week 6 Flashcards
what do microtubules do
- they act as tracks for transport
what do you see when you look at microtubules
- 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.
squid axons
- model system
radio active amino acids assay
- 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)
what does the gel protein assay teach us
which proteins are travelling together (i.e. they remain together at the different time frames)
Kinesin
- motor protein
- there many types, 14 known classes coded by 45 genes in humans
in what direction does kinesis move
moves along microtubules towards plus end
composition of kinesin
- 2 heavy chains
- 2 light chains
what are the kinesis heavy chains composed of
- head, neck/linker, stalk/tail
kinesin head
microtubule binding domain that has ATPase activity (is able to hydrolyze ATP while moving towards plus end)
kinesin neck/linker region
- flexible linker region
kinesin stalk/tail
stalk region that goes into the tail leads to light chains.
kinesin light chains
- 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
what would happen if u ran the heavy and light chain regions
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)-
kinesin 1
- 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
kinesin 2
- 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)
roles of kinesin 1 and 2
organelle transport
kinesin 5
- 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
kinesin 13
- 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
kinesin 13 function
depolyermizataion
kinesin 5
microtubule sliding
how does cargo bind to light chain of kinesin
- cargo needs the right receptor that can be recognized by a specific light chain
kinesin movement
- usually anterograde
(uses atp hydrolysis to move head towards + end)
how is kinesin 1 regulated
- inactive when folding (no ATPase activity) but will be active once bound to receptor
how far does kinesin 1 go
- 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
cytoplasmic dynein
- retrograde transport
- minus end directed motor protein
- found in cytoplasm
composition of cytoplasmic dynein
- 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
heavy chains. of dynein
- 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
what is a difference between kinesin and dynein
- stem domain that leads to the tail, does not directly recognize cargo
- for dynein to recognize cargo, Neds dynastic hetero complex of proteins
dynactin hetero complex
- contains componetsn like action, dynamitin
dynamitin
releases cargo once delibered
- moves cargo away form dynein
p150
binds to MT, and DHC, and helps stabilize the whole complex, not a motor protein just holds things close to the mT
high levels of dynamtini
dynactin and dynein explode apart
dynein tail ends
- bind to dinazin heterocmoplexes which bind to cargp
kinesin and dyeing working together
- 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
tubulin stablitt
- must be stable in order to have motor protein movement
cilia + flagella
- two versions of same thing
- cilia = shorter (2-10 um)
- flagella = 10-2000 u
Flagella
properly cellscil
- bending
cilia
sweeps material across tissue and there are many of these working together
- beating
axoneme
- 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
how are doublets in the axoneme held in place
- by necin proteins. in between
- radial spokes that hold doublets in place
the dynein attached to the A tubule…
dyneien head reaches out to B tubule, while the tail is stuck to the A tubule
what makes up the base of cilia and falgella
- absa l body
basal body
** is the tip of the cilia made up of paired singlets or doublets?
-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.
axoneme bending
- micortubles sliding against each other powered by ax dynoein
how does sliding occur
- 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
why is theree no sliding in axonome
- bending bc no sliding
- bc nexin in between and basal body present at bottom
intraflagellar transport
- material is moved up and down
- movement is not related to bending
- uses cytoplasmic dynein
difference between centrosome MT and mitotic apparatus MT
- 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
how is the mitotic apparatus formed
duplication of centrosome
- ecntorsome facilities novel mitotic MT dynamics
what are the components of the mitotic apparatus
- 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
kinetochore MT
captures kinetochore
polar MT
misses chromosome but is moving from pole to pole, overlap with other polar in antiparallel arrangements
spindle
all MT between poles (so the kinetochore and polar mTs)
centromere
attachment site for microtubules
- kinetochore proteins miediate attachment of chromosomes to MTs
what happens at kinetochore
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
spindle formation involves…
- kinesin 7, plus end directed motor
- need to capture chromosomes on bothb sides
- kinesin 13
-dynein
+ end directed motor, kinesin 7 in spindle formation
- movingg towards + end, pushes the chromsomes towards the right
kinesin 13 role in spindle formation
- 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
dynein role in spindle formation
- can pull towards right to move the chromosomes right
- while depolyermization happens at that side to move towards right
why must the chromosome be captured from both sides
- allows for tension
- cell must know that the chromosome is attached on both sides
NDC80 and tension
- 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
dynein
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