Cell-substrate interactions 2 Flashcards
What makes the ECM
Proteases - endopeptidases (cleaves proteins)
What is the main protease type in the ECM
Matrix metalloproteinases - 24 of them
Where are matrix metalloproteinases found
Outside and transmembrane
What ion do matrix metalloproteinases need to
Zinc
What do metalloproteinases break down
Anything - not very specific
Are MMPs secreted in their active form and why
No not secreted in an active form - to prevent autodigestion
How are MMPs activated
Pro-peptide cleavage is required for MMP activation
Pro-peptide covers active side.
What inhibits metalloproteases
TIMP - tissue inhibitors of metalloproteinases
What resulted from a TIMP2 knockout
Expected more MMP2 activation but got inactive MMP2 instead.
Why - TIMP is an inhibitor and also needed for activation. MMP2 is activated by MMP14 (via cleavage), TIMP2 links MMP2 and MMP14 on the cell surface.
However too much of TIMP2 is inhibitory by binding to MMP14 which prevents cleavage of MMP2
What activates MMP14
Pro-protein convertases (furin)
pro-MMP2 –> MMP2
What inhibits furin
Alpha-1-antitrypsin
How does cancer use the ECM
Degrades it which helps it metastasise
Where does cancer usually grow
Behind basal lamina - it degrades this to metastasise –> degrades the endothelial basal lamina and spread via the blood
What is zymography
Method to measure MMP activity by denaturing them.
MMPs are active even when completely denatured - this is because it changes MMP shape and exposes the active site.
How does zymography work
Take the cell medium –> concentrate it –> use SDS PAGE –> get staining
The gel may contain MMP substrate so the assay isnt very specific as MMPs arent very specific but can tell MMP type via molecular weight
Give some pros of zymography
Easy
Cheap
Shows all the forms
Quantitative
Give some cons of zymography
Works best for secreted proteins
Requires millions of cells
Not cell-specific
Time consuming
How does fluorescent based work
Put a thin layer of cells on fluorescent matrix with a cover plate - use microscope to look down - can see size, distribution and changes overtime
Give some pros of fluorescent based
Cell specific
Quantitative
Provides localisation
Live
Give some cons of fluorescent based
Requires microscope skill
Doesn’t identified proteases/MMPs - just identifies activity
Fluorescent matrix is expensive
How does quenched based work
Flourescent dye is bound to peptide which MMPs cleave –> when cleaved the dye is quenched and becomes fluorescent –> can quantify how active the MMP is
Give some pros of quenched based
Cheap
Easy
Fast
Quantitative
Give some cons of quenched based
Require plate leader
Dont identify specific proteases
Not cell specific
Limited substrate available
What is the transwell assay
A cell invasion type assay - simulate the process of mets -
Structure -
Cells and matrix
FILTER
Medium containing chemo-attractant
If cells can degrade matrix they will get through into chemo attractant - gives indication of ability and timeframe in vivo
Pros of transwell assay
Easy to preform
Easy to analyse
Cheap
Quick
Cons of transwell assay
Low reproducibility - too busy with too many variables to reproduce
Require chemo-attractant
Not cell specific
Lack of control of process
What is 3D migration and how does it work
Method of looking at cell invasion - looking at cells while they degrade matrix.
Can add MMPs etc to look at result
Give some pros of 3D migration
Very informative
Similar to vivo
Give some cons of 3D migration
Complex to preform and analyse
Require instrumentations and skills
Expensive
Limitations on the possible matrix
What other role do MMP have
WIDER READING - Sternlicht 2000
Cleave cell surface molecules to regulate cell behaviour - wound repair, inflammation and cancer
What role do MMPs have in cancer
WIDER READING - Pozzi et al 2000
May initially increase the initial development of the cancer but overall decrease the severity.
What role do MMPs have in arthritis
WIDER READING - (Mudgett et al. 1998).
Worsen it - stromelysin (MMP) deficient mice.
How many MMPs are there
WIDER READING - Lohi et al 2001
22 in humans
Give an example of MMP regulation regarding collagen
WIDER READING - Vogel et al 1997
Type I collagen acts as a ligand for discoidin domain-containing receptor-like tyrosine kinases that induce MMP1 expression when they are activated by intact collagen and become inactive when they bind MMP1-cleaved collagen
How are MMPs released
WIDER READING - Raza et al 2000
Mostly automatically secreted once translated but In macrophages, plasmin and thrombin induce the secretion of MMP12, but do not alter its rate of transcription
How is E-cadherin important in cancer
WIDER READING - Vleminckx 1991
E-cadherin is a calcium dependent transmembrane CAM and methylation of this occurs which silences it in cancer - this results in loss of cell adhesion allow mets
What is the role of plakoglobin in cancer cells
WIDER READING - Aceto N et al 2014
CAM junction protein that when knocked-down in cancer resulted in less metastatic spread due to stopping cancer cell cluster formation
How was zmyography used to determine laminin and MMP interactions in cancer cells
WIDER READING - Koshikawa 2000
Showed taht MT1-MMP is the trigger for laminin 5 migration as was shown to both be distributed over same area in the colon and breast cancer tissue specimens
What is important to check for transwell assays
WIDER READING -F Trepat et al 2012
essential to check previously if cells are capable to invade the membrane and the Matrigel coating, as there exist cell types which can migrate horizontally very fast but they cannot invade a pore membrane
Why cant prolonged transassays be done
Wider reading
Test agent will equalise between membrane.
