Microtubules

Question 1

Functiosn of mt’s

Answer 1

Make up the mitotic spindle

Arranging cellular organelles during interphase

Serving as tracks for celllular constituents to be transported

Cilia & flagellar movement

Question 2

Taxol as cancer therapy

Answer 2

Stabilizes mt’s so that cells can’t perform mitosis (no anaphase)

Question 3

The end ringed by _tubulin is the negative end

The end ringed by _tubulin is the positive end

Which holds the ATP that gets hydrolyzed?

Answer 3

a-tubulin -> negative

B-tubulin -> positive ; holds the ATP that gest hydrolyzed

Question 4

MTs fom 3 distinct ring structures- what are their functions?

Answer 4

Single ring- interphase & mitosis

Doublet - cilia & flagella

Triplet- basal bodies and centrioles involved in mt organizing centers (MTOCs)

Question 5

Explain this graph

Answer 5

MT assembly depends on dimer pool concentration

At low [monomer], dimers form spontaneously.

As [monomer] increases, a critical conc (Cc) is reached; at or above the Cc, dimers spontaneously polymerize into MTs and dimer conc no longer increases.

Question 6

Why does assembly and disassembly occu rmore rapidly at the (+) end than the (-) end?

Answer 6

The (+) end has a lower critical concentration - less dimers are needed to stimulate polymerization from the (+) end

Question 7

The B tubulin added to the (+) end must

Answer 7

be GTP-bound

Question 8

Catastrophe- what is it and how do you rescue?

Answer 8

Catastrophe: rapid depolymerization of the (+) end because the GTP-bound dimer pool is far below Cc(+).

Simply increase [GTP-dimer]

Question 9

When does treadmilling occur? What is it?

Answer 9

Treadmilling: dimers are added to the (+) end but removed at the (-) end

[dimer] is greater than Cc(+) but less than Cc(-)

Question 10

What happens as more MTs are polymerized and [GTP-dimer] drops?

Answer 10

Assembly slows until [GTP-dimer] is less than Cc(+) –> GTP cap is hydrolyzed, producing an unstabel GDP cap and catastrophe

Question 11

MT associated proteins (MAPs)

Answer 11

Stabilize the MT because

• they contain positive amino acids in their MAP basic domain that bind to negatively charged amino acids in tubulin –> reduces charge repulsion between neighboring tubulin subunits
• They have a negative acidic projection domain that maintains the distance between MTs

Tau, MAP2, MAP4

Question 12

How do you inactivate MAPs to destabilize to MTs?

Answer 12

Phosphorylation of their positive basic domain to create negative charges

Question 13

MT destabilizing proteins: Kinesin 13, Stathmin, Katanin

Answer 13

Kinesin-13: Family of proteins that binds to the end of the MT and bends the protofilaments to promote catastrophe and

Stathmin/OP18: also binds to the end and bends, but removes two dimers at once; may also promote GTP hydrolysis; inactivated by phosphorylation

Katanin: sever or induce breaks in the MTs to expose new GDP-caps that promote catastrophe at the newly formed (+) end

Question 14

Colchicine

Answer 14

Binds and sequesters tubulin dimers to bring [GTP-dimer] below Cc(+), preventing polymerization and promoting catastrophe.

Used to reduce WBC migration and inflammation in gout

Question 15

Vinblastine , Podophyllotoxin, and Nocodazole

Answer 15

cancer treatment by inhibiting mitosis (like taxol)

Cells are arrested in G2 (Nocdazole used in the lab to synchronize cells in culture)

Question 16

Alzheimer’s disease and Tau

Answer 16

Tau is a MAP that stabilizes MTs.

In AD, it is hyperphosphorylated and dissociates from the MT –> forms neurofibrillary tangles that lead to neuronal cell death

Question 17

Significance of a phosphothreonine-proline motif in Tau and APP

Answer 17

If the proline is in a cis-configuration, then phosphorylation is accelerated and promotes the formation of amyloid plaques from APP and neurofibrillary tangles from Tau

Question 18

Pin1

Answer 18

Converts spontaneously formed cis-prolines in phosphothreonine-proline back into trans -

-> dephosphorylation of both APP and Tau

–> prevents deposition of amyliod plaques and neurofibrillary tangles

Question 19

MT organizing centers- what does it do and what are otehr names?

Answer 19

Nucleate the initial polymerization of MTs; has the (-) pole embedded and stabilized in its matrix

Centrosome in interphase when it helps organize the spatial arrangement of organelles

Spindle poles when organizing the mitotic spindles

MTOC for the mts of axons

Basal bodies for cilia and flagella

Question 20

MTOCs either possess or are derived from

Answer 20

Centrioles: triplet-ringed MT structures

Question 21

MTs transport vesicles and organelles.

How is bidirectional cargo transport possible?

Answer 21

• Kinesin motor protein binds cargo and B-tubulin subunits –> walk across subunits to transport cargo toward the (+) end
  
  • Anterograde
  • Uses ATP hydrolysis
• Dynein motor protein: retrograde toward (-) end
  
  • Interact with dynactin, which binds the cargo
  • Head domain provides ATPase fxn; hydrolysis moves the head with respect to the stem
  • Stalk domain binds MT and moves when ATP hydrolysis causes the power stroke

Question 22

When do MTs use their track funciton?

