MCB 2210 L26: Mitosis Part I - Prophase, Prometaphase, Metaphase

What kind of embryos exhibit coordinated mitosis?

Fly embryos

Describe the cell cycle

Interphase = G1 → S → G2

• G1 = initiation of DNA synthesis

• S = DNA synthesis

• G2 = period b/w DNA synthesis

M phase = Mitosis + Cytokinesis

• Mitosis = process of chromosome segregation

Name the 3 major subcellular changes of M phase

1. Chromosome condensation = organized into nucleosomes by histones

   1. Condensin = protein that compact in M phase

2. Mitotic spindle assembly

3. Formation of the contractile ring = actin + myosin II

   1. Ring contraction

Overview of stages of mitosis

• Interphase = chromosome duplication + cohesin + centrosome duplication

• Prophase = Breakdown of interphase MT display + Mitotic aster separation + Chromosome condensation = visible + Kinetochores begin to assembly

• Prometaphase = nuclear envelop breakdown, chromosomes

• Metaphase = chromosome aligned @ metaphase plate

• Anaphase = ACP/C activated + cohesin degraded

  • Anaphase A = chromosome movement to poles

  • Anaphase B = spindle pole separation

• Telophase = Nuclear envelope reassembly + assembly contractile ring

• Cytokinesis = interphase MT array reforms + contractile ring = cleavage furrow

Describe the role of S-phase and G2 on mitosis

S phase = completed

• 1 duplicated chromosome = 2 sister chromatids

G2 phase

• Chromosome condensation

• Condensin = compact chromosomes

Describe how sister chromatids are attached

• Centromere = region of chromosome where chromatids = constricted + attached

• Cohesins = proteins that glue sister chromatids together

• Kinetochores = specialized protein complexes that assemble on centromere of each sister chromatids

Describe transition through interphase

G1 = 1 centrosome

S + G2 → centrosome = duplicated but @ 1 side of nucleus

Describe what happens during prophase

• Prophase = condensed chromosomes = seen by light microscopy

• Interphase MTs = disassemble

• 2 centrosomes separate → each centrosome nucleates aster

• Asters move apart to opposite sides of nucleus → begin forming mitotic spindle

What does mitotic spindle formation organizational activity involve?

• Centrosomes

• ChromosomesZ

What happens to MTs during prophase of mitosis?

Increase in # of MTs nucleated from centrosome

Interphase array of long stable MTs → prophase array of short, more dynamic MTs

Prometaphase

• Nuclear envelope breakdown

• Kinetochore MTs captured by kinetochores

• Chromosomes move back + forth b/w poles

Purpose of nuclear envelope breakdown

2 mechanisms of nuclear envelope breakdown

What happens to cells with mutant human lamin A?

• Gives spindle MTs access to chromosome kinetochores

1. Cytoplasmic dynein = binds to proteins outside of nuclear envelope

   1. Walk to (-) ends → rip hole in nucleus

2. Lamins of nuclear lamin cytoskeleton → nuclear envelope breakdown

Mutant lamin A → MTs cannot bind properly to chromosomes

What are the 3 types of motor proteins involved in spindle assembly?

Kin-N Kinesin = Kinesin 5 → (+) end directed

• Push 2 poles apart

Kin-C Kinesin = Kinesin-14 → (-) end directed

• Pull 2 poles together

Dynein = anchored to cell cortex → (-) end directed

• Walk toward astral MT (-) end

• Pull poles apart

Explain how spindle organization can happen by chromosomes + the experiment that was conducted to prove this

• Mitotic spindle formation → absence of centrosomes

• No centrosomes → spindle organized by chromatin

• Cytoplasmic extracts w/o centrosomes → Xenopus frog egg extract w/o centrosomes

  • Beads coated w/ chromatin

  • Beads added to frog egg extract

  • Mitotic spindle formation around beads

  • Results = chromatin = sufficient to direct spindle assembly

Describe the mechanism behind chromatin-based spindle self-organization

• Ran-GEF binds to chromatin (in nucleus)

  • Ran-GDP → Ran-GTP

• Ran-GAP binds to cytoplasm

  • Ran-GTP → Ran-GDP

• Importin cargo = MT nucleating proteins + MT stabilizing proteins

• Importin binds to Ran-GTP → MT nucleating proteins = released → allow MT nucleation + polymerization near bead

  • Beat coated w/ Ran-GEF or Ran-GTP works as well

Identify the three motor proteins associated with kinetochores

Explain the name of the model and the mechanism of how MTs are bound to kinetochores

Kinetochores = Kin-N Kinesin (CENP-E) + Dynein + Kin-I Kinesin (Kin-13) + Ndc80 complex = mediate binding to MTs in spindle

• Chromosomes = NO centrosomes → CANNOT interact w/ spindle MTs = CANNOT correctly separate during mitosis

Search-and-Capture Model

• Search = prometaphase MTs = dynamic instability

• Capture = some MTs encounter kinetochore + bind through motor proteins

• Side of MT binds to kinetochore → Kin-N Kinesin motor move chromosome to (+) MT end

  • Sister chromatids = bi-oriented when both kinetochores are captured by MTs from opposite poles

• Ndc80 complex = stabilizes attachment of kinetochore to MT (+) end

What phase does chromosome congression occur in?

Chromosome congression = captured chromosomes → equator of spindle + back-and-forth movement of bi-oriented chromosomes near equator

• Polymerization + depolymerization @ (+) ends of kinetochore MTs of bioriented chromosomes

• Kin-13 = depolymerizing

• Kin-7 = CENP-E = polymerizing

What are the 2 parts of chromosome congression during prometaphase?

• Poleward MT flux = treadmilling = addition to (+) ends of spindle MTs by Kin-7 + (-) ends depolymerized by Kin-13

• Polar Ejection Force = move chromosomes away from spindle poles = Chromokinesins (Kin-N kinesins on chromosome arms)

  • Move to (+) ends of polar MTs

  • AWAY from spindle poles

Explain what happens dynamically during metaphase

Forces = balanced @ metaphase

• (+) end polymerization = (-) end depolymerization

• Constant spindle length

• TREADMILLING

What happens during metaphase?

Metaphase = Point when chromosomes aligned in one plane halfway b/w 2 spindle poles

• Chromosome held under tension @ metaphase plate

  • Metaphase plate = region where chromosomes align

• Paired kinetochores + kinetochore MTs