7: Actin Microfilaments Flashcards
actin is usually found – because it has
beneath the PM because it has mainly cortical function for cell shape and movement of organelles beneath PM
actin responds to
extracellular signals
Actin based structures include:
- epithelial cells: microvili, cell cortex, adherens belt
- migrating cells: filopodia, lamellipodia, stress fibers
- muscle and non-muscle functions: phagocytosis, moving endocytic vesicles, contractile ring
actins can form
networks and bundles
actin structure includes
- monomers ie G-actin/globular actin with 4 domains and ATP binding cleft (polar)
- alpha actin in muscle
- beta actin in cortex
- gamma actin in stress fibers
actin polmer is
- F-actin (filamenous) = microfilaments with ATP binding cleft at minus end
- double helix structure 36nm for g-actin to end in same spot
- polarity
myosin is
actin motor protein that binds to actin with arrow pattern where pointed end is (-) end and moves towards + end
stabilizes actin = can’t depolymerize
can use myosin coated actin as nucleus
which in is faster for polymerization of actin
(+) end has faster polymerization because of increased polymerization speed and the critical concentration is 0.12µM
(-) end Cc is 0.60µM
for actin polymerization, there are 3 requirements?
- nucleus + above critical G-actin concentration
- all actin in ATP form
- uncapped
depolymerization occurs when
G-actin below critical conc
if the g-actin critical conc is greater than 0.6
polymerize (+) and (-) end
if g-actin critical conc is less than 0.12µM
depolymerization at both ends
if g-actin critical conc is between 0.12µM and 0.6µM
treadmilling occurs: growth @ plus end and depoly @ minus end
what can treadmilling do?
can push something in front or pull something from behind
minus end of microtubule is different from actin because
MT: (-) end capp = not usually poly/depoly
actin: (-) end poly and depoly slower bc slow rate + high Cc
cellular conc of G-actin is — so we need –
400µM
so we need regulation of actin poly. so that we don’t always poly
What is thymosin?
sequesters most available actin by binding to it and releases g-actin when needed
What is profilin?
protein that promotes actin polymerization by charging
G-ADP into G-ATP actin (no profilin activation = regulate excess g-actin)
What is cofilin?
protein enhancing depolymerization
actin regulating proteins?
- thymosin
- profilin
- cofilin
- capZ
- tropomodulin
- formins
- Arp2/3
What is CapZ?
actin capping protein for (+) end to stop poly and depoly
What is Tropomodulin?
actin capping protein for (-) end to stop poly and depoly
Actin-disrupting drugs?
cytochalasin: depoly actin microfilaments
phalloidin: stabilizes actin microfilaments for no depoly
What are formins?
- nucleating proteins that regulate polymerization at + end for unbranched filaments (- end grows away)
- activity is regulated by Rho-GTP at the PM
- if Rho is in GDP form = no formin activity
also still need above Cc, G-actin in ATP form and profilin activation
how is actin different from MT in structure?
MT rarely branches
actin commonly branches
What is Arp2/3
- nucleating protein for filament branching on minus end (+) end grows away
- requires nucleation promoting factor (NPF) like WASp or WAVE
- Cdc40 and Rac are cell surface g-proteins
- WASp gets activated by Cdc42
- WAVE gets activated by Rac
How does listeria use actin?
listeria - parasite that uses branching to project actin out of cell
- has act A protein which activates Arp2/3 causing rapid branching of actin microfilaments helping listeria parasite move around quickly = burst through PM + infect next cell
Arp2/3 provides nucleus for branching filaments and can also
aid in endocytosis (pull PM by growing at + end)
aid in phagocytosis (push PM)
What is an actin bundle?
many unbranched microfilaments held together by fimbrin and alpha actinin proteins
Fimbrin is
actin bundling protein for microvili, filopodia and focal adhesions
What is alpha-actinin?
actin bundling protein for stress fibers, filopodia, muscle Z line
What is actin networks?
complexities of actin MT with branching held together by spectrin and filamin proteins to strengthen PM
What is spectrin?
protein in cell cortex for actin networks
What is filamin
protein in leading edge/lamellipodia, stress fibers and filopodia for actin networks
How is actin involved in RBCs?
