L9 mitosis part 2 Flashcards
(13 cards)
microtubule polarity?
Made from dimers of α and β tubulin, which bind to GTP.
α tubulin is exposed at the minus end (can’t hydrolyze GTP), while β tubulin is exposed at the plus end (can hydrolyze GTP).
Time lag is required for GTP hydrolysis (time to exchange gtp and GDP)
The structure of the filament is different in the GTP-bound vs GDP-bound form.
When GTP is released, the filament kinks, causing disassembly. The straight structure induces polymerization.
microtubule dynamic instability?
Microtubules and Dynamic Instability
Microtubules typically form rows of 13 filaments and come together laterally, forming tubules.
As GTP is lost, the core structure disassembles, causing kinks.
When growing rapidly, microtubules keep a GTP-capped end to allow continued polymerization.
If the GTP cap is lost, the microtubule may rapidly shrink.
Dynamic instability: fast-growing microtubules can either grow or shrink, which helps them explore cellular compartments.
Microtubules can bind to cargo, stabilizing when they find something.
In mitosis, instability is high, crucial for spindle function.
MAPs (Microtubule-Associated Proteins) help stabilize microtubules.
Catastrophe factors promote rapid disassembly.
Slowing dynamic instability during mitosis can affect spindle function and chromosome alignment.
motor proteins?
Motor Proteins Organize the Mitotic Spindle
Motor proteins are ATP-binding molecules that bind to the cytoskeleton and transport cargo.
In mitosis, their cargo is another microtubule.
Antiparallel microtubule arrangement:
Kinesins move toward the plus end, pushing microtubules apart.
Other kinesins and dynein move toward the minus end, focusing the spindle poles.
These proteins work together to make the mitotic spindle highly dynamic, forming a self-organizing structure that segregates chromosomes.
Microtubules Attach to Kinetochores on Chromosomes
Centromere: A specialized chromosomal region that specifies the kinetochore, which binds to microtubules. Chrosomes constrict in the middle where the centromere is.
Kinetochore: A protein complex that attaches microtubules to the chromosome, ensuring proper segregation.
To ensure accurate chromosome segregation, microtubules must form end-on attachments at their plus ends to both kinetochores of a pair of sister chromatids
kinetochore?
Kinetochore is a layered protein structure that assembles on the centromere.
Inner kinetochore: Proteins that bind directly to centromeric DNA.
Outer kinetochore: Proteins that interact with microtubules, allowing chromosome attachment and movement.
how do motor proteins organise the mitotic spindle?
- nucleation
antiparallel cross-linking by kinesin-5
outward pushing by kinesin-4,10
focusing of poles by dynein and kinesin-14
kinetochore and microtubules?
Kinetochore-Microtubule Interactions
The kinetochore remains attached to the microtubule even as it shrinks.
Instead of being a static attachment at the plus end, the kinetochore acts like a dynamic interface, allowing it to stay connected while the microtubule depolymerizes.
This enables chromosome movement, as the kinetochore is “dragged” towards the spindle pole.
Microtubules Capture Kinetochores
As microtubules grow and shrink, they explore the cell and search for chromosomes.
Kinetochores are small structures compared to the entire chromosome, making it challenging to establish a stable attachment.
Unnatached chromosome at prometapahase.
Initially, (astral) microtubules form lateral attachments with kinetochores, which are not very stable.
Motor proteins help move/slide the chromosome towards the minus end of the microtubule (towards the spindle pole).
Eventually, this lateral attachment converts into an end-on attachment, which is more stable and allows for proper chromosome segregation. Microtubule now called kinetochore microtubule.
Converts unipolar to bipolar attachment.
The density of microtubules increases closer to the spindle pole.
microtubules, kinetochore and the spindle pole?
Microtubules coming from one pole are not stable, as they would cause both sister chromatids to be pulled toward the same side, leading to mis-segregation.
Once a stable bipolar attachment is formed, microtubules exert tension on kinetochores, pulling apart sister chromatids, which are still held together by cohesin rings at the centromere.
