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20 Cards in this Set
- Front
- Back
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What is the Arp2/3 complex?
Why is it important? |
complex formed by 7 Actin Related Proteins
(similar sequence to actin) normally the barbed end is pointed towards the cell membrane It is a pointed-end nucleator have a similar barbed end to actin subunits, bind to the pointed end of actin while the barbed end of the nascent filament is free to extend. branched filaments occur by having the complex sit on existing filaments. branches are normally of 70 degrees |
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Who showed that treadmiling takes place in cells?
And how? |
Yu-Li Wang in the mid-80s
Microinjected gerbil fibroma cells with labelled actin (rhodamin, fluorescent) That got incorporated into existing actin structures. He then photobleached an area near the membrane edge, marking a region on the filament. The black spot was shown to move backwards at a speed of ~1 micro meter per minute. The black spot also recovered fluorescence as it moved, showing that the filament ends are in exchange with the monomer pool. This was alot faster than the predicted speed, which was predicted by multiplying the rate of subunit addition by the subunit length, which gives 0.18 micro meters per minute. This is due to actin binding proteins. Observation: treadmilling does not exert force against the membrane in this case, instead the filaments are just being pushed back. to do so the filament has to be coupled with the surface somehow, to force the membrabe to move forward with respect to the filament |
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reflection interference microscopy:
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can be used to highlight areas in which cells are in close contact with the the substratum.
fluorescent phalloidin can label actin filaments, which are shown to associate with these contacts used to identify focal contacts |
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Focal contacts
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points of very close contact between the membrane and the substratum, shown to associate with actin filaments.
also coincide with integrins |
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I N T E G R I N S
what are they and how were they discovered |
Transmembrane proteins which bind the extracellular protein F I B R O N E C T I N
and enhance the crawling rate of cells Discovery: -make monoclonal antibodies for cell surface proteins -assay for clones that make antibodies which prevent cells from attaching to fibronectin coated tissue culture wells. - one of these antibodies was used to purify integrins by affinity chromatography - obtain sequence - use cytoplasmic peptides of the protein to do more affinity chromatography in order to identify actin binding proteins like talin, alpha-actinin (involved with focal point formation) |
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Myosins
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-Motor proteins (actin)
- pointed -> barbed - hydrolise ATP -> conf. change in the globular head biases random diffusion towards the barbed end. - many classes, most important: Myosins I + II |
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Myosin II
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-similar to muscle myosin
in that they form bipolar filaments, which can contract actin gels - different in that they are controlled by phosphorylation: -> unphosphorylated: alpha-helical coiled coil folds back and prevents bipolar filament formation -> phosphorylation causes spontaneous bipolar filament formation (vital for cytokinesis) |
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what is a lamellipodium
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an 2D actin quasi-mesh that causes a protrusion on the cell. responsible for cell crawilling
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Where in the lamellipodium are myosin II
and myosin I localised? |
immunifluoresence indicates Myosin II at the periphery of the actin rich lamellipodium
Knockout of Myosin II cells lose polarised morphology though still exhibit peripheral ruflfing Knocking out Myosin I affects phago/pinocytosis |
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What is Fluoresence Speckle Microscopy and what was illustrated by it
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->fluorescent phaloidin becomes incorporated into filaments, when microinjected to motile cells.
-> this gives a speckled appearance -> observe under video microscopy -> allows tracking of cytoskeleton movement - cytoskeleton shown to move backwards in respect to the membrane. - speckels disappear towards the center of the cell, illustrating dissasembly. (net pol at edge, net depol at center) - Myosin II overalps with depol site, thought to speed up depol. -> Blebbstatin (MyoII inhibitor) shown to decrease depol rate. |
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Cell movement
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four processes acting in concert.
1) actin polymerisation forces lamellipodium forward 2) new focal points made to adhere the newly advanced membrane 3) contraxion of the cortex by bipolar myosin arrays to drag the lagging cell forward (or depol in the rear) 4) focal points in the rear release |
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why is treadmilling an order of magnitude faster in vivo than in vitro?
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profilin and thymosin sequester much of the unpolymerised actin.
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Profilin action
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1) binds ATP-G actin + accelerates the addition to barbed ends
2) promotes dissociation of ADP to be replaced by ATP |
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Arp2/3 action
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-nucleates branched filaments from existing ones
-nucleation occurs at the pointed end, allowing the faster barbed end to extend - barbed end is subsequently capped. as older filaments are moved away from the membrane edge, capping allows for subunits to be directed to the leading end of the cells. |
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Cofilin action
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destabilises pointed ends (ADP) -> dissociate 25 times faster
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Cell signalling to Cytoskeleton
How do environmental cues control cell motility |
via transmembrane receptors, many of which involve small G proteins
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What are small G-proteins
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GTPases
when inactive they cannot release GDP when activated, can release GDP for GTP-> conf change activates downstream receptors may control cytoskeleton |
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exoenzyme C
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comes from Clostridium botulinum
inactivates Rho (G protein) by ADP ribosylation causes cell to round up and lose focal contact - attaching actin cables **first evidence that G proteins can affect the cytoskeleton |
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Two other examples of G-protein controlled cytoskeleton
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constitutively active mutants of G-proteins--> induce formation of actin cables (stress fibers)
Rac when constitutively expressed will increase membrane ruffles |
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CRIB domain
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Cdc42/Rac interactive binding motif
example: N-WASP adaptor protein contains a CRIB motif and an Arp2/3 complex binding site Arp2/3 complex is hidden in an acidic domain kept hidden by internal association with a basic domain when G-protein binds to CRIB domain -> cryptic Arp2/3 binding region is revealed |
