BSC 300 Exam 4 (pt. 2) - Microfilaments +

cytoskeleton fn

maintain & change cell shape, cell motility, intracellular tx, location of organelles, cell division

components of cytoskel

intermed filaments, microtubules, microfilaments

changes in cytoskel are in response to

signal transduction pathways

microfilaments (actin filaments)

can be organized into many structures within cells, including cell cortex, adherens belts, phagocytosis, and contractile rings

G-actin

single actin (globular actin) - not in a filament; has a cleft that binds atp/adp

F-actin

polymerized actin (microfilament)

minus end (aka pointed end) of microfilament

end with exposed binding cleft (+ end / “barbed end” is opposite)

phases of G-actin polymerization into F-actin filaments

nucleation → elongation → steady state

nucleation - G-actin polymerization into F-actin filaments

“lag phase” - inefficient formation of 3 ATP + G-actin initiates filament formation (skipped if short actin filament added to start)

elongation - G-actin polymerization into F-actin filaments

actin subunits rapidly assemble onto each end of filament

steady state - G-actin polymerization into F-actin filaments

G-actin monomers exchg w/ subunits @ filament ends, but no net change in total length

critical concentration (Cc) for polymerization

concentration of free actin (G-actin) where the rate of filament assembly equals the rate of disassembly

if conc. of free actin is above critical conc,

will be assembling filament

if conc of free actin is below critical conc

disassembling microfilament

if conc of free actin is at critical concentration

rate of assembly & disassembly are equal (see graph @ 13:00 lecture 20)

critical concentration at + en of actin filament is ____ than Cc at - end

lower

result of difference in critical concentration on ends of actin filament (+ end < - end)

“treadmilling”

actin treadmilling

ATP-actin subunits assemble faster at + end while being disassembled from - end; can recharge removed subunits by adding ATP & put them back on + end; position of indiv. subunits doesn’t change, filament changes around it

if actin treadmilling is confusing

18:00 in lecture 20 (thanks)

Profilin - regulation of filament turnover by actin-binding proteins

binds to side of G-actin opposite cleft, catalyzing ADP→ATP & preventing addition of G-actin to - end

Cofilin - regulation of filament turnover by actin-binding proteins

increases rate of disassembly by fragmenting ADP-actin filament regions and making more - ends

Thymosin-beta4 - regulation of filament turnover by actin-binding proteins

provides reservoir of ATP-G-actin: binds to actin when conc. high and releases actin when conc. low

filament capping proteins

block assembly and disassembly at filament ends

+end capping proteins - CapZ

limits actin assembly and disassembly dynamics to that at the - end

+end capping proteins: Gelsolin

severs actin filaments by inserting self btwn subunits & blocks new + end. some activated by rise in Ca2+

-end capping protein: Tropomodulin

blocks end where disassembly normally occurs, stabilizing filament

actin nucleation by formin FH2 domain

FH2 domains from two formins form a dimer → take turns stepping up to allow assembly of one actin subumit onto + end → PROTECTS + END FROM BEING IMMEDIATELY CAPPED

what does formin FH2 prevent

immediate capping of + end by end capping prot

regulation of formin activity

Activation by 1) unfolding by Rho-GTPases (signal transduction) or 2) FH1 domain bonding to profilin complexes; inactive when folded in on self

what do formins assemble

linear filaments

actin nucleation by Arp2/3 complex

two nucleation promoting factor (NPFs) domains each bind an actin monomer → bind & activate Arp2/3 → Arp2/3 binds - end of monomers to the side of an existing actin filament → new filament assembles on side

in actin nucleation by Arp2/3 complex, what end of the monomers is bound to the existing actin filament?

-

In actin nucleation by Arp2/3 complex, what angle is the new filament constructed at?

