Topic 5: The Cytoskeleton

Question 1

What is the structure and function of the cytoskeleton?

Answer 1

movement: subcellular movement, organismal movement (muscles)

organization: polarity, nuclear organization

shape: organellar shape, cellular shape

Question 2

What are the three filament types in the cytoskeleton?

Answer 2

microfilaments: muscle, cell polarization, cytokinesis

intermediate filaments: nuclear lamino, desmosomes

microtubules: vesicle movement, chromosomal movement

Question 3

What is the structure of microfilaments?

Answer 3

organize into many subcellular structures: microvilli, cell cortex, adherens belts, stress fibers, contractile ring

composed of actin monomers: many different isoforms that are expressed differentially depending on cell type

G actin: globular actin, monomer
F actin: filamentous, polymer

F actin has structural and functional directionality (positive favors polymerization, negative favors depolymerization)

Question 4

What are the kinetics of polymerization?

Answer 4

because of structural polarity of the actin filament, the minus end ATP-cleft is exposed to solution and can hydrolyze into ADP, this hydrolysis causes destabilization and depolymerization of the actin filament

Question 5

How is the actin filament both polymerized and depolymerized?

Answer 5

the actin filament is both polymerizing at the plus end and depolymerizing at the minus end at the same time

G actin can bind ATP or ADP

ATP bound is polymerization favoured

ADP bound is depolymerization favoured

Question 6

What is actin polymerization in vitro?

Answer 6

ATP-G actin is added to the plus end: if in excess –> polymerization

requires nucleation of polymerization, short oligomers of G-actin are needed to nucleate filament formation

filament formation is must faster at the positive end because of the shielding of the ATP cleft

polymerization is possible at both positive and negative ends with excess ATP-G actin but at steady state occurs at positive end

steady state = treadmilling: balance of pol at positive end and depol at negative is sufficient to generate force

Question 7

What is actin-associated protein modulate treadmilling?

Answer 7

e.g. actin sequestering proteins: bind available G actin to prevent polymerization

e.g. profilin: enhances the loss of ADP off ADP-G actin, promotes polymerization

Question 8

What are the motors of microfilaments?

Answer 8

actin dynamics can cause cellular motion but most actin-based movement is caused by myosin motor movement

Question 9

What is myosin II?

Answer 9

myosins are ATP dependent proteins that can bind actin, undergo a conformational change and move against the rigid actin CSK

all myosins contain: actin binding head, heavy chain (motor unit), flexible neck region, cargo binding tail

type II myosins are hexamers

typical type II myosin consists of: 2 heavy chains that associate by dimerization of tails, 2 essential light chains, 2 regulatory light chains

Question 10

What is the contractile mechanism?

Answer 10

head domains can bind actin and ATP

neck region can bend in response to the chnaging conformation of the head domain

head domain changes conformation in response to ATP hydrolysis

mechanism of myosin II movement

Question 11

What is the cross bridge cycle?

Answer 11

release of Pi allows myosin to bind actin

release of ADP that drives “power stroke” = contraction

if not ATP or ADP is bound, the myosin is in a bound and contracted state

myosin is not bound to actin when myosin bonds to ATP, ATP associates releases myosin off actin

ATP hydrolysis allows myosin cocking (opening of myosin)

Question 12

What are contractile ring in cytokinesis?

Answer 12

non-muscle type II myosin-based constriction during cytokinesis

actin is bundled into a circumferential array at the cortex

pure-string model of contraction

Question 13

What are microtubules?

Answer 13

structure MT’s are hollow polymers of alpha and beta tubulin dimers

each tubulin subunit also binds GTP and beta tubulin has a GTPase function

the protofilaments have intrinsic polarity (positive and negative ends)

composed of polymers of tubulin dimers

dimers polymerize into protofilaments

tubulin monomers can be GTP bound (pol favoured) or GDP bound (depol favoured)

13 protofilaments assemble into a hollow tube

Question 14

What is a microtubule organizing centers?

Answer 14

microtubules are anchored in structures called MTOCs in the cell with the negative end anchored into the MTOC (centrosome) which is usually located centrally in the cell close to the nucleus

negative ends are centered near middle of cell

MTOC sits near the interphase nucleus

positive radiate to periphery of cell

negative end of MT are anchored in centrosomes/MTOC

centrosomes contain centrioles and pericentriolar material

Question 15

What are the kinetics of microtubules?

Answer 15

MTs are continuously assembled and disassembling at the ends and can facilitate CSK-based movements

in vitro: polymerization can occur at both ends (negative end not anchored in MTOC in vitro)

balance of pol/depol is sufficient to generate force

Question 16

What is treadmilling?

Answer 16

treadmilling in MTs has a greater cellular role than with MFs for cellular movements

less common in MT because negative end is stabilized by centrosome/MTOC

treadmilling generates forces for chromosomal movement in mitosis

Question 17

What is dynamic instability?

Answer 17

refers to the difference in growing and catastrophe phases in the normal growth of MTs, it is not as simple as just treadmilling

growth of microtubules can occur rapidly at the positive end

growth of MT is fast at positive end as long as concentration of GTP-tubulin is high

polymerized GTP-tubulin forms straight protofilaments

polymerized GDP-tubulin forms curled protofilaments

tubulin has GTPase activity in beta subunit

dynamic instability occurs because of the GTP hydrolysis that occurs in beta tubulin

Question 18

What are the steps of dynamic instability?

Answer 18

addition of GTP tubulin forma a “GTP cap”

if concentration of GTP tubulin increases, then polymerization at the positive end

if keep adding GTP-tubulin then MT is stabilized because GTP tubulin forms straight protofilaments

if concentration of GTP tubulin decreases then cap is not present and protofilaments curl away = rapid depolymerization = catastrophe

after significant catastrophe, MT can recover, if pool GTP-tubulin is replenished

Question 19

What are the two types of motors associated with microtubules?

Answer 19

kinesin: positive end directed MT motor

dynein: negative end directed MT motor

Question 20

What is kinesin?

Answer 20

dimers of heavy chains with association to light chains

monkey-bar movement

Question 21

What is dyenein?

Answer 21

6 subunits (2 large, 2 intermediate, 2 small)

negative end directed

inch worm movement

Question 22

How do kinesin and dynein cooperate?

Answer 22

facilitate vesicular transport

coordination of anterograde and retrograde transport

melanophores in fish

allows distribution of pigmentation that is neuronally controlled

camouflage or mating displays

Question 23

What is the structure of intermediate filaments?

Answer 23

biochemically heterogenous, no true monomer

no associated motor proteins

not involved in cell movement

provide structural integrity to the cell

no intrinsic polarity

Question 24

What is the function of intermediate filaments in the nuclear lamina?

Answer 24

network of lamin intermediate filaments that forms a meshwork of extending on the inner surface of the nuclear envelope

composed of lamin A, B, and C

provides structural support to the nucleus

sits on the inner face of nuclear envelope

must be disassembled upon mitotic entry (nuclear envelope breakdown)

NEBD is regulated by the phosphorylation of lamins