BIOL 2520 - Lecture 6 (Mitosis and Cytokinesis)

M phase characteristics

1.) It includes mitosis and cytokinesis

2.) It takes place in a relatively short amount of time

3.) It is the most dramatic phase

4.) Its stage and its events are set up by the earlier cell cycle phases

How long does the M-phase take in mammalian cells

One hour

How interphase prepares for M-phase

1.) The mitotic spindle is assembled, which is what is used to segregate the duplicated chromosomes

2.) The contractile ring is assembled, which is what is used to divide the cell into two.

What structures mediate M-phase in animal cells

It is mediated by transient cytoskeletal structures, specifically the microtubules of the mitotic spindle and the actin and myosin filaments in the contractile ring.

What drives entry into mitosis?

Activation of the M-cdk complex

Before the M-phase

1.) Cells increase in size during G1 and G2

2.) DNA of the chromosomes are replicated during S-phase

3.) The centrosome is duplicated

What is a centrosome

It is the primary microtubule-organizing centre in animal cells, where the microtubules of the mitotic spindle originate from

Stages of M-phase

1.) Prophase

2.) Prometaphase

3.) Metaphase

4.) Anaphase

5.) Telophase

6.) Cytokinesis

Prophase

1.) The centrosomes are already duplicated and the mitotic spindle is starting to form

2.) The DNA is condensed and duplicated

3.) The nuclear envelope is still intact

Prometaphase

1.) The centrosome has travelled to the opposite poles of the cell

2.) The microtubules are starting to capture the DNA, specifically their kinetochore proteins

3.) The nuclear envelope has dissolved/dissintegrated

Metaphase

The kinetochore of the chromosomes have now all been captured by the microtubules, allowing the microtubules to align them midway of the cell to form the metaphase plate

Anaphase

The microtubules shorten, pulling apart the sister chromatids in the process

Telophase

1.) Each side of the cell now has its own set of DNA and centrosome

2.) The nuclear envelope is starting to reform around the DNA on both sides of the cell

3.) The contractile ring is starting to form

Cytokinesis

1.) The cytoplasm has divided

2.) The nuclear envelope has fully surrounded the DNA

3.) The contractile ring contracts, forming the cleavage furrow, until it completely pinches off into 2 cells.

Which of the following events occurs in mitotic metaphase?

A. Nuclear envelope breakdown

B. Nuclear envelope reassembly

C. Chromosome attachment to spindle microtubules for the first time

D. Chromosome alignment at the spindle equator

E. Mitotic spindle assembly

D. Chromosome alignment at the spindle equator

Microtubules

1.) They are the cytoskeleton structure that forms the mitotic spindle.

2.) They contain tubulin subunits

Different types of microtubules

1.) astral microtubules

2.) Kinetochore microtubules

3.) Non-kinetochore microtubules

Astral microtubules

1.) They radiate from the centrosome

2.) And helps anchor/position the spindle apparatus, which determines where the cleavage plane is

Kinetochore microtubules

1.) They attach to the kinetochore proteins in the centromere of the chromosomes.

2.) They are essential to chromosome segregation, as it pulls the chromosomes onto opposite poles

Non-kinetochore (interpolar) microtubules

1.) They interdigitate (interlock) with opposing pairs of microtubules by sliding past each other, with help from microtubule-associated proteins

2.) They support the basic framework of the mitotic spindle

3.) And helps push the centrosomes apart using motor proteins

Microtubule polarity

Microtubules have a cylindrical structure made up of tubulin dimers and exhibits polarity, such that it has a (+) end and a (-) end.

(+) end of microtubules

1.) They point away from the centrosome and are closer to the kinetochore proteins.

2.) They have a higher rate of assembly, which is why they’re known as the growing end

(-) end of the microtubule

1.) They point towards the centrosome and point away from the kinetochore proteins (i.e. it is the end attached to the centrosome)

2.) It has a lower rate of assembly

(+) end vs. (-) end

The two ends of the microtubules differ from each other based on their rates of assembly. It is high at the (+) end and low at the (-) end.

Two key events in spindle assembly

1.) Formation of the poles, i.e. when spindle microtubules attach/anchor to the poles

2.) Capture of chromosomes, i.e. when the spindle microtubules attach to the chromosomes

Formation of the poles

1.) During prophase, the centrosomes begin to move apart and travel towards the opposite poles for the orientation of the mitotic apparatus.

2.) This process is achieved mainly by the use of motor proteins

Motor proteins used for pole formation

1.) The two main ones are dynein and kinesin.

2.) They walk on the microtubules. Whichever direction they’re walking towards, they pull the microtubules the opposite way.

Dynein motor proteins

They walk towards the (-) end, which pulls the astral microtubules towards the cell cortex, helping anchor the mitotic apparatus.

Kinesin motor proteins

1.) They walk towards the (+) end and cross-links the non-kinetochore microtubules.

2.) This helps push the centrosomes apart, thereby establishing their position at the two opposite poles

Altered DNA sequence effect on kinetochore

1.) Kinetochores recognizes the DNA sequence that forms the chromosome centromere.

