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ANAT 262 > Cytoskeleton: Actin > Flashcards

Cytoskeleton: Actin Flashcards

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1
Q

What is function actin

A

Cell motility
Contractiliy

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2
Q

What are actin formed form

A

G-actin subunits (globular)
2 twisted protofilaments

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3
Q

Actin diameter

A

80 angstroms

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4
Q

Is actin polar

A

Yesss
Barbed end and pointed end

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5
Q

Is actin flexible

A

Very flexible - 17um persistence length
More than micortubules

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6
Q

Actin often…

A

Cross linked into thicker bundles

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7
Q

Where is actin usually

A

At cortex
Associated with pm

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8
Q

Describe actin polymer growth

A

Dictated by on and off rates
On rate = m^-1,s^-1, depends on concentration free subunits - higher concentration = faster addition
Off rate = s^-1 concentration independent - subunits fall off at a constant rate

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9
Q

Describe critical concentration actin

A

The concentration of free monomer where kon and koff are balanced
Rate of addition=rate of loss in filament

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10
Q

When actin polymer grows

A

Conc free monomer > critical concentration (adds on both ends but diff rates)

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11
Q

When actin polymers shrink

A

Concentration free monomer <Cc

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12
Q

When actin monomer - no net growth

A

[Free monomer]=Cc

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13
Q

Describe ends of actin filament

A

Plus end = barbed
Minus end = pointed end
Addition at one End is not equivalent to addition at other bc Rates - kinetics bc shape of filaments, adds quicker to plus end

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14
Q

Describe actin subunit addiing to filament

A

Structural changes occur in subunits on incoporation into filament
Actin subunit changes conformation on incorporation into file ant
Has to change conf - atp added to monomer so can get into filaments
ATP state monomer conformation and assembly linked

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15
Q

When does actin polymerize

A

ATP binding drives actin polymerization
Subunits add in atp state
Time to takes for actin filament to get hdyrolyzed = time filament stays stable

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16
Q

When does actin depolymerize

A

ATP hydrolysis drives depolymerization
Dissociate in adp state

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17
Q

What does atp do to actin

A

ATP increases affinity fo free subunits for polymer

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18
Q

What does atpase do for actin

A

Atpase rate increases on incorporation to polymer hydrolysis to adp destabilizes filament contacts

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19
Q

What happens when subunit adds

A

Delay between subunit addition and atp hydrolysis
The longer a subunit is in polymer the more likely it will have hydrolzyed atp to adp
Cc neg end > cc pos end - diff on/off rates

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20
Q

What is treadmilling

A

Cc- > free monomer > cc+
Filament reaches constant length but subunits flux through

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21
Q

Name actin binding proteins

A

Regulate filament assemble
Nucleation and anchoring
Sequestration
Destabilization
Cross linking
Growth acceleration
Stabilization

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22
Q

Describe actin filament growth

A

Filament growth ncuelation limited
Lag phase = time to achieve nucleation

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23
Q

NAME ACTIN NUCLEATION COMPLEXs

A

Most common = arp2/3 complex
Formins

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24
Q

DESCRIBE arp2/3 complex

A

Actin homologous arp2 and arp3 form nuclei
Complex arp2/3 activity conformationally regulated
Ncuelation site for actin filaments
ARP2/3 with activating factor = they come together and then in presence actin monomers = forms ncuelation actin filament = monomers stick to it (makes neg end)
Complex FLEXIBLE = can stick to actin and do branched ncuelation fo actin