The force sensed by the centromeres stabilizes the microtubule attachment. This is how the cell ensures it is safe to proceed with chromosome segregation.
The cell must ensure every pair of sister chromatids is correctly attached before anaphase begins.
Depolymerization of Microtubules Pulls the Kinetochore Toward the Spindle Pole
The plus end of kinetochore microtubules depolymerizes, which pulls the kinetochore toward the spindle pole.
This movement is essential for proper chromosome segregation.
Intra-Kinetochore Deformations and Sister Kinetochore Movements
The inner kinetochore (attached to DNA) and the outer kinetochore (bound to microtubules) experience tension.
As microtubules pull in opposite directions, kinetochores deform slightly.
This deformation ensures that chromosomes are under proper tension, confirming correct attachment before segregation.
Aurora B kinase?
Error-Correction Mechanism and Aurora B Kinase
The error-correction mechanism is thought to be regulated by a spatial gradient of Aurora B kinase.
Aurora B kinase is localized to the centromeric chromatin, where it binds to DNA.
Aurora B Kinase and Its Role in Attachment Regulation
The centromeric region consists of a “stretchable” matrix made up of centromeric chromatin and the two sister kinetochores.
Aurora B kinase undergoes autophosphorylation, leading to its self-activation.
Aurora B phosphorylation weakens or disrupts incorrect attachments between microtubules and kinetochores.
High Aurora B activity → Destabilized (incorrect) attachments
Low Aurora B activity → Stabilized (correct) attachments
Tension-Dependent Regulation
Improper attachments (low tension):
The centromere remains unstretched, keeping Aurora B kinase concentrated near kinetochores.
This high local activity leads to phosphorylation of kinetochore components, breaking weak or incorrect attachments.
Proper attachments (high tension):
When kinetochores experience pulling forces, the centromere stretches, spreading Aurora B activity over a larger area.
At the outer edges of the kinetochore, Aurora B activity is lower, allowing microtubule-kinetochore attachments to stabilize.
capturing chromosomes and kinesin?
Capturing Chromosomes by the Spindle
During prometaphase, the spindle microtubules capture the chromosomes at their kinetochores.
Experiment with laser cutting the chromosome:
If the chromosome is cut with a laser, the arm without a kinetochore moves away from the spindle pole.
The arm with the kinetochore moves toward the pole, which suggests that the kinetochore is pulling the chromosome toward the spindle pole.
Polar Reduction Force and Kinesins
Polar reduction force: This is the force that reduces the distance between sister chromatids during mitosis.
Kinesins (motor proteins) bind to the arms of chromosomes and generate forces that can push the chromosome arms away from the spindle poles, facilitating proper alignment and segregation.
metaphase and anaphase?
Spindle Assembly Checkpoint (SAC) and its Role in Metaphase/Anaphase Transition
Opposing Forces Position the Kinetochores at the Metaphase Plate:
Kinesin-4 and Kinesin-10 are plus-end directed motor proteins that push the chromosomes, helping position the kinetochores along the metaphase plate.
Spindle Assembly Checkpoint Monitors Unattached Kinetochores:
The Spindle Assembly Checkpoint (SAC) ensures that chromosomes are properly attached to the spindle before proceeding to anaphase.
Mad2 is a key protein involved in the SAC and localizes to unattached kinetochores.
If kinetochores are unattached, Mad2 helps signal that the cell should delay entry into anaphase.
Principles of the Spindle Assembly Checkpoint:
The Mitotic Checkpoint Complex (MCC) consists of C-Mad2, Cdc20, Bub3, and Rubr1.
The MCC blocks the activation of the anaphase-promoting complex (APC) to prevent premature chromatid separation.
Cdc20 is an important activator of APC/C, a ubiquitin ligase.
APC/C (Anaphase-Promoting Complex) and Transition to Anaphase:
APC/C is an E3 ubiquitin ligase that triggers the separation of sister chromatids by ubiquitinating specific proteins.
Securin, a protein that keeps separase inactive, is ubiquitinated and degraded by APC/C.
