Slide set 11

Question 1

cytoskeleton

Answer 1

• protein polymers composed of actin subunits, tubulin subunits, and intermediate filaments

Question 2

microtubules are made up of

Answer 2

tubulin subunits

Question 3

microtubule growth

Answer 3

microtubules continually grow from the centrosome added to a cell extract

Question 4

dynamic instability

Answer 4

some microtubules suddenly stop growing and then shrink back rapidly (rapid disassembly)

constant polymerization and depolymerization cycle

Question 5

actin filaments aka

Answer 5

microfilaments

Question 6

actin filaments

Answer 6

• helical polymers of actin
• flexible
• organize into linear bundles, 2D networks, and 3D gels
• actin filaments are dispersed in the cell but most highly concentrated in the cortex (right below the plasma membrane)

Question 7

where do you find actin filaments?

Answer 7

microvilli, striated msucle

Question 8

microtubules

Answer 8

• long, hollow cylinders made of tubulin
• more rigid than actin
• long, straight
• usually have one end attached to a microtubule-organizing center (MTOC) called a centrosome

Question 9

where do we find microtubules and what are they made of

Answer 9

tubulin!

cilia!

Question 10

intermediate filaments

Answer 10

• ropelike fibers
• made of intermediate filament proteins

Question 11

intermediate filament example

Answer 11

forms meshwork called nuclear lamina beneath the inner nuclear membrane

extend across cytoplasm

gives mechanical strength!

in epithelial tissue, they span cytoplasm from one-cell junction to another (strengthening epithelium)

Question 12

cytoskeletal polymers are….

Answer 12

DYNAMIC!

• cells can rapidly reorganize cytoskeletal organization
  
  • it uses subunits of polymers to build new structures
• cytoskeletal polymers assemble from subunits AND undergo self-assembly

Question 13

cytoskeletal polymers determine…..

Answer 13

cell polarity and internal organelle organization!

image is small intestine

actin increases SA for food absorption

Question 14

actin monomers bind together to form….

Answer 14

a polymer/filament!

• actin monomers have ATP binding pocket (ATP can be hydrolyzed to ADP)
• actin monomers have plus and minus end
• subunits bind together head-to-tail
• subunits are added to the end of growing polymer (NOT inserted in the middle)

Question 15

polarity of actin filament image

Answer 15

barbed = plus end

pointed = minus end

2 Ps don’t go together!

Question 16

blocking actin filament assembly/disassembly has what effect on cells

Answer 16

toxic!

Question 17

actin chemical: Latrunculin

Answer 17

effect: depolymerizes
mechanism: binds actin subunits
source: sponges

L in latrunculin bc larry the lobster is in spongebob

Question 18

actin chemical: Cytochalasin B

Answer 18

effect: depolymerizes
mechanism: caps filament plus ends
source: fungi

Question 19

actin chemical: Phalloidin

Answer 19

effect: stabilizes
mechanism: binds along filaments
source: Amanita mushroom

Question 20

actin assembly graph

Answer 20

A. polymerization of pure actin subunits into filaments occurs after a lag phase

B. polymerization occurs more quickly in presence of preformed fragments of actin filaments (act as nuclei for filament growth)

% of free subunits after polymerization reflects critical concentration (C_c) at which there is no net change in polymer

Question 21

how do we study actin polymerization?

Answer 21

observe change in light emission from a fluorescent probe (pyrene)

fluorescent probe covalently attaches to actin

pyrene-actin fluoresces more brightly when incorporated into actin filaments

Question 22

nucleation

Answer 22

• a helical polymer is stabilized by multiple contacts between adjacent subunits
• in actin, 2 molecs binds weakly to each other, but a 3rd actin (forms trimer) makes the whole group more stable
• once further monomer addition occurs, this now is a nucleus for polymerization. (tubulin nucleus is larger)
• assembly of nucleus is slow (explains the lag phase seen during polymerization)
  
  • lag phase can be reduced or abolished entirely by adding premade nuclei (EX: fragments of already polymerized microtubules or actin filaments)

Question 23

how do reduce or eliminate lag phase of actin polymerization?

Answer 23

lag phase can be reduced or abolished entirely by adding premade nuclei (EX: fragments of already polymerized microtubules or actin filaments)

Question 24

on rate vs off rate

Answer 24

polymerization = assembly

depolymerization = disassembly

A linear polymer of protein molecs (ex: actin filament or microtubule) assembles and disassembles by addition and removal of subunits at the ends of the polymer

rate of addition of these subunits (called monomers) is given by rate constants of kon or koff

Question 25

Plus end

Answer 25

* fast growing end * difference in rate of growth is bc the changes in conformation of each subunit as it enters polymer * ratio of koff/kon is the same at both ends for simple polymerization (no ATP or GTP hydrolysis

Question 26

minus end

Answer 26

slow growing end

Question 27

nucleotide hydrolysis

Answer 27

* each actin carries a tightly bound ATP molecule that is hydrolyzed to a tightly bound ADP soon after its assembly into the polymer * each tubulin has a GTP converted to a tightly bound GDP * hydrolysis of bound nucleotide reduces the binding affinity of subunit for neighboring subunits and makes it more likely to dissociate from each end of the filament * T form adds to filament and D form leaves * this is steady state, not equilibrium * bc ATP or GTP that is hydrolyzed must be replenished by a nucleotide exchange rxn of the free subunit

Question 28

ATP and GTP caps

Answer 28

rate of addition of subunits to growing actin or microtubule can be faster than rate at which their bound nucleotide is hydrolyzed in these conditions, end has a "cap" of subunits containing the nucleoside triphosphate ATP cap for actin filaments GTP cap for microtubules

Question 29

treadmilling graphs

Answer 29

A. explains diff critical concentrations (Cc) at plus and minus ends * subunits with bound ATP polymerize at both ends of a growing filament and then undergo nucleotide hydrolysis within the filament. * in this plus end, terminal subunits are in T form bc elongation is faster than hydrolysis * at minus end, hydrolysis is faster than elongation so terminal subunits are in D form B. treadmilling at diff concentrations * critical concentration is T form is lower than for D form * if conc is between these two, plus end grows while minus end shrinks (this is treadmilling!)

