Actin and microfilaments part 3

inter-strand and intra-strand contacts between actin monomers are … and …

extensive and noncovalent

the use of lebelled phallotoxins helped with … of F-actin

visualisation

phallotoxins are … … isolated from the … … mushroom

bicyclic heptapeptides, death cap

Phalloidins

bind to … … and … them

phallotoxin derivatives are relatively small so many … … … can still bind to actin after treatment with … phallotoxins

one limitation of phalloidins is that they are not easily … … by the cells and require … or … of cells

a similar toxin from … … called … is membrane permeable and can be used insteas

actin filaments, stabilise

actin binding proteins, fluorescent

taken up, injections or permeabilization

sea corals, jasplakinolide

analysis of … phalloidin and … … phalloidin stabilised F-actin filaments reveals the … of the …

native, gold tagged, spacing of the subunits

the native phalloidin can either be … or … …

unstained or negatively stained

in gold tagged PHD the single gold clusters appear every … along the two … … helical strands

5.5nm, long pitched

… labelled phalloidin is …

alexa, fluorescent

DNase I

bovine pancreatic … binds to … …

it is used to obtain … of … for … analysis

very useful for … and … the proportion of … actin

can be used in conjugation with … to visualise both … and … actin

it is not permeable and requires … and … of cells

deoxyribonuclease, monomeric G actin

crystals, G actin, structural

detecting and quantitating, unpolymerised

phalloidin, F and G

fixation and permeabilization

… are inhibitors of actin polymerisation

latrunculins

latrunculins are inhibitors of … …

actin polymerisation

latrunculins

toxins isolated from a … …

specifically binds … in a … ratio at … concentrations

binds to the … between the major … and inhibits … and hence …

i.e. prevents … … from occuring

shifts the equilibrium towards …

a major advantage is that it is … …

disadvantages: very … and requires … amounts

widely used to study the effect of … of … structures on cellular processes such as …, … … and …

sea sponge

G actin, 1:1, submicromolar

cleft, subdomains, rotation, polymerisation

conformational changes

monomers

membrane permeable

expensive, stoichiometric

loss of F actin, endocytosis, cell division and movement

list 3 traditional ways to measure actin polymerisation

1. DNase I inhibition assays

2. viscosity measurements

3. spin down assays

most recent method for measuing actin polymerisation

flurescence enhancement of pyrene conjugates

measuring actin polymerisation with a continuous assay

principle: enhancement of … upon assembly of … labelled … into filaments

flurescence after polymerisation is increased … fold

the continuous assay is performed by …

the assay follows the classic …, … and … … phases

the upper limit of F-actin can be measured in the presence of …

the assay is very … and even small amount of … can be distinguished from coexisting …

fluorescence, pyrene, G actin

25

fluorimetry

lag, polymerisation and steady state

phalloidin

sensitive, F actin, G actin

all regulatory mechanisms of actin filaments include a huge array of … and … … that are often linked to … … …

binding and regulatory proteins, cellular signalling pathways

regulatory mechanisms must exist for

1. … of … … of the G-actin pool

2. quick … for formation of new filaments

3. … of existing filaments

4. … or … existing filaments

5. regulation of … of filaments

1. inhibition of spontaneous polymerisation

2. nucleation

3. elongation

4. breaking or severing

5. organisation

the … complex a conserved … subunit protein machine that builds … actin chains by … new "daughter" filaments off the sides of existing "mother" filaments at a characteristic … degree angle

Arp2/3, seven, branched, nucleating, 70

… is a small protein that binds to … actin and plays a central role in … the actin cytoskeleton

profilin, monomeric, regulating

… is a protein that … … filamentous actin into orthogonal, web-like … networks rather than tight, parallel bundles

filamin, cross links, gel

… and … are key proteins that work together to … … and organize actin microfilaments into … and … bundles inside cell structures like intestinal …

villin and fimbrin, cross link, tight and parallel, microvilli

list 2 G-actin binding proteins and 5 F-actin binding proteins and 2 that bind to both

1. profilin

2. Arp2/3 complex

3. myosin

4. filamin

5. villin

6. fimbrin

7. capping proteins

8. cofilin

9. ADF (actin depolymerising factor)

… … are specialized regulatory molecules that bind to the ends of actin filaments to halt the … or … of actin subunits

capping proteins, addition or loss

… and … are actin binding proteins that drive the rapid … and … of the actin cytoskeleton

cofilin and adf, turnover and remodelling

actin bindsing proteins regulate actin …, … and …

function, polymerisation and organisation

name the actin binding proteins involved in the following

1. monomer binding

2. nucleating

3. contracting

4. gelating

5. bundling

6. capping

7. fragmenting

1. profilin

2. Arp2/3 complex

3. myosin

4. filamin

5. villin and fimbrin

6. capping proteins

7. cofilin and adf

name the functions of the following

1. profilin

2. Arp2/3 complex

3. myosin

4. filamin

5. villin and fimbrin

6. capping proteins

7. cofilin and adf

1. monomer binding

2. nucleating

3. contracting

4. gelating

5. bundling

6. capping

7. fragmenting

besides actin binding proteins in nucleation there are also many … … … (…) many of which are associated with … conditions

nucleation promoting factors, NPFs, pathological

bundles and orthogonal networks of actin can regulate … … of other proteins and so positions them in … locations

spatial organisation, polarised

… serves as a molecular … linking … receptors to the … cytoskeleton

FLN A, adapter, dopamine, actin

adapter proteins are important for … the receptors at the right place and concentration

positioning

the immune synapse happens between … … … and … such as …, … and … cells

antigen presenting cells, lymphocytes, T, B and NK

in this image a … cell is interacting with a … cell (a professional APC) through a molecular … … described as an immunological …

T, dendritic, polarised junction, synapse

the immunological synapse is dependent on … of the actin cytoskeleton of the … cell which in turn … … and … to the synapse

reorganisation, T, localises receptors, scaffolds

in the immunological synapse polarisation of actin is triggered by interactions between the … and … with … and small …

as actin … it promotes clustering of … and …

TCR and CD3, integrins, GTPases

reorganises, TCRs and integrins

in the immune synapse actin filaments facilitate … … and orchestrate the … …

overall they establish protein … …

TCR triggering, signalosome assembly

spatial organisation

example of an integrin

lymphocyte function associated antigen 1