cell bio exam 1 - actin, myosin, and muscle contraction

cytoskeleton

a scaffold or skeleton within the cytoplasm (bones and muscles of the cell)

• drives cell shape change and movement w/ coordinated forces

• provides structure and organization to the cell

• resists/transmits stress

• connects the cell physically to the external environment

the three types of cytoskeletal filaments are

• actin filaments - helical, cell migration, cell shape, cytokinesis

• microtubules - hollow cylinders, organelle position, intracellular traffic, chromosome movement

• intermediate filaments - ropelike fibers, mechanical strength

cytoskeletal components have supporting proteins with different functions:

• regulators of cytoskeletal dynamics - nucleators, capping proteins, severing proteins

• crosslinkers

• proteins that link the cytoskeleton to membranes

• motor proteins

what does the actin cytoskeleton do?

• cell shape

• cell polarity

• cytokinesis

• endocytosis

• motility within cells, whole cell migration

• sense and transmit mechanical forces

f-actin actin filaments are

polymers of the protein actin

cels can assemble and regulate diverse actin structures like

• microvilli - epithelial cell

• stress fibers - contractile bundles

• filopodial lamallopodia - leading edge of migrating cell

• contractile ring - dividing cell

actin monomers called (?) are polymerized to form actin filaments called (?)

G-actin; F-actin

each actin monomer has a binding site for ATP and can hydrolyze ATP to ADP, making it an

ATP synthase

actin monomers are held together by

noncovalent interactions, which allows for rapid asembly and disassembly

actin filament polarity has an effect on filament growth rates

• plus end - addition of actin monomers is fast

• minus end - addition of monomers is slow

actin polymerization includes

• nucleation/lag phase: rate limiting step where individual monomers come together

• elongation: filament grows at both ends, steep part of growth

• steady state: point at which rate of addition of new subunits to both ends equals the rate of dissociation

what is Cc?

the concentration of monomers at steady state, where there is no net association or dissociation; growth will continue until it reaches this concentration

nucleation is a slower process because

when only two monomers bind together, their binding is weak; the addition of a third monomer to form a trimer (or seed), makes the entire group stable

• this allows progression into the fast addition of monomers

if an actin filament is polymerized. with pre-formed seeds,

the lag phase is eliminated and growth can start right away

why is plus end addition more fast than minus end?

the plus end holds ATP-bound monomers that are hydrolyzed to ADP-bound monomers closer to the minus end; most free actin monomers are ATP-bound

when ATP is hydrolyzed to ADP with an actin monomer, a conformational change occurs which

reduces strength of binding between actin monomers and promotes depolymerization

“treadmilling” occurs when

the rates of monomer addition and loss are equal; causes the filament to appear to maintain a constant length even t hough there is a flux of actin subunits through the filament

actin-binding proteins control the

structure and behavior of actin filaments in cells

actin monomer-binding proteins include

• profilin - binds opposite of ATP cleft, increases binding, binds actin monomers, speeds elongation

• thymosin-beta4 - blocks monomer addition, blocks nucleotide exchange

formin

dimeric complex that nucleates actin filaments

• “whiskers” can bund to profilin actin

• associates with plus-end and stays there

• gathers free unbranched actin monomers

• can be sped up by profilin actin

arp 2/3

• complex made up of actin related proteins (arps), including arp 2 and 3

• nucleates branched actin filaments, and has a similar structure to actin

• mimics the plus end of actin after binding to nucleation promoting factor; able bind to the side of a “mother filament” and grows a branch at 70 degrees

• actin monomers are added to the plus end of arp 2/3 complex, which now becomes the minus end of the branch

examples of actin-based cellular structures that depend on formin or arp 2/3

formin → contractile ring

arp 2/3 → meshwork at a leading edge

capping proteins

• accessory protein to actin, regulates how higher-order actin structures are created

• binds to and stabilizes plus ends

cofilin

• actin-binding protein the regulates how higher-order actin structures are created

