Mitosis and Cytokinesis - Bi Flashcards

1
Q

How does a branched network of actin filaments get formed in the example of a neutrophil chasing a bacteria?

A

chemoattractant –> receptor and trimeric G protein –> small G protein (CDC42/Rac) –> WASP/Wave –> Arp2/3 –> dynamic actin –> cell migration

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2
Q

How long is mitosis ? how long is cytokinesis?

A

Mitosis is an hour. Cytokinesis is 20 minutes.

Constant across most cell types, G1 is what varies.

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3
Q

What 2 things have morphologies that we are interested in during cell division cycle?

A
  • spindle

- chromosome

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4
Q

Three types of microtubules associated with the spindle?

A
  1. Astral microtubules
  2. Kinetochore microtubules: attach to chromosome
  3. Interpolar microtubules: Microtubules that come from both poles and interdigitate.
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5
Q

Which direction are microtubules pointing?

A

Plus ends point outward, minus ends are toward the spindle pole.

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6
Q

MTOC

A

Microtubule organizing center - the centrosomes.

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7
Q

How are centrosomes duplicated?

A

In a semi-conserved manner, initiated in G1 and finished in G2. Each has a pair of centrioles.

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8
Q

Are centrioles required for microtubule nucleation? Is the pericentriolar matrix? Wat about the gamma tubulin ring complex?

A

Centrioles are not required
Pericentriolar matrix - is required
Gamma tubulin ring - is required.

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9
Q

What percent of gamma tubulin complexes are MTOCs?

A

20%

The remaining 80% of gamma tubulin complexes do something else.

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10
Q

What is a gamma tubulin ring complex?

A
Located in the pericentriolar matrix
blocks minus end growth
binds to MT minus ends
does not block plus end growth.
involved in initial seeding and formation

It interacts with a beta subunit to seed microtubule formation at the centrosome. It associates with microtubule plus ends… It can cause microtubule nucleation on existing microtubules

80% of gamma-tubulin complexes at non-centrosome locations and function to produce new MT nucleation along existant MT bundles.

Abbreviated gamma-TuC

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11
Q

How many motors are there?

A

Multiple motors in each organism and each is distinct in sequence.. They can share essential functions. Proper spindle assemby requires motors that push in opposite directions. So you need overlapping/antagonistic motors.

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12
Q

How do you coordinate multiple motors?

A

Transient steady states depends on balance of forces generated by multiple complementary / antagonistic motors
Tipping balance by up or downregulating a set of motors drives specific mitotic movements.

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13
Q

What happens during prophase?

A
  1. Centrosomes separate
  2. Chromosomes condense
  3. Interphase microtubules break down.
  4. Nuclear envelope is not apparent.
    • end directed c terminal kinesins (pole-pole: inward) - minus end directed kinesin pulls the poles closer together.
    • end directed dynein (pole-pole: outward) - minus end directed, connecting it to the cell membrane, so cell body moves out.
  5. Polymerization at plus ends.
    Net action is to pull poles toward the center.

Good piture of this.

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14
Q

Prometaphase to metaphase

A
  1. Nuclear envelope breakdown
  2. Polar microtubules interdigitate
  3. Chromosomes are captured and align on the metaphase plate. Basically during metaphase the chromosomes align.
    • end directed c terminal kinesins (pole-pole: inward)
    • end directed dynein (pole-pole: outward)
    • end directd bipolar kinesins - Cin8 and kip1 in yeast. - push the poles away from one another. pole-pole outward. Pushes poles farther away, now things elongate.

Overexpression of CIN8P will elongate faster.

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15
Q

What happens if you delete one motor in yeast? What about multiple motors?

A

1 - cell is viale. Multiple - cell is dead.

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16
Q

What is chromosome capture and alignment

A

I think these are experiments where you use a laser to cut parts of the chromosomes and you can observe when you do this polar wind blowing the chromosome away (this is due to a kinesin causing mvmt toward the plus end.

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17
Q

Kinetochore

A

A centromere (DNA sequence) and microtubules and other proteins. 20-25 microtubules per kinetochore. Conserved.

In yeast - complex of a small centromere 120 bp + proteins, 1 microtubule per kinetochore

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18
Q

Metaphase

A

?

