Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Dynamic Cell > 1 (Cell motility+ actin) > Flashcards

1 (Cell motility+ actin) Flashcards

1
Q

Describe the the different types of cell protrusion

A

Lamellipodia
* found on fibroblast and epithelial cells
* Behind the highly
dynamic lamellipodium is a more stable
lamella, which contributes to cell migration by coupling the actin
network to myosin II-mediated contractility and substrate adhesion
* extension via actin polymerisation by arp2/3

Pseudopodia
* found in neutrophils + amoeba

Filopodia
* More important as sensors, looking for guiding information/sites to attach
* Neurons use filopodia
* Has a structure called a growth cone which guides the filopodia to make sure it goes in the right direction
* Filopodium forms from a denser rod of parallel actin filaments
* Filopodia is often used by viruses as a bridge between cells

Invadopodia
* Actin polymerization couples with the
extracellular delivery of matrix-degrading metalloproteases to clear a path for cells through the
extracellular matrix. Aiding invasion through tissues.
* frequent in cancer metastasis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Differentiate between G-actin and F-actin, with context.

A

G actin are monomeric actin proteins, whereas F-actin is actin that has been polymerisied into a chain.

Polymerisation of G actin into f actin is found at the leading edge.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What molecule binds to f-actin but not G-actin

A

Phalloidin, from the death cap toadstall

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What would be a good molecule to test if actin filaments are needed for protrusion assembly?

A

Cytochalasin D is used, which blocks actin polymerisation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How is the treadmilling nature of the lamellipodium shown in the lab?

A

Photobleaching using UV light, with polymerisation occuring at the leading edge, and shifting back.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What attach cells to the extracellular matrix, and what are they made from?
How would you show this?
What happens if focal adhesions don’t stick properly?

A
  • Cells use focal adhesions to attach to extracellular matrix
  • Focal adhesions are made from proteins and integrins
  • you could use GFP-paxillin, a molecule which binds to focal adhesions
  • Ruffles are created, where adhesions haven’t stuck down properly
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are stress fibres made from?
How are they used in cell motility?
How would you show that this is how they work?

A

Actomyosin cables, actin + myosin 2 that walk in opposite directions. They are anchored to the ECM via focal adhesions, the focal adhesions at the rear release, the actomyosin cables pulls the rear in.

You could use a myosin inhibitor like blebbistatin to show that it is myosin that pulls the rear in.

Contain:
* Long unbranched actin filaments
* Myosin 2, a motor protein
* A-actinin, crosslinks actin to stabilise
* Tropomyosin, prevents depolymerisation of actin

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

How do amoeboids migrate/form protrusions?

A
  • Has a layer of actomyosin around the membrane creating pressure
  • Actomyosin breaks forming a protrusion similar to lamellipodium but lacking in actin
    Actomyosin then reforms inside the bleb
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Describe Nucleation, elongation and steady state of acti in relation to rate.

What is the critical concentration?

A

Nucleation
* Formers of dimers and trimers during nucleation is kinetically unfavourable so is slow
* doesn’t occur in the cell without a trigger

Elongation
* Elongation rate is proportional to the amount of actin monomers

Critical concentration:
Where monomer addition = rate of monomer dissociation.
Usually due to low concentration of actin monomers, doesn’t really happen outside of vitro

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What does profilin do?

A

Profilin binds to actin monomers, but not filaments.

  • this makes polymerisation polar as profilin bound monomers can only bind to the plus ends
  • profilin monomers swap ADP for ATP
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Describe the role of ARP2/3 in actin polymerisation.

A

ARP2/3 binds to the side of existing actin filaments and nucleates

  • Inactive Arp2/3 involves separated arp2 and arp3
  • This makes it easier, as forming a dimer is often the hard part
  • In the Active state the two come together
  • Profilin-bound actin is the incorporated
    Filament grows with the arp at the minus end
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What stops actin filaments growing indefinetly after nucleation?

How are filaments then depolymerised?

A
  • Capping proteins
  • They are depolymerised by ADF/cofilin (actin depolymerising factor)
  • ADF/Cofilin binds and severs ADP bound Actin, but not ATP actin
  • Meaning the old actin molecules are targeted, so depolymerization occurs at the minus end
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What do filopodia do?
Describe how filopodia form.

