Mitosis

Chromosome Segregation

• Chromosomes must be bioriented to segregate properly
• Biorientation creates tension on the kinetochores, allowing the cell to monitor attachments
• Microtubules remain dynamic and chromosomes oscillate even after biorientation
• Outer kinetochore proteins bind to the sides of microtubules, allowing microtubules to dynamically grow and shrink without detaching from kinetochore

Cohesin Removal and Separase

• For sister chromatids to seperate, cohesin must be removed
• Separase is the protein that cleaves cohesin rings

Anaphase

• Two mechanisms drive chromosome segregation during anaphase: Anaphase A and Anaphase B
• Anaphase A: chromosomes move to spindle poles driven by microtubule depolymerization at kinetochore
• Anaphase B: spindle poles separate driven by kinesin-5 and dynein

Dynein Activity in Anaphase B

• Astral microtubules extend towards the cell cortex
• Dynein at the cell cortex walks towards the minus ends of astral microtubules
• This generates a pulling force that reels in the astral microtubule and pulls the centrosome closer to the plasma membrane

Interphase vs. Mitosis Chromosomes

• Individual chromosomes are visible in mitosis due to increased condensation.
• It is harder to distinguish individual chromosomes in interphase because they are less condensed.

Condensins in Chromosome Condensation

• Condensins are protein complexes that form rings that function to condense DNA in mitosis.
• They help organize the DNA into loops which results in full condensation of mitotic chromosomes.

Spindle Assembly

• Centrosomes nucleate microtubules
• Centrosomes duplicate during S phase
• Centrosomes separate along the outside of the nuclear envelope
• A bipolar spindle forms with a centrosome at each pole nucleating microtubules
• The nuclear envelope breaks down to allow microtubules to capture chromosomes

Microtubule Dynamic Instability

• Microtubules undergo phases of growth and shrinkage by addition/loss of tubulin subunits
• Dynamic instability increases in mitosis compared to interphase
• This increased dynamics facilitates breakdown and rearrangement of the interphase microtubule array and attachment of chromosomes to the spindle

Microtubule Dynamics in the Mitotic Spindle

• While microtubules are dynamic in mitosis, specific populations are stabilized.
• Microtubules growing out of the centrosome are highly dynamic and form asters; these are astral microtubules.
• Some astral microtubules become stabilized by cross-linking proteins and become interpolar microtubules.
• Interpolar microtubules help form the football shape of the spindle
• The overlap zone between interpolar microtubules is where kinesin-5 walks to generate outward force.

Kinesin-5 in Spindle Assembly

• Kinesin-5 is a tetrameric kinesin with two heads on each side.
• Both sets of heads can walk to MT plus ends.
• Each set of heads associates with a different microtubule.

Kinesin-5 Function in Spindle Assembly

• Kinesin-5 localizes to the overlap zone of interpolar microtubules.
• It walks towards the plus end of each antiparallel microtubule.
• This outward force pushes the poles apart, promoting centrosome separation during spindle assembly.

Chromosome Capture and Alignment

• After spindle formation, chromosomes begin attaching to microtubules.
• After attachment, captured chromosomes are moved to the middle of the cell, called chromosome congression

Kinetochore Organization

• Kinetochores are protein structures that mediate attachment of microtubules to the centromere.
• Multiple microtubules attach to each kinetochore.

Kinetochore Structure

• Core kinetochore proteins form a platform on the centromere region.
• Outer kinetochore proteins extend and bind to the microtubules.

Search and Capture Process

• Microtubules emanating from centrosomes grow and shrink.
• When a microtubule hits a kinetochore it forms an attachment, known as microtubule capture.
• Eventually, chromosomes become bioriented, with one sister attached to each spindle pole.

Biorientation of Sister Chromatids

• Both sister chromatids must be attached to opposite spindle poles.

M Phase Events

• Chromosome duplication and condensation
• Spindle assembly
• Chromosome capture and alignment
• Chromosome segregation
• Cytokinesis

Chromosome Duplication and Condensation

• DNA duplicates prior to mitosis, resulting in sister chromatids.
• Chromosomes condense to fit the long DNA molecules into the nucleus.

DNA Replication and Sister Chromatids

• Chromosomes duplicate during S phase.
• Sister chromatids are connected by the cohesin complex.

Cohesin Complex

• Cohesin forms a ring around duplicated strands during S phase.
• Responsible for keeping sister chromatids attached until anaphase.

Stages of Mitosis

• Prophase: chromosomes condense, centrosomes move apart.
• Prometaphase: nuclear envelope breaks down, spindle forms, chromosomes captured, chromosome congression.
• Metaphase: chromosomes line up at the metaphase plate.
• Anaphase: sister chromatids move to opposite spindle poles.
• Telophase: contractile ring forms, nuclear envelope reforms.
• Cytokinesis: contractile ring constricts, cell pinches in two, chromosomes decondense.