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25
DESCRIBE arp2/3 complex - binds do
Existing actin filaments Nucleates actin at leading edge of cell that’ wants to crawl Fixed angle = 70 degrees, most stable conformation,highest angle to get force in this direction Creates dense, branched network at cell periphery
26
Describe formins
Bundle - like in mts Forms nucleates and remain attached to plus end - formin dimer here, moves side to side = makes spot for new actin monomer Unbranched filaments - can be readily cross linked
27
Describe regulating effective actin concentration - gen
Cc of actin = 1uM In cells - [actin] = 50-200uM SO CELL SHOULD BE FULL OF ACTIN FILAMENTS
28
What limits actin filament assembly
Thymosin binds actin subunits and prevents incorporation into filaments Effectively reduces free actin concentration Regulation!!! Keep from adding to + end - barbed end
29
How do you ever get any filament assembly?
Profiling displaces thymosin and binds the actin + end -pushed off thymosin Prevents binding at minus end, allows binding at + end Conformational change in actin on incorporation to filament displaces profilin
30
Describe cooperation of formin and profilins
Formin = like hands = grabs profilin Profilin = bound to + end g actin and pushes in - locks monomers into polymer Some formins have multiple profilin bindin sites - accelerates growth
31
name filament stabilization - capping and side binding
Tropomysoin Capping protein s
32
describe tropomyosin - filament stabilization - capping and side binding
Binds multiple actin subunits along filament length to stabilize it - length wise Will stop treadmilling Muscles - block myosin binding side, cannot contract until move tropomysoin
33
Describe capping proteins - filament stabilization - capping and side binding
Capping proteins bind ends = restricts growth to one direction + end cap = capZ - end cap= tropomodulin
34
Name proteins for filament destabilization
Cofilin gelsolin
35
Describe cofilin
Binds along length of filament = over twists it = makes it shorter and causes it to fall apart Strain of over twisting results in more rapid depolymerization
36
Describe gelsolin
Severs actin filaments and caps end Simailr to katanin and spastin in mt severing - chops it up
37
Describe actin cross linking
Individual actin filaments are relatively thin and flexible - frequently strengthened by cross linking - each have diff classes actin Stress fibres - contractile bundles Cell cortex - gel liek network Filopodium = tight parallel bundle
38
Name actin cross linkers
Made up up actin biding domains and spacers Spectrin Fimbrin Alpha actinin Filamin
39
Name branched cross linkers
Filamin Spectrin
40
NAME parallel to anti parallel cross linkers
Fimbrin Alpha actinin
41
Describe spectrin
Actin binding linker at ends and tons of spacers = 4 come together = huge distance between actin filaments - cross links them Tetramer
42
Describe fimbrin
Monomer No spacers No contarctility
43
Describe alpha actinin
Dimer Surrounds cells Myosin can fit between and contraction possible
44
Describe filamin
V shaped dimer molecule
45
Describe contractile bundle actin
Actin filaments and alpha actin Myosin can come - loose packing allows mysosin ii to enter bundle Straight, stiff connections - more rigid like structures
46
Describe parallel bundle actin
Actin fimbrin Tight packing prevents myosin 2 from entering bundle Actin cannot move or slide Straight, stiff connections - more rigid like structures
47
What do parallel bundles do
Support microvilli - lateral myosin bundles =use myosin to slide a bit
48
What do filamin and spectrin do
Crosslink actin mesh work Gel like mesh- flexible and strong
49
Describe linking actin cytoskeleton to plasma membrane
Inactive folded conformation of erm linker proteins —> phosphorylation induced by signals —> active extended conformation of erm protein —> erm mediated cross linking = link tm proteins to actin cytoskeleton (Erm= ezrin, radixin, moesin)
50
Name nucleators
Arp2/3 complex (branched networks) formins (straight filaments, bundles)
51
Name growth modulators
profilin (adds subunits only at + end)
52
Name stabilizers
tropomyosin (side-binding) CapZ (+ end cap) tropomodulin (- end cap)
53
Name destabilizers
gelsolin (severing) cofilin (twisting strain)
54
Name cross linkers
α-actinin (contractile bundles) fimbrin, villin (non-contractile bundles) filamin, spectrin (mesh-like networks)
55
Name anchors
ERM-1 (link to membranes)
56
Describe myosin
Myosin’s convert energy of atp hydrolysis to directed movement along actin filaments Coiled coile of 2 alpha helices, 2 heads Mysosin 2 - contraction in muscle and contractile ring Large fam with diff domain organizations All plus end directed except myosin vi (reverse - negative motro)
57
What is myosin similar to
Kinesins Dimeric motor protein, atpase, provides energy, diverse fam Core atpase structures preserved But DIFF STEPPING MECHANISMS= myosin not processive, kinesin = processive - always one foot on mt
58
Compare myosin and kinesin structures
Motor domains Linker Coiled coil Cargo binding domains
59
Describe myosin motor cycle
Attached = no atp, strongly bound Released = atp binding causes motor head to disengage Cocked = atp hydrolysis causes larger conformational change, forward head displacement Force generating = pi release - weak binding to actin causes backwards head displacement Attached =adp released, string attachement to filament again
60
How does myosin walk - compared to kinesin
Myosin takes short works on actin filaments - not processive High atp concentration in cell mean motors are almost always in atp state ATP state = unbound Most time spent unbound= why not processive Motor heads not coordinated - bipolar filament of myosin - head on each side all doing own thing Step size depends on linker/neck region
61
How does kinesin walk - compared to myosin
ATP state= tightly bound Most time spent bound Motor heads coordinated Processive - long walks
62
What is step size dictated by
Length of lever arm Depends on how long arm moving forwards Myosin ii = 5-10nm Myosin v = 30-40nm swing lever arm
63
What generates contarctility
Actin myosin arrays
64
Describe what myosin forms
Large buckets with motor heads sticking out Force generated by sliding between bundles myosin and actin filamentts - during power stroke Linked coiled could domains - bipolar myosin bundle
65
Describe muscle organization
Myosin = thick filament Actin fialemnts = capped stable, no treadmilling Length determined by nebulin - side binding protein
66
What does calcium do
Ca signalling triggers contraction Response to nerve signal opens release Channels in sarcoplasmic reticulum flooding cytoplasm with calcium
67
What is calcium response due to
Tropomyosin movement Calcium binds to troponin = causes tropomyosin to slide out of way of myosin binding
68
Name 2 other locations fo actin myosin based contraction
Stress fibres Contractile ring
69
Describe stress fibres
Myosin bundles pulling on actin filaments produces tension and tends to generate paralle actin arrays
70
Describe contractile ring
Myosin bundles pulling on actin filaments produces tension and tends to generate paralle actin arrays Contractile ring = cytokinesis ring Highly regulated, based in gtpase, postulated contractile unit = actin and myosin = bind together = ring that pinches cell in 2
71
Describe what rapid reorganization of myosin 2 depends on
Phosphorylation Inactive state = light chain not phosphrulated- myosin light chains - holds in a inactive state (at neck myosin molecule) Phosphorylation by myosin light chain kinase = flips open actin binding sites Active state = Light chains phosphorylated -spontaneous self assembly
72
What does actin underly
Cell crawling on glass Actin polyemriztaion at plus end protrudes lamellipodium (causes protrusion) Attachment and traction Adhesion Motor driven by actin polymreization at leading edge

ANAT 262 (24 decks)

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