Cytoskeleton: Actin Flashcards
What is function actin
Cell motility
Contractiliy
What are actin formed form
G-actin subunits (globular)
2 twisted protofilaments
Actin diameter
80 angstroms
Is actin polar
Yesss
Barbed end and pointed end
Is actin flexible
Very flexible - 17um persistence length
More than micortubules
Actin often…
Cross linked into thicker bundles
Where is actin usually
At cortex
Associated with pm
Describe actin polymer growth
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
Describe critical concentration actin
The concentration of free monomer where kon and koff are balanced
Rate of addition=rate of loss in filament
When actin polymer grows
Conc free monomer > critical concentration (adds on both ends but diff rates)
When actin polymers shrink
Concentration free monomer <Cc
When actin monomer - no net growth
[Free monomer]=Cc
Describe ends of actin filament
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
Describe actin subunit addiing to filament
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
When does actin polymerize
ATP binding drives actin polymerization
Subunits add in atp state
Time to takes for actin filament to get hdyrolyzed = time filament stays stable
When does actin depolymerize
ATP hydrolysis drives depolymerization
Dissociate in adp state
What does atp do to actin
ATP increases affinity fo free subunits for polymer
What does atpase do for actin
Atpase rate increases on incorporation to polymer hydrolysis to adp destabilizes filament contacts
What happens when subunit adds
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
What is treadmilling
Cc- > free monomer > cc+
Filament reaches constant length but subunits flux through
Name actin binding proteins
Regulate filament assemble
Nucleation and anchoring
Sequestration
Destabilization
Cross linking
Growth acceleration
Stabilization
Describe actin filament growth
Filament growth ncuelation limited
Lag phase = time to achieve nucleation
NAME ACTIN NUCLEATION COMPLEXs
Most common = arp2/3 complex
Formins
DESCRIBE arp2/3 complex
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
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
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
Describe regulating effective actin concentration - gen
Cc of actin = 1uM
In cells - [actin] = 50-200uM
SO CELL SHOULD BE FULL OF ACTIN FILAMENTS
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
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
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
name filament stabilization - capping and side binding
Tropomysoin
Capping protein s
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
Describe capping proteins - filament stabilization - capping and side binding
Capping proteins bind ends = restricts growth to one direction
+ end cap = capZ
- end cap= tropomodulin
Name proteins for filament destabilization
Cofilin gelsolin
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
Describe gelsolin
Severs actin filaments and caps end
Simailr to katanin and spastin in mt severing - chops it up
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
Name actin cross linkers
Made up up actin biding domains and spacers
Spectrin
Fimbrin
Alpha actinin
Filamin
Name branched cross linkers
Filamin
Spectrin
NAME parallel to anti parallel cross linkers
Fimbrin
Alpha actinin
Describe spectrin
Actin binding linker at ends and tons of spacers = 4 come together = huge distance between actin filaments - cross links them
Tetramer
Describe fimbrin
Monomer
No spacers
No contarctility
Describe alpha actinin
Dimer
Surrounds cells
Myosin can fit between and contraction possible
Describe filamin
V shaped dimer molecule
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
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
What do parallel bundles do
Support microvilli - lateral myosin bundles =use myosin to slide a bit
What do filamin and spectrin do
Crosslink actin mesh work
Gel like mesh- flexible and strong
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)
Name nucleators
Arp2/3 complex (branched networks)
formins (straight filaments, bundles)
Name growth modulators
profilin (adds subunits only at + end)
Name stabilizers
tropomyosin (side-binding) CapZ (+ end cap) tropomodulin (- end cap)
Name destabilizers
gelsolin (severing)
cofilin (twisting strain)
Name cross linkers
α-actinin (contractile bundles)
fimbrin, villin (non-contractile bundles) filamin, spectrin (mesh-like networks)
Name anchors
ERM-1 (link to membranes)
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)
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
Compare myosin and kinesin structures
Motor domains
Linker
Coiled coil
Cargo binding domains
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
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
How does kinesin walk - compared to myosin
ATP state= tightly bound
Most time spent bound
Motor heads coordinated
Processive - long walks
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
What generates contarctility
Actin myosin arrays
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
Describe muscle organization
Myosin = thick filament
Actin fialemnts = capped stable, no treadmilling
Length determined by nebulin - side binding protein
What does calcium do
Ca signalling triggers contraction
Response to nerve signal opens release Channels in sarcoplasmic reticulum flooding cytoplasm with calcium
What is calcium response due to
Tropomyosin movement
Calcium binds to troponin = causes tropomyosin to slide out of way of myosin binding
Name 2 other locations fo actin myosin based contraction
Stress fibres
Contractile ring
Describe stress fibres
Myosin bundles pulling on actin filaments produces tension and tends to generate paralle actin arrays
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
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
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
