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Dr Millard's lectures L2-L5 Flashcards

1
Q

What are lamellipodia?

A

flat 2d actin protrusions

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2
Q

What are pseudopodia?

A

thick 3D protrusions on neutrophils

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3
Q

What are filopodia?

A

sensors to explore environment. Often found within lamellipodia and pseudopodia.

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4
Q

Phalloidin

A

Helps visualise actin. Binds F actin but not G actin so if fluorescently labelled, shows up filaments.

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5
Q

Cytochalasin D

A

Blocks actin polymerisation. Stops lamellipodia forming so shows they need actin for protrusions.

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6
Q

Photobleaching (FRAP)

A

Laser on GFP stops it fluorescing. At PM fluorescence goes and comes back showing actin is added tot he front right under PM.

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7
Q

Stress fibres

A

Actin and myosin II contractile cables.

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8
Q

alpha-actinin

A

Cross’links actin filaments to stabilise cable

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9
Q

Blebbistatin

A

Myosin inhibitor. Treat with this, cells fail to move, shows need myosin.

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10
Q

Amoeboid migration

A

Blebbing.
Actomyosin creates pressure inside PM. Actomyosin breaks at 1 point. Pressure squeezes cytoplasm out, with PM. Actomysoin reforms

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11
Q

Stages of actin pol in vitro

A
  1. Nucleation- spontaneous but slow.
  2. Filament growth- protportional to monomer conc.
  3. Steady state- Equilibrium. approx 0.1uM conc of monomers
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12
Q

Stages of actin pol in vivo in lamellipodia

A
  1. Nucleation- not spontaneous but very rapid when triggered.
  2. Filament Growth- Rapid but only one end. makes branched network.
  3. Steady state- 100uM. Not at equilibrium. High conc.
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13
Q

Cell adhesions

A

Attach back of cell down. Need to detach for back of cell to move

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14
Q

Profilin

A

Binds to actin monomers. Changes monomers’ behaviour:Profilin bound monomers:

  1. They CANNOT incorporate into minus end, only +
  2. They exchange ADP for ATP
  3. They CANNOT spontaneously nucleate new filaments
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15
Q

Arp2/3 complex

A

Protein complex with 7 subunits including Arp 2 and Arp3. When activated, arp 2 and 3 come together. Arp2/3 binds to side of existing filament. associates with minus end of new filament. New profilin-bound monomers incorporated to produce new filaments at 70 degrees. Branched network good for lamellipodia.

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16
Q

Capping protein

A

Binds to plus end of filament. High concentration in lamellipodia. Caos within 1 second of filament growth.

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17
Q

Ena/VASP

A

Protein family “anti-cappers”. Prevent capping fo plus ends. Promote filament elongation. High conc= long and bendy filaments. Low conc= shorter, stronger push force.

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18
Q

ADF/cofilin

A

Binds to old sections and severs adp-actin. (not ATP actin)

Adf= actin depolymerising factor

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19
Q

Fluorescent speckle microscopy

A

can see actin filaments treadmilling

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20
Q

Filopodia tip complex

A

Filopodia need to be long unbranched actin filaments tightly bundled. Tip complex associates with actin filaments in filopodia. Stops capping. contains Ena.

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21
Q

Actin-bundling protein

A

Cross links the filaments in convergent elongation of filopodia. Have at least 2 actin binding sites.

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22
Q

alpha- actinin

A

an actin-bundling protein. cross links contractile bundle in stress fibre. antiparallel

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23
Q

Filamin

A

an actin-bundling protein. cross links gel-like network in cell cortex.

24
Q

Fascin

A

an actin-bundling protein. cross links tight parallel bundle like in folopodia.

25
Q

DAAM

A

a formin protein. Needed for filopodia formation if no ARP2/3/
arp2/3 usually needed to nucleate lamellipodial network first

26
Q

Formins

A

Family of proteins including DAAM and mDia. Can nucleate actin filaments. unlike ARp23, formind bind to plus ends and promote elongations as well as nucleation. Formins make long, unbranched chains. Can form filopodia de novo without lamellipodial network and important in making stress fibres.

