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EMC > Cell Migration Kate Nobes L1-3 > Flashcards

Cell Migration Kate Nobes L1-3 Flashcards

1
Q

What structures of actin are associated with cdc42, rac1 or RhoA

A

cdc42-PAK WASP IQGAP - filopodia, polarity, adhesion formation
Rac1-WAVE IQGAP - lamellipodia, adhesion formation
RhoA-mDIA1 ROCK - stress fibre formation, cell contraction, adhesion turnover

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2
Q

RhoA is a type of GEF from the Rho family of GTPases. It can be blocked by what toxin?

A

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

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3
Q

If RhoA is inhibited what will the shapes of cells look like?

A

They often exhibit long tails incapable of retraction and detachment

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4
Q

RhoA induces formation of stress fibres through binding mDia1. Is mDia1 auto-inhibited or activated in the cell?

A

Auto-inhibited (folded on itself)
Rho-GTP partially activates it
Phosphoinositides are required for full activation

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5
Q

What is the role of Rho Kinase?

A

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

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6
Q

Lamella

A

Devoid of organelles

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7
Q

Contact inhibition of locomotion behaviour

A

In normal cells, contact causes the lamella to stop moving and the cells switch direction to move away from the point of contact

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8
Q

Why do cells migrate?

A

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

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9
Q

Cell migration-tissue repair

Migration is often directional-chemotaxis

A

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

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10
Q

Cell motility in disease/cancer

A

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

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11
Q

How big are the 3 types of protein filaments that form the cellular cytoskeleton?

A

microfilaments/actin= 7-9nm
intermediate filaments= 10nm
microtubules= 24nm

IFs = keratin/vimentin
give cells mechanical strength
down regulated when cells want to move

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12
Q

What end of actin is favoured for growth?

A

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

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13
Q

What is the mechanism for membrane protrusion?

A

“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

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14
Q

What accessory proteins regulate cytoskeletal dynamics?

A

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

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15
Q

What are type of actin cross-linking proteins?

and bundlers
Organising actin filaments

A

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

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16
Q

Adhesions

VINCULIN is a component of an adhesion complex and associated to the stress fibres

A

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

17
Q

What angle does actin polynucleation occur?

A

70degrees

18
Q

How many proteins are in Arp2/3

A

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

19
Q

Why does actin need a capping protein?

A

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

20
Q

What is the VCA domain?

A

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

21
Q

What are filopodia?

A

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

22
Q

What protein prevents capping of actin filaments?

A

Tip complex
VASP (ena family of proteins)
prevents capping and allows continuous elongation of actin filaments

23
Q

What protein crosslinks the filaments in a filopodia?

A

FASCIN
Bundling protein
The bundles of actin in filopodia are able to push the p.memb forward occurring at the tip.

24
Q

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

A

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

25
Q

What are WASp family of proteins?

A

Involved in transduction of signals from receptors on the cell surface to the actin cytoskeleton-activate the Arp2/3 complex to promote actin polymerisation
WASp/N-WASp - autoinhibited
WAVE
ActA(listeria)

26
Q

What activates the WASp complex?

WASp regulation

A

Cdc42 (a Rho family GTPase)

Binds to the CRIB part of the WASp complex so it opens up

27
Q

How is WAVE/Scar regulated?

A

No CRIB domain
SHD binds a complex of proteins
Abi1, Nap1, Pir, HSC300

Regulation by Rac1

Extracellular stimuli>GTPases + PIP2>Rac>WAVE/Scar>Arp2/3

28
Q

What inhibits ADF/Cofilin?

A

LIM-kinase
LIM kinase is activated by PAK
PAK=serine/threonine kinase which phosphorylates LIM-kinase that phosphorylates and inhibits ADF/cofillin

29
Q

Actin stress fibres

A

Thick bundles of microfilaments
Bundled by alpha-actinin
Span cell and anchor p.memb (focal adhesions)
Contain myosin II-so contractile
Allow mechanical force to be applied to cell
Myosin II forms biopolar filament
ATP hydrolysis leads to a power stroke that pulls the actin filaments past each other.

30
Q

Cells integrate extracellular signals to modulate cytoskeletal dynamics via:

A

Rho family GTPases -activated at p.memb > WASp/Scar
Phosphoinositides
Protein kinases egPAK

31
Q

What are the best characterised Rho family GTPases?
Small GTPases
>20 genes in humans

A

cdc42
Rac1
RhoA

Each has several effectors
Effector binding dependent on GTP binding to Rho GTPase

32
Q

For a Rho GTPase what codon is mutated to make it dominant negative?

A 
T17N
Codon 17
Locked into GDP from
N17cdc42
N17 Rac1
N19 RhoA

These can bind GEFs but can’t do anything else-cant become activated - sequester GEFs - inhibit GTPase activation

33
Q

mDia1

A

Leaky cap and binds to barbed end to the growing filament

Helps/regulates actin molymerisation

34
Q

How does RhoA regulate contraction of stress fibres through myosin activity

A

RhoA—>ROCK—|MLC phosphatase—>myosin light chain—>myosin—>stress fibre contraction

ROCK can also directly phosphorylate MLC

35
Q

What can Rho be activated by?

A

LPA-lysophosphatidic acid

36
Q

Why is the MTOC located in front of the nucleus towards the leading edge?

A

MT growth is towards the leading edge
Important for trafficking receptors and cell memb components to the front where most of the dynamic activity is occurring in this migrating cell
We want the vesicle trafficking to occur in direction of migration

37
Q

cdc42, IQGAP, CLIP 170, Nucleus

A

Cdc42 is activated at the leading edge (filopodia formation)
IQGAP is the effector and establishes polarity
CLIP170 is recruited and associated with the growing end of the MT. It captures and stabilises the MT at the leading edge of the migrating cell, therefore there is more vesicle trafficking to the front end

38
Q

What happens in the absence of cdc42?

A

MTs become spontaneously stabilised in all directions
Lamellipodia protrusion is not focussed in one part of the cell.

cdc42 has a role in establishing polarity through MT stabilisation by downstream effectors (IQGAP/CLIP170)

EMC (12 decks)

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