A
  • Used by cells as sensors to explore the environment
  • contain long unbranched actin filaments

Convergent Elongation
- Tip complex associates with a subset of actin filaments preventing them from being capped
- filaments grow and coalesce
- cross linked by actin bundling proteins, like Fascin

Tip Complex
- Ena/VASP proteins
- anti-capping

Actin bundling proteins
- Contain at least 2 f-actin binding sites
-

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is the role of Ena/VASP proteins?
What difference does the concentration of Ena have?

A

They form the tip complex and prevent capping.
-They regulate lamellipodia actin network

  • Low Ena means shorter but stronger so it can push through a more viscous environment
  • High Ena means longer filaments but weaker pushing force so can’t push through a viscous environment
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What are formins, and what does it mean for actin filaments?

A

A family of proteins including DAAM and mDia. These can nucleate plus ends of actin filaments, meaning filopodia can form without Arp2/3 complex or even a lamellipodia network.

  • formins make long unbranched actin filaments
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are stress fibres made from?

A
  • Long unbranched actin filaments
  • Myosin 2, a motor protein
  • A-actinin, crosslinks actin to stabilise
  • Tropomyosin, prevents depolymerisation of actin
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

See if you can remember what these all do.

Profilin
Arp2/3
Capping protein
ADF/Cofilin
Ena
Fascin
Formins
Myosin Il
Tropomyosin
a-Actinin

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Check you can do these
(correlate to colour)

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What is the role of Rho GTPases in actin network regulation?

A

Rho GTPases are molecular on/off switches

The active state is GTP bound, the inactive GDP bound.

IN the active state it can bind and regulate other proteins.

GEF (guanine exchange factor) is responsible for swapping GDP to GTP.
GAP (GTPase activating proteins) convert GTP to GDP.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Desrcibe the role of FRET in sensing when GTPases are active

A

FRET (fluorescent resonant energy transfer) as a biosensor

  • These constituents form a singular protein
  • If Rac is activated, GDP is swapped to GTP and binds to a protein
  • This brings GFP and RFP close together which allows FRET so red fluorescence given off
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Describe the process by which listeria move within cells

A

They induce actin polymerisation and get pushed around.

  • uses ActA, a transmembrane protein in listeria
  • it induces confirmation change bringing ARp 1 and Arp2 together to create the active Arp2/3 that nucleates actin polymerisation.
  • aided by anti-capping proteins recruited by ActA

ActA has a:
* Proline rich domain that binds Ena proteins
* WH2 domain that binds actin monomers
* Acidic domain that binds Arp2/3

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Describe the role of Rho GTPases as co ordinators of the actin cytoskeleton

A

Rho, Rac and Cdc42 are Rho GTPases.

  • They regulate the activity of many binding proteins
  • Rac and Cdc42 dominate the leading edge whereas Rho dominates at the rear
  • Rac activates actin assembly and inactivates filament disassembly

Cdc42
* Cdc42 activates filopodia formation via both formin and Arp2/3 dependent pathways
* binds to formins mDia and reveals nucleation site
* Inactivates ADF/cofilin: Activates PAK -> activates Lim kinase -> inactivates ADF/cofilin

Rho
* stress fiber formation
* Rho activates ROCK which activates the motor activity of myosin 2

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

How do invadopodia differ from filopodia or lamellipodia in cell migration.

A

Actin polymerization couples with the
extracellular delivery of matrix-degrading metalloproteases to clear a path for cells through the
extracellular matrix. Aiding invasion through tissues.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Describe Wiskott-aldrich syndrome

A

It is recessive x linked disorder that causes immune deficiency and eczema.

This is caused by a mutation in the WASP gene. WASP family of proteins cont WH2 domains and acidic domains similar to that of ActA and can activate the Arp2/3 complex. This causes actin polymerisation.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

How does WAVE activate Arp2/3?

A

WAVE is a human gene that is normally bound to an inhibitory complex.

Binding of Rac GTP releases WAVE from the inhibitory complex, allowing it to activate Arp2/3.

It is able to activate Arp 2/3 because it contains a WH2 domain which binds actin monomers and a acidic domain which binds ARp2/3.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

How does Rac promote lamellipodia formation?

A

It can inactivate ADF/Cofilin (which depolymerises).