Once securin is degraded, separase is activated. Separase then cleaves the cohesin complex, which holds sister chromatids together, leading to their separation during anaphase.
Cyclin D and APC Activation:
Cdc20 also activates APC/C but only after the checkpoint complex is not assembled, signaling the cell to move into anaphase.
Cyclin D is involved in regulating cell cycle progression, but its role here is not entirely clear unless you’re referring to its interaction with other cyclins in G1 phase.
anaphase?
Anaphase A:
Chromosomes move to opposing spindle poles:
During Anaphase A, kinetochore microtubules shorten, pulling the sister chromatids towards opposite poles of the cell.
Forces at the kinetochores:
The forces that drive this movement are primarily at the kinetochores, where microtubules are attached.
Anaphase B:
Spindle poles move apart:
In Anaphase B, the two spindle poles move farther apart.
Sliding force:
There is a sliding force generated between interpolar microtubules from opposite poles. These microtubules push the poles apart, contributing to the elongation of the spindle.
Elongation and pulling forces:
Interpolar microtubules also elongate during this phase, and there is a pulling force directly acting on the poles, further separating them. Additionally, microtubule growth at the plus ends of the polar microtubules helps elongate the spindle.
telophase?
The nuclear envelope re-forms around each set of chromosomes.
Membrane sheets form, reticulons are removed, and membranes close around the chromosomes.
The nuclear lamina assembles, and nuclear pore complexes are re-formed to allow for transport across the newly-formed nuclear envelope.
order of events
Prometaphase: Microtubules Capture Kinetochores
Unattached chromosome at prometaphase
At the start of prometaphase, chromosomes are not yet attached to the mitotic spindle, and microtubules begin their search.
Microtubules Capture Kinetochores
As microtubules grow and shrink, they explore the cell and search for chromosomes.
Kinetochores are small structures compared to the entire chromosome, making it challenging to establish a stable attachment.
Microtubules from the spindle poles (astral microtubules) begin to interact with kinetochores.
Initial Lateral Attachments
Initially, (astral) microtubules form lateral attachments with kinetochores, which are not very stable.
Motor proteins help move/slide the chromosome towards the minus end of the microtubule (towards the spindle pole).
Kinesins and other motor proteins play a role here, sliding chromosomes along microtubules.
Conversion to End-On Attachment
Eventually, this lateral attachment converts into an end-on attachment, which is more stable and allows for proper chromosome segregation. Microtubule now called kinetochore microtubule.
Converts unipolar to bipolar attachment.
The chromosome shifts from being attached to microtubules from one pole (unipolar) to both poles (bipolar), setting the stage for proper segregation.
Capturing Chromosomes by the Spindle
During prometaphase, the spindle microtubules capture the chromosomes at their kinetochores.
Experimental evidence: If the chromosome is cut with a laser, the arm without a kinetochore moves away from the spindle pole, while the arm with the kinetochore moves toward the pole, suggesting that the kinetochore is pulling the chromosome toward the spindle pole.
Aurora B Kinase and Error Correction
The error-correction mechanism is thought to be regulated by a spatial gradient of Aurora B kinase, localized to the centromeric chromatin where it binds to DNA.
Aurora B kinase undergoes autophosphorylation, leading to its self-activation.
Aurora B phosphorylation weakens or disrupts incorrect attachments between microtubules and kinetochores.
High Aurora B activity → Destabilized (incorrect) attachments; Low Aurora B activity → Stabilized (correct) attachments.
This ensures only proper bipolar attachments persist as prometaphase progresses.
Tension-Dependent Regulation
Improper attachments (low tension): The centromere remains unstretched, keeping Aurora B kinase concentrated near kinetochores, leading to phosphorylation of kinetochore components and breaking weak or incorrect attachments.
Proper attachments (high tension): When kinetochores experience pulling forces, the centromere stretches, spreading Aurora B activity over a larger area. At the outer edges of the kinetochore, Aurora B activity is lower, allowing microtubule-kinetochore attachments to stabilize.
