BIOMG 1350 Lecture 13 - Mitosis

What two events in interphase set the stage for M phase?

Genome replication and centrosome duplication.

Genome Replication

• The first event in interphase that sets the stage for M phase.

• During S phase the genome is replicated by DNA polymerase. During replication cohesion proteins bind to the genome and form rings that hold the duplicated sister chromatids together.

• Sister chromatids are two exact copies of the same chromosome. They each consist of DNA and histone proteins (chromatin).

• Sister chromatids or chromosomes before the M phase are loose and uncoiled rather than coiled hence the need for cohesion.

Centrosome Duplication

• The second event in interphase that sets the stage for M phase.

• During S and G2 phases: The interphase centrosome duplicates, which will later become the poles of the mitotic spindle.

• Microtubules play a critical role in chromatid segregation.

• Typical cell pre S phase only has 1 centrosome.

M-Phase Stages

M phase consists of two distinct, but overlapping parts.

• Mitosis is the first part of M phase which is the precise and equal segregation of the duplicated chromosomes by the microtubule based mitotic spindle, resulting in two nuclei.

• Cytokinesis is the second part of M phase and is where the division of the cytoplasm in two halves to generate two cells occurs. It’s the actual division of the two nuclei into two cells via cell plate or cleavage furrow formation.

5 Stages of Mitosis (in M-Phase)

Mitosis consists of 5 phases: Prophase, prometaphase, metaphase, anaphase, and telophase.

Prophase

• During prophase the mitotic spindle begins to form or extend and chromosomes condense (chromosomes condense via condensin).

• Prophase is driven by the activation of M-CDK (activated in the G2/M checkpoint by dephosphorylation by Cdc25).

• Duplicated centrosomes are pushed apart by kinesin 5 to end up on opposite sides of the nucleus. They are now called spindle asters with their associated microtubules.

• The mitotic spindle begins to form outside the nucleus branching from the centrosome.

• The kinetochore also assembles during prophase.

Condensin

• Condensin proteins form rings that condense chromosomes to facilitate segregation during mitosis.

  • Condensin is similar to, but not the same as, cohesin. Both proteins form rings, but they have different functions. (Cohesion is used during S phase whereas Condensen is present during prophase).

  • Condensin organizes chromatin into loops, resulting in compaction.

Mitotic Spindle

• The mitotic spindle is the protein machinery that segregates sister chromatids into separate nuclei.

• Mitotic spindle is made of microtubules, motors, and the 2 centrosomes.

• It also has a built in quality control system (the spindle assembly checkpoint which checks if all the chromosomes are properly attached to the mitotic spindle).

• Contains 3 classes of microtubules: astral, interpolar, and kinetichore microtubules.

• Mitotic spindle comes from the spindle poles (2 of them are present after S phase due to replication of spindle pole along with other organelles in preparation for cell division).

Spindle Poles

Two opposite ends of the mitotic spindle where microtubules are organized during cell division. In other words spindle poles refer to the opposing ends of the mitotic spindle apparatus or the centrosome microtubule complex.

Bi Orientation

• Bi-orientation is favored because microtubule attachments to just one kinetochore, or to inappropriate kinetochores, are unstable. When correctly bi-oriented, tension develops in the microtubules, which stabilizes the interaction between the kinetochore and microtubules.

• Chromosomes are bi-oriented during prometaphase. A correctly bi-oriented chromosome has microtubules linking each spindle pole to each of its two kinetochores. The chromosome then moves to a point equidistant from the two poles (ie the ‘metaphase plate’).

Prometaphase

• During prometaphase, the nuclear envelope breaks down and microtubules begin attaching to chromosomes.

• Each chromatid has a kinetochore – a specific region associated with the centromeres to which the plus end of kinetochore microtubules can attach.

• Phosphorylation of nuclear lamins (intermediate filament proteins) and nuclear pore proteins, both of which form the nucleus, causes the nuclear envelope to break down into vesicles.

• Microtubules begin attaching to kinetochores. Kinetochores are like harnesses attached to the centromere of the sister chromatid. Cohesins are still holding the chromatids together.