1) Activates PAK
2) Which activates LIM kinase (phosphorylates)
3) Which inactivates ADF/Cofilin

It can activate WAVE (which activates Arp2/3)
1) Binding of Rac-GTP to the inhibitory complex releases WAVE allowing it to activate Arp2/3

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

Describe the ways in which Cdc42 leads to filopodia formation

A

Activates N-WASP (which activates Arp2/3)

  • N-WASP is usually in an autoinhibited conformation
  • Cdc42 binds, which changes it into its active conformation, revealing WH2 site and acidic domain
  • allows activation of ARP2/3, which nucleates filopodia formation

Activates Formin mDia

  • Cdc42 binds to autoinhibited conformation of formin mDia, turning to active confirmation
  • reveals nucleation site

Inactivate ADF/Cofilin (same way as RAC does)
1) Activates PAK
2) Which activates LIM kinase (phosphorylates)
3) Which inactivates ADF/Cofilin

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

What is Rho?

A

A family of gtpases.

They are master regulators of the actin. Molecular on/off switches. Rho, Rac and Cdc42 are all members of of the Rho family.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

How does Rho promote stress fibre formation?

A

Rho activates ROCK which activates the motor activity of myosin 2.

Activate forming mDia and inactivate adf/cofilin also helps.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

How do GEFs aid Rho activity?

A

They catalyse the exchange of GDP for GTP, which is the thing that activates them.

31
Q

Why do cells need to move?

A
  • Morphogenesis
  • Wound healing
  • Immunity
32
Q

What is a good model organisms for studying cell motility?

A

Drosophila
* Transparent, view cell movements
* Lots of processes that require cell motility
* Easy to use genetics to interfere with protein functions

33
Q

What is morphogenesis?
Name some examples.

A

The process by which an organisms and its tissues develop shape.

Epithelial closure
* Tissue morphogenesis, neural tube closure, palate fusion
* dorsal closure (i.e in drosphilla)
* Wound healing
* Failure: Birth defects, spina bifida, cleft lip/palate

  • Tissue lengthening
  • Branching in lungs
34
Q

Name a good experimental technique for visualising epithelial closure.

A

Expressing GFP-actin, so that the actin is fluorescent green, so the filopodia can be seen.

35
Q

How does epithelial closure occur and how could this be demonstrated?

A

Filopodia reach across and pull together tje and actomyosin ring contracts to create a taught and organised edge. In a wound, this actomyosin ring contracts and cell cell adhesions join the sides together.

  • You would need to make sure any knockouts/flourcent tagging is specific
  • knocking out Ena as that is only found the filopodia
  • tagging with GFP-fascin as that is also only found in filopodia
  • knocking myosin 2 so that can determine importance of actomyosin ring
36
Q

Why are filopodia important in dorsal closure?

A
37
Q

In wound healing, an actomyosin ring contracts to join the wound edges. How would we show this experimentally?

A

We would need to block Myosin 2, to show the processes dependence on it.

Rho is a GTPase which helps form the actomyosin cable via myosin 2 and mDia. Blocking Rho therefore blocks actomyosin cable formation.

* No longer joins neatly
* Back up plan of using lamellelpodium to reach across but only works in small wounds
38
Q

How do the different Rho gtpases affect haemocyte migration? ( Rho, Rac, Cdc42)

A

Rho
* activates ROCK which activates the motor activity of myosin 2. Without the contraction of myosin 2, the cell cant retract the rear. Rho is found mainly at the rear.

Rac
* Rac activates actin assembly and inactivates filament disassembly, without then no actin filaments to push forwards on the lamellipodia

Cdc42
* Cdc42 activates filopodia formation via both formin and Arp2/3 dependent pathways
* No filopodia means cant sense environment, so not sure which way to go eh

39
Q

Describe Chemotaxis in:

  • neutrophils
  • bacteria (Dictyostelium)
  • Development
  • Cancer
A

Neutrophils
* Prokaryotes begin protein synthesis with N-formylmethionine, neutrophils detect tiny amounts of N-formylated peptides
* They use GPCR called formyl peptide receptor 1

Dictyostelium
* soil amoeboid that has GPCRs that detect cAMP

Development
* collective migration of cells
* i.e. lateral line primordium in zebrafish

Cancer metasitis
* varied chemoattractant and receptor pairs
* govern their movement
* interact with macrophages

40
Q

Compare the models cells use for chemoattraction in high gradient and low gradient.

A

Strong gradient

  • Compass model
  • Leading edge follows the closest part of chemoattractant
  • Is due to high receptor occupancy at the leading edge

Weak Gradient
Bifurcation and bias model

  • Moves towards the chemoattractant gradually
    But low receptor occupancy means lots of noise and the pseudopod cell splits at times trying to go a different direction but the one closest to the chemo… prevails
41
Q

Explain the role of GPCRs in chemoattraction

A

GPCRs get activated by the chemoattractant.

