mbs 320 exam 3

AHHHH AHHHH

how do proteins move forward?

move along microtubule using motor proteins that grab the vesicle, walking with it to the golgi

what is the distribution of all organelles inside the skeleton regulated by?

cytoskeleton

how do actins interact with the cell membrane?

membrane is originally very floppy, so actins help provide shape

how does actin polymerization work (brief overview)?

actin monomers (g-actin) form soluble subunits that dissociate with each other to form f-actin

how doe the solubility vary between g-actin and f-actin?

g-actin is soluble, but as f-actin gets larger, it becomes “insoluble” since it can be precipitated

how does flexibility vary with actin filaments?

the less rows there are, the less sturdy the filament is, but it is more flexible

what causes the polarity in the actin filaments?

specific and asymmetric interactions between monomers; g-actins are positioned in the exam same direction in a filament with a plus and minus end

t/f: the structure of the actin filaments is also true for microtubules and intermediate filaments

false; it applies to microtubules but does not apply to intermediate filaments

how does actin filament assembly/disassembly work (6)

1. actin monomer is an atpase that with a deep binding pocket that can hydrolyze on its own

2. monomer has a poor rate of hydrolysis but has a strong affinity for actin filament if it has atp

3. actin subunits required salt and atp for polymerization

4. actin subunits are added onto the ends of the filament. the plus side has a higher affinity for actin polymerization so there will be a higher rate of assembly, but the negative side can still have some assembly

5. atp hydrolysis happens after polymerization

6. hydrolysis causes slight changes in the actin structure, allowing for slightly easier depolymerization via destabilization

how does spontaneous nucleation of actin filaments occur (2)?

actins must bind in the same orientation while free-floating and stick by chance

• rate of disassembly is mediated by subunit attachment

describe the growth curve of actin filament assembly

nucleation, elongation, steady state

what is the critical concentration in polymerization?

concetration at which the rate of assembly = rate of disassembly; concentration of actin in equilibrium WITH A FILAMENT END

what is steady state?

NOT equilibrium! the overall changes in the actin filaments leads to a constant concentration of products and reagents (monomers can still be added/removed from both ends)

what is actin polymerization dependent on?

dynamic and dependent on the concentration of soluble g-actin (number of monomers in the solution)

is the rate of dissociation independent or dependent on the concentration of g-actin?

independent

how to determine the rate of polymerization?

(concentration of g-actin * kon) - koff

how do we represent/calculate the critical concentration?

koff/kon

is the critical concentration the same for the plus and minus ends of actin filaments?

no; the Cc(-) should be higher compared to the Cc(+) due to a lower rate of assembly

what happens to an actin filament if it is surrounded by a concentration of acitn monomers higher than the Cc(+) but lower than Cc(-)?

it will grow from the (+) end and shrink from the (-) end

what is treadmilling for actin filaments?

actin filament grows form one end and depolymerizes from the other, maintaining a constant rate for Cc and the length remains unchanged

what does the composition of acitn filaments look like as it continues to stay in the treadmilling range?

old filaments are eventually completely replaced by new actin monomers while the length of the filament is maintained

can microtubules treadmill?

yes

how does a % actin subunits in filaments vs. time after salt addition & a [g-actin] vs. time after salt addition graph compare?

they will be mirrored version of each other (similar to an s-shaped curve)

how does steady state differ from equilibrium?

• equilibrium: reaction assembly & disassembly is at the same rate at one end

• steady state: plus side adds subunits while minus side depolymerizes, so the rate of assembly for the filament appears constant but is actually actively changing

what nucleating protein complexes nucleate actin filaments?

arp2/3, formins

describe the arp2/3 complex (4)

• arp2 & arp3 “identical” to the plus end of an actin monomer

• rapidly elongate actin filament by adding actin monomers onto the arp plus end

• activation required, in the cytoplasm

• cannot form or incorporate with filaments, so must associate with other proteins first

describe how actin filaments are nucleated by the arp2/3 complex (4)

• activating factor (WASP) binds to the inactive arp2/3 complex, which has arp2, arp3 and other ptoeins

• g-actin monomers can bind to the complex, which mimics the plus end of an actin filament by binding to the minus end of subunits

• minus end is capped/remains bound, so the complex won’t dismantle as actin monomers are continuously added and form a filament

• arp complex randomly binds to the lateral side, creating branches by crosslinking filaments

at what angle does arp2/3 complex bind to the actin filaments to form a tree-like network?

