Flashcards in exam 3b Lecture 32 Mitotic Spindle Deck (71):
Is intraflagellar motility happening during flagellar beating?
Yes. They happen simultaneously.
What happens to cells during prophase?
Cells round up, chromosomes condense, centrosomes separate, asters form. Golgi disperse.
What happens to cells during prometaphase?
Nuclear envelope breaks down. Microtubules attach to kinetochores, chromosomes move to metaphase plate.
What happens to cells during metaphase?
Chromosomes are aligned at metaphase plate – sister chromatids under tension.
What happens to cells during anaphase?
Paired kinetochores separate to poles
What happens to cells during telophase?
Nuclei reform as chromosomes decondense.
What happens during cytokinesis?
Daughter cells form.
What kind of cells round up in prophase?
Cells without walls. Animal cells.
Are cells still connected to surface during prophase?
Yes, with retractor fiber.
Why is prophase important for mitotic spindle?
The round up happens to create space for spindle so it can form and function.
What fiber system causes rounding in prophase?
What happens to sister chromatids after DNA replication in S phase?
The sister chromatids are linked together by cohesins.
What are cohesins and condensins?
Members of the SMC (structural maintenance of chromatin) family. They are large protein machines that hydrolyze ATP to manipulate chromatin.
What kind of domain do cohesins have?
Coiled coil domain.
How do condensins work with sister chromatids in prophase?
In prophase, condensins are phosphorylated and begin to disentangle sister chromatids, compacting them tightly by coiling them around.
Structure of condensing
Five subunit protein complex. ATPase head comains of its two major subunits, Smc2 and Smc4, are held together by three additional subunits.
What protein event does chromosome condensation depend on during prophase?
Where can centrosomes only be found?
How are centrioles similar to DNA?
They both have semiconservative replication.
When do centrioles separate?
When do centrioles replicate?
During S phase. The mother and daughter cell each have a baby.
Which centrioles can assemble primary cilium?
Where are centrioles found?
When in cell cycle do centrosomes move apart?
When do centrosomes form asters and polarize?
What are asters?
Radial array of microtubules.
When do asters form mitotic spindles?
During metaphase, when the nuclear envelope breaks down.
What do centrosomes do during mitosis?
Increase microtubule assembly and dynamics.
What is polarity of mitotic spindle?
Minus at center. Plus at distal ends.
What are kinetochore microtubules?
Microtubules that reach out and connect with kinetochore.
What are interpolar microtubules?
Microtubules that contact microtubules on other side of kinetochore.
What are astral microtubules?
Radiate out from the poles into the cytoplasm
If spindles lack centrosomes, are there astral microtubules?
What do microtubules do in mitotic spindle?
Grow and shrink. As they grow out they may contact kinetochore or microtubule from other side of kinetochore.
What happens to microtubule if it reaches kinetochore or microtubule from other side?
This is stabilizing for microtubule.
What happens to microtubules when they are stabilized in mitotic spindle?
They set up array of microtubules.
What is the mechanism for spindle assembly in cells with centrosomes?
a. Minus end directed kinesin 14 cross-links microtubules from opposite poles and slides poles together. B. Kinesins 4 and 10 (chromokinesins) pull chromatin cargo to plus ends of microtubule (away from the poles) c. plus-end directed kinesin 5 (bipolar kinesin) cross-links microtubules from opposite poles and slides them apart. D. cytoplasmic dyneins at plasma membrane “pull” on astral microtubules.
How does Kinesin 14 work? Towards what end is it directed?
Directed towards minus end. Binds to microtubule as its cargo. Walks toward minus end of a microtubule (so towards aster). Pulls microtubules and poles together.
Plus end directed motors that associate with chromosome arms and push the attached chromosome away from the pole.
How do kinesin 4 and 10 work? Towards what end do they work?
These are the chromokinesins. They work towards the plus end. They connect to chromatin and generate polar wind. Chromosomes move away from the poles.
How does kinesin 5 work? Towards what end does it work?
Works toward plus end. Forms dimer. It is a bipolar filament like myosin 2. It cross links microtubules and pushes poles apart.
How does cytoplasmic dynein work? Towards what end does it work?