  • activated GPCRs promote local PIP3 generation (by dissociating G beta gamma from the heterotrimeric g protein allowing it to turn PIP2 into PIP3)
  • PIP3 helps recruit GEFs such as p-rex1 in mammals, which aid Rac-GTP loading
  • Rac GTP promotes lamellipodia formation via SCAR/WAVE (Arp2/3…)
  • Experiments showed it was more complicated
    Could recognise chemoattractant without PIP3 but with less efficiency
42
Q

What is a good way of measuring Rho gtpases? (Rho/Rac/Cdc42)

A

FRET

  • encode the gtpase alongside a protein that binds to the activated gtpase, sandwiched between two probes (GFP + RFP)
  • When activated the protein will bind to the gtpase, causing conformational change bringing the two probes next to each other
  • this enables measuring of activity via how far the probes are from each other.
43
Q

Describes the roles of Steering, Engine and retraction in relation to the Rho Gtpases Rho, Rac and Cdc42.

A
44
Q

Describe Haptotaxis and Durotaxis

A

Haptotaxis refers to cells moving in a direction as a response to matrix ligands.

High ligand density makes it easier to form CMACs, so move in that direction over time. If a cell has fibronectin-binding integrins then it will move towards fibronectin but not laminin or vitronectin.

Bell curve response to rate of cell migration against ligand density

Durotaxis
* Moving from soft matric/surfaces to stiffer ones
* Can stabilising CMACs on stiffer substrate easier, can mature them
Allows pertrusions in one directions
* pulling repeadtly on the ECM from the CMACs allows cells to determine stiffness gradient

45
Q

What is required for successful directional migration?

A
  • Large, mature adhesion complexes
  • successful turnover of adhesion complexes
  • levels of adhesion high enough to generate traction but not too high so they cant turnover
46
Q

How are cells linked to the ECM?

A

Via cell matrix adhesion complexs;

  • Integrins bind to the ECM extracellularly and to adaptor molecules intracellularly
  • Adaptor molecules like talin and vinculin bind to the integrin cytoplasmic tails and connect to F-actin,
  • Paxillin and FAK are other adaptor molecules and these recruit signalling molecules, such as Rho gtpase activors/inactivators
47
Q

How are integrins activated?

A

Talin binds to the integrin tails causing a conformational change to the primed conformation.

To fully activate it needs to bind to an ECM ligand (which stabilises the alpha helix domain) and receive some force.

48
Q

What are the purposes of integrins?

A
  • The connect the ECM to the intracellular cytoskeleton
  • they form receptors for a range of different extracullar matrix proteins such as collegen and lamins and any tri peptide motifs
49
Q

What causes ligands to form adhesion complexs

A

Nucleated by integrin ECM binding and ligand induced clustering of integrins promotes assembly of adhesion complexs via recruitment of adapters.

50
Q

What are the 3 intermediates in adhesion complex maturation?

A
  • Nascent adhesions: less dense adhesions, appear as a line at the leading edge
  • Focal contacts are the dark spots at leading edge
  • Some mature into Focal adhesions

Focal adhesions are matured and arefound further to the centre of the cell

51
Q

How do adhesions mature?

A

Mainly by Paxillin. Paxillin recruits beta-pix, a Rac, that leads to actin polymerisation.

Once matured into a focal complex, Paxillin
can recruit RhoA which recruits ROCK leading to myosin RLC causing contraction within the actin creating tension to mature into focal adhesion.

52
Q

Whats the difference in adhesion maturation of nascent adhesion/focal contacts and that of focal adhesions.

A

Nascent adhesions/focal contacts
promote signals to Rac and Cdc42 to
promote actin polymerisation (cell
front)

Mature focal adhesions signal to RhoA
to promote contractility and retraction
(cell rear)

This compartmentilises it.

53
Q

Whats responsible for CMAC turnover?

A

Focal adhesion kinase (FAK)

FAK recruits calpain. Calpain cleaves talin which severs the link between integrins and the cytoskeleton.