Answer 22

To organize organelles in teh cell

To move vesicles (e.g. secretory vesicles to the cell surfacE)

Question 23

In exocytosis of a secretory vesicle, the secretory vesicle is brought to the cell surface along MTs using ___. The vesicle is then exchanged from the MT to the ___ so __ can bring the vesicle the rest ofthe way to the membrane for exocytosis.

Answer 23

Use kinesins to get to the actin cytoskeleton, so that myosin motors can bring teh vesicle the rest of the way

Question 24

During endocytosis, molecules are brought into the cell in ____, which are transported to their final destination in teh cell along the __ using ___

Answer 24

Enter as endosomes

Transported to their final desitnation via MTs using dynein

Question 25

What is this?

Answer 25

Doublet MTs –> it’s cilia and flagella

Each doublet consists of A and B tubules

Question 26

The outer and inner dynein of a cilia/flagella connect to the _ tubule as cargo and then motor along the _tubule of the adjacent double toward the __pole at the base.

Answer 26

The dyneins connect to the A tubule as cargo and then motor along the B tubule of the adjacent doublet toward the (-) pole.

Rhythmic sliding of adjacent mt filaments due to these axonemal dyneins move the cilia or flagella

Question 27

Ciliary dyskinesia

Answer 27

immotile ciliary syndrome arising from dynein dysfunction.

50% of people with this have a mirror-image reversal of their internal organs (situs inversus totalis) –> Kartagener syndrome

Poor mucociliary clearance, affected hearing, infertility due to fallopian tubes or sperm

Question 28

MTs in early interphase

Answer 28

Centrosome (later called the “spindle pole”) begins to duplicate and new daughter centrioles bud from the centrosomal matrix of the MTOC.

Question 29

MTs in prophase

Answer 29

The interphase MTs begin to degrade and get replaced by mitotic spindle MTs, all of which originate from the replicated spindle pole and separate to opposite sides of the nucleus.

First one is the Aster MTs, which separate to opposite sies o the nucleus.

Chromsomes are condensing

Question 30

Prometaphase

Answer 30

• Nuclear membrane breaks down
• Central spindle region is established and the kinetochore & polar MTs extend toward the central spindle region
• Condensed chromsomes are captured

Question 31

Metaphase

Answer 31

Condensed chromosomes are aligned equidistant between the centrosomes at the metaphasic plate

Question 32

Anaphase

Answer 32

Separation of chromosomes.

• Cohesin holding the sister chromatids together at the centromere is broken down.
• Kinetochore MTs diassemble, pulling the sister chromatids to the opposing centrosomes
  
  • Meanwhile, the centrosomes are pulled farther apart from one another.

Question 33

Telophase & cytokinesis

Answer 33

• Nuclear membrane reforms around each diploid set of chromosomes
• Myosin contractile ring condenses
• Mitotic MTs break down and are reorganized back into the interphase MT arrangement

Question 34

Three types of mitotic MTs

Answer 34

• Aster/astral MTs run from the spindle poles toward the cell cortex; orients the spindle poles w/ respect to the axis of cell division
• Kinetochore MTs bind the kinetochores to pull apart the chromatids
• Polar MTs:
  
  • ​push duplicated centrosomes apart in prometaphase
  • maintain orientation of the central spindle
  • push the spindle poles furhter apart in late anaphase

Question 35

Centrosome (spindle pole) migration

Answer 35

Aster MTs connect the spindle poles to the cell cortex

Dyneins (green) on aster MTs attached to the cortex motor toward the (-) end, pulling the spindle poles apart.

Kinesins (yellow) on polar MTs motor toward the (+) end of the opposing polar MTs, pushing the poles apart.

Question 36

Chromosome kinetochore end capture

Answer 36

1. Kinetochore MTs bind the kinetochore
2. Kinetochore-associated kinesins move along the kinetochore MT toward the (+) end until the chromosome is end-captured.
3. End-captured MTs are stabilized by their interactions with the chromosome’s kinetochore.

Question 37

Chromosome alignment of metaphase- how do they get aligned perfectly equidistant between the two spindle poles?

Answer 37

To push it forward, kinesin-7 can hold onto the kinetochore and motor toward the growing (+) end.

To pull i tback, kinesin-13 is an MT destabilizing protein; as MT depolymerizes, a dynein will pull it toward the (-) pole

Question 38

Anaphase: the kinetochore MTs disassemble to drag the chromatids back toward each othe spindl epoles.

Anaphase A vs B

Answer 38

Anaphase A: kinetochore MTs disassemble at both (+) and (-) ends by kinesin-13

Anaphase B:

• Kinesin motors on opposing polar MTs, pushing the polar MTs and spindle poles apart.
• Sister dynein proteins attached to the cortex motor toward the (-) end of the aster MTs, pulling the spindle poles apart.

Question 39

What is the structure of the tubulin dimer sin regard to the status of the GTP

Answer 39

MTs are repeating a-tuulin and b-tubulin monomers, which form ab-tubulin heterodimers. There’s a GTP in both, but the GTP in a-tubulin is never hydrolyzed but the GTP in the B-tubulin dimer can be hydrolyzed

Question 40

At low tubulin concentrations, tubulin monomers

Answer 40

spontaneously assemble into ab-tubulin heterodimers.

As conc increases to critical concentration, the dimers then form into protofilaments and microtubules.