• binds to side of actin filaments

• severs the filaments

cross-linking proteins

• actin-binding proteins that regulate how higher-order actin structures are formed

• ssembles networks and bundles of actin filaments

drugs that affect filaments

• phalloidin - binds and prevents depolymerization (“freezes”)

• cytochalasin - caps plus ends, depolymerization at minus ends

• latrunculin - binds and prevents polymerization (monomers can’t join)

motor proteins

• bind to f-actin, microtubules

• have a head domain and tail domain

• generate force to move by coupling ATP hydrolysis to conformational change

what do head domains of motor proteins do?

bind to filaments and hydrolyzes ATP

what do tail domains of motor proteins do?

carry cargo, mediates dimerization

myosin

• motor protein on actin

• move towards plus-end

• n terminal, head domain - hydrolyze ATP

• c terminal - associates with other myosin motor proteins to form myosin filaments (thick filaments in muscle cells, bipolar filaments in non-muscle cells)

myosin nucleotide binding states include

• myosin + no nucleotide (strongest), rigor configuration

• myosin + ATP (weakest)

• myosin + ADP (strong)

• myosin + ADP + Pi (weak)

how does the myosin motor mechanism work?

1. myosin + ADP + Pi weakly associates with actin filament (head docks)

2. docking causes Pi dissociation

3. myosin + ADP has strongest affinity for actin; rapid conformational change leads to lever arm swings and generates motion (power stroke)

4. ADP dissociates; myosin + no nucleotide state, rigor state

5. ATP binds to myosin; ATP + myosin has very weak affinity to actin and dissociates → myosin head above filament

6. ATP hydrolyzes into ADP + Pi; returns to first state

muscle contraction depends on

• coordinated behavior of f-actin and myosin ii

• synaptic input from neurons

each muscle fiber is made up of (?) which are made up of (?)

myofibrils; sarcomeres

thin filaments are composed of

actin filaments and proteins; plus ends are capped and attached to a z disc; minus ends are capped and extend towards the midline

thick filaments are composed of

myosin and associated proteins; bipolar arrays

myosin heads walk towards which end of actin filaments?

plus ends

microtubules

• most rigid

• rapid polymerization/depolymerization

• organizing centers (MTOCs) coordinate behavior

• direct intracellular traffic; help w cell division as mitotic spindle; form stable cores of cilia and flagella

microtubule monomers are

tubulin dimers

• bind GTP

• have an alpha and beta end and stack to form protofilaments

• stack together with noncovalent bonds

how many protofilaments are in one hollow tube microtubule?

13

what is the difference between plus and minus end of a microtubule?

• plus - beta ends, fast growing, bind GTP and cannot hydrolyze

• minus - alpha ends, slower, anchored in MTOC, bind GTP and can hydrolyze

MTOCs

• control location, number, orientation of MT

• centrosome, spindle poles, basal body

centrosome

• MTOC of interphase cells

• matrix of proteins organized by pair of centrioles

• ring-shaped structures made from gamma tubules (gamma tubulin ring complex

gamma tubulin ring complex

• nucleation site for the growth of MTs

• eliminates lag phase *(spontaneous polymerization)

• anchors minus ends

• centrosome MTOC

dynamic instability

period of slow growth and then rapid disassembly

• allows MTs to search and find targets

• random exploration, selective stabilization

• driven by GTP hydrolysis

how is dynamic instability achieved?

GTP hydrolysis change conformation of individual subunits - protofilaments are curved w/ weakened lateral bonds

GTP cap is removed

GDP is more weakly bound and protofilaments peel away from the plus end

followed by fast depolymerization until the GTP can be reformed

drugs that stabilize and depolymerize microtubules

• taxol - binds and prevents depolymerization (freezes)

• colchicine, colcemid - forms complex that binds to end of microtubule and blocks polymerization (freezes)

• nocodazole - binds and blocks tubulin polymerization (no more added)