week 6 Flashcards

1
Q

what do microtubules do

A
  • they act as tracks for transport
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2
Q

what do you see when you look at microtubules

A
  • see things moving in. both directions on microtubules at different s[eeds
  • can be seen when looking at in vitro experiments using axons of giant squids.
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3
Q

squid axons

A
  • model system
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4
Q

radio active amino acids assay

A
  • injecting radioactive amino acids into the cell body of a large axon.
  • dividing axon into segments, and then collect different bits of the axon at different distances from the injection site.
  • these isolated proteins are then run on a gel
  • the transport of proteins that are made from the amino acids is not random (not just simple diffusion)
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5
Q

what does the gel protein assay teach us

A

which proteins are travelling together (i.e. they remain together at the different time frames)

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6
Q

Kinesin

A
  • motor protein
  • there many types, 14 known classes coded by 45 genes in humans
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7
Q

in what direction does kinesis move

A

moves along microtubules towards plus end

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8
Q

composition of kinesin

A
  • 2 heavy chains
  • 2 light chains
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9
Q

what are the kinesis heavy chains composed of

A
  • head, neck/linker, stalk/tail
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10
Q

kinesin head

A

microtubule binding domain that has ATPase activity (is able to hydrolyze ATP while moving towards plus end)

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11
Q

kinesin neck/linker region

A
  • flexible linker region
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12
Q

kinesin stalk/tail

A

stalk region that goes into the tail leads to light chains.

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13
Q

kinesin light chains

A
  • variable light chains
  • there are lots of different types present that bind to different types of cargo
  • light chains are located at the end of tail regio
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14
Q

what would happen if u ran the heavy and light chain regions

A

you would get three bands (1 thick band ofr the heavy regions because same size and therefore occupy large molecular weight)
- and then each of the two light chains would be different sizes and have a small molecular weight (hard to indeitify how heavy they are)-

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15
Q

kinesin 1

A
  • most important
  • conventional kinesin, found all over cytoplasm
  • head domains bind to microtubules
  • does most of the work and is made of two heavy chains and two variable light chains
  • light chains are variable and depend on cargo
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16
Q

kinesin 2

A
  • heterotrimeric
  • has two different heavy chains (not identical)
  • head domains bind to the microtubules
  • has different kinesin family member that is sort of like a light chain
  • made up of three different molecules
  • three different banding patterns on SDS page (i.e. you would see the three bands travelling together on SDS page)
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17
Q

roles of kinesin 1 and 2

A

organelle transport

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18
Q

kinesin 5

A
  • bipolar (both sides are the same)
  • does not bind cargo
    • head domains bind to microtubules
  • binds to stalk domain of two other heavy chains
  • there are four heavy chains that come together in this bipolar chain
  • head domains on both sides, both ends of the kinesin can bind to the microtubule
  • causes microtubule sliding
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19
Q

kinesin 13

A
  • does not bind cargo (not for transport)
    • head domains bind to microtubules
  • uses atp hydrolysis to remove dimers off of the ends of microtubules (uses ATP to cause depolymerization)
  • no stalk or tail domain
  • primarily works at the + end because - end is usually capped by gamma tubulin ring complex
  • however, it is possible to have depolyermization at either end
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20
Q

kinesin 13 function

A

depolyermizataion

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21
Q

kinesin 5

A

microtubule sliding

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22
Q

how does cargo bind to light chain of kinesin

A
  • cargo needs the right receptor that can be recognized by a specific light chain
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23
Q

kinesin movement

A
  • usually anterograde
    (uses atp hydrolysis to move head towards + end)
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24
Q

how is kinesin 1 regulated

A
  • inactive when folding (no ATPase activity) but will be active once bound to receptor
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25
Q

how far does kinesin 1 go

A
  • when hydrolyzed, the head moves 16 nm
  • before that, the kinesin heads are 8 nm apart when kinesin is not mobing
  • the behind head steps out in front of the other head (one head remains stationary and bound)
  • in total, moves 16 nm
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26
Q

cytoplasmic dynein

A
  • retrograde transport
  • minus end directed motor protein
  • found in cytoplasm
27
Q

composition of cytoplasmic dynein

A
  • 2 heavy chains that work together
  • has a head domain that is an atpase
  • hydrolysis of atp results in shape changes that drive movement
  • stalk that is part of the head
  • linker and stem/tail interact with dynactin hetero complex to recognize and bind cargo
28
Q

heavy chains. of dynein

A
  • 2 heavy chains that are head domains that bind to the microtbutles and move to the - end
  • the heavy chains have stalk sticking out (stalk is part of the head domain in the middle)
  • stalk contains the micortuble binding domain
  • head domain moves to - end and binds to microtuble
29
Q

what is a difference between kinesin and dynein

A
  • stem domain that leads to the tail, does not directly recognize cargo
  • for dynein to recognize cargo, Neds dynastic hetero complex of proteins
30
Q

dynactin hetero complex

A
  • contains componetsn like action, dynamitin
31
Q

dynamitin

A

releases cargo once delibered
- moves cargo away form dynein

32
Q

p150

A

binds to MT, and DHC, and helps stabilize the whole complex, not a motor protein just holds things close to the mT