70

in which direction do actin filaments tend to orient their plus ends?

towards the membrane

describe how actin polymerization causes membrane protrusion

• arp2/3 activated near the plasma membrane by activation factors, with their plus ends near the membrane

• activated arp2/3 nucleates more actin filaments in the tree-like structure that push the plasma membrane forward

• actin depolymerization occurs away from the plasma membrane, so the arp complex is eventually lost overtime

t/f: arp/2/3 can individually bind to actin

false; they must move together in the right orientation

you start a polymerization reaction with g-actin, atp, nacl and a nucleating factor (arp/23). which graph best describes this reaction?

exponential/rapid growth (minus end capped; nucleation factor allows for skipping of the lag phase) before plateauing by reaching the Cc of the plus end

what is a formin?

nucleating factor at the plus end of of the actin filament that promotes fast growth without capping; pushes the actin filament towards the cell interior

how does formin increase actin filament formation (4)?

• formin ring binds to the plus end of the actin filament; swing around to give room for the new monomer to come in

• formin whiskers capture g-actin with the help of profilin to bring to end of filament

• profilin also is an adp/atp exchange factor that enhances filament assembly

• depolymerization decreases slightly, balanced by whiskers bringing monomers

how fast is formin nucleation compared to a true free plus end?

not as fast; grab g-actin with whiskers to reduce random collision rate & reduces koff from plus ends by allowing new actin monomers to arrive but not to leave

does formin crosslink acitn filaments?

no, only arp2/3 complex does

briefly describe how actin can “move”

myosin grabs the filament, changes configuration upon binding, and takes a step and releases to walk along the actin filament

describe the basic general structure of myosins (3)

1. head domain - motor atpase that is actin binding

2. neck/lever arm - rigid alpha helix with light chains that provide strength to the arm by binding to the alpha helix

3. tail domain - involved in dimer formation, cargo binding, and filament assembly

how does the myosin ii structure differ from the myosin v structure?

ii: 2 heads with 2 light chain lever arm that can form its own filaments and assist in muscle contractions, cell movement, and other energetic reactions connected to an alpha helical coiled tail

v: 2 heads with 6 light chain lever arm and a cargo-binding domain attached via alpha helical coiled tail

what is unique about myosin ii, and how does its structure allow it to do so?

only myosin that can form bipolar filaments; possible due to long tails that combine two tails from other myosins via ionic interactions and h-bonds (form myosin ii filament)

describe how conformational changes in the myosin head drive the power stroke (4)

1. rapid equilibrium between free and bound states of myosin head with adp + pi

2. pi dissociates and light chain domain rotates (uses energy of hydrolysis to crank the arm up into the high energy pre-stroke position)

3. adp dissociates and atp binding occurs

4. head dissociates from actin filament due to hydrolysis causing a conformation change in the myosin

in which direction do most myosins move?

most are plus end directed motors, so the actin will move to the minus direction as the myosin moves towards the plus end of the actin filament

how does a sliding motility assay work for myosin motors (3)

• microscope slide allows for myosin to stick

• heads stick up and allow for actin binding. without atp actin remains bound, and with atp the myosin heads will walk and omve the actin

• myosin can be labeled fluorescently

what is step size and strength of the power stroke proportional to?

