1. Mechanisms of Anaphase and Telophase Lecture 41BSCI 420,421,620Dec 9, 2002 “They paved paradise and put up a parking lot” - Joni Mitchell 1.Metaphase 2.Metaphase-to-Anaphase Transition 3.Anaphase Mechanisms 4.Telophase

2. 1.Metaphase Metaphase may look static, but chromosomes are held at the metaphase plate by a dynamic balance of forces on kinetochores. Also, MTs are treadmilling, not tied up at ends, but held by motor proteins. Ev. for treadmilling: 1. Movement of tubulin speckles

3. 2 nd evidence for treadmilling in metaphase MTs: Photoactivation of “caged” fluorescence on tubulin moves toward poles.

4. 2. Metaphase-to-Anaphase Transition Mitotic cells wait in metaphase until they pass the spindle-attachment checkpoint, which asks: Are all kinetochores attached to the spindle and under tension? Evidence: a. Colchicine-treated mitotic cells – Chromosomes condense but no spindle forms, so sister chromatids never separate. b. When one chromosome is detached from the mitotic spindle, all the other chromosomes wait until it re-attaches & moves to the midpoint of the spindle. How is M -> A controlled? Central is the Anaphase-Promoting Complex (APC), a ubiquitin ligase that targets certain metaphase proteins for destruction, Especially securin and cyclin B.

5. Anaphase trigger mechanism Chromosomes wo (the anaphase inhibitor) tension ?

6. How do cells detect that all chromosomes are under equal tension? Nicklas et al at Duke discovered that only unattached kinetochores bind certain proteins. Eg Mad2 (Mitotic arrest defective) shown by red fluorescent anti-Mad2. (below) Mad2 inhibits activation of APC only when attached to k-cores. Hypothesis: Changes in kinetochore proteins under tension activate a protein kinase that phosphorylates APC so Cdc20 binds and activates the anaphase promoting complex.

7. 3. Anaphase Mechanisms. Two components of Anaphase: Movement of chroms to polesMovement of poles apart

8. Kinetochores move to poles by following depolymerizing (+) Ends of kinetochore MTs. Evidence: Photobleached (stays the same)(shortens)

10. Anaphase B – separation of the poles by MT sliding, Powered by kinesin like motors (+) end directed.

11. Degradation of M-cyclin and removal of phosphates by phosphatases allows TELOPHASE (reversal of the events of prophase) and CYTOKINESIS.

12. Destruction of M-cyclin is not the anaphase trigger, and is not required for anaphase. But inactivation of M-Cdk is required for the events of telophase : 4. Telophase events a.Decondensation of chromatin b.Reformation of nuclear envelope c.Breakdown of MA & formation of interphase MT array d.Formation of the contractile ring for cytokinesis