• The chromosomes are then bi-oriented. A correctly bi-oriented chromosome has microtubules linking each spindle pole to each of its two kinetochores. The chromosome then moves to a point equidistant from the two poles (ie the ‘metaphase plate’).

Kinetochore

A protein complex that links microtubules to the centromere of each chromatid. Kind of like a medium or harness for the chromosome to correctly attach to the microtubule. The kinetochore has little ropes (connecting protein complexes) that attach to the plus end of the kinetochore microtubules.

A sister chromatid has 2 centromeres and thus 2 kinetochores.

Centromere

A special region of DNA within each chromosome that the kinetochore attaches to.

A sister chromatid has 2 centromeres and thus 2 kinetochores.

Metaphase

• During metaphase, the chromosomes are arranged on the metaphase plate (midway between spindle poles).

• How does this occur?

  • Tension creates a stable bipolar attachment. Tension stabilizes the microtubule kinetochore attachments.

  • Unattached kinetochores send out a biochemical signal that stops progress into anaphase – this is the molecular basis of the checkpoint in mitosis.

  • Only the correct bipolar attachment has tension across the kinetochores.

  • If the kinetochore microtubule attaches to the kinetochore in any way but the most ideal orientation for proper mitosis by random chance, then it would be unstable or there would be a lack of tension.

• Once all chromosomes are appropriately attached to the spindle, the Anaphase Promoting Complex (APC/C) is activated, which leads to degradation of M-cyclin and the cohesins holding the chromatids together. The cell now progresses into anaphase.

How does the cell ensure all the sister chromatids are attached to the kinetochore microtubules properly?

• Tension creates a stable bipolar attachment. Tension stabilizes the microtubule kinetochore attachments.

• Unattached kinetochores send out a biochemical signal that stops progress into anaphase – this is the molecular basis of the checkpoint in mitosis.

• Only the correct bipolar attachment has tension across the kinetochores.

• If the kinetochore microtubule attaches to the kinetochore in any way but the most ideal orientation for proper mitosis by random chance, then it would be unstable or there would be a lack of tension.

Spindle Assembly Checkpoint

• The spindle assembly checkpoint occurs during metaphase and it basically asks whether all the chromosomes are attached to the kinetochore in the proper orientation.

• All chromosomes must be bi oriented (meaning the kinetochores of sister chromatids must be attached to microtubules extending from opposite spindle poles during cell division).

• Activation of the Anaphase Promoting Complex (APC/C) leads to the degradation via ubiquitylation of M Cyclin and thus the inactivation of M-CDK.

• Activation of the APC/C leads to the degradation of M-cyclin and cohesion proteins.

  • Usually the seperase (proteolytic enzyme) is attached to the inhibitory protein (securin) preventing it from performing its function. APC/C degrades M-cyclin and also degrades the securin allowing the separase to be initiated.

  • Separase helps to initiate anaphase by degrading cohesins which keep the sister chromatids together.

Summary of the Spindle Assembly Checkpoint

• When properly connected to both spindle poles, the kinetochore microtubules pull on the kinetochores and the cohesins pull the chromosomes together.

• Any unattached kinetochore sends out a single preventing exit from metaphase by preventing activation of APC/C.

• When all kinetochores are attached to appropriate microtubules, this satisfies the checkpoint, which leads to the activation of the Anaphase Promoting Complex (APC/C).

• Activation of APC/C leads to both the degradation of M cyclin (inactivating M-CDK) and also to the degradation of securin (allowing separase to degrade the cohesins holding the chromosomes together, initiating anaphase).

Separase

An enzyme that breaks down cohesin proteins holding sister chromatids together during cell division. This allows sister chromatids to separate and move to opposite poles of the cell during anaphase.

Securin

A protein that binds to and inhibits separase, preventing sister chromatids from separating too early. During anaphase, securin is broken down, releasing separase to separate the sister chromatids.

Cohesin Degradation

The process in which separase breaks down cohesin proteins that hold sister chromatids together. This occurs at the start of anaphase, allowing sister chromatids to separate and move to opposite poles of the cell.