Tension stabilizes the correct attachments as chromosomes align.
Polar Reduction Force and Kinesins
Kinesins (motor proteins) bind to the arms of chromosomes and generate forces that can push the chromosome arms away from the spindle poles, facilitating proper alignment and segregation.
This helps position chromosomes as they prepare for metaphase.
Metaphase: Alignment and Checkpoint Control
Stable Bipolar Attachment and Tension
Once a stable bipolar attachment is formed, microtubules exert tension on kinetochores, pulling apart sister chromatids, which are still held together by cohesin rings at the centromere.
The force sensed by the centromeres stabilizes the microtubule attachment. This is how the cell ensures it is safe to proceed with chromosome segregation.
The density of microtubules increases closer to the spindle pole.
Chromosomes are now aligned at the metaphase plate.
Opposing Forces Position the Kinetochores at the Metaphase Plate
Kinesin-4 and Kinesin-10 are plus-end directed motor proteins that push the chromosomes, helping position the kinetochores along the metaphase plate.
These forces balance the pulling from kinetochore microtubules.
Spindle Assembly Checkpoint (SAC)
The Spindle Assembly Checkpoint (SAC) ensures that chromosomes are properly attached to the spindle before proceeding to anaphase.
Mad2 is a key protein involved in the SAC and localizes to unattached kinetochores.
If kinetochores are unattached, Mad2 helps signal that the cell should delay entry into anaphase.
The Mitotic Checkpoint Complex (MCC) consists of C-Mad2, Cdc20, Bub3, and Rubr1, blocking the activation of the anaphase-promoting complex (APC) to prevent premature chromatid separation.
The cell must ensure every pair of sister chromatids is correctly attached before anaphase begins.
Anaphase: Chromosome Segregation
APC/C Activation and Cohesin Cleavage
APC/C (Anaphase-Promoting Complex) is an E3 ubiquitin ligase that triggers the separation of sister chromatids by ubiquitinating specific proteins.
Securin, a protein that keeps separase inactive, is ubiquitinated and degraded by APC/C.
Once securin is degraded, separase is activated. Separase then cleaves the cohesin complex, which holds sister chromatids together, leading to their separation during anaphase.
Cdc20 activates APC/C after the checkpoint complex is disassembled, signaling the cell to move into anaphase.
Anaphase A: Chromosomes Move to Poles
During Anaphase A, kinetochore microtubules shorten, pulling the sister chromatids towards opposite poles of the cell.
The plus end of kinetochore microtubules depolymerizes, which pulls the kinetochore toward the spindle pole.
The kinetochore remains attached to the microtubule even as it shrinks.
Instead of being a static attachment at the plus end, the kinetochore acts like a dynamic interface, allowing it to stay connected while the microtubule depolymerizes.
This enables chromosome movement, as the kinetochore is “dragged” towards the spindle pole.
The inner kinetochore (attached to DNA) and the outer kinetochore (bound to microtubules) experience tension, deforming slightly to ensure proper segregation.
Anaphase B: Spindle Elongation
THERE WAS NO Q12 IN THE ORIGINAL TEXT SO I ASSUMED THAT Q11 WAS ANAPHSE AND THAT Q12 IS TELOPHASE AND THAT THE ORIGINAL AUTHOR MESSED UP THE NUMBERING AND MEANT TO WRITE Q13 HERE INSTEAD OF Q12
In Anaphase B, the two spindle poles move farther apart.
There is a sliding force generated between interpolar microtubules from opposite poles, pushing the poles apart and contributing to the elongation of the spindle.
Interpolar microtubules also elongate during this phase, and microtubule growth at the plus ends of the polar microtubules helps elongate the spindle.
Telophase: Nuclear Reformation
Nuclear Envelope Reformation
The nuclear envelope re-forms around each set of chromosomes.
Membrane sheets form, reticulons are removed, and membranes close around the chromosomes.
The nuclear lamina assembles, and nuclear pore complexes are re-formed to allow for transport across the newly-formed nuclear envelope.