• step size is proportional to the length of the lever arm

• strength of the power stroke is inversely proportional to the length of the lever arm

describe the movement of myosin ii (3)

• NOT a processive motor; runs and loses contact and travels faster

• short arm resists more power (short but strong steps)

• one head binds and relases from the actin filament BEFORE second head binds, caused by FAST ADP release

describe the movement of myosin v (3)

• processive motor that takes long steps made possible by a longer lever arm and having SLOW ADP release step from the cycle

• to let go, the myosin must keep one of its heads attached to the actin filament before the next head in front of it can engage

• moves organelles along actin filaments

• cannot bind on the side or else it will rotate around the filament; must have certain points that allow for walking without rotation)

what would happen if you slow down the release of adp from a myosin head?

release of myosin from actin will slow down. adp will remain bound to actin, so atp will bind slower

how are actin and myosin ii filaments involved in muscle contraction (3)?

• each bundle stimulated by independent neurons

• actin and myosin ii filaments interdigitate forming a sarcomere

• thin and thick filaments will slide along each other to form contractions as the myosins walk along the actin and the actins move closer to eahc other

describe the structure of a sarcomere (actin + myosin ii filaments)

• i-band - variable length; only actin at the ends of the sarcomere, connected to the z-disk (plus ends are embedded and capped)

• a-band - constant & spans the length of the myosin filament; has myosin thick filaments and actin plus ends that are capped

• h-zone - variable length; portion of the sarcomere with only myosin

what is the relaxed state of the sarcomere?

• NOT stretched or elongated

• little myosin/actin interactions

• muscles may be elongated by contracting the opposite muscle

what role do troponin and tropomyosin paly in muscle contraction?

• tropomyosin blocks access to the actin filament

• calcium binds to the troponin complex bound to the actin, causing a shift in the position of tropomyosin exposing myosin binding sites, which allows for contraction of the sarcomere

• as muscles contract, calcium is released within the muscle cell cytoplasm

how are troponin complexes distributed?

proportional to the length of the actin filament regardless of activity (complex every x distance)

the body of a dead animal becomes very stiff in rigor mortis. which of the following is NOT a factor in the development of rigor mortis?

a. calcium released from calcium stores inside the muscle cells

b. lack of atp synthesis causes a failure of muscle contraction

c. tropomyosin proteins don’t block myosin binding sites along actin filaments

d. myosin heads remain bound to actin fialments after their power stroke

b; lack of atp synthesis causes an inability to relax the muscle instead

how is the movement of cells mediated by the actin cytoskeleton and cell adhesion?

• actin cortex placed under tension as actin polymerization at the plus end protrudes and unpolymerized actin moves towards the plus end

• contraction with myosin ii occurs behind the cell, while focal contacts with integrins experience rapid actin polymerization that will physically attach to the plasma membrane

• at the back, removal of attachment sites causes a squeezing action of actin myosin

which statement is correct?

a. beta-tubulin can bind to both alpha-tubulin and beta-tubulin proteins

b. alpha and beta tubulins are encoded by the same gene

c. alpha-tubulin can hydrolyze gtp

d. you can form dimers with two alpha or two beta tubulins

e. the alpha/beta dimer can easily dissociate into monomers

a

what is the structure of the alpha-beta tubulin dimer like, and what is it a monomer for? (4)

• alpha & beta tubulin bind to each other as soon as they are made and remain associated through the life of the protein, forming microtubules

• dimer assembled with specialty chaperones that have gtp bound

• beta tubulin can bind gtp and hydrolyze it (plus end)

• alpha tubulin can only bind gtp and cannot hydrolyze it (minus end)

t/f: alpha-beta dimers cannot hydrolyze gtp as a dimer but must be in a microtubule form to do so

true

t/f: microtubules have polarity

true

describe the assembly of a microtubule

13 protofilaments arranged in an imperfect helix with a seam (one protofilament slightly higher than the previous one)

how does microtubule assembly occur (3)?