2.) Therefore, if this sequence is altered, the kinetochores fail to form.

3.) And it they fail to form, the microtubules have nothing to recognize and so the chromosomes are not pulled apart

Capture of chromosomes (steps 1-2)

1.) The microtubules are constantly extending and drawing back into the centrosome, trying to search for the kinetochore proteins.

2.) They make first contact with the chromosomes (not necessarily fully attached) during prometaphase, when the nuclear envelope starts to dissolve.

Capture of chromosomes (steps 3-4)

3.) After a few initial unstable attachments, they eventually capture the kinetochore and become more stable

4.) Once attached to the spindle poles, the chromosomes are tugged back and forth until they align at the equator of the cell and become equidistant from the poles, thereby forming the metaphase plate.

What triggers chromosome separation

Separation of the sister chromatids during anaphase is triggered by proteolysis, specifically the cohesin linkage between the sisters that is broken by the enzyme, separase.

Separase

The enzyme that breaks the cohesin linkage between sister chromatids once it’s no longer being inhibited by securin

Securin

1.) The inhibitor that inhibits separase.

2.) It gets ubiquitinated by APC/C, resulting in its degradation.

Chromosome separation steps

1.) APC/C taggs securin for degradation via ubiquitination

2.) The degradation of securin then signifies the start of anaphase

3.) Since securin is no longer inhibiting separase, separase can now break down the cohesin linkage

4.) Resulting in the separation of the sister chromatids

Cohesin

The protein complex that holds the sister chromatids together. It is broken by the separase enzyme.

Where else is the APC/C seen?

It not only degrades securin, but it also degrades M-cyclin at the end of M-phase

2 different events of anaphase

Anaphase A and Anaphase B

Anaphase A

The initial poleward movement of the chromosomes that pulls the chromosomes apart and pulls them towards opposite poles, which is done by the shortening (i.e. depolymerization) of the kinetochore microtubules

Anaphase B

The separation of the spindle poles themselves, as they are pulled and pushed apart by the astral and non-kinetochore microtubules, with help from motor proteins

What microtubules do in Anaphase B

1.) A sliding force between the non-kinetochore microtubules from opposite poles pushes the spindle poles apart

2.) A pulling force at the cell cortex by the astral microtubules then drags the two poles apart

3.) The growth (polymerization) of the (+) ends of the nonkinetochore microtubules also pushes the poles apart

Microtubules at metaphase

They grow longer and shorter via polymerization and depolymerization to position the chromosomes at the equator to form the metaphase plate

What happens when not all the chromosomes are properly attached

1.) The kinetochores on the unattached chromosomes sends a stop signal to the cell-cycle control system, specifically the spindle assembly checkpoint

2.) This stop signal prevents the APC/C from being activated.

3.) This means that securin can’t be degraded and separase remains inhibited, therefore they can’t break the cohesin and the chromosomes remain together.

What are the consequences of blocking APC/C

In general, it delays mitosis, because blocking it prevents the chromosomes from being separated and it prevents M-cyclin from being degraded

What happens when not all of the chromosomes are properly attached AND it’s not sensed by the cell-cyle control system

The cell cycle continues and completes cell division, resulting in one progeny having more chromosomes and the other to have less chromosomes

The breakdown and assembly of the nuclear envelope

1.) The phosphorylation of nuclear pore proteins and lamins triggers the disassembly of the nuclear envelope during prometaphase.

2.) Dephosphorylation of these same proteins reverses this process and reforms the nuclear envelope during telophase.

Mitosis is complete with…

It is complete with formation of the nuclear envelope

What is cytokinesis

It is the process that physically separates the cytoplasm and cell into two daughter cells

Initiation of cytokinesis

1.) It requires the formation of the contractile ring, a structure made of actin and myosin filaments.

2.) The first signs of the formation of this structure is seen during anaphase.

Where is the plane of cleavage going to be

It is perpendicular to the long axis of the mitotic spindle. If the mitotic spindle moves, the cleavage furrow moves with it.

What happens if we dispace the mitotic spindle

The cleavage furrow develops perpendicularly to it, corresponding to the new spindle location and orientation

Cytokinesis in animals

It has a contractile ring made of actin and myosin filaments, which pinches the cell membrane into two cells

Cytokinesis in plants

Plants have a rigid cell wall that would be hard to pinch off. Therefore, they have to construct a new cell wall, which they do by using golgi derived vesicles and phragmoplasts.

Cytokinesis in plants steps

1.) Non-kinetochore microtubules at telophase form a structure called phragmoplasts, which guides the golgi derived vesicles that contain cell wall material to the equator after chromosome separation

2.) The vesicles fuse together to form the new cell wall at the equator, separating the cytoplasm and cell into two daughter cells

Mitosis vs meiosis

Mitosis: It duplicates all of the chromosomes and produces genetically identical diploid cells.

Meiosis: It produces 4 haploid cells, specifically gametes. It has two cell divisions but only replicates the chromosomes once.