Cytoskeleton Pt 2 Flashcards
Functions of Actin
Provide strength and shape to plasma membrane
Cell surface projections (filopodia) used to explore territory and move around
Muscle contraction (association with myosin(motor protein))
Microvilli (intestinal epithelial cells): enhance nutrient absorption
Whole cell locomotion (movement)
Cell cortex is
the area under the plasma membrane that interacts with actin
Cytoskeletal changes in cell division
Crawling fibroblast-dynamic actin cytoskeleton
Leading edge: site of actin assembly at filopodia
Cell division-rearrangement of MT
Actin-contractile ring (also myosin filaments)
The Actin subunit is
asymmetric (Globular Actin or G-actin)
G-actin is
bound to ATP or ADP
F-actin is
Actin subunits assemble head to tail to form a tight right handed helix (8nm wide)
called Filamentous or F-actin
___ end favors elongation
plus
flexibility of actin subunit
more flexible than microtubule
Arrowhead appearance of the plus end is due to
interaction between actin and motor protein myosin.
The rate limiting step of actin formation is
Nucleation
The regulation of actin filament formation is how cells control
their shape and movement
Nucleation of actin
Small oligomers (3 monomers - trimer - stable enough to promote growth) of actin subunits can assemble spontaneously (but unstable)
Actin subunits can spontaneously bind one another, but the association is unstable until subunits assemble into an initial, oligomer, or nucleus, that is stabilized by multiple subunit–subunit contacts and can then elongate rapidly by addition of more subunits.
Critical Concentration
Cc=As the polymer grows, subunits are used up. Concentration (C) of subunits is observed to drop until it reaches a constant value (Cc).
At this concentration, rate of subunit addition =rate of subunit loss
for C > Cc, both ends grow; for C < Cc, both ends shrink.
The lag phase (nucleation) of actin formation can be eliminated with
preformed filament (crosslinked actin)
Actin can catalyze
ATP hydrolysis (Occurs slowly in free subunits, faster in filaments)
T and D forms of actin
T form (bound ATP)
D form (bound ADP)
Nucleotide hydrolyzed (energy stored in polymer), with free energy change for dissociation of _____ form polymer more negative than ____ form
D
T
(D form polymers will therefore shrink, T form polymers grow)
The critical concentration (Cc) marks the level at which G-actin monomers are
in equilibrium with the actin filaments.
Actin filaments are only formed at monomer concentrations above theCc
Actin filaments are only formed at monomer concentrations
above theCc
Actin filament at Equilibrium
Rate of subunit addition=Rate of subunit loss
Treadmilling occurs when
the rate of addition is faster than hydrolysis at plus end, but slower than hydrolysis at minus end.
Plus end will be in T form, Minus end in D form.
During treadmilling the plus end is in ___ form and the minus end is in ____ form
Plus end will be in T form,
Minus end in D form.
Plus and minus ends in actin are determined by
growing rate
the fast-growing end is called the plus end, whereas the slow-growing end is called the minus end
During treadmilling the Polymer maintains a
constant length, even though there is a net flux of subunits through the polymer
Cc at plus and minus end
CcD> CcT
CcD is higher because D promotes shrinkage - subunits are removed (not added)
During treadmilling, the __ end grows and the ___ end shrinks
This requires___
Plus end grows while minus end shrinks (at same rate)
Requires a constant consumption of energy in the form of ATP hydrolysis
Actin Inhibitors
Cytochalasins
Latrunculin
Phalloidins
Cytochalasins
Fungal products that prevent actin polymerization (bind to plus end). (promotes depolarization)
Application: Can change cellular morphology, induce apoptosis, inhibit cell division. potential use to treat cancer
Latrunculin
Prevent actin polymerization (bind to actin subunits), from sponges
Phalloidins
toxins isolated from Amanita mushroom,
bind tightly to side of filament, stabilize against depolymerization, imaging application (can no longer depolymerize)
Sequestering monomers
Profilin and Thymosin
Nucleation and Elongation
Arp 2/3 complex, Formin
F actin crosslinking protein
Filamin
Profilin
binds to actin causing a conformational change that promotes addition
Thymosin
locks actin so it can’t associate with + end, preventing growth, “locking it in place”
Arp 2/3 complex causes
a caping so there is no growth at the minus end
this accelerates polymerization
ARP2/3 Complex as Microfilament Nucleator:
- Caps Pointed (associate with minus end)
- Analogous to GammaTRC for MTs
The Arp 2/3 complex can bind to the side of a filament causing
pointed nucleation or branched actin
Formins have a __ that attaches to ___
dimer attaches to the plus (barbed) end of a growing filament -> promoting nucleation and additional growth
(there is no branching)
Filamin
is a dimer, cross linking actin, loos arraignment causes a gel like state
is important for membrane extensions ex. filopodia
clamps together pairs of actin filaments roughly at right angles, thereby promoting the formation of a loose and highly viscous gel.