The integrins are then endocytosed. FAK is responsible for recruiting microtubes and clatherin and dynamic 2 which then enable integrins to be moved and taken away.
For focal adhesions, Rab21 is important as it binds directly to the integrins and promotes internilisation/endocytpsis

54
Q

What is the role of Rab21

A

Rab21
When focal adhesions don t mature and want to dissassemble instead, Rab21 will be bind to promote the internilisation of the integrins.

55
Q

How do migrating cells generate force?

A

Actomyosin system

56
Q

What is a catch bond?

A
  • Opposing force from matrix
  • Cytoskeleton pulling otherway
  • Catch bond grows stronger the more you pull
    Increases force of attachment
57
Q

How does non-muscle myosin 2 enable contractility? How is it activated?

A

It is a motor protein and bound to the f-actin filament, they walk in opposite directions. It is a Hexomer, two heavy chains, two essential light chains, 2 regulatory light chains.

  • RhoA-rock activates the light chains of myosin 2 through its effector kinase ROCK
  • Phosphorylation then leads to changes in conformation allowing filaments to form/activation of ATPase
58
Q

How is contractility important to mature adhesions? How does it work?

A

Pulling on adhesions promotes recruitment of further adaptors and signalling molecules.

* Talin binds integrins and links to actin cytoskeleton
* A Force applied, a change in confirmation in talin reveals binding sites
* Early step is the recruitment of vinculin to the stretched talin
59
Q

What do cells use to the ‘feel’ the properties of their environment?

A

CMACs and actyomyosins

  • allows force to be generated and CMACs to mature (or not mature)
60
Q

How is retraction of the rear of the cell controlled?

A

RhoA rock phosphorylates the myosin 2 lightchain. This allows formation of the myosin filaments and contractility to occur.

61
Q

How does contractile force lead to the growth and maturation of CMACs?

A

Stretching induces conformational change in CMAC proteins such as talin.

62
Q

What is F-actin retrograde flow?

A

The flow of actin filamentback away from the lamellipodium. Occurs due to the leading edge polymerisation applying pressure on the the cell cortex. If CMACs can form then cell will move instead.

  • In nascent adhesions and focal complexes, provide links to actin, the nascent adhesions talin starts to get stretched and provides a binding site for vinculin, and they can mature further.
63
Q

Why do cells preferentially move on stiff matrix?

A

Can generate more force

64
Q

How do cells migrate through barriers in a 3D matrix?

A
  • Degradation via invadipodia
  • Squeezing through gaps, aided by blebbing
65
Q

How do Invadipodia and blebs help migration in a 3D environment?

A

Invadopodia coordinate delivery of proteases
Cut through collagen fibres
Allow efficient migration in 3D

66
Q

How does epithelial-mesenchymal transitions enable cancer metastisis?

A

Enables migration and invasion into blood stream to migrate around the body.

This is the cells plasticity that allows them to change state.

67
Q

What are the functions of filopodia when migrating in a 3D matrix?

A
  • sensing chemoattractants
  • Interacting with ECM fibrils
  • generating protrusion force
68
Q

What happens when a cell moves quickly in 3D matrix?

A

It accumulates membrane at the rear, creating low membrane tension.

Caveolae are allowed to form as a result of the low tension

Low membrane tension acts as a signal.

Cavoelae recruit Ect2, a RhoA GEF. Ect2 activates RhoA and actomyosin. This means low membrane tension drives contractility at the cell rear via cavolae and ect2

69
Q

What happens when a cell moves quickly in 3D matrix?

A

It accumulates membrane at the rear, creating low membrane tension.

Caveolae are allowed to form as a result of the low tension

Low membrane tension acts as a signalW

70
Q

What is the role of caveolae in cell migration?

A

It activates Rho A

As they are brought about by low surface membrane tension (increasing tension or inhibiting caveloin 1 inhibits RhoA).

They activate Rho Specific GEF, Ect2, which activates RhoA and actomyosin.

Meaning low membrane tension activate RHoA, which will then cause contractility via its effector kinase ROCK and pulls the rear in.

71
Q

How does the Nuclear piston aid migration in a 3D matrix

A

The Nuclear piston

  • Actomyosin contractility drives nucleus forwards which Increases pressure at the front, driving protrusions, causing blebbing.

Protrusion here are call lobopodial protrusions.

72
Q

Ameoboid migration is useful for squeezing in a 3D matrix. What is needed for the bleb to be effective?

A

F-actin to migrate back in

73
Q

What is a lobopodial protrusion?

A

Protrusions caused by the nuclear piston.

Dynamic Cell (3 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions