Lecture 24 Cell polarity 2 Flashcards by Rosie Rudin

How are epithelial cells polarised?

Because they have membrane domains:

apical
basal

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What does the very lateral domain interact with? How?

ECM 
Via integrins (TM receptors)

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How do we visualise apical and basolateral domains

Immunofluorescence

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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

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How are the epithelial cells used in immunofluorescence studies generated?

Tissue culture cell lines

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What is meant by planar cell polarity

This means that different sides of the cell have asymmetrical distribution of proteins

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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

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What do defects in planar cell polarity give rise to?

Spina bifida

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Name 3 types of spina bifida

Myelomeningocele
Meningocele
Spina bifida occulta

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Which is the most severe type of spina bifida

Myelomeningocele

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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

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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

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Which is the most common type but mildest type of spina bifida

• Spina bifida occulta

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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

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What diseases result from defects in polarity proteins

Cancer cell metastasis

Wound healing

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Which species has provided evidence on MET and EMT

Drosophila

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What is AmotL2 role in cancer

It disrupts apical-basal cell polarity and promotes tumour invasion

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What is apical basal polarity essential for

Functional epithelia

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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’