- actin network binding proteins to support cell membrane + provide shape (ankrin and spectrin)
- ankrin achnors actin and spectrin cortical network to PM
How is actin involved in epithelial microvilli?
- bundles of actin with fibrin and ezrin protein
- exrin links PM to actin
- increases stability and decreases sliding movement
How is actin involved in muscles?
- muscle cells work by pulling on other muscle cells/tendons for movement by pulling on extracellular matrix
- actin linked to membrane by dystrophin
What is the structure of myosin?
heavy chain and light chain
* head is ATPase with actin binding sites
* neck binds to lights chains and determines how fast myosin can move/bend
* tail heavy chain binds to cargo
what is myosin 1?
step size, structure, use
- 10-14nm step size
- head, neck and tail
- tail binds to membrane phospholipid
- use: membrane association and endocytosis
what is myosin 2?
structure, function, step size
- 2 thick filament tails that overlap and bind to each other
- 2 heads
- muscle contracion
- many myosin heavy chain tails align making bipolar structure between actin
- step size 8nm
- most abundant in muscle
what is myosin 5?
- 2 heavy chains, tain region has variable domain to bind to different vesicles
- long neck region and a lot of light chain
moves fastest bc step size is 36 nm
what is the sliding filament assay for?
detect myosin-powered movement
how is sliding filament assay working?
- take fragments of actin by digestion and add to glass coverslip
- add flurescently labelled actin added
- add different things to see what is required for movement (ex. ATP)
- use fluorescent light microscope
what determines rate of mysin movement of actin
length of myosin neck
- longer neck = more ligh chains bind = help bending and speed = faster movement
What are the steps of ATP Driven Myosin Movement along actin filaments?
- rigor state myosin head in ADP form attaches to actin microfilament
- ADP is displaced by ATP = head releases actin
- ATP hydrolyzed to ADP + Pi= myosin head rotates into cocked state moving towards + end
- myosin head binds to another actin filament farther to + end
- power stroke release Pi - straighten myosin
- ADP released and ATP binds again
rigor moritis?
muscle stiffness in death when ATP runs out
What is a sarcomere?
basic contractile subunit of muscle fiber containing actin, myosin and stabilizing + scaffolding proteins between 2 Z disks
What is the structure of a sarcomere?
- z disk on either side
- actin microfilaments are + to minus from outside to inside
- myosin 2 heads on outside tails on inside between actin
- **CapZ **and tropomodulun caps on actin
- titin protein holds myosin thick filaments in centre and bend when contracting
- nebulin coating protein wraps and stabilizes actin
What are A bands and I bands?
A bands=actin+myosin in middle of sarcomere and don’t change size in contraction
I bands = actin + z-disk on either side of sarcomere and decrease during contraction bc Z disks move closer together
sarcomere opening is induced by
other muscles contracting
contraction of sarcomere requires
ATP and Ca2+
muscles cells are different bc they
are multinucleated, PM = sarcolemma, has sarcoplasmic reticulin
Ca2+ is stored and regulated in
sarcoplasmic reticulum
The sarcolemma has
projections that go into cell called Tranverse (T) tubules which reach into cell and are in close contact with sarcoplasmic reticulum
How does Ca2+ release occur>
- T tubules bring PM close to SR membrane
- nerve impulse required for coluntary muscle contraction
- electric changes in PL
- oens calcium release channels from SR and are quickly pumped back
why is there quick recovery of Ca2+ in muscle cells
↑ control of muscles Ca2+ doesn’t interact with phosphate to make precipitate
what are troponin and tropomyosin? how are they regulated?
proteins that work together and coat actin to inhibit myosin binding sites of actin
* regulated by Ca2+ presents
* Ca2+ binds to troponin and tropomyosin to shift their position to expose myosin binding sites for a short period of time so few steps taken
