Lecture 4

Question 1

What is catastrophe good for

Answer 1

It’s good for
chromosome segregation
during cell division.

Question 2

When is the mitotic spindle formed

Answer 2

During pro metaphase

Question 3

Where do Mts attach to chromosomes

Answer 3

MTs in the spindle attach to chromosomes at their kinetochores.
Target area: ~1 μm2
Search volume: ~1000 μm3

Question 4

What we know about MTs

Answer 4

Tubulin dimers polymerise to form Mts. Dynamic instability at the + end depends on GTP hydrolysis.
The rate of polymerisation > GTP hydrolysis = GTP cap and rapid polymerisation.
When the rate of GTP hydrolysis begins to catch up to the rate of polymerisation, then will have catastrophe = the rapid depolymerisation. Then, rescue.

Question 5

Impact of GTP hydrolysis on MTs

Answer 5

Destabilises the MT lattice. Protofilmanets can assemble linearly if beta-tubulin binds GTP. If it doesn’t protofilaments are curved, a conformational change that strains the lattice. Protofilaments curve away from MT w/ loss of GTP cap.

Question 6

Role of centrosome

Answer 6

Stabilises the negative end and + end radiates out. So in the middle of cell during mitosis, have region of overlap. (w/ two centrosomes).

Question 7

gamma-tubulin ring complexes

Answer 7

nucleate microtubule
polymerization at the centrosome

Question 8

Centrosome duplication

Answer 8

precedes formation
of a bipolar spindle

Question 9

Summary

Answer 9

*Microtubules exhibit dynamic instability
*Centrosomes organize MTs during interphase and mitosis
*MTs reorganize into a spindle structure during mitosis, emanating
from two centrosomes on either side of the cell

Question 10

Search and capture model

Answer 10

• MTs radiate from centrosomes on either
  side of the cell, polymerizing at their plus
  ends
• MTs switch from polymerizing to rapidly
  depolymerizing (catastrophe!)
• After catastrophe, MTs re-grow in a
  different direction
• Kinetochore-MT interactions stabilize MTs
  (no catastrophe!)
• MTs keep growing and shrinking until all
  kinetochores are captured

Search - Mts can dynamically search by polymerising and depolymerising (MTs regrow in different direction after catastrophe).
Capture - when Mts interact with kinetochore, this stabilises Mts, preventing catastrophe.

Dynamic instability at the MT plus end allows MT to search
the cell for kinetochores. Binding to kinetochores stabilizes
MT plus ends.

How to decide if this is realistic - compare to cell cycle scales.

Question 11

If you stabilize MT (i.e. prevent dynamic
instability), how should that affect MT-
kinetochore attachment and chromosome
segregation? in the search and capture model

Answer 11

Less reliable MT-kinetochore
attachment/chromosome segregation

Question 12

In the search and capture model, How should the number of MTs
radiating from the centrosome relate
to the length of time it takes to
capture all kinetochores?

Answer 12

Time to capture decreases as the
number of MTs increases

Question 13

Unbiased search and capture

Answer 13

Is not fast enough.

Bacterial cell life cycle duration = 150 mins. Cell division lasts about 15 mins.

Human cells, cell cycle about 20 hours. Mitosis is approx 1 hour in human cells. (G2 is 2-3 hours, G1 is about 10, S about 6-7).

Time for search and capture with computer model and within reasonable estimates of Mts showed a mean of between 500 mins to 125 mins.

Question 14

Chromosomes and search and capture

Answer 14

Chromatin itself nucleates microtubules. Depends on Rann GTPase, which are molecular switches. When bound to GTP, Ran is “on” and can bind effectors. When it hydrolyses GTP, “off”, cannot bind effectors. Must release GDP, bind another GTP. This release is slow. Accessory proteins affect the speed. GAPs promote GTP hydrolysis (switch off), GEFs promote GDP release (switch on).

Question 15

Accessory proteins and Ran GTPase

Answer 15

When bound to GTP, Ran is “on” and can bind effectors. When it hydrolyses GTP, “off”, cannot bind effectors. Must release GDP, bind another GTP. This release is slow. Accessory proteins affect the speed. GAPs promote GTP hydrolysis (switch off), GEFs promote GDP release (switch on).

Question 16

2 types of molecular switches

Answer 16

Signalling by phosphorylation
Signalling by GTP hydrolysis.

Question 17

Ran-GTP

Answer 17

promotes MT nucleation near
chromatin via a reaction-diffusion mechanism.

• RanGEF binds to chromosomes, activating
  RanGTP
• RanGAP diffuses throughout the cell,
  inactivating RanGTP
• Active RanGTP recruits gamma-TuRC

Question 18

Astral MT

Answer 18

capture K-fibers growing from the
kinetochore in a dynein-dependent fashion

Question 19

Biased search-and-capture

Answer 19

is fast enough to
account for observed rates of spindle assembly.

• MTs radiate from centrosomes on either side of the cell, polymerizing at their plus ends
• MTs switch from polymerizing to rapidly
  depolymerizing (catastrophe!)
• After catastrophe, MTs re-grow in a
  different direction
• Kinetochore-MT interactions stabilize MTs (no catastrophe!)
• A gradient of RanGTP results in MT
  nucleation near chromosomes
• MT emanating from the kinetochores bias the search
• MTs keep growing and shrinking until all kinetochores are captured

Question 20

Biased search and capture - If you remove RanGTP, how should
that affect the time it takes to capture
all kinetochores?

Answer 20

Time to capture should increase.

Question 21

Biased search and capture - If you constitutively activate RanGTP,
how should that affect the time it
takes to capture all kinetochores?

Answer 21

Time to capture should increase

Question 22

Biased search and capture - If you constitutively activate RanGTP,
how should that affect the time it
takes to capture all kinetochores?

Answer 22

Time to capture should increaseDynamic instability at the MT plus end allows MT to search
the cell for kinetochores. Binding to kinetochores stabilizes
MT plus ends. RanGTP gradient biases search towards the
chromosomes.