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19
Q

Anaphase

A

Anaphase A - chromosomes start to move toward pole but spindle size is the same. Breakdown of sister chromosome cohesion proteins… I think this means that the sister chromosomes move away from each other but the poles are the same distance apart.
Anaphase B - chromosomes move toward the pole but spindle size is elongating. Forces are generated at the kinetochore (minus-end-directed) and differential microtubule shortening at both ends. I think the poles are farther apart.

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20
Q

Two models for microtubule destabilization? (and movement? in anaphase?)

A
  1. Pacman model: There are proteins chewing away the microtubule plus ends.
  2. Poleward flux model: if you label tubulin subunits, you can see movement.
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21
Q

What provides the force during anaphase A to drive chromosome movement toward the spindle pole?

A

Disassembling microtubules, apparently. Disassembling microtubules at the plus end along with an Ska1 complex next to the kinetochore creates force which drives movement??

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22
Q

What increases distance between spindle poles?

A

2 possible forces:

  1. Plus end directed bipolar kinesins act at interdigitating zone of interpolar MTs
  2. Minus end directed motors in the cell cortex (dynein).
23
Q

Spindle middle zone

A

Mircrotubules in the center, begin to form at interphase

Middle body is 1.7-2 ug larg, and it is larger than the yeast division site.

24
Q

Function of cytokinesis

A
  1. Increase cell number

2. Asymmetric cell division increases cell diversity and cells with different orientations.

25
Q

How does cytokinesis occur?

A
  1. actomyosin ring contraction - involves F-actin ring, type II myosin, profilin, formins, septins. ESCRT-III mediated abscission
  2. Targeted membrane deposition - increase surface area at the division sit, deliver synthetic and hydrolytic enzymes for ECM remodeling
  3. ECM remodeling - spatiotemporal coordination of AMR function and membrane trafficking/ECM remodeling.

Occurs during late anaphase / telophase.

26
Q

ESCRT-III

A

Located on the shoulder of the middle body; causes constriction force to constrict the membrane.

27
Q

Actinomyosin ring contraction.

A

involves F-actin ring, type II myosin, profilin, formins, septins. ESCRT-III mediated abscission

28
Q

What method for cytokinesis is used in animal and fungal cells?

A

A contractile actomyosin ring.

29
Q

How does cytokinesis work in animal and fungal cells?

A

There is targeted membrane deposition at the cleavage site
Chondroitin and glycosaminoglycan synthesis is essential for embryonic cytokinesis as well. ECM proteins.
Actomyosin ring contraction and targeted membrane deposition have to coordinate in time and space during cytokinesis.

30
Q

How does one study cytokinesis?

A

Live imaging and quantitiatve analysis
Loss of function studies
RNAi screen 0 this was done in 2004 and identified 7000 dsRNAs/conserved genes that affect cytokinesis.
They also purified the midbody from mammalian cells and mass-spec-ed all the proteins. Most of the proteins they found functioned in membrane trafficking and actin organization.
You could also do a kymograph to map constriction rate.
They tried to make a contracting machine and made a “cell-ghost” system in fission yeast but region of constriction is very large.

31
Q

How does actomyosin ring assembly occur?

A
  1. type II myosin
  2. formns
    Both are required
    We have not actually crystallized a structure or seen one.
    Prevailing model: Purse string model.
32
Q

Where is cytokinesis controlled spatially?

A

2 models

  1. Astral stimulation model - astral bodies stimulate cytokinesis
  2. Central spindle stimulation model - central spindle stimulates cytokinesis.
  3. Astral relaxation model
33
Q

Experiment used to determine role of astral microtubules in division plane determination

A

Take fertilized sand dollar eggs
Add glass beads to displace the central spindle.
Furrow forms only on one side of a cell, producing a binucleate egg.
Then both nuclei enter mitosis
Cleavage occurs both between centrosomes linked by mitotic spindles and between 2 centrosomes that are simply adjacent. and four daughter cells are formed.
That’s why we tink it’s the asters - there’s no central spindle involved in the second half of this expt.