70 degrees

regulation of Arp2/3 complex

requires two input signals for activation (coincidence detection) - Arp’s Basic domain (B) binds regulatory phospholipid; Arp’s Rho-binding domain (RBD) binds active rho family GTPase

example of signal convergence

Arp2/3 complex

Arp2/3 dependent actin assembly during clathrin-mediated endocytosis

endocyt assembly fx recruit NPFs that activate Arp 2/3; a burst of Arp2/3 dependent actin assembly drives endocytic vesicles away from the PM

phagocytosis & actin dynamics

leukocytes’ Fc receptors activate Arp2/3 complexes, which assemble an actin filament network that moves the cell membrane around what’s being endocyted

opsonization

process of foreign pathogens being tagged for elimination

actin cross-linking proteins

varying structure can change flexibility and motility

myosin motor proteins

move along actin filaments by using ATP for mechanical work

myosin I mcs

SINGLE HEADED; one heavy chain w/ head & neck domain; variable # of light chains assoc w/ neck domain; some assoc directly w/ mb thry tail-lipid interxns

myosin 1 fn

membrane assoc, endocytosis

myosin step size smallest to largest

class II < class I < class V

myosin II mcs

two heavy chains - each w/ head and neck domain that binds two diff light chains; heavy chain has long, helical tail

which class of myosins can assemble into bipolar filaments thru tail interxns?

II

myosin II fns

contraction

myosin V mcs

two head domains & 6 light chains per neck; end of tails interact w/ specific receptors on organelles, which they transport along actin filament tracks

all three classes of myosin move toward which end of actin filaments

positive (+ end directed motors)

myosin V fn

organelle transport

myosin VI

only negative (-) end directed motor

myosin II motor protein

assoc w/ antiparallel actin filament; when activated, pull actin filaments in opposite directions; long neck domain = more force; makes contractile force of muscles & contractile rings of dividing cells

atp driven myosin movement along actin

myosin binds ATP & releases from actin → atp hlyzed, head into cocked state → head binds actin → “power stroke” myosin straightens and moves actin left → repeat

when is ATP used (=hydrolyzed) in myosin II movement??

when moving head into “cocked” state (think of a catapult and cranking lever)

lack of ATP by myosin means

it can’t be released from actin (think rigor mortis & muscle stiffness)

the length of the myosin II neck domain dets the rate of movement

longer neck → faster movement

sarcomeres

discrete contractile units in muscles; combine to form myofibrils; have a banding pattern that gives a striated appearance

sarcomere banding pattern

thick filaments → myosin (H,A bands); thin filaments → m=actin (I,A bands)

directionality of sarcomere contraction

myosin moves toward plus end, but it is anchored in place and actin pos ends are toward outside. thus, myosin pulls actin in and shortens entire segment (56:30 lecture 20)

accessory prot in skel muscle - for actin

CapZ caps + end @ Z disc; Tropomodulin caps - end; nebulin binds subunits & dets length

accessory prot in skel musc - for myosin

titin attaches to Z disk & M band to center myosin & prevent overstretching

muscle contraction regulated by Ca2+

sarcoplasmic reticulum releases Ca2+ via voltage gated ion channels & allows contraction to occur

why can’t actin & myosin interact w/o ca2+?

troponin & tropomyosin block actin binding sites. Ca2+ binds to troponin, which moves tropomyosin & exposes binding sites

which are “thick” filaments?

myosin

which are “thin” filaments?

actin

where does ca2+ come from for muscle contraction?

sarcoplasmic reticulum

myosin II in cytokinesis

forms contractile ring to separate one cell into two during cytokinesis

myosin V regulation

activated when cargo present - tail straightens, binds to cargo & head activity activated

cell locomotion (whole cell moving)

focal adhesions to ECM → new adhesion in desired direction → translocation (actin-myosin II contraction) → de-adhesion of old adhesions & repeat (1:07:10 lecture 20) “like a rock climber”

actin-based structures involved in cell locomotion

stress fibers, leading edge, focal adhesions

what proteins regulate/control microfilament assembly?

Rho family GTPases