Cell 6

Question 1

Microtubules: Tracks for Transport

Answer 1

Vesicle Transort (Retrograde + Anterograde)

Motor proteins (ATP)

Question 2

Axonal Transport

Answer 2

Squid Axons are a model system.

Labelled protiens travel at different speeds in cells

Question 3

Axonal Steps

Answer 3

Inject radioactive amino acids into squid axons.

they are incorporated into proteins in the axon.

these proteins are potentially moving along the microtubules (not coloured. they are radioactive)

Wait some time and then start collecting bits of the axon at different sections away from the injection site.

Isolate proteins from the different segments and run them along the SDS-PAGE gel.

Wait a few minutes and repeat the process.

Match the banding patterns of identical protiens on different blots.

Different proteins are traveling in complexes.

Isolate these bands and determine what these protein complexes are.

Question 4

Kinesin

Answer 4

Microtubule plus end directed motor protein.

Many types (14 known classes; 45 genes in humans)/Splicing

2 heavy chains: Head + flexible neck (linker) and stalk (tail)

2 light chains (variable)

Question 5

Heavy chain heads have _____ ____ and _ _ ______ ability.

Answer 5

ATPase activity

MT binding

ATP hydrolysis moves the kinenin to the plus end.

Flexibility is required to move along in the cell,

Light chains bind to the cargo.

Question 6

Types of Kinesin + Their Function

Answer 6

K1: Two heavy + Two light chains (common)

K2: Two heavy + 1 kinesin family member (heterotrimeric protein) involved in cargo binding (3 proteins)

K5: Bipolar, both sides are the same; 4 heavy chains get together (tails from either chain overlapping forming one head domain on either side) Both ends try to move to the plus end forcing these microtubules to slide past one another.

K13: Doesn’t bind cargo, just two heavy chains that have only the head and neck domains. It binds microtubules and uses ATP hydrolysis to remove dimers from the microtubule ends (usually works at the (+) end; can work at both ends)

Question 7

Movement of Kinesin

Answer 7

Anterograde

ATP-hydrolysis causes conformational change in kinesin.

ATP is hydrolysed as each head moves 16 nm.

K1 regulated as it is inactive when folded (no ATPase activity) and active upon receptor binding

Question 8

Cytoplasmic Dynein

Answer 8

MT minus end directed motor proteins

Involved in retrograde transport.

Heavy chain heads have ATPase activity and stalk. Linker and stem in turn interat with dynactin (hetero) complex to recognize and bind cargo.

ATP hydrolysis results in shape change that drives movement.

Stem domain does not directly recognize cargo

Question 9

Dynactin Complex

Answer 9

Links dynein to cargo.

Dynactin complex contains many components (actin; dynamitin) and links dynein to cargo and regulates movement.

its association with dynein regulated in part by dynamitin.

P150glued binds microtubules but is not a motor; it stabilizes the complex.

Dynamitin is responsible for releasing the cargo.

Question 10

Kinesin and Dynein cooperate in anterograde and retrograde transport of cargo

Answer 10

Motor proteins can be cargo.

Question 11

Posttranslational modification of tubulin affect microtubule stability and transport

Answer 11

Acetylation of a lysin residue of the alpha tubulin both stabilized the MT and promotes kinesin-1 movement.

Question 12

Centrosome has microtubules

Answer 12

polymerizing away; all the plus ends are leading outside fo the cell.

Question 13

Motor proteins are also the…

Answer 13

Cargo themselves, when kinesin has moved to the end of the cell it needs dynein to tranport it back to the middle of the cell.

Dynein can hitch a ride with the mitochondiran and kinesin, both proteins work together to move things both ways,

Motor proteins are influenced by the stability of microtubules.

Question 14

Cilia and Flagella

Answer 14

Two versions of the same thing.