Question 30

C_c of minus vs plus end

Answer 30

Cc(minus) \> Cc(plus)

Question 31

treadmilling

Answer 31

* nucleotide hydrolysis that comes along with polymer formation is to change the critical concentration at the 2 ends of the polymer * if both ends of polymer are exposed, polymerization proceeds until concentration of free monomer reaches a value that is above Cc for the plus end but below Cc for the minus end * at this steady state, subunits undergo net assembly at the plus end and disassembly at the minus end at an identical rate * polymer maintains a constant length even though a net flux of subunits through the polymer

Question 32

actin binding proteins

Answer 32

actin binding proteins regulate where and when actin polymerizes or depolymerizes - different cells have diff collections of these proteins - these proteins can respond to form different arrays of actin filaments

Question 33

actin monomer binding proteins

Answer 33

* when thymosin is bound to actin monomers, they cannot be added to the plus end of an actin filament * profilin can bind actin monomers and rapidly add the monomer to the filament * profilin competes with thymosin for binding to actin monomers (they can't both bind!) * promotes assembly! * profilin is faster

Question 35

formin

Answer 35

nucleates assembly and remains associated with the growing plus end

Question 36

thymosin

Answer 36

binds actin subunits, prevents assembly

Question 37

Arp 2/3 complex

Answer 37

nucleates assembly to form a web and remains associated with the minus end (actin subunits)

Question 38

profilin

Answer 38

binds actin subunits, speeds elongation

Question 39

tropomodulin

Answer 39

prevents actin filament assembly and disassembly at minus end

Question 40

cofilin

Answer 40

binds ADP-actin filaments, accelerates dissassembly

Question 41

gelsolin

Answer 41

severs filaments and binds to plus end gel = not glueing together

Question 42

capping protein

Answer 42

prevents assembly and disassembly at plus end

Question 43

tropomyosin

Answer 43

stabilizes actin filament

Question 44

actin nucleating proteins

Answer 44

determine where polymerization occurs actin, arp2, arp3 when an activating factor binds the complex, Arp2 and Arp3 are brought together into a new configuration that resembles the plus end of an actin filament actin subunits can assemble onto this structure, bypassing the rate-limiting step of filament nucleation

Question 45

what does Arp2/3 bind

Answer 45

existing filaments result is a branching array of filaments Arp2/3 nucleates actin filaments growth at 70 degree angle to original filament results in treelike web of actin filaments

Question 46

Formins

Answer 46

formins ride along on growing plus ends and promotes assembly by binding actin monomers - formins promote growth of linear filaments, not branched networks - formins can interact with profilin and actin - formins form a dimeric complex that nucleates formation of a new actin filament and stays stuck to plus end as it elongates

Question 47

other actin binding proteins

Answer 47

* some proteins bind the sides of filaments to stabilize them * proteins can bind the ends of filaments, which can stabilize the filament by preventing assembly or disassembly * proteins can destabilize filaments

Question 48

cofilin

Answer 48

binds actin filaments and twists them to make them less stable and prone to rapid disassembly cofilin binds best to ADP actin

Question 49

shape and dynamics of actin filaments is determined by....

Answer 49

which regulators are active and when they are active

Question 50

stress fibers

Answer 50

contractile, exert tension

Question 51

actin cortex

Answer 51

underlies the plasma membrane and consists of gel-like networks or dendritic actin networks that enable membrane protrusion at lamellopodia

Question 52

filiopodia

Answer 52

spike-like projections of the plasma membrane that allow a cell to explore its environment

Question 53

myosin

Answer 53

mechanochemical ATPases couple ATP hydrolysis to force generation

Question 54

force can be generated by....

Answer 54

coupling ATP hydrolysis and shape changes myosin II cycle the head remains bound to the actin filament for only about 5% of the entire cycle time, allowing many myosins to work together to move a single actin filament

Question 55

how cells move

Answer 55

1. the actin-polymerization-dependent protrusion and firm attachment of a lamellipodium at the leading edge of the cell move the edge forward and stretch the actin cortex 2. contraction at the rear of the cell propels the body of the cell forward to relax some of the tension 3. new focal contracts are made at the front, and old ones are disassembled at the back as the cell crawls forward 4. this can occur quickly because all steps can be tightly coordinated

Question 56

studying locomotion

Answer 56

fish keratinocytes

Question 57

moving the plasma membrane

Answer 57

the whole branched array of actin treadmills and pushes the plasma membrane forward advancement of lamellipodium nucleation by Arp 2/3 complex at front newly nucleated actin filaments are attached to sides of preexisting filaments filaments elongate, pushing plasma membrane forward at a steady rate, actin filament plus ends become capped after newly polymerized actin subunits hydrolyze their bound ATP in the filament lattice, the filaments become susceptible to depolymerization by cofilin this causes spatial separation between net filament assembly at the front and disassembly at the back so the actin filament network as a whole can move forward (even though individual filaments within it remain stationary)