CSB331H1 Lecture Notes - Lecture 9: Small-C, Fiberglass, Fibronectin
8 Jun 2018
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CSB311 – Lecture 9: Cell Migration in Vivo: Wound Repair and Dorsal Closure in Drosophila
Metabolic tool called talin – cytoskeleton protein concentrated at regions of cell-substratum contact
Found in high concentrations in focal adhesions
Capable of linking integrins to the actin cytoskeleton either
directly or indirectly by interacting with vinculin and alpha-
actinin
Has a high affinity for vinculin – another cytoskeletal
protein concentrated at points of cell adhesion
Autoinhibited
Vinculin – it can be at adherence junctions
How you inactivate talin and bring in another scaffold called vinculin.
Steps of image:
Rho factor signaling to activate trimeric-GTP
End up with IP3 diacylglycerol (DAG) and calcium is
released from the ER
o Diacylglycerol stays in the membrane
Results in the activation of a GEF that activates RAP1
RAP1 in activate state as result of this GEF, binds to this molecule RIAM, and it tethers to RAP1
When RIAM opens up – it’s job as an adaptor will take up the autoinhibited talin
Talin will interact with PI-2 (red dot) on the plasma membrane and cause it to unfold
Talin’s job is to start activating integrins – interacts with
the beta strand of the integrin
You end up bringing in vinculin
Endogenous tensional forces are very important
The force-dependent interaction between talin and vinculin plays a
crucial role in the initiation and growth of focal adhesions
1. No force added (use magnetic tweezers) – there’s still interaction with adaptors (get a complex), but nothing happens (silent event)
2. All of the sudden, your pulling event on the membrane is causing unfolding of talin – the FERM domain starts to interact with the
integrin results in the unfolding of the entire complex - Now with the unfolding, you have sites exposed for vinculin binding
3. Talin, actin filaments and vinculin brought into the equation
4. If you keep pulling, you can lose association with the GEF and displace the adaptor molecule – so now vinculin and talin get
concentrated around there and it involves more than one integrin. If you
pull too much you start to dissociate it all
Don’t forget about tensional forces!
FAK and talin at nascent and mature focal adhesions
We used to think that talin brought FAK (focal adhesion kinase) to the integrin
engagement site, but it appears to be the other way
When you FAK stain, you see protrusions – when you put it on FN (sticky
molecule that loves integrins) after 15 minutes you see extensions
Eventually you get elongation and stabilizing as a result of clustering of
integrins – as time goes on, mature focal adhesions
If you mutate FAK, you lose talin
Nascent adhesion focal complexes (a little more stable) focal adhesions
fibrillar adhesions (on FN)
Focal complexes observed in vivo; Fibrillar adhesions may not exist in vivo
Migrating cells form initial sensing environment for migration, as they
mature, they increase in complexity of these sites (recruit more proteins)
Scaffold brings in more molecules, their phosphorylation creates docking sites, target signal transduction pathways and affect Rho
When FAK is mutated (second image) there is a loss of talin (not recruited)
History of Wound Repair in a Chick Embryo
A) Cross-section through a closing wound on dorsal surface of a wing bud
Epithelium sweeps forward (left to right) to cover the naked wound mesenchyme – the basal
lamina gets pulled along by epithelium
In the right cortex, epithelial sheets can move (doesn’t have to be EMT)
The major difference between us and the fruit fly is the adaptive immune system – they do not
have an innate immune system
When you’re young, changes are, when you get a cut, it’ll heal without a trace of a scar – the
goal of tissue regeneration is healing with scars (would be an issue if it was heart tissue)
B) Scanning EM of a similar wound
Reveals the superficial layer of the epithelial cells (the periderm) as the sheet moves forward
(upwards) to cover the wound mesenchyme
C) Immunofluorescence confocal microscopy: optical section through basal layer of epithelium
Reveals a bright actomyosin cable in the leading edge cells stained with an anti-myosin
antibody
RhoA inhibitors prevent wound healing.
Drosophila melanogaster (fruit fly): Large number of proteins are structurally & functionally conserved
between Drosophila and humans
GAL4/UAS technique for targeting gene “misexpression” in Drosophila
From yeast – not naturally in Drosophila
GAL4 is a transcriptional activator (very selective for its target sequence)
There is no GAL4 expressed in the fruit fly (no sequence equivalent)
Figure: a transgenic animal is created with two separate constructs inserted in its genome
One insert has an upstream activating sequence (UAS = a yeast specific regulatory
sequence) coupled to a copy of the coding sequence gene G (gene of interest)
The other insert contains the coding sequence of the yeast GAL4, whose product is
a yeast-specific gene regulatory protein that binds to the UAS element
Wherever gene H is normally expressed, GAL4 protein is also made and drives
transcription of Gene G
This GAL4 insert is placed next to, and controlled by the regulatory region of gene H
- GAL4 will be expressed wherever actin is expressed
Clone in your gene of interest (Eg: -GFP-actin or a dominant neg protein) – it will be expressed according to your chosen tissue
specific regulatory sequence
Now you have coexpression – GAL4 will bind to the UAS element(green fluorescent protein) and promote expression of your
protein of interest - Your fly will glow green since actin expression is all over the place
Drosophila melanogaster development
Embryo: dorsal closure occurs near end of gastrulation
(our focus)
Drosophila gastrulation
1. Gastrulation begins
2 to 6. Germ band extension begins
7, Germ band retraction begins – once it retracts it
leaves an opening
8. Leaves amninoserosa exposed (extraembryonic
epithelium)
8 to 10. Note the progression of the dorsal closure of the dorsal aspect of the embryo
10. Dorsal closure complete
Document Summary
Csb311 lecture 9: cell migration in vivo: wound repair and dorsal closure in drosophila. Metabolic tool called talin cytoskeleton protein concentrated at regions of cell-substratum contact. Capable of linking integrins to the actin cytoskeleton either directly or indirectly by interacting with vinculin and alpha- actinin. Has a high affinity for vinculin another cytoskeletal protein concentrated at points of cell adhesion. Vinculin it can be at adherence junctions. How you inactivate talin and bring in another scaffold called vinculin. End up with ip3 diacylglycerol (dag) and calcium is released from the er. Rap1 in activate state as result of this gef, binds to this molecule riam, and it tethers to rap1. Results in the activation of a gef that activates rap1. When riam opens up it"s job as an adaptor will take up the autoinhibited talin. Talin will interact with pi-2 (red dot) on the plasma membrane and cause it to unfold.