Cilia 2-10 micrometers

Flagella 10-2000 micrometers

Flagella: propel cells

Cilia: sweeps material across tissue

Question 15

Axoneme Components

Answer 15

Underlying structure of cilia and flagella

Over 250 proteins

9+2 array of microtubules (9 doublet ring surrounding two singlet MT)

Doublet ring has no particular function

Axonemal dynein

Nexin

Radial Spoke Head

Question 16

Axonemal Dynein

Answer 16

Bound permenantly to the alpha tubule (at the stem) teh head of the dynein is reaching towards the adjacent B tubule.

Dynein can bind to and move along the B tubule.

Question 17

Basal Body

Answer 17

Underlies the microtubule.

The Basal body is similar to the centriole.

Triplet microtubules in the basal approach the cell surface, ABC microtubules approach the cell surface, the C microtbuble will stop and A and B will protrude out of the cell surface.

9 triplet microtubules lead to 9 doublet microtubules.

90 degree basal bodies in chlamydomonas.

Basal body actually does contribute microtubules to the axoneme

Question 18

Axoneme Bending

Answer 18

Generated by sliding of the microtubules against each other; powered by axonemal dynein.

If there is no nexin A tubule of one doublet walks along the neighbour B towards the (-) end.

MTs slide pas each other

But nexin is present, as is basal body. So sliding cannot occur. Bending does.

Question 19

Axoneme Bending Occurs as a ____. It is very well _______ in _____ _____ of the flagella.

Answer 19

Wave
regulated
different regions

Not every dynein is binding to every microtubule at the same time, only one side is bending at a time, its like corkscrewed bending, there is a regulation of the movement

Question 20

Intraflagellar transport moves material “up and down”

Answer 20

Movement is not related to bending.

Cytoplasmic dynein utilized. May be related to stability and signalling events.

Cytoplasmic dynein and kinesin present allows antero and retrograde transportation, cilia and flagella are used to transport signals.

Question 21

Many interphase cells contain a non-motile primary cilium.

Answer 21

No axonemal dynein.

Important roles in cell-cell signaling.

In embryos this signaling is important. Many interphase cells contain cilia, the cell can send out one cilium with an axonemal structure that is not used for bending, it is just used for signaling (no dynein)

Primary cilia are crucial for embryonic signaling event, their absence or mutations results in issues in development.

The tubulin a acetylated to stabilize them

Question 22

Karyokinesis

Answer 22

The division of the chromosomes.

Question 23

Interphase MT vs Mitosis MT

Answer 23

Interphase MT are very different from the ones found in the mitotic apparatus.

Question 24

During Mitosis Centrosomes…?

Answer 24

Centrosomes replicate and form the mitotic poles

Question 25

Key difference in MT/Centrosomes during cell cycle

Answer 25

Interphase microtubules from the centrosome last longer (5min half life).

Centrosomes duplicate to form mitotic poles, the poles are MTOC.

MT are much more dynamic (15s half life).

K13 activity remains constant (decreasing stability), XMAP215 activity drops (can no longer provide stability)

Question 26

Mitotic Apparatus Components

Answer 26

Polar and Kinetochore microtubules (make spindle)

Astral microtubules

Question 27

Structure of mitotic poles vs centrosomes

Answer 27

Identical structure.

Two triplet microtubules that are 90 degrees from each other.

mitotic poles form the mitotic apparatus.

Question 28

Kinetochore MTs

Answer 28

Grow from the plus end away from the pole towards the chromosome, it capture the kinetochore.

Question 29

Polar MTs

Answer 29

Microtubules can polymerize towards the other pole but it misses a chromosomes, as its going from pole to pole its called a polar MT.

MT overlap, they’re ovarlapping in an antiparallel fashion

Question 30

the spindle is all the microtubules from one pole to other pole, all the microtubules ______ the poles.

Answer 30

between

Question 31

Astral MTs

Answer 31

Microtubules can go away from the other pole forming a star like structure, astral microtubules.

Question 32

Centromere

Answer 32

Attachment site for microtubules.

