AC Lecture 37: Intracellular architecture

Question 1

List the cytoskeleton framework filament types.

Answer 1

microtubules
intermediate filaments
actin/microfilaments (smallest)

Question 2

Describe IF assembly.

Answer 2

monomer (elongated, alpha helical with a globular N-terminus and C-terminal tail) combine to form dimers (coiled coil) which associate to form tetramer which associates with 7 other tetramers to form ropelike filaments (associate head to tail so there is no polarity)
most are not reversible

Question 3

What is the function of IFs?

Answer 3

protect the cell from stretching forces
surround nucleus to form a network towards the cell periphery
interact with desmosomes or hemidesmosomes (junction proteins)

Question 4

What are the classes of IFs?

Answer 4

keratins
vimentins
nucleofilaments
nuclear lamins

Question 5

What allows for the breakdown of the nuclear membrane during mitosis?

Answer 5

phosphorylation of nuclear lamins allowing for reversible assembly of intermediate filaments

Question 6

What is the function of microtubules?

Answer 6

intracellular organization and intracellular transport of organelles and vesicles throughout the cell
form distinct structures such as the mitotic spindle, cilia, and flagella

Question 7

What is the difference between cilia and flagella?

Answer 7

cilia- present on the exterior side of the plasma membrane to help move materials over the cell surface
flagella- larger and used for locomotion of whole cells (e.g. sperm)

Question 8

Describe the structure of microtubules.

Answer 8

grow from a tubulin dimer (one alpha and one beta, each bound to GTP) forming linear protofilament that grow out of centrosomes from minus end (which binds to structures composed of alpha tubulin) to plus end (where there is a free beta to bind incoming alphas)
have polarity

Question 9

Describe the dynamic growth of microtubules.

Answer 9

1. alpha subunit binds to centrosome to start building
2. under conditions where there are plentiful* free dimers, beta tubulin hydrolyzes its bound GTP as a free alpha subunit binds to it (minus to plus end)
3. hydrolysis from GTP to GDP stabilizes structure
   * note that under tubulin limited conditions, the GDP form is at the cap and hydrolyzes without the addition of another subunit to its end, destabilizing the structure

Question 10

What drugs impact polymerization of microtubules?

Answer 10

prevent polymerization: colchicine/vinblastine

prevent depolymerization: taxol (chemotherapeutic)

Question 11

What are microtubule binding proteins?

Answer 11

proteins that regulate the spacing between long tubules

Question 12

What are the different kinds of microtubule-organizing centers (MTOCs)?

Answer 12

in interphase cells: centrosome

in cilia and flagella: basal body

Question 13

What is dynamic instability?

Answer 13

the growing and shrinking of microtubules in a GTP hydrolysis dependent manner
when GTP is hydrolyzed before a new dimer can bind the end the structure is destabilized (dependent on the concentration of present dimers)

Question 14

What motor proteins use microtubules as tracks?

Answer 14

kinesin (anterograde; plus end directed motors)- normally associated with synaptic vesicles and axonal growth materals
dyein (retrograde; minus end directed motors)- normally associated with recycling of membranes to lysosome in cell body

Question 15

How are microtubles essential to cilia and flagella?

Answer 15

facilitate the beating motion via specific arrangements

also dependent on the action of motor proteins (dyneins induce slide of one microtubule against another)

Question 16

What is primary cilia?

Answer 16

non-motile (due to lack of central pair of microtubules) sensory organs found in terminally differentiated cells

Question 17

What diseases result from mutations in the function of cilia?

Answer 17

polycystic kidney disease

Question 18

How are microtubules involved in cell division?

Answer 18

microtubules existing in interphase cells reorganize into the mitotic spindle which attaches to the centrioles
in prophase, centrioles divide and separate to opposing sides of the nucleus by kinesin motors (asters)
in prometaphase these become attached to centromeres via kinetochores to help organize and align the chromosomes in a congression during metaphase
during anaphase the chromosomes move apart under tension from teh kinetochore microtubules

Question 19

What are the kinds of microtubules in the spindle.

Answer 19

kinetochore- microtubules that bind directly to the chromosomes at the centromeres
overlap/polar- bind each other in an overlapped fashion
astral- bind to the cell surface

Question 20

How are microtubules in the spindle shortened through the cell cycle?

Answer 20

disassembly at both the kinetochore and spindle ends (motor proteins push and pull the spindle to move the poles further apart; dynamic instability constantly shrinks the kinetochore)

Question 21

What is actin?

Answer 21

monomer protein that binds ATP and polymerizes to form actin filaments in a helical arrangement
have polarity (plus end with G-actin binding; nucleotide facing minus end)

Question 22

What roles do actin filaments play in human cells?

Answer 22

function dependent on acting binding proteins it interacts with:
form structural component of microvilli (seen in epithelial cells)
found around cell membrane (cell cortex)
form structural belt around the cell (adherens belt)
form structures used to promote cell movement or crawling (e.g. lamellipodia or filopodia) which can also promote endocytosis
form part of the contractile ring used in cell division

Question 23

What are the steps of actin filament formation?

Answer 23

1. nucleation
2. elongation
3. steady state

Question 24

What is treadmilling?

Answer 24

where the rate of binding at the plus end of a growing actin filament equals the rate of dissociation at the minus end

Question 25

Describe the dynamics of actin filament formation.

Answer 25

1. actin-ATP binds to plus end
2. ATP hydrolyzes
3. filament contains mostly actin-ADP which has a weaker affinity for being in filament and dissociates from the minus end

Question 26

What can regulate actin filament formation?

Answer 26

stabilization of actin filament: CapZ (in muscle fibers)

stimulation of actin filament growth: formins (during stress leading to movement) and Arp2/3 (filament branching)

Question 27

Describe the role of actin filaments in cell crawling.

Answer 27

1. cells attach to surface at integrins that bind actin

2. cell is pushed forward via actions of contractile bundles of actin in association with myosin (motor protein)

Question 28

Provide an example of a non-human cell that uses human actin for motility.

Answer 28

Lysteria monocytogenes (bacteria)
hijacks actin machinery via ActaA which recruits actin and machinery that promotes actin branching and assembly  resulting in comet tail that "shoots" the bacteria around

Question 29

How is actin activity regulated?

Answer 29

growth factors (e.g. EGF and PDGF) bind to receptors on the cell surface, leading to changes in cell movement via the actions of small GTP binding proteins (Rac, Rho, and Cdc42) which are activated by signals from outside the cell

Question 30

What do each of the small GTP binding proteins that signal for actin do individually?

Answer 30

Cdc24- stimulates filopodia formation, establishes cell polarity and direction of movement
Rac- stimulates membrane ruffles/lamellipodia, establishes leading edge
Rho- stimulates stress fibers that contract, moves via contraction

Question 31

Describe the composition of skeletal muscle.

Answer 31

formed by teh fusion o fmuscle cells to form myofibril which is composed of many repeating sarcomeres (which can shorten up to 70% upon contraction)

Question 32

What types of filaments do sarcomeres have?

Answer 32

thick filaments- correspond to a polymerized form of myosin II (bipolar motor protein)
thin filaments- comprised of actin and actin binding proteins

Question 33

How are the filaments of sarcomeres arranged?

Answer 33

plus ends associated with Z-disks

Question 34

How do muscles contract?

Answer 34

1. action potential at a neuromuscular junction
2. release of Ca2+ stores from sarcoplasmic reticulum
3. alteration of binding of tropomyosin and troponin on actin to allow myosin II binding which slides the z-disks towards the center of the sarcomere