Works towards minus end. Pulls apart poles.
So, in what directions do motor proteins all work?
Kinesin 14 pushes toward the poles. Kinesin 4 and 10 push away from the pole. Kinesin 5 pushes poles apart and cytoplasmic dynein pulls apart poles.
How are spindles assembled in cells without centrosomes?
In these cells chromatin is focal point for microtubule growth and spindle assembly.
How does Ran work with chromatin to form spindle?
Ran-GEF has a high affinity for chromatin, so it binds to chromatin, and keeps a high gradient of Ran-GTP closest to chromatin. When nuclear envelope breaks, Ran-GTP imports assembly factors for spindle and regulates these factors.
What two roles does GTPase Ran play in mitotic spindle assembly?
a. nucleation and stabilization of microtubules around chromatin. B. localization of motor proteins to orient microtubules
What roles do motor proteins play in mitotic spindle assembly without centrosomes?
After Ran-GTP nucleates microtubules, kinesin-5 does antiparallel cross-linking of microtubules. Kinesin 4, 10 push the microtubules outward. Cytoplasmic dynein and kinesin-14 focus poles.
Are mitotic spindles in cells without centrosomes focused at minus ends?
No. they have wider ends and are barrel shaped.
What experiment shows role of ran in mitotic spindle assembly?
Experiment with GEF missing or with mutated Ran-GTP. They form a multipolar spindle if Ran-GTP not there. Disrupts mitosis.
What happens during prometaphase?
Nuclear envelope breaks and chromosomes attach to the mitotic spindle.
What are kinetochores?
Proteins that bind to centromeric DNA and interact with microtubules.
What do kinetochores do to microtubules in prometaphase?
Kinetochore capture cycle.
Late prophase, microtubules form outside of nuclear envelop. In early prometaphase, lateral kinetochore attachments, chromosomes arms pushed outwards. In mid prometaphase, there is end-on attachment. During metaphase, there is bi-orientation.
What is bi-orientation?
Chromosomal pair attach to each end of kinetochore evenly.
What does bipolar attachment to opposite poles generate?
High levels of tension at kinetochores.
How does kinetochore bind to microtubule?
Binds to side microtubule. Attaches to plus end.
Can the microtubule while attached to kinetochore?
Yes, it can grow and shrink because the ndc80 complex that attaches kinetochore to microtubule slides along microtubule.
Why do metaphase chromosomes encounter opposing forces generated by spindle machinery?
a. kinetochore microtubule disassembly at plus ends pulls chromosome to poles. B. microtubule assembly at plus ends, disassembly at minus ends is called “treadmilling”. C. polar ejection force (polar wind) is generated by kinesins 4, 10 (pushes chromatin away from pole).
How do cells ensure bi-orientation during metaphase?
They have a mechanism for sensing tension.
How do cells sense tension?
A “spindle assembly checkpoint” mechanism requires bi-orientation of all chromosomes (dependent on both tension and microtubule attachment) before metaphase-to-anaphase transition.
Which kinase is at work to sense tension?
Aurora-B kinase. It is activated under low tension.
What does aurora-B kinase do when there is high tension?
Nothing because it cannot reach its target to phosphorylate it.
What is spindle assembly checkpoint?
Mechanism that detects proper attachment of all kinetochores to spindle microtubules. Improper attachment of even one kinetochore prolongs metaphase.
How does spindle assembly checkpoint work?
a. spindle microtubule disassembly with nocodazole prolongs metaphase. B. Mad2 protein localizes to unattached kinetochore, sensing lack of tension. When spindle microtubules are disassembled with nocodazole, Mad2 appears at every kinetochore. Mad2 binds and inhibits Cdc20, a protein required for initiation of anaphase.
What prolongs metaphase?
What protein binds at every kinetochore to inhibit Cdc20?
What is Cdc20?
A protein required for initiation of anaphase.
What happens at anaphase?
Sister chromatids separate.
What are two components of anaphase?
Anaphase A and anaphase B
What is anaphase A?
Disassembly of kinetochore microtubules at both ends. Shortening of kinetochore microtubules; movement of daughter chromosomes to poles; forces generated mainly at kinetochores