How does Listeria use actin to infect a cell
Has receptors for Arp2/3, nucleates actin, tail is then like a motor, is mobile through a cell, then infects another cell
Myosin is a ___, using ___
is a motor protein, ATPasse
uses energy from ATP hydrolysis to move (very similar to transport proteins)
Myosin II is composed of
two heads with a long tail.
Composed of 2 heavy chains (green) and four light chains (blue)
The heavy chains form coiled coils
In Myosin II ___ binds to ATP
the head domains, is the area of conformation change (what binds to ATP)
Tail-tail interactions of myosin form
large, bipolar thick filaments, with several hundred myosin heads
The head domains are pointed outward - bind to actin
The bare zone of the myosin II bipolar thick filament is composed of
myosin tails
Each myosin head binds to and hydrolyzes ATP, causing it to walks towards ___
the plus end of actin filament
inches towards end, actin slides in opposite direction
Steps of the myosin actin cycle
- Myosin (lacks bound nucleotide)-binds tightly to actin
- ATP binds to myosin (conformational change in actin binding site, reduced binding affinity of myosin head to actin)
- Bound ATP triggers movement of lever arm (myosin head displaced along actin filament). ATP hydrolysis
- Weak binding of myosin head to new site on actin causes release of Pi, myosin head binds tightly to actin.
- Release of Pi triggers power stroke (release of ADP)-original conformation of myosin head
(4/5 - same step)
Skeletal muscle cells (muscle fibers) anatomy
Huge, multinucleated cells (formed by fusion of many muscle cell precursors).
Myofibrils: cylindrical structure found in cytoplasm (consists of contractile units called sarcomeres)
Dynamics of the sarcomere
not as much dynamics because it is capted at both sides
Thin vs thick filaments of sarcomere
Thin filaments: actin and associated proteins, attached to Z disc (+ end). Capped (- end) of actin extend towards middle of sarcomere (overlap with thick filaments)
Thick filaments: bipolar assemblies of myosin II
Organization of accessory proteins in a sarcomere
(Image)
Z disk end of actin - Plus end
Capted end of actin- Minus end
M line - myosin
Muscle cell features make rapid contraction possible
- Myosin motor heads spend a fraction of ATP cycle time bound to thin filament, which allows many myosin heads to act in rapid succession on same actin (not interfere with each other)
- Specialized membrane system relays signal rapidly throughout cell
(during contraction - overlap but actin and myosin did not changed in length)
Neuromuscular junction activation and Ca2+ Channels
VGCa 2+ channel - close to Ca 2+ release channel -
T tubules and the sarcoplasmic reticulum
The T tubules are formed from an invagenation of the plasma membrane - they cover the myofibril - they allow for rapid contraction
Calcium dependence of skeletal muscle contraction is due to
accessory proteins that are closely associated with the actin thin filaments
Ca in muscle contraction
Ca2+ floods into cytosol, initiating contraction of each myofibril.
Increase in calcium is short-lived, Ca2+ pump actively transports calcium ions back to SR (restored concentrations within 30ms, allowing myofibrils to relax)
Two energy requiring steps: ATPase of myosin motor domain and Ca2+ pump
troponin
three polypeptide chains that are Ca sensitive
binding of Ca causes a change in tropomyosin, causing it to move away from binding sites so they can bind to actin