• concentration-dependent to dimers in solution and requires gtp-tubulin

• must have gtp bound to beta subunit (alpha subunit will ALWAYS have gpt)

• assembly/disassembly occurs only at the ends

how are actin filament and microtubule assembly similar vs. different (3 similar, 2 different)?

similar: assembly/disassembly only at the ends of the filaments; addition & loss is faster at the plus end than minus end; hydrolysis occurs after assembly

different: actin polymerization requires salt; growth rate is less drastic for microtubule assembly

what is the gtp cap for microtubules?

growing end of a microtubule in which hydrolysis has not occurred yet for the alpha-beta dimers; caps the less stable region of microtubule with gdp-tubulin dimers

what is the role of gtp in microtubule stability (3)?

• protofilaments with gtp have a straight configuration

• hydrolysis causes a preferential configuration at a slight angle, weakening the bond in the polymer

• as the protofilament continues to curve, depolymerization occurs and gtp-gdp exchange occurs

what happens if gtp hydrolysis reaches the end of the tubule?

rapid depolymerization due to the angle, causes protofilaments to shoot out

what is catastrophe and rescue in terms of microtubule dynamic instability?

catastrophe - loss of gtp cap

rescue - regaining of gtp cap

what are the uses of microtubule dynamic instability?

allows the cell to “explore” the cytoplasm with a “blind” system of filaments

if you have a small dimer concentration, how does that affect the chances of catastrophe?

higher chances of catastrophe due to hydrolysis catching up

what is the nucleating factor for the microtubule?

gamma-tubulin ring complex (gamma-turc)

how does gamma-turc nucleate microtubules (2)?

• mimics a plus end that allows for alpha subunit docking

• remains bound/caps the minus ends of the microtubules

where is the gamma-turc found and how does that influence microtubule formation (3)?

• found on the surface of the centrosome, which is the microtubule organizing center of the cell

• centrosome eminates microtubules that are anchored at the minus end (capped by gamma-turc) and spread out

• microtubules grow/shrink to push hte centrosome to the center by bumping into things around the cell

what are centrioles made of?

tubulin

what are the two main microtubule motor proteins?

kinesins, dyneins

in what direction do kinesins move vs. dyneins?

kinesins: cytosolic (+), spindle (±)

dyneins: (-)

describe the structure of a cytosolic kinesin (2)

• globular head of heavy chains acts as the engine by binding microtubules and performing atp hydrolysis

• coiled coil alpha helix connected to light chains that can bind to transported vesicle

describe the structure of a cytoslic dynein (4)

• 2 copies of large heavy chain polypeptide that rotates to allow for dynein movement along microtubule

• heavy chain = motor domain + intermediate + light chains

• motor domain = atpase & stalk that has a microtubule binding site

• ring homologous to nsf (aaa atpases)

which of the following statements about the gamma-turc is FALSE?

a. gamma-turc has 14 copies of gamma tubulin protein

b. gamma-turc is not a component of the centrioles

c. gamma-turc mimics plus end of a microtubule

d. gamma-turc is mostly found in the centrosome

e. gamme-turc caps the minus ends of microtubules

f. gamma-turc binds to the beta subunit of tubulin dimers

f

which microtubule motor would be used if an organelle was moving to the interior (ex: copii vesicle) vs. moving out to the golgi (copi vesicle)

interior: dynein

moving out: kinesin

• if bidirectional, can have both motor types and activate/deactivate their movement

describe how microtubule motility assays work, including their requirements

• requirements: stabilized & marked microtubules, purified motor protein, atp

• microtubules are frozen to prevent dynamic instability

• label motors that are bound onto glass slide with fluroescence to see individual molecules

• beads attach to microtubule with the motor which allows for landing

vesicles can move along microtubules in the presence of _____

atp

what allows bidirectional movement of organelles along a single microtubule?

microtubules are very large, so there is room for multiple organelles that have different motors moving them along the microtubule

how does kinesin move (4)?

