Advanced Molecular Cell Biology - Lecture 23

integrins start to ________ after activation

cluster

what follows after integrin clustering?

focal adhesion maturation: connection to the stress fibres (which contain filamentous actin and motor protein myosin)

they connect to F-actin via talin

what follows Focal adhesion maturation?

Fibrillar adhesion formation, where Talin is replaced via tensin, which are much more stable structures than focal adhesion structures

what are focal adhesions?

The microstructure allowing the connection of integrin to stress fibres

talin has ___________ functions

mechanosensory

What force-generating system is a result of the level of stiffness of the ECM?

• Stress fibres already pull on the ECM

• If they sense more stiffness, then they pull harder on the ECM and vice versa

(Newton’s 3rd law)

What happens to the superstructure of Talin after pulling F-actin?

talin has different domains due to its secondary and tertiary structure

pulling on talin causes the domains to unfold, the more it is pulled the more domains that are unfolded

within the talin structure there is a hidden binding site for _________ which stabilises focal adhesions making them stronger, lead to full _________ to allow _________ adhesion formation

vinculin, maturation, fibrillar adhesion

myosin use _____ to be able to pull on ___-_______

ATP, F-actin

what does FAK (focal adhesion kinase) do?

When activated, it activates small GTPase Rho, which leads to activation of YAP, a transcriptional regulator which is regulated in terms of localisation by mechanical signals

the ECM produced by CAFs (cancer activating fibroblasts) is recognised by ______ __________, which then activates _____ GTPase leading to increased _________, _________ and _________ which are all hallmarks of cancer progression

focal adhesions, Rho, migration, survival, proliferation

what are inhibitors that inhibit ECM production by CAFs?

TGF-beta inhibitors (Pirfenidone, fresolimumab), MMP inhibitors and losartan

What are inhibitors that can reduce stiffness by crosslinking of different polymers of the ECM done by lysyl oxidases?

LOXL2 inhibitors (simtuzumab) (blocks lysyl oxidases)

TGF-beta inhibitors (Pirfenidone, fresolimumab), MMP inhibitors, losartan and LOXL2 inhibitors (simtuzumab) can be used for what diseases?

fibrosis and cancer

What are inhibitors other than LOXL2 inhibitors (simtuzumab), TGF-beta inhibitors (Pirfenidone, fresolimumab), MMP inhibitors and losartan that can be used to treat cancer and fibrosis?

integrin inhibitors (anti-alphavbeta6 and anti-alphavbeta1 antibodies), Rho inhibitors (e.g statin), HA inhibitors (e.g PEGP20) and FAK inhibitors (e.g defactinib)

describe Caveolae:

invaginations of the plasma membrane of cells (i.e endothelial), containing Cavin molecules (this is connected to stress fibers)

What happens to caveolae upon shear stress/increase in membrane tension?

they flatten, providing excess membrane

What do Cavin proteins do upon caveolae flattening?

cavin proteins dissociate from the caveolae, go into the nucleus and modulate transcriptional activity by interacting with other proteins

what is the equivalent of YAP in drosophila?

yki

describe the principal Hippo pathway:

• Membrane regulators and cytoplasmic regulators activate Mst1/2, which is a kinase

• this activates Lats1/2, which phosphorylates YAP

• Scenario 1: YAP becomes ubiquitinated and is destined for degradation

• Scenario 2: 14-3-3 molecules interact with phosphorylated YAP, meaning YAP can not enter the nucleus

• Stress fibres are connected directly to the nucleus via focal adhesions

• on soft ECM, there is little stress force on the nucleus, it will be round and less permeable not allowing YAP to enter

• if force is exerted on the nucleus via stress fibers the nucleus becomes flattened and nuclear pores are stretched open allowing YAP in

• Large actomyosin contractility leads to YAP being able to enter the nucleus; depending on cell type, there are different outcomes: can lead to increased proliferation or stem cell differentiation into osteoblasts

• If the ECM is soft or on elastic pillars or the cell size is small, YAP remains in the cytosol, which can lead to growth arrest, apoptosis or differentiation into an adipocyte.

name cellular functions regulated by the Hippo pathway:

• proliferation

• cell survival

• cell competition

• stem cell maintenance

• metastasis

• regeneration

name cell or tissues properties that regulate the hippo pathway:

• apical-basal polarity

• mechanotransduction

• cell-cell adhesion

• contact inhibition

what is a downstream target of YAP?

CTGF

Latromine A is an inhibitor of?

F-actin

NSC is an inhibitor of?

Rac

Noco is an inhibitor of?

microtubule polymerisation

C3 is an inhibitor of?

Rho

_____ inhibitor and ____ inhibitor have been shown to reduce nuclear localisation of YAP/TAZ

Rho, Rac

when using an inhibitor for Rho kinase which is activated by Rho and myosin light chain kinase inhibitor what could be concluded?

activity of myosin, the regulation of actomyosin contractility is important for nuclear localisation of YAP/TAZ

rigid pillars mimic ___ ECM and elastic pillars mimic ____ ECM, rigid pillars promote ________ ________ of _____/____

stiff, soft, nuclear localisation, YAP/TAZ

if you knock out mechanotransduction in mesenchymal stem cells and place them on a stiff matrix, what happens?

they differentiate into adipocytes