Cell Migration Kate Nobes L1-3 Flashcards
What structures of actin are associated with cdc42, rac1 or RhoA
cdc42-PAK WASP IQGAP - filopodia, polarity, adhesion formation
Rac1-WAVE IQGAP - lamellipodia, adhesion formation
RhoA-mDIA1 ROCK - stress fibre formation, cell contraction, adhesion turnover
RhoA is a type of GEF from the Rho family of GTPases. It can be blocked by what toxin?
C3 toxin produced by clostridium botulinum which also produces botox (botulinum toxin). C3 toxin acts to prevent stress fibre formation and cell migration which has the same effects as dominant negative Rho
If RhoA is inhibited what will the shapes of cells look like?
They often exhibit long tails incapable of retraction and detachment
RhoA induces formation of stress fibres through binding mDia1. Is mDia1 auto-inhibited or activated in the cell?
Auto-inhibited (folded on itself)
Rho-GTP partially activates it
Phosphoinositides are required for full activation
What is the role of Rho Kinase?
Regulates the bundling and contractive activity of stress fibres
It is a downstream effector of Rho and binds to GTP
Phosphorylates and inactivates myosin light chain phosphatase
Therefore myosin light chain is highly phosphorylated and activates myosin which leads to stress fibre contraction
Lamella
Devoid of organelles
Contact inhibition of locomotion behaviour
In normal cells, contact causes the lamella to stop moving and the cells switch direction to move away from the point of contact
Why do cells migrate?
1) embryonic development-body plan/tissues
2) axon guidance-formation of synapses, lamella guides axon to its final destination
3) tissue repair-migration of epithelial cells and fibroblasts
4) immune defence-in response to bacteria/viruses immune cells will migrate
Cell migration-tissue repair
Migration is often directional-chemotaxis
Not just WBCs/leukocytes migrate into wound site but also fibroblasts do and they convert into a cell called a MYOfibroblast-these cells are CONTRACTILE and draw the edges of the wound together
Endothelial cells migrate as a sheet rather than individual cells or the integrity of the wound would be lost
Extend lamellapodia and migrate by pulling forward
Cell motility in disease/cancer
Inflammatory dieases eg arthritis
immune cells migrate to the inflammatory site and don’t resolve/leave
EMC transition-cancer cells move away from tumour mass migrating through the extracellular matrix, squeezing through the epithelial layer of the lymphatics/capillaries/blood vessels
They use the actin cytoskeleton to squeeze through the endothelial cell layer
How big are the 3 types of protein filaments that form the cellular cytoskeleton?
microfilaments/actin= 7-9nm
intermediate filaments= 10nm
microtubules= 24nm
IFs = keratin/vimentin
give cells mechanical strength
down regulated when cells want to move
What end of actin is favoured for growth?
Barbed + end
pointed - end is non-growing end
Filament has molecular polarity which is key for cell migration
Actin filaments are arranged with their growing end just beneath the p.memb
Actin monomers are NT binding proteins
Energy released by hydrolysis destabilises the filaments, ADP actin are preferentially depolymerised
Treadmilling
What is the mechanism for membrane protrusion?
“Brownian Ratchet” mechanism
Growth is preferential at + barbed end and this pushes the p.memb out
For an actin subunit to bind the end of the growing filament-the filaments ‘wobble’ which allows another subunit to bind on
What accessory proteins regulate cytoskeletal dynamics?
Can sever filaments
COFILIN/ADF
GELSOLIN
target ADP actin within the filament
THYMOSIN:actin prevents a monomer of acting from binding to an actin filament - sequestered
PROFILIN:actin allows acting to bind to the + end of the growing filament - polymerisable
binds to actin monomer and catalyses the replacement of ADP for ATP
What are type of actin cross-linking proteins?
and bundlers
Organising actin filaments
SPECTRIN (tetramer) actin filament binding domains
FIMBRIN (monomer) 2 actin filament binding domains v.close to one another. Holds filaments PARALLEL to one another in tight bundles-involved in FILOPODIA
ALPHA-ACTININ (dimer) holds actin filaments in an ANTI PARALLEL way - distance is important since allows myosin motors to sit inb/w and slide the actin filaments
involved in actin stress fibres/actin bundles
Key for pulling up rear of cell
FILAMIN (dimer) - binds actin filaments and holds them at 90degrees to one another-helps form a 2D network of actin filaments in the LAMELLA of a migrating cell
Adhesions
VINCULIN is a component of an adhesion complex and associated to the stress fibres
Formed at base of lamellipodium
contain transmembrane integrins
traction control
adhesions form stress fibres
Adhesions stay where they are as the cell migrates
Adhesion complexes remain stationary with respect to substrate. New complexes form
-focal complexes move to rear or are turned over
Lost at rear of cell and made at front of cell
What angle does actin polynucleation occur?
70degrees
How many proteins are in Arp2/3
7
ARPC1-5 support structure/arrangement/positioning of Arp2/3
Arp2
Arp3
Arp=Actin related protein
This protein is activated at the p.memb
Capping means that actin polymerisation can be focused at the p.memb where Arp2/3 is activated therefore actin polymerisation can be focussed here
Why does actin need a capping protein?
Important as prevents these actin filaments from getting too long and too flexible in order to push the p.memb. forward
It maintains optimal filament length for force generation
What is the VCA domain?
VCA domain binds Arp2 and Arp3 plus an actin monomer to create an actin trimer equivalent that is required for spontaneous polymerisation and growth of the actin filament
It is attached to NPF (nucleation promotion factor)
V-binds actin monomer
A-binds Arp2/3
C-contributes to binding both
What are filopodia?
Thin finger like actin rich protrusions
Highly dynamic
Arise by reorganisation of lamellipodial dendrtic network
Arise from parts of actin that are not capped and become associated with a tip complex-privileged status
These barbed ends drift together and elongate together
What protein prevents capping of actin filaments?
Tip complex
VASP (ena family of proteins)
prevents capping and allows continuous elongation of actin filaments
What protein crosslinks the filaments in a filopodia?
FASCIN
Bundling protein
The bundles of actin in filopodia are able to push the p.memb forward occurring at the tip.
What proteins are needed to recapitulate Listeria motility?
Listeria are a type of bacteria that hijack the actin organisation in cells in order to move around the cells
1) Listeria Act A (surface protein) -activates Arp2/3 complex
2) Actin
3) Arp2/3 complex - nucleates actin filament formation
4) Capping protein - caps barbed ends
5) Cofilin - mediates filament depolymerisation
VASP (anti capping protein) and profilin not needed but greatly enhance listeria motility
