G2/M: Chromosome condensation

SMC family of proteins structural maintenance of chromosomes large coiled coil proteins with ATPase

domain interact in complexes

Cohesin - maintains sister-chromatid cohesion

Condensin - mitotic chromosome condensation

Cohesion and condensation

Cohesins and condensins

13S Condensin SMC2 and SMC4, with xCAP-D2, xCAP-G, xCAP-H

(Drosophila barren) 13S condensin induces positive writhe and supercoiling

in DNA Association of 13S with chromosomes occurs

exclusively in mitosis 13S in mitotic extracts: binds DNA, is phosphorylated, induces

supercoil in presence of ATP and TOP1 13S in interphase extracts: binds DNA, is not phosphorylated,

does not induce supercoil Immunodepletion of cdc2 decreases P of 13S and leads to loss

of condensin activity; adding cdc2 back increases P of 13S in condensin activity

Histone H3 phosphorylation An initiating event of chromosome condensation

is phosphorylation of histone H3 on ser-10 When site is mutated in Tetrahymena, there is

aberrant chromosome condensation and segregation defects (but not in budding yeast!)

H3 Kinase? Not MPF, may be NIMA, or aurora kinase RNAi depletion of aurora B decreases P of H3,

decreases chromosome condensation, and leads to failure to recruit barren

Condensin, histone H3 and the cell cycle

Mitosis: Overview

Stages Prometaphase, metaphase, anaphase,

telophase Purpose: equational and equal

segregation of chromatids to daughter cells

Checkpoints

Mitosis: Overview

Mitosis: Prometaphase

Spindle morphogenesis MTs, MT polarity and dynamic instability MT motors

• kinesin (+ end directed)• dynein (- end directed)

Centrosome and spindle assembly Centromeres and kinetochores “Dance” of the chromosomes

Bipolar orientation & importance

The mitotic spindle

The centromere and kinetochore

The centromere and kinetochore

MT dynamic instability

Mitosis: Metaphase

Stable bipolar orientation achieved Role of tension

• Cohesin Tension-sensitive checkpoint

Anaphase signal

How do cells get out of metaphase?

Via anaphase by action of the anaphase promoting complex (APC)

Injection into Xenopus oocytes of RNA encoding non-degradable cyclin B leads to sustained high MPF and metaphase arrest

Ubiquitination of chromosome cohesins and cyclin targets them to the proteasome

Synthesis - Destruction Cycle

Cohesion release at M/A

Control of anaphase entry

Mitosis: Anaphase

Anaphase A - separation of chromosomes Mechanisms Where is the motor?

• Class, 04.23.02

Anaphase B - elongation of spindle

Anaphase B