34
Q

Chromosome passenger complex

A

Associates with the central spindle, includes
INCENP
Aurora B
Survivin
Borealin
These proteins walk from chromosome arms / centromere in prophase to spindle midzone in late anaphase/telophase.

35
Q

InCENP

A

Inner centromere protein, associates with central spindle

36
Q

Aurora B / AIM-1

A

Aurora and Ipl1-like midbody associated protein kinase, associates with central spindle, part of the chromosome passenger complex.
Phosphorylates MKLP to control its localization and function at the central spindle. Defects in this phosphorylation event cause defects in cytokinesis.

37
Q

Survivin

A

INhibitor of apoptosis - has a specific mitotic role of protection against apoptosis

38
Q

Borealin

A

May be involved in chromosome segregation and cytokinsis

39
Q

Profilin

A

Actin monomer binding protein - when you have a profilin knockout you get a very disorganized central spindle.

40
Q

What is P-ASS?

A

Posterior asymmetric severing - a cytokinesis furrow is positioned by 2 consecutive signals - first by astral microtubules and then by the central spindle.

41
Q

What was the experiment they did to show P-ASS?

A

They took the single off center or ablated an aster with a laser, and they found when they did this there were 2 furrows.

42
Q

What the hell is a phragmoplast?

A

In plants, this instructs cell wall deposition - directs ECM remodeling and deposition

43
Q

Primary MTOCs

A
  • Centrosome - pair of centrioles plus pericentriolar matrix

- Basal body - cilia and flagella

44
Q

Centrosome

A

pair of centrioles plus pericentriolar matrix
centrioles are at right angles
Surrounded by pericentriolar matter
Each daughter centrosome contains one of the parent centrioles - they replicate in a semi-conservative manner. Replicate in late G1, Early G2

45
Q

Pericentriolar matter

A

Lattice of microtubule and associated proteins
Gamma tubulin ring nucleates microtubule assembly.
Pericentriolar matrix NOT CENTRIOLES are involved in MT nucleation.
Centrioles do not make direct contact with the ends of cytosolic microtubules.

46
Q

Push pull model for mitotic spindle shape

A

Transient steady state spindle structures depend on balance of forces generated by multiple complementary and antagonistic motors. These are used to shape the spindle.

MT flux - polymerization at plus ends and depolymeriztaion at minus ends also generates forces

47
Q

What are kinesins usually?

A

Plus end directed motors, but in the context of mitosis/cytokinesis they are sometimes minus end motors, or have 2 motor domains that slide antiparallel mts past each other in opposite directions.

48
Q

New motors

A
  • kinesin minus end motors
  • kinesins with 2 motor domains (help to slide antiparallel MTs past each other in opposite directions to move poles outward). Plus end directed motor.
  • kinesin like proteins with depolymerase activity
  • dyneins to move + to -, toward the centrosome. Plus is leading edge?
  • kinesins to move - to + (away from MTOC). Minus is leading edge?? All major kinesin organelle motors are - to + anyway.

There’s a lovely figure that explains everything. look at that.

49
Q

Polar flux model

A

Continuous tubulin addition generates force toward the spindle pole

50
Q

Chromosome capture

A
  1. Chromosome is unattached at prometaphase
  2. One kinetochore binds laterally to astral microtubule
  3. Chromosome slides toward spindle pole
  4. Lateral attachment converts to end on unipolar attachment - now this is a kinetochore microtubule
  5. Free kinetochore captures microtubule from opposite spindle pole to convert unipolar to bipolar attachment.
51
Q

Polar ejection force/polar wind

A

Interpolar/astral microtubule has plus end directed kinesin 4,10 which pushes chromosome arm away.

52
Q

In vitro filament sliding assay

A

Isolate chromosomes and attach to glass slides
Perfuse microtubules which are captured by kinetochores
perfuse in ATP - see minus end directed movement (dynein like)
Phosphorylate kinetochores, then perfuse in ATP (see plus end directed - kinesin like)
both are associated with kinetochores.

53
Q

Pac-man model vs poleward flux model

A

Chewing up microtubule tracks at the plus end - force pulls kinetochore to the pole. Depolymerization of plus end of kinetochore microtubules.
Chromatids are pulled towards the poles through depolymerization of kinetochore microtubules at the minus ends.