Lecture 24 Cell polarity 2 Flashcards
How are epithelial cells polarised?
Because they have membrane domains:
- apical
- basal
What does the very lateral domain interact with? How?
ECM Via integrins (TM receptors)
How do we visualise apical and basolateral domains
Immunofluorescence
How does immunofluorescence work?
- use antibodies to label apical/basolateral proteins
- 1) stain with antibody
- 2) stain with secondary antibody
- 3) sec antibody has fluorescent label
How are the epithelial cells used in immunofluorescence studies generated?
Tissue culture cell lines
What is meant by planar cell polarity
This means that different sides of the cell have asymmetrical distribution of proteins
What is planar cell polarity key for
- Neural tube closure (defects can give rise to spina bifida)
- Co-ordinated cellular movement during development
- Cochlear stereociliary orientation in the ear
What do defects in planar cell polarity give rise to?
Spina bifida
Name 3 types of spina bifida
- Myelomeningocele
- Meningocele
- Spina bifida occulta
Which is the most severe type of spina bifida
Myelomeningocele
What occurs in Myelomeningocele
- Baby’s spinal canal remains open along several vertebrae in the back, allowing the spinal cord and protective membranes around it to push out and form a sac in the baby’s back
What occurs in Meningocele
- Protective membranes around the spinal cord (meninges) push out through the spine; the spinal cord usually develops normally so surgery can often be used to remove the membranes without damaging the nerves
Which is the most common type but mildest type of spina bifida
• Spina bifida occulta
What occurs in Spina bifida occulta
- 1 or more vertebrae don’t form properly but the gap in the spine is very small
- Doesn’t usually causes any problems and most unaware they have it
What diseases result from defects in polarity proteins
Cancer cell metastasis
Wound healing
Which species has provided evidence on MET and EMT
Drosophila
What is AmotL2 role in cancer
It disrupts apical-basal cell polarity and promotes tumour invasion
What is apical basal polarity essential for
Functional epithelia
When is cell polarity lost
In advanced tumours leading to invasive and malignant properties
How is the maintenance of cell polarity described and why?
Highly dynamic
Because interfering with membrane trafficking disrupts polarity
Majority of human cancers are…
Epithelial in origin
80%
What is a hallmark of cancer? Why?
Loss of polarity
It leads to malignancy
The AmotL family are described as … proteins
scaffold proteins
What domains do AmotL contain?
Binding domains for ZO-1 and other junctional proteins
What do AmotL interact with
Actin
Members of the AmotL family are important for what?
Blood vessel formation as endothelial cells are also polarised
What are members of the AmotL family postulated to integrate? why?
Apical polarity
Junctional formation
Actin cytoskeleton
By integrating these signals, they allow the actin cytoskeleton to establish polarity and maintain it too
Expression of AmotL2 in normal breast tissue
No expression of this protein
What is the expression of AmotL2 in early breast and colon cancer?
Very little expression as no staining of AmotL2, cells organised and polarised despite being cancerous
What is the expression of AmotL2 in advanced breast and colon cancer?
high expression of AmotL2 as extensive brown staining, higher resolution shows more disorganised epithelium with lots of little brown spots
What does the first staining experiment suggest?
Suggests AmotL2 correlates with a more invasive phenotype
What does the first staining experiment not suggest?
It does not tell you the function of AmotL2
Why is there a loss of polarity when AmotL2 is overexpressed?
AmotL2 sequesters Par3 (junctional complex protein) and Crb3 (apical protein) to large intracellular large vesicles and so causes these proteins to be in the wrong subcellular location
What are the correct locations of Par3 and Crb3
Par3: Junctional complex protein
Crb3: Apical protein
How was it shown why a loss of polarity occurs when AmotL2 was overexpressed?
In the absence of Dox (no AmotL2 expression) there is apical staining of Crb3 and junctional staining of Par3
If Dox added (AmotL2 expressed), you lose the staining of Crb3 and Par3
This shows you AmotL2 is normally a scaffold protein and an integrator of actin and trafficking of junctional proteins
How would we see if overexpression of p60 affects Par3 dynamics?
Measure the kinetics of Par3 in the presence and absence of AmotL2 using FRAP
Define FRAP
Fluorescence recovery after photobleaching
What does FRAP show about Par3 in normal locations and in vesicles?
Par3 in normal location, it recovers quickly
Par3 is in a vesicle, it recovers slower
What is transepithelial resistance used to measure?
How good/leaky the epithelial layer is
What would happen to TER value if AmotL2 expressed
TER value low
Epithelium leaky
What occurs in absence and presence of AmotL2 if calcium has been added once disassembled
- Recovery is quick in normal conditions
- Recovery is less when overexpressing of AmotL2 poor polarity when the epithelium is leaky
Time for cells to repolarise (visually) in in absence and presence of AmotL2
11hours vs 18 hours
Is there a general disruption of polarity when AmotL2 is overexpressed? How?
Yes
Measuring peaks of fluorescence across the cell showed that proteins (e.g. e.g. ezrin is an apical membrane protein, E-cadherin is a junctional protein, beta-catenin is a basolateral protein and c-Met which is a lateral protein) were disorganised when AmotL2 was overexpressed due to loss of polarisation.
The Golgi complex is also dispersed instead of being located between the nucleus and the apical membrane. This means the rate of which polarity proteins are delivered is disrupted
Are the in vitro effects of AmotL2 mirrored in vivo?
YES
• In A and C, you can see when the cells are not expressing AmotL2, the tumours still form but you still get a lumen so the polarised phenotype is maintained
• When cells overexpressing AmotL2, you get a strongly cancerous phenotype, loss of polarity and you get lots of invasion into the surrounding tissue
What is similar/different about in vivo mice and in vitro
• Rate of tumour growth in both cases are very similar (shown in E)
• What is different is the degree to which they are invasive
- In D, they invade into the tissue and this is quantified in H (amount of DNA from tumour cells in blood stream which is an indication of metastases
CONCLUSION
AmotL2 ‘controls tissue architecture (polarity), responsiveness to invasive cues and, consequently, tumour invasion and tumour growth’
