1. How can extracellular signals mediate inside-out signaling and activation of integrins?
??
A inside out signal lets the integrins reach out + recruitment of Kindlin and Talin -> they find something to bind and through a conformational change they send an outside-in signal back -> that makes actin filaments attatch
sensation environment & reaction to environment
integrins can regulate their affinity for extracellular ligands. by undergoing conformational changes in their extracellular domains
2. Which important molecular principle allows for the detection of forces by cells? What kind of protein domains are involved?
mechanosensing / mechnotransduction
-> unfolding of Talin -> opens binding site for
vinculin to attatch to it -> vinculin recruits actin fibers -> actin
forec-dependent unfolding of a-catenin:
opens up new vinculin binding sites 6 reinforces linkage between E-Cadherin & actin cytoskeleton
3. What happens to junctional complexes and actin linkage when cells experience tension (i.e. ‘being stretched’)?
How and when does positive strain arise in tissues?
more force -> more actin attatchment
α-Catenin Conformational Change: Under tension, α-catenin associated with cadherins unfolds, exposing binding sites for vinculin.
Vinculin Recruitment: Vinculin binds to the exposed sites on α-catenin, linking the cadherin-catenin complex more strongly to the actin cytoskeleton. This reinforcement helps to stabilize the junction under mechanical stress.
positive strain by stretching, elongation
in cell migration, growth, gastrulation usw..
4. Explain how Src may regulate E-cadherin adhesion and what important consequences bimodal regulation of E-cadherin adhesion by Src has for E-cadherin-dependent tissues.
-> constituively active Src Kinase -> EMT, invasiveness
-> too little and too much Src is bad!
too little : no active recruitment, decreased adhesion, death
too much : Src oncogene, gain of invasiveness
Positive Regulation:
Phosphorylation of Adhesion Complex Components: Src can phosphorylate components of the E-cadherin/catenin complex, such as β-catenin and p120-catenin, which can enhance the stability of E-cadherin at the cell membrane. Phosphorylation by Src can promote the interaction of catenins with the actin cytoskeleton, thereby strengthening cell-cell adhesion.
Actin Cytoskeleton Dynamics: Src can modulate the actin cytoskeleton through various downstream effectors, promoting the assembly and stabilization of adherens junctions, where E-cadherin is located.
Negative Regulation:
Disassembly of Adhesion Complex: Src-mediated phosphorylation can also lead to the disassembly of the E-cadherin complex. For instance, excessive phosphorylation of E-cadherin or catenins can reduce their binding affinity, leading to the internalization and degradation of E-cadherin.
Induction of Epithelial-Mesenchymal Transition (EMT): Src activation is associated with EMT, a process where epithelial cells lose their adhesion properties and gain migratory and invasive characteristics. Src can induce transcription factors like Snail and Twist that repress E-cadherin expression, contributing to the disassembly of adherens junctions.
5. How does the phenomenon of contact-inhibition explain regulation of proliferation by Hippo/YAP/TAZ signaling?
Sensing of open space -> cells need to proliferate!
Cell Density and Contact: When cells are at low density and not in contact, the Hippo pathway is inactive. YAP and TAZ are active and translocate into the nucleus where they promote gene expression that supports cell proliferation and survival.
Cell-Cell Contact: As cells proliferate and come into contact with each other, this triggers the activation of the Hippo pathway. Key upstream regulators, such as the membrane proteins FAT, NF2 (Merlin), and other cadherins, sense the cell density and contact -> phosphorylation of YAP/TAZ binding of 14-3-3 protein = inhibition, Cytoplasmic Sequestration and Degradation
6. Which component of junctions or junctional signaling pathways may be mutated in cancer to uncouple contact-inhibition and proliferation?
YAP/TAZ oncogene always in nucleus
Hippo inhibits YAP/TAZ -> tumor suppressor
adhesion molecules and Src mutation -> signal cell still has contact -> EMT / invasiveness
Cadherins, Integrins, Src, FAK
7. What needs to happen to adhesive junctions when cells undergo apoptosis? How could this feed back into proliferative signals in neighboring cells to maintain tissue homeostasis?
-> loss of attatchment -> 1st mode of action of apoptosis master regulators “caspases” = cut of adhesion -> leading to signal towards neighbouring cells to proliferate and fill the space ASAP also ERK signals survival to the cells so they dont die aswell!
Downregulation of cell-cell contact molecules in apoptotic cells
-> neighboring cells sense lower cell density due to fewer cell-cell contacts
-> lower cell density results in inactivation of Hippo (fewer junctional complexes) and therefore nuclear translocation of yap/taz and binding to tead —> transcription of genes promoting proliferation
8. How does the availability of beta-catenin (think back to your Wnt signaling lecture) and G-actin affect different cellular signaling pathways?
Much ß-catenin cut mean the destruction complex in WNT cant destroy enough -> active WNT gene expression
Much G-actin -> serum response factor -> much force stress -> much actin needed -> release TF
Shifting ß-Catenin from mechanical to biochemical pathways
9. Which techniques allow measurements of mechanical properties or of forces generated by cells and proteins?
hydrogel with fluorescent beads: measure displacement of particles under cellular traction forces
Micropillars: altered rigidity but not surface area of the cell -> measure force per focal adhesion by measuring pulling force on pillars
FRET sensor: elastic silk between with 2 fluorescent proteins at the end, low force no stretching -> high FRET due to proximity, stretching -> low FRET
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