Slide set 11 Flashcards

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1
Q

cytoskeleton

A
  • protein polymers composed of actin subunits, tubulin subunits, and intermediate filaments
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2
Q

microtubules are made up of

A

tubulin subunits

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3
Q

microtubule growth

A

microtubules continually grow from the centrosome added to a cell extract

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4
Q

dynamic instability

A

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

constant polymerization and depolymerization cycle

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5
Q

actin filaments aka

A

microfilaments

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6
Q

actin filaments

A
  • 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)
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7
Q

where do you find actin filaments?

A

microvilli, striated msucle

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8
Q

microtubules

A
  • 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
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9
Q

where do we find microtubules and what are they made of

A

tubulin!

cilia!

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10
Q

intermediate filaments

A
  • ropelike fibers
  • made of intermediate filament proteins
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11
Q

intermediate filament example

A

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)

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12
Q

cytoskeletal polymers are….

A

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
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13
Q

cytoskeletal polymers determine…..

A

cell polarity and internal organelle organization!

image is small intestine

actin increases SA for food absorption

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14
Q

actin monomers bind together to form….

A

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)
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15
Q

polarity of actin filament image

A

barbed = plus end

pointed = minus end

2 Ps don’t go together!

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16
Q

blocking actin filament assembly/disassembly has what effect on cells

A

toxic!

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17
Q

actin chemical: Latrunculin

A

effect: depolymerizes
mechanism: binds actin subunits
source: sponges

L in latrunculin bc larry the lobster is in spongebob

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18
Q

actin chemical: Cytochalasin B

A

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

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19
Q

actin chemical: Phalloidin

A

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

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20
Q

actin assembly graph

A

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 (Cc) at which there is no net change in polymer

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21
Q

how do we study actin polymerization?

A

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

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22
Q

nucleation

A
  • 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)
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23
Q

how do reduce or eliminate lag phase of actin polymerization?

A

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

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24
Q

on rate vs off rate

A

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

25
Q

Plus end

A
  • 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
26
Q

minus end

A

slow growing end

27
Q

nucleotide hydrolysis

A
  • 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
28
Q

ATP and GTP caps

A

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

29
Q

treadmilling graphs

A

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!)
30
Q

Cc of minus vs plus end

A

Cc(minus) > Cc(plus)

31
Q

treadmilling

A
  • 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
32
Q

actin binding proteins

A

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
33
Q

actin monomer binding proteins

A
  • 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
34
Q
A
35
Q

formin

A

nucleates assembly and remains associated with the growing plus end

36
Q

thymosin

A

binds actin subunits, prevents assembly

37
Q

Arp 2/3 complex

A

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

38
Q

profilin

A

binds actin subunits, speeds elongation

39
Q

tropomodulin

A

prevents actin filament assembly and disassembly at minus end

40
Q

cofilin

A

binds ADP-actin filaments, accelerates dissassembly

41
Q

gelsolin

A

severs filaments and binds to plus end

gel = not glueing together

42
Q

capping protein

A

prevents assembly and disassembly at plus end

43
Q

tropomyosin

A

stabilizes actin filament

44
Q

actin nucleating proteins

A

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

45
Q

what does Arp2/3 bind

A

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

46
Q

Formins

A

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
47
Q

other actin binding proteins

A
  • 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
48
Q

cofilin

A

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

cofilin binds best to ADP actin

49
Q

shape and dynamics of actin filaments is determined by….

A

which regulators are active and when they are active

50
Q

stress fibers

A

contractile, exert tension

51
Q

actin cortex

A

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

52
Q

filiopodia

A

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

53
Q

myosin

A

mechanochemical ATPases

couple ATP hydrolysis to force generation

54
Q

force can be generated by….

A

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

55
Q

how cells move

A
  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
56
Q

studying locomotion

A

fish keratinocytes

57
Q

moving the plasma membrane

A

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)

58
Q
A