33
Q

high levels of dynamtini

A

dynactin and dynein explode apart

34
Q

dynein tail ends

A
  • bind to dinazin heterocmoplexes which bind to cargp
35
Q

kinesin and dyeing working together

A
  • both are attached to a molecule that is being transported so that for example if travelling to + end with kinesin, dyenein is there to bring back kinesin
36
Q

tubulin stablitt

A
  • must be stable in order to have motor protein movement
37
Q

cilia + flagella

A
  • two versions of same thing
  • cilia = shorter (2-10 um)
  • flagella = 10-2000 u
38
Q

Flagella

A

properly cellscil
- bending

39
Q

cilia

A

sweeps material across tissue and there are many of these working together
- beating

40
Q

axoneme

A
  • composed of doublet micortubles
  • nine doublet micortubles (9+2, +1 or +0, depending on how many singlet microtubules in the middle)
  • singlets are stable unlike cytoplasmic singlets
41
Q

how are doublets in the axoneme held in place

A
  • by necin proteins. in between
  • radial spokes that hold doublets in place
42
Q

the dynein attached to the A tubule…

A

dyneien head reaches out to B tubule, while the tail is stuck to the A tubule

43
Q

what makes up the base of cilia and falgella

A
  • absa l body
44
Q

basal body
** is the tip of the cilia made up of paired singlets or doublets?

A

-triplet micortuble.
- nine triplets in basal body, (MTOC)
- 9 triplet micortublues reach transitional zone, loses singlet and become doublet mcirotbules to create axoneme, and gains back doublet instead of singlet to form axone
- at tip, u end up with 9+2 arrangement.

45
Q

axoneme bending

A
  • micortubles sliding against each other powered by ax dynoein
46
Q

how does sliding occur

A
  • a tubule has ax dynein permanently attached
  • the head reaches towards B tubule of next doublet microtubule, while the tail remains attached to the A micortuble
  • this causes the head to move towards the positive end, generating a rightward movement
47
Q

why is theree no sliding in axonome

A
  • bending bc no sliding
  • bc nexin in between and basal body present at bottom
48
Q

intraflagellar transport

A
  • material is moved up and down
  • movement is not related to bending
  • uses cytoplasmic dynein
49
Q

difference between centrosome MT and mitotic apparatus MT

A
  • the centrosome micorotubles are used during interphase and the mitotic ones are used during mitotis
  • interphase MT = 5 min half life
  • mitosis MT = 15 min half life
50
Q

how is the mitotic apparatus formed

A

duplication of centrosome
- ecntorsome facilities novel mitotic MT dynamics

51
Q

what are the components of the mitotic apparatus

A
  • polar MTs, Astral MTs, Kinetochore MTs
  • the 2 MTOCs are the two mitotic poles, still consists of 2 triplet icortubels that are90 degrees to each other
52
Q

kinetochore MT

A

captures kinetochore

53
Q

polar MT

A

misses chromosome but is moving from pole to pole, overlap with other polar in antiparallel arrangements

54
Q

spindle

A

all MT between poles (so the kinetochore and polar mTs)

55
Q

centromere

A

attachment site for microtubules
- kinetochore proteins miediate attachment of chromosomes to MTs

56
Q

what happens at kinetochore

A

the microtubules extend from poles to capture the chromsomes
- microtubules stick to kinetochore proteins
- plus end is not capped or antything is still free, but it is the plus end that is moving away from he MTOC to the kinetochore

57
Q

spindle formation involves…

A
  • kinesin 7, plus end directed motor
  • need to capture chromosomes on bothb sides
  • kinesin 13
    -dynein
58
Q

+ end directed motor, kinesin 7 in spindle formation

A
  • movingg towards + end, pushes the chromsomes towards the right
59
Q

kinesin 13 role in spindle formation

A
  • ove chromsomes by depoylermzing microtubule on one side (o.e. depolyermize on left side to move right)
  • polyermization happening on left side, depolyemrizaiton on right side
60
Q

dynein role in spindle formation

A
  • can pull towards right to move the chromosomes right
  • while depolyermization happens at that side to move towards right
61
Q

why must the chromosome be captured from both sides

A
  • allows for tension
  • cell must know that the chromosome is attached on both sides
62
Q

NDC80 and tension

A
  • when there is no tension, there is phosphorylation of NDC80 proteins at the kinetochore (by Aurora B) which results in weak microtubule interactions with the kinetochore.
  • when there is not tension, Aurora b does not phospylrate and Ndc80 remains bound to mT
  • nDC80 briefly holds on - must hold on to allow other microtubule to attach, but to do this must make decision
  • nDC8- on both sides must not be phosphorylated, no tension (no microtubule on other side,), ndc80 lets go
63
Q

dynein

A

-