slide set 19

Question 1

how does microtubule dynamic instability change in mitosis

Answer 1

active Cdk1 protein

Question 2

what drives the change in microtubule dynamics in mitosis

Answer 2

Allows microtubules to find chromosomes
plus end

Question 3

MT-based motors contribute to…

Answer 3

motion and the spindle structure/organization
some push MT away, some pull away
kinesins that bind to chromosome and push them out toward metaphase plate

Question 4

when do MT-based motors contribute to motion and spindle structure

Answer 4

during times when mitotic spindle is forming
prometaphase, metaphase
helps move chromosomes to center and avoid having them out near the spindle poles

Question 5

Chromosomes reach metaphase…

Answer 5

forces are balanced here!

• Even in metaphase, the chromosomes aren’t static: they oscillate

Question 6

How can we measure chromosome oscillation?

Answer 6

Chromosome oscillation can be measured by fluorescently labeling the kinetochore and the centromere and measuring the distance between the 2 for each sister chromatid

At any given time there is a certain distance the kinetochore and centrosome will be apart (this distance changes over time)

Focus on red and blue lines!

when one is high, the other is low (forces are balanced on either side)

when one is pushed the other is pulled

Question 7

What kinetochore orientations are most stable?

Answer 7

correct kinetochore orientations are the most stable (proper bi-orientation: 1 kinetochore binds to 1 sister chromatid on each side)

you need both attachments on both sides

unstable orientations have weaker MT binding

Question 8

tensions across sister kinetochores detects…

Answer 8

proper attachments

Question 9

low tension across sister kinetochores

Answer 9

• When both sister kinetochores are NOT properly attached to MTs, there is no tension or space between the kinetochore layers
• A kinase in the inner layer (Aurora-B) can reach its target (Ndc80 complex) and phosphorylate it.
  
  • These phosphorylations prevent strong attachment of MTs

Question 10

high tension across sister kinetochores

Answer 10

• When both sister kinetochores are properly attached to MTs, there is tension that creates a space within the kinetochore itself.
• A kinase in the inner layer (Aurora-B) can no longer reach its target (Ndc80 complex) and phosphorylate it.
  
  • Therefore, microtubules can bind

Question 11

How do we know where forces act to move a chromosome?

Answer 11

• Mark a set of kinetochore microtubules
  
  • Could do so using fluorescently labeled tubulin and FRAP!) and watch in which direction the photobleached region of microtubule moves
    
    • toward the centrosome or toward the kintetochore
• If the mark moves toward the pole with the kinetochore, the force is produced at the centrosome (by motors)
• If the kinetochore moves toward the mark, the force is produced at the kinetochore

Question 12

Slow poleward flux

Answer 12

• Movement of tubulins within the MT: the entire MT moves toward a pole

Question 13

Two sources of movement

Answer 13

1. force produced at the kinetochore
2. pulling force at the pole to drag microtubules and the kinetochore toward the pole

Question 14

Potential drivers of minus-end directed movement acting at the kinetochore

Answer 14

• motor proteins associated with the kinetochore
  
  • kinesins
  • Dyneins
• microtubule depolymerization
  
  • Pacman
    *

Question 15

Potential drivers of minus-end directed movement acting at the centrosome

Answer 15

• To generate poleward flux:
  
  • Treadmilling, where tubulin subunits are removed at the minus end and added at the plus end
  • Motor proteins anchored at the spindle pole that pull on the MTs by walking to the minus end
• Poleward flux

Question 16

A third force is generate by non-kinetochore MTs and kinesin on chromosome arms…

Answer 16

this pushes chromosomes away from the pole

Question 17

Ejection forces do what

Answer 17

Ejection forces prevent a mono-oriented chromosome from getting too close to one pole (right after it makes its first attachment)

Question 18

At metaphase, forces balance

Answer 18

Question 19

sister chromatids during anaphase

Answer 19

sister chromatids oscillate up through anaphase, then each moves toward its pole

Question 20

Unattached kinetochores generate…

Answer 20

• Unattached kinetochores generate a “wait” signal that stops cells from moving from metaphase to anaphase
• A single unattached kinetochore is sufficient to stall the metaphase to anaphase transition

Question 21

Metaphase to Anaphase transition via APC/C to destroy cohesins (and cyclins)

Question 22

How does a single unattached kinetochore block APC/C?

Answer 22

1. Mad 2 binds unattached kinetochores
2. Bindings changes Mad 2 conformation
3. In this conformation, Mad2 can bind and inhibit APC/C-Cdc20 complex
4. No anaphase can occur until all chromosomes are attached to the spindle and under tension

Question 23

movement types during anaphase

Answer 23

Anaphase includes both chromosome to pole movement (Anaphase A) and movement of the two spindle poles further away (Anaphase B)

Question 24

Cytokinesis

Answer 24

Cytokinesis splits the cell in two, generating two daughter cells

Each daughter cell gets one copy of the genome, one centrosome, but also come ER, Golgi, lysosomes, etc.

Question 25

Ring of actin filaments and myosin motors forms where?

Answer 25

just under the plasma membrane

myosin-based forced pull the attached membrane in to eventually pinch off the plasma membrane between the two daughter cells