16 - the cytoskeleton

Question 1

Function of the cytoskeleton

Answer 1

provides structure and mechanical support in cells without cell walls.
provides capacaty for movement
helps organize and move internal structures like organelles and vesicles

Question 2

components of the cytoskeleton

Answer 2

actin filaments, microtubules, intermediate filaments

Actin filaments determine the shape of the cell’s surface and are necessary for whole-cell locomotion, and are important for cytokinesis.

Microtubules determine the positions of membrane-enclosed organelles, direct intracellular transport, and form the mitotic spindle that segregates the chromosomes during mitosis.

intermediate filaments provide mechanical strength.

motor proteins convert the energy of atp hydrolysis to mechanical forces that can move organelles or filaments.

Question 3

microtubules

Answer 3

highly conserved

bind GTP/GDP

made up by tubulin hetero-dimer - one alpha and one beta tubulin. beta = + end, alpha = - end. beta-tubulin is a GTP-ase (hydrolyses GTP to GDP)

subunits linked by non-covalent bonds.

Question 4

actin filaments

Answer 4

highly conserved

3 isoforms of actin make up the filaments; alpha, beta, gamma.

bind ATP/ADP - polymer favors ATP hydrolysis

actin filament assemble head-to-tail. the asymmetrical actin subunits all point in one direction, so the ends are different; minus end (slow growing) and plus end (faster growing). minus end = pointed end, plus end = barbed end

Question 5

What is the advantge of having non-covalent

bonds linking the elements of the protofilaments

Answer 5

easier to assemble or disassemble quickly by loss or addition of monomers at the ends. this allows greater flexibility than if assembly and disassembly were more difficult.

Question 6

what is nucleation

Answer 6

for a new filament to form, it must grow on something. That is the assembled “nucleus” of subunits that can elongate by addition of other subunits.

nucleation is induced by changes in salt concentration or temperature. Actin trimer is quite stable = nucleus. for tubulin, the nucleus is larger (13 or more subunits).

the rate of filament assembly depends on the concentration of the free subunit. At the critical concentration, the rate of subunit addition = rate of subunit loss.

Three phases: lag phase (nuclation happens), growth phase (subunit addition to exposed ends), equlibrium phase (no net change in polymer)

if concentration of free subunits is more than critical concentration -> growth. Vice versa.

Question 7

nucleotide hydrolysis in actin and tubulin

Answer 7

actin has ATP, tubulin GTP. Both are hydrolyzed in the filament.

T-form (bound by ATP or GTP) is added to plus side, D-form is made upon addition, and D-form is lost from minus side

Question 8

treadmilling

Answer 8

filament maintains constant length while net flux of subunits happens - addition on plus side is equal to loss on minus side, so length is constant but the filament appears to move.

Question 9

catastrophe

Answer 9

change from growth of filaments to shrinkage of filaments. Opposite of rescue

Question 10

accessory proteins:

Answer 10

thymosin and profilin regulate monomer availability

formin and Arp2/3 complex regualte filament nuclation

CapZ and tropomodulin regulate filament stability

gelsolins and cofilin regulate filament depolymerization

fibrim and alpha-actinin regulate bundling

filamin and spectrin regulate gel forming.

Question 11

regulation of actin filament extension by thymosin and profilin

Answer 11

thymosin binding prevents actin monomer from association with plus ends of the filaments
profilin binding prevents binding to minus end of filament -> selection for binding to growing end.

These two compete with each other for binding to local actin monomers, thus regulating filament extension.

Question 12

regulation of actin nucleation

Answer 12

actin-nucleating proteins bring several monomers together to form a !seed”.

Nucleation is usually catalyzed by either formins or the Arp 2/3 complex.

formin:
mediate nucleation of straight/unbranched filaments -> controlled nuclation of actin filaments.
Remain associated with plus end of microfilament and facilitate addition of new actin monomers -> increased elongation rate.

Arp 2/3 compelx:

• two binding sites allowing filament network formation: one for actin, one to the side of a filament.
• If Arp 2/3 stays bound to nucleation start (minus end), then stabilization occurs.

Question 13

refualtion of actin nuclation by formins

Answer 13

formins can have “whiskers” which can facilitate interaction with profilin and increase subunit addition rate at the plus end under certain condition (compared to simple diffusoin-based addition)

Question 14

regulation of filament branching by the Arp 2/3 complex

Answer 14

Arps (actin related proteins) cannor form filaments on their own or incorporate in actin filaments.

they are inactive until they are bound by an activation factor.

active configuration resembles the plus end of an actin filament - provides the site for nucleation of a new filament at a 70 degree angle relative to the original filaments.

Question 15

regulating actin depolymerization

Answer 15

cofilin (actin depolymerization factor) facilitates filament breakdown through interactions with F-actin (ADP bound) and creation of mechanical stress.

Critical for polarized directed growth of the actin network

Question 16

lamellipodia

Answer 16

epithelial cells and fibroblasts

Arp 2/3 and actin are localized at the leading edge, and cofilin and actin are localized behind the leading edge.

Question 17

How is the unidirectional motion of lamellipodia maintained?

Answer 17

unidirectional treadmiliing.

Filament nucleation (regulated by Ark 2/3) and PLUS end polymerization occurs at the leading edge - pushing the membrane forward.

ATP hydrolysis in the filament convrts the actin from T to D form

cofilin preferentially binds D form, and destabilizes the filaments predisposing them to depolymerization.

This leads to a spatial separation between net filament assembly at the front and net disassembly at the rear.

Question 18

accessory proteins regulate microtubules

Answer 18

Filament nucleation is regulated by MTOC and gamma-TuRC

Filament destabilization is regulated by :
1. stathmin - regulates subunit availibility
Kinesin-13: plus end destabilization
3. katanin - MT severing proteins.

Filament stabilization regulaed by:

1. • TIPS
2. XMAP215
3. structural MAPs

Bundling and cross-linking by:
- tau, MAP2 (MAP = microtubule associated protein 2)

Question 19

microtubule nucleation

Answer 19

microtubules are nucleated from a mictorubule organizing center (MTOC), in aminals it is the centrosome.

The nucleation proceeds slowly, and addition of preformed nuclei bypasses the lag phase and causes rapid polymerization.

MTOCs are enriched in y-tubulin, and nucleation depends on a complex between y-tubulin and accessory proteins (y-TuRC (RC= ring complex))

Question 20

stathmin

Answer 20

binds to two unpolymerized tubulin molecules, preventing their addition and decreadsing the effective concentration of tubulin subunits available for polymerization and thus favors filament shrinkage.

stathmin is inhibited by phosphorylation.

Question 21

XMAP215

Answer 21

plus end binding protein, delivers tubulin subunits to the plus end to increase growth rate and suppress catastrophes.

inhibited by phosphorylation - very important during mitotic spindle construction

Question 22

motor proteins

Answer 22

move cargos (vesicles, organelles, molecules) around cells or create forces used to generate movement

differ in the type of filament they bind (actin or tubulin) and direction of movement and cargo transported.

Question 23

Motor proteins mediate transport and organization of different cargos

Answer 23

Dynein and kinesin interact with microtubules

Myosin interact with actin.

Motor proteins have the ability to transform chemical energy into movement (hydrolysis of nucleotides)

motor proteins consist of a head/motor (track selection and directionality), and tail/stalk (cargo secrion) domains.

Question 24

How does myosin generate force?

Answer 24

1. attached: myosin is bound to actin filament in a locked configuration.
2. Relased: ATP binds to a large “cleft” on the back of the haed, reducing affinity for actin and triggering release
3. cocked: ATP hydrolysis induces conformational change causing displacement of myosin head in relation to actin by 5nm. ADP and Pi remain bound.
4. force-generatin: weak binding of myosin II to actin induces Pi release, which causes both tight binding of myosin II to actin and power stroke (force generating shape change). During power stroke, ADP is released.

repeat.

Question 25

filament organization in the sarcomere

Answer 25

CapZ and tropomodulin prevent depolymerization from plus and minus ends respectively.

Question 26

Regulation of muscle activity by intracellular Ca++

Answer 26

Action potential arrives at neuro-muscular junctions, and induce:

• membrane potential change on the plasma membrane of muscle cells
• releae og Ca++ from the T-tubules
• release of Ca++ from SR, which initiates contraction of myofibril
• rapid pumping of Ca++ back into the SR by “Ca++ ATPase”
• return to steady state

In skeletal muscle, Ca++ regulates myosin activity via tropomyosin and troponin interactions. Tropomyosin binds along the groove of the actin filament, and troponin is a complex of 3 proteins (toponins T, I and C (tropomyosin binding, inhibitory and Ca00 binding).

In resting muscles, troponin I binds to actin and troponin T. This complex pulls tropomyosin out of its resting groove, to where it interferes with myosin head binding, and thus prevents force generation.

During action potentials:
Troponin C causes troponin I to release from actin, allowing myosin head engagement and force generation.

In other muscles Ca++ dependent phosphorylation regulates myosin II. Phosphorylation og myosin light chain leads to contraction.

Question 27

Myosin V

Answer 27

involved in organelle transport along actin filaments

ATP hydrolysis derived energy used to move part of the protein lever -> moving the myosin bundle relative to the actin filament = WALKING

Question 28

Motor proteins movign along microtubuli

Answer 28

Two types of motor proteins move along microtubules: kinesins and dyneins

the major function of microtubule motors is the transport and positioning of membrane-enclosed organelles

Question 29

Kinesin superfamily

Answer 29

2 light and 2 heavy chains, heavy mediate tubulin binding and have ATPase domains, light are required for organelle/cargo attachment.

Most walk toward plus ends of microtubules

Question 30

kinesin/microtubule interaction differs from myosin II /actin interaction

Answer 30

before step 1: Lagging domain bound tightly to microtubule because of ATP, leading domain is loosely bound to microtubule because of ADP

Step 1: hydrolysis of ATP

Step 2: Exchange of ADP for ATP in front causes tight binding to microtubule and change in “neck linker” conformation from rearward pointing to forward pointing. Shift in linker conformation drags lagging ADP bound domain forward.

After step 2: previous lagging domain is now leading, and vicec versa (leading = ADP, lagging = ATP)

Basically, ADP in from and ATP in back, then ATP id hydrolysed (now 2 ADPs), the new ADP moves forward and the old one is phosphorylated to ATP. ADP still facing foreward, but they have swapped

Question 31

Dyneins

Answer 31

largest motor proteins

two major types: cytoplasmic and axonemal.

Cytoplasmic dynein 1 is in most euks, minus end directed, golgi localization and vesicle trafficking, centrosome positioning, mitotic spindle construction. Cytoplasmic dynein 2 is for intraflagellar transport.

axonemal dyneins are specialized for rapid and efficient sliding movement of microtubules driving the beating of flagella and cilia.

Question 32

Intermediate filaments

Answer 32

Not found in animals with exoskeleton

preferentially found in cytoplasm of cells subject to mechanical stress, like skin cells.

Easily bent, kan be stretched over 3x their lengths.

Rapid turnover, in division/migration/dfferentiation.

ropelike

Question 33

epithelial intermediate filament

Answer 33

has kreatins (type I kreatins = acidic, type II = neutral/basic)

important for mechanical stability and tissue integrity.

One keratin filament consists of one type I and one type II, forming dimers that combine to tetrameres.

Keratin networks are held together by disulfide bonds