Kinetochore proteins mediate attachment of chromsomes to MTs

Question 33

Kinetochore

Answer 33

A huge complex of proteins found in this replicated pair of the chromosomes.
It is at the kinetochore the MTs is going to capture the chromosome.
Microtubules come from one pole and microtubules coming form the other pole at the other side.

Question 34

Even though the microtubules capture these proteins at the kinetochore they always have the _____ _____ _____.

Answer 34

plus end free

the plus end is not capped

The microtubule plus end is coming in and the kinetochore proteins come and capture the microtubule but the plus end is free, the end is not being held it is free to polymerize and and depolymerize.

Question 35

Spindle Formation

Answer 35

All chromosomes must be captured.

All must be aligned during metaphase.

Requires motors and MT dynamics.

Polymerization and Depolymerization is used to move this chromosome pair to the right or to the left to get it to the center of the cell.

Kinesin; dynein and K13 are present.

To move chromosomes to the right depolymerize the microtubules to the right hand side.

dynein is going to be moving to the minus end pulling the chromosome to the right.

kinesin on the left can push the chromosome to the right

Checkpoints at the cytoskeleton ensure all the chromosomes are in line at the metaphase plate.

Question 36

How does the cell know the microtubules have captures the chromosomes form both sides?

Answer 36

It uses tension.

Ndc80 protein is part of the kinetochore complex; it briefly holds the MT; the decision to let go is based on whether another MT holds on at the other end.

If there wasn’t a capture at the other side we can’t seperate the chromosomes; tension indicates capture on both sides, no tension Ndc80 lets go because Aurora B phosphorylates Ndc80 causing it to let go.

Aurora B inactivated when tension occurs.
When ndc80 is not phosphorylated it binds and hold on allowing the chromosome to be moved to the metaphase plate.

Question 37

Dynein-Dynactin Center and Orient the spindle

Answer 37

Astral MTs can sense where the middle of the cell is because on either side are active cortical anchors, proteins on the outside of the cell.

Activity of the cortical anchors enables the cell to position the spindle right in the middle. The astral MTs are projecting away form the poles, they can be associated with dynein.

If the cortical anchor is inactivated then the MTs can’t do anything.

The active cortical anchor activates dynein and dynactin, they are present in the cortical anchor. If this cortical anchors is activated the astral microtubules pull the spindle pole to the right because of the active cortical anchor.

They are used with astral microtubules and dynein, dynein pulls the pole to the right or the left, the activity of the cortical anchor determines where the pulling occurs.

Cortical anchor pulls the mitotic apparatus to the right. You want activity on the side you want to move towards.

Question 38

Anaphase A

Answer 38

Anaphase A is the movement of the chromosome towards the pole, the kinetochore microtubules shorten so that the distance between chromosome and the pole shortens.

The chromosome gets closer to the pole because the kinetochore microtubule is shrinking at the kinetochore there is activity that cause the microtubule to shrink, K13. at the spindle pole there is also depolymerization, the kinetochore is shrinking from both sides and is its shrinking the chromosome is moving towards the pole. Its driven by disassembly of the microtubules.

Question 39

Anaphase B

Answer 39

In anaphase B the poles are separating, the reason this is happening is because cytokinesis is happening in the middle, you don’t want the chromosomes to be caught in the middle as this is occurring. Overlap of anitparallel microtubules, in between them is kinesin 5, which is involved in microtubule sliding. Kinesine 5 reduces the overlap of the microtubules ultimately pushing the poles apart.

Dynein and the astral microtubules in the cortical anchor is activated, dynein is bound to the cortical anchor in the membrane and is going to move towards the minus end the microtubule, as its moving towards the mitotic pole its pulling the mitotic pole towards it. The poles are being pulled apart by dynein and pushed apart by kinesin 5.

This astral microtubule has to depolymerize or else it will go right through the membrane, so it has to shorten through depolymerization.

Question 40

Polar microtubules are associated with the position of _______, they position and activate them.

Answer 40

RhoGtp is responisble for the formation of the active actin myosin contractile ring.

This ring is as far way as you can get from where the new nuclei is forming.