Lecture 4 Flashcards
What is catastrophe good for
It’s good for
chromosome segregation
during cell division.
When is the mitotic spindle formed
During pro metaphase
Where do Mts attach to chromosomes
MTs in the spindle attach to chromosomes at their kinetochores.
Target area: ~1 μm2
Search volume: ~1000 μm3
What we know about MTs
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.
Impact of GTP hydrolysis on MTs
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.
Role of centrosome
Stabilises the negative end and + end radiates out. So in the middle of cell during mitosis, have region of overlap. (w/ two centrosomes).
gamma-tubulin ring complexes
nucleate microtubule
polymerization at the centrosome
Centrosome duplication
precedes formation
of a bipolar spindle
Summary
*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
Search and capture model
- 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.
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
Less reliable MT-kinetochore
attachment/chromosome segregation
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?
Time to capture decreases as the
number of MTs increases
Unbiased search and capture
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.
Chromosomes and search and capture
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).
Accessory proteins and Ran GTPase
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).
2 types of molecular switches
Signalling by phosphorylation
Signalling by GTP hydrolysis.
Ran-GTP
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
Astral MT
capture K-fibers growing from the
kinetochore in a dynein-dependent fashion
Biased search-and-capture
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
Biased search and capture - If you remove RanGTP, how should
that affect the time it takes to capture
all kinetochores?
Time to capture should increase.
Biased search and capture - If you constitutively activate RanGTP,
how should that affect the time it
takes to capture all kinetochores?
Time to capture should increase
Biased search and capture - If you constitutively activate RanGTP,
how should that affect the time it
takes to capture all kinetochores?
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.