APC/C (Anaphase-Promoting Complex)

A protein complex that triggers the start of anaphase by marking securin for degradation, allowing separase to become active and separate sister chromatids. It also breaks down M-cyclin to help the cell exit mitosis.

Anaphase

• During anaphase the sister chromatids are pulled apart.

• Two processes occur simultaneously in anaphase and they are named Anaphase A and Anaphase B.

  • Anaphase A is when the chromosomes are pulled poleward and then Anaphase B is when the poles are pushed and pulled apart.

  • Interpolar and astral microtubules are involved in anaphase B whereas kinetichore microtubules are involved in anaphase A.

Anaphase A

• In Anaphase A the chromosomes are pulled poleward.
  The shortening of kinetochore microtubules occurs. Forces are generated at the kinetochores to move the chromosomes toward their spindle pole.

• Microtubule depolymerization at the kinetochore pulls the chromosome to the pole.

• The kinetochore is attached to the microtubule via a ring on the microtubule which has strands extending to the kinetochore. When the microtubule dissociates from the plus end, that harness drags the kinetochore and by extension the sister chromatid.

Anaphase B (Interpolar microtubules)

• Anaphase B involves interpolar microtubules and the Kinesin 5 motor along with the astral microtubules and dynein motor

• A sliding force is generated between interpolar microtubules from opposite poles to push the poles apart;  a pulling force then acts directly on the poles to move them apart 

• Interpolar microtubules must keep growing on their plus ends to continue to interact with kinesin 5 and thus interpolar microtubules become longer during anaphase.

• Kinesin 5 is a double headed motor which attaches to interpolar microtubules from both centrosomes. Since kinesin 5 cannot grow but the interpolar microtubules can, as they grow in opposite directions kinesin push them allowing the centrosomes and thus sister chromatids (now separated) to get further apart.

Anaphase B (Astral microtubules)

• Anaphase B involves interpolar microtubules and the Kinesin 5 motor along with the astral microtubules and dynein motor

• Astral microtubule plus ends are connected to Dynein anchored to the cell cortex which move towards the minus end. Dynein pulls the astral microtubules and the attached centrosomes toward the opposite sides of the cell membrane.

• Astral microtubules can depolymerize at their plus ends giving more space for Dynein to move toward the minus end of the astral microtubule and pull the microtubule and centrosome closer to the cell membrane/cortex. However, dynein doesn’t require microtubule depolymerization.

Telophase

• During telophase the nuclear envelopes reassembles around the chromosomes, chromosomes decondense, and the contractile ring (in animal cells) begins to form, and cytokinesis begins.

• Nuclear envelopes form around each set of chromosomes to produce two nuclei.

• Cytokinesis begins near the end of telophase.

• Main distinction between telophase and cytokinesis is that telophase involves the reformation of 2 nuclei whereas cytokinesis involves the physical division of the cell into two daughter cells.

Cytokinesis

• Main distinction between telophase and cytokinesis is that telophase involves the reformation of 2 nuclei whereas cytokinesis involves the physical division of the cell into two daughter cells.

• During cytokinesis, the cell completes the division process by separating the cytoplasm.

  • In animal cells a completed nuclear envelope surrounds the chromosomes which begin to decondense, a contractile actin ring begins to create a cleavage furrow, and reformation of interphase array of microtubules nucleated by the centrosome also occurs.

  • Animal cell cytokinesis involves the contraction of an actin based contractile ring (ring is made of actin and myosin filaments) till two daughter cells are no longer conjoined.

  • Plant cell cytokinesis involves vesicle trafficking (Golgi derived vesicles) between the two nuclei which forms a plasma membrane between two cells and thus a cell wall.

  • Vesicle trafficking delivers membrane material to the center of the cell to form new plasma membranes that separate the two daughter cells. The cell plate eventually connects the existing plasma membrane.

Animal vs Plant Cell Cytokinesis

In animal cells, an actin-myosin contractile ring tightens to form a cleavage furrow, pinching the cell into two daughter cells.

In plant cells, Golgi-derived vesicles gather at the center to form a cell plate, which develops into new plasma membranes and a cell wall separating the daughter cells. Both processes occur after nuclear division during telophase and cytokinesis.