27
Q

Tropomyosin

A

stops actin filaments from depolymersisin. wraps around filaments holds it together.

28
Q

GEFs

A

switch GTPases into active GTP bound state

29
Q

GAPs

A

switch GTPases into inactive, gdp bound state

30
Q

Rho

A

stress fibres

31
Q

Rac

A

Lamellipodia

32
Q

Cdc42

33
Q

FRET

A

get fret then red fluorescence when rac is active. Because GFP and RFP are close enough using the RAc biosensor fusion protein.
changes conformation when rac is active, get FRET

34
Q

Listeria bacteria

A

Induces formation of actin comet tails containing Arp2/3 and ena proteins. Uses hosts own actin.

35
Q

ActA

A

TM proteins needed for Listeria movement. Binds Ena, actin monomers (by WH2 domain) and Arp2/3 (by acidic domain).
Activates Arp2/3 complex, changes arp2/3 conformation, brings actin monomer, filament growth helped by ena. attaches to all these things.

36
Q

WASP

A

Wiskott-aldrich syndrome protein. Contain WH2 and acidic domain like ActA and can activate Arp2/3 complex. Recessive X-linked disorder causing immune deficiency caused by mutation.

37
Q

WAVE

A

similar structure to ActA and WASp. RAc-gtp activates WAVE, allowing it to activate ARP2/3.

38
Q

Rac promoting lamellipodia

A

Rac inactivates ADF/cofilin and activates WAVE (which activates ARP2/3.

39
Q

filopodia formation

A
  1. arp2/3 dependent. convergent elongation.
  2. formin-dependent.

cdc42 can activate both.

40
Q

N-WASP

A

activated by cdc42. changes conformattion from auto-inhibited conformation when cdc42 binds. the activates arp2/3 - get filopodia formation

41
Q

mDia

A

activated by cdc42. it binds on and changes mdia’s confomration from auto-inhibited to active. So nucleates new filaments- forming filopodia.

42
Q

ADF/cofilin inactivation

A

Active PAK, activates LIM kinase by phosphorylation. Lim kinase phosphorylats ADF/cofilin by phosphorylation (inactivates it.)

43
Q

Rho making stress fibres

A

Rho activates the formin mDia. Rho inactivates ADF/cofilin by activating LIM kinase. Rho activates myosin II via ROCK.

44
Q

PDGF

A

PLatelet-derived growth factor-> activate Rac and cdc42.

45
Q

LPA

A

Lysophophatidic acid- activates Rho

46
Q

neuronal growth cone

A

if it senses Ephrins on some cells, recruits a tyrosine kinas, recruits a rho-gef which activates Rho, inducing growth cone collapse so goes in different direction away.

47
Q

Ephrins

A

Expressed on some cells. Has receptor- Eph no growth cone.

48
Q

Ephexin

A

A rho-GEF recruited by tyrosine kinase, activates Rho so growth cone retreats.

49
Q

Photoactivatable Rac

A

Rac inhibited unless shine laser. Shows all you need for cell to move is rac.

50
Q

Focal adhesion

A

First row= border between front and back of cell. Contain alpha and beta integrins that bind to ECM. Focal adhesions allow bidirectional signalling from cell to ECM.

51
Q

alpha and beta integrins

A

TM proteins. Many types in different combos.

52
Q

Adhesion maturation

A

enlarge, change protein composition and signalling. caused by tension, stretching

53
Q

Nascent adhesions

A

small with few integrins. Promot Rac activity. Positive feedback as rac promotes these nascent adhesions

54
Q

Bigger focal adhesions

A

Long structures and stable. primarily have alpha5 beta1 integrins.

55
Q

Talin and p130cas

A

Promote Rho activity. Positive feedback. Proteins in the adhesions that change conformation when stretched, Exposes protein binding sites, recruiting proteins to adhesion -> leading to growth.

Dynamic Cells (5 decks)

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