes them unfold and go into a high affinity conformation and become sticky
This is important in the immune system and blood clotting
- Platelets have integrins on their surface, but they are inactive - this is good because you don’t want them to stick to everything
- When we do need to activate the platelets, inside-out signals will activate the integrins so they become high affinity and start to stick
Outside-in signalling:
- A cell can receive information about its surroundings from its adhesion to ECM
- E.g. the composition of ECM will determine which integrin complexes bind and which signals it receives
- This can alter the phenotype of the cells
10
Q
What are the conformational changes that occur during the activation and deactivation of integrins?
A
- The integrin on the left is in the low affinity state
- They can be switched on, into the high affinity conformation, by an inside-out signal (this can be due to an external factor such as a growth factor or hormone)
- Once they bind to the matrix, you get other changes taking place
- The ligand binding also causes a change in conformation
- The legs separate and cytoplasmic signalling molecules can then bind, and that binding will then allow signalling to take place - this is outside-in
- When the integrins cluster, the molecules act on one another and you get signalling
11
Q
What is the importance of the ECM on the phenotypes of the cells, how can you test that?
A
- Mammary epithelium was cultured in a gel matrix (rather than on a flat surface)
- When they cultured the epithelium in a medium containing type 1 collagen - the cells formed balls of cells that were clumpy and loosely associated with each other
- When they did the same thing in a medium with basement membrane proteins (e.g. laminin) - the cells organised themselves into organoids that had a polarised epithelium
- There was an empty space in the middle and the cells had polarity (apical and basement membranes)
- The cells were well differentiated, and they even produced milk proteins
NOTE: all of the other stimuli it required for milk production (e.g. prolactin) was present in both instances, but the cells couldn’t respond in the type 1 collagen but it could respond in the laminin
- This shows that the ECM has a profound effect on the phenotype of the cells
12
Q
Why do cells stop dividing when they reach a high density in a constricted space (how is that called)?
A
- For most normal cell types, when you grow them in culture they require a surface to stick to
- As the cells become densely packed, the rate of proliferation starts to slow down
- When they fill up the space provided, they tend to stop division or it becomes very minimal
- It used to be thought that this cessation of proliferation was due to the cells running out of space
- Then an experiment was conducted where a fresh medium containing all the necessary factors for growth was spread across part of the culture surface and the cells that were exposed to these extra growth factors continued to proliferate
- This showed that it wasn’t the contact with neighbouring cells that was preventing cell division, it was actually the availability of growth factors
- The density of cells was too high so there wasn’t enough growth factor available for cell division - DENSITY-DEPENDENCE OF CELL DIVISION
- This is a competing theory for contact inhibition – that when cells meet each other in a mono-layer, they stop proliferating due to the contact of the cells.
13
Q
Which are the Signals controlling proliferation of tissues, how so we call this relationship?
A
- Growth factors trigger the ERK cascade and cause cell division
- There is cross-talk between ECM and growth factor signalling
- It is the signalling from growth factors and signalling from ECM come together to produce proliferation
- So there are two signals that interact to produce proliferation:
- Growth factor (density dependence)
- ECM (anchorage dependence)
- So growth factor receptors and integrin signalling complexes can each activate identical signalling pathways (e.g. MAPK)
- Individually, this activation is weak and/or transient
- Together, this activation is strong and sustained
- These separate pathways act synergistically (together)
14
Q
What are the different contact interactions between cells, in which cells does it happen?
A
Short-term: transient interactions between cells that do NOT form stable cell-cell junctions (bumping)
- Non-epithelial cells:
- When most non-epithelial** cells collide, they do **NOT form stable cell-cell contacts
- They do not like touching each other
- They actually REPEL one another by paralysing motility at the contact site
- This promotes the formation of a motile front at another site of the cell so the cell moves away in the opposite direction
- This is CONTACT INHIBITION OF LOCOMOTION
- It prevents multi-layering of cells in culture and in vivo
Long-term: stable interactions resulting in the formation of stable cell-cell junctions
- Epithelial, endothelial and neuronal cells:
- Upon contact, some cell types strongly adhere and form specific cell-cell junctions (adherens junctions, desmosomes, tight junctions, gap junctions)
- It also occurs for neurons forming synapses and myocardial tissue
15
Q
Cell junctions revision
A
Junctions are usually arranged as continuous belts (zonula) or discrete spots (Macula).
16
Q
Describe the process of contact-induced spreading of epithelial cells
A
- This property of epithelial cells was observed in culture
- When the cells made contact, there was a mutual induction of spreading
- They would form stable junctions between the cells
- This is CONTACT-INDUCED SPREADING OF EPITHELIAL CELLS
- This allows epithelial cells to be able to promote the type of behaviour that will enable them to form a coherent epithelium
17
Q
How can the cell-adhesion affect proliferation?
A
Cell-cell contact in epithelia can have a strong effect on cell proliferation
- This experiment used cell in culture in normal levels of calcium (physiological levels) and in low calcium (enough to survive but low)
- The reason for removing the calcium is that many of the cell-cell junctions are calcium-dependent
- When you remove the calcium, the cells are still at high density and they are forced to touch one another but they are NOT forming junctions - the junctions have broken down
- If you reintroduce the calcium, the junctions will reform
When the junctions were absent:
- MAPK much more activated
- Decreased level of p27KIP1 (inhibitor of proliferation in the cell cycle)
- HIGH proliferation
When calcium was reintroduced and the junction reformed:
- MAPK inactivated
- Increased level of p27KIP1
- Low proliferation
The calcium switch caused a change in proliferation
- Because calcium is such an important physiological ion and it is important in signalling, it was thought that calcium was responsible for this effect (rather than the change in cell-cell junctions)
- The same experiment was then performed using adhesion blocking antibodies instead
- They got the same effect by adding the adhesion blocking antibodies
18
Q
Describe the Molecular organisation of adherens junctions
A
- These are sort of like the master junctions of cells - they control the formation of other types of junctions
- It consists of a Cadherin (calcium dependent, homophilic cell adhesion molecule) which binds to similar molecules on the adjacent cell
- Intracellularly it binds to b-catenin, which is associated witha-catenin, which, in turn, links to the actin cytoskeleton
- b-catenin was found to be much more important than just being a molecule linking adherens junctions to the actin cytoskeleton
19
Q
What is Adenomatous Polyposis Coli (APC) and which molecule does It interact with?
A
- APC is an inherited form of colon cancer - there are a number of familial forms
The APC gene-product is a protein that is involved in the degradation of beta-catenin
20
Q
Where can the Beta-catenin be found in the cell, what is their role with which mutation can it be linked and how?
A
- What we first knew was that beta-catenin is a molecule that is found at cell junctions and is associated with cadherins so it was assumed that it was all sequestered at the cell membrane
- In the cytoplasm, it was found that beta-catenin is rapidly degraded in a complex that involves the molecule APC (the gene-product that is mutated in APC)
- Normally, any free beta-catenin in the cytoplasm is rapidly degraded but if it accumulates in the cytoplasm, it can associate with LEF-1 to form a complex that acts as a transcription factor
- This beta-catenin/LEF-1 complex then goes into the nucleus and influences gene expression and proliferation
- In adenomatous polyposis coli, the APC mutation reduces the efficiency of degradation of beta-catenin (as the APC isn’t functioning properly)
- This leads to an accumulation of beta-catenin in the cytoplasm, which then led to more beta-catenin associating with LEF-1 –> INCREASED PROLIFERATION
Other cadherin-associated signalling pathways are known to influence contact-induced inhibition of proliferation
- When cadherins are clustered together, you get changes in the activation of some of the small GTPases including Rac and Rho
- They are affected by GTP and they act as switches in signalling
- Changes in these small GTPases can influence proliferation
- You also get association of many growth factor receptors into cell-cell contacts and that can prevent their activation
- So established cell-cell contacts might actually prevent growth factor receptors from being able to respond to soluble growth factors
21
Q
What happens when cells lose their social skills?
A
Under certain conditions cells will lose their behavioural restraints and, as a result, they will:
- Proliferate uncontrollably (lose density dependence of proliferation)
- Are less adherent and will multilayer (lose contact inhibition of locomotion and lose anchorage dependence)
- Epithelia break down cell-cell contacts
- Not hayflick limited - they express telomerase and become immortal
Loss of Contact Inhibition in Cancer Cells
- Normal cells usually form a nice monolayer
- Cancerous cell lose contact inhibition of locomotion and they will pile up on each other
24
Q
Oncogenes and Proto-oncogenes
A
- Oncogene = mutant gene which promotes uncontrolled cell proliferation
- Proto-oncogene = normal cellular gene corresponding to the oncogene
- Examples:
- Ras is a particularly important oncogene - it is mutated in 30% of all cancers
25
Q
Uncontrolled Proliferation of Tissue Cells
A
- In cancer you have usually lost anchorage dependence (ECM binding) and density dependence (growth factor)
- A normal cell would not be able to proliferate if it loses both these signals
- However, a cancer cell doesn’t require either of these signals because one of the signals that is further downstream, has been permanently turned on
- The pathways has been ‘short-circuited’ (uncontrolled proliferation and loss of GF dependency)
26
Q
Benign vs Malignant
A
- Benign - there is too much proliferation, but it is contained in one place
- Malignant - the cells are usually more poorly differentiated, and they invade surrounding tissues
27
Q
Describe local Invasion and Metastasis
A
Cancer
- In addition to deregulated proliferation, a major feature of cancerous tumours is the ability to spread.
- Most human cancers are carcinomas (of epithelial origin).
- To spread, the cells must break away and form a secondary tumour, distant from the primary.
- Mechanism of metastasis:
- Cell-cell adhesion broken down-regulated.
- Cells must be motile.
- Degradation of ECM (via MMPs) to migrate.
- The degree of cell-cell adhesion is an indicator of how differentiated the primary tumour is and indicates its invasiveness and the prognosis.
28
Q
How does a primary carcinoma cell metastasise?
A