• processive motor; moves continuously along a microtubule

• moves along microtubule axis without lateral displacement (moves along single protofilament)

• moves from one tubulin dimer to the next (takes 8 nm steps)

• every step requires atp

what do intermediate filaments do?

provides strength to anything that experiences mechanical stress, such as skin

what is the nuclear lamina?

network of intermediate filaments formed by the protein lamin

describe the epithelial tissue and ecm matrix (3)

• epithelial tissue - single cell layer that secretes proteins in the ecm; connect intermediate tissue

• basal lamina - sticky cells

• connective tissue that make up the ecm; include fibroblasts that synthesize and remodel proteins in the matrix

describe intermediate filament formation (3)

• alpha helical monomer pairs with another to form coiled-coil dimer

• staggered tetramer forms between two dimers (soluble)

• 8 tetramers laterally associate to a growing filament

describe the loss/formation of intermediate filaments, and the polarity (3)

• no polarity (perfectly symmetrical)

• no motors can walk on intermediate filaments

• loss/addition of subunits can occur anywhere

what facilitates the assembly/disassembly of intermediate filaments?

phosphorylation

what allows for intermediate filaments to be very strong?

staggered long subunits with lateral contacts dominating; allow for rope-like properties

describe the proteins and general composition of the extracellular matrix (4)

• network of many secreted glycosylated proteins

• includes collagen, fibronectin, laminin, etc. with many different binding locations that allow for protein network formation

• precise composition of ecm depends on the tissue function

• proteins made via secretory pathway (gene with erss and no transmembrane segments; not membrane-bound)

what do fibroblasts do?

secrete collagen fibrils and organize them into bundle with other collagen-binding proteins

what is collagen (4)?

• protein found in the ecm

• prolines modified in golgi via hydroxylation

• 3 alpha-helical subunits elongate in the structure that can be homotrimers or heterotrimers

• long proteins can bind laterally, forming a narrow/wider diameter

describe the layers of the skin

• epidermis - stratified epithelial tissue layer

• cells attached by desmosomes to each other and by hemidesmosomes to the dermis

• dermis - connective tissue layer with collagen fibers

• fibroblasts attached to matrix via focal adhesions

• germinal layer includes cells actively dividing & migrating to upper levels

examples of cell-cell junctions vs. cell-extracellular matrix adhesion (2)?

• cell-cell - cadherin in desmosomes and adherin junctions

• cell-ecm/substrate - integrins (most integrins binds to ecm proteins in hemidesmosomes & focal adhesions)

what are cadherins?

integral membrane proteins that can bind cells together by “homophilic” interactions

how are cadherins structured to bind cells together?

• must be bound to calcium

• n-terminus in the extracellular space grab onto each other using adherins, c-terminus in the plasma membrane of the cell (1 transmembrane segment)

• constant distance between the two cells determined by protein length

• velcro interaction (sturdy adhesion overall but individual points can be broken off)

how can cadherins mediate cell sorting during development (3)?

• remove calcium to separate cells based on the cadherin expressed

• cells may be separated based on having a high affinity for the same kind of cadherin but weaker affinity for others

• allow cells to mix and spread again by providing calcium

describe integrin assembly

• dimers of alpha and beta integrin proteins that are tightly bound

• alpha helix cleaved in two but linked via disulfide bond

• n-terminus in the extracellular space (erss with transmembrane segment), c-terminus in the cytosol

• calcium required for integrin binding

what and how do integrins bind and where can they be found (3)?

• connect ecm with intermediate or actin filaments

• use adaptor proteins to mediate actin-binding on the cytosolic side

• found in hemidesmosomes and focal adhesions

how do integrins bind to ecm proteins?

bind to proteins with the aa sequence arginine-glycine-aspartate (rgd); must be exposed so that they can be bound to integrins

how are epithelial cells structured (3)?

• apical side - in contact with lumen and has a lot of endocytosis, secretion

• basal side - contact with ecm proteins and integrins

• polarity contributes to function