D103 Discovery of cyclins using biochemistry (Video 35, ALS 22)

mitosis

portion of cell cycle when a fully grown cell segregates the duplicated chromosomes to opposite ends of a microtubule scaffold (spindle)

cytokinesis

process of cleavage between fully grown cell to produce 2 daughter cells

interphase

the portion of the cell cycle during which cells grow and replicate their dna

hypothesis for mitosis control

factor in a dividing cell that are responsible for cell division can promote division of a resting cell

• early exp evidence for the existence of an inducer of the G2 → M transition

exp method for mitosis control

1. fuse mitotic (M) cell with a cell that is in interphase (G1)

2. shortly afterwards, fix cells and stain DNA (chromosomes)

3. examine heterokaryons (fused cells) using microscope

results of mitotic control exp

• nuclear envelope of interphase G1 cell disassembles

• chromosomes of interphase G1 cell try to condense

interpretation of mito control exp

a factor in mitotic cell causes interphase cells to disassemble their nuclear envelope, condense their DNA, and enter mitosis prematurely, regardless of which stage the interphase cells are at in the cell cycle

• we now know the identity of this factor to be mitotic cyclin

biochem appraoch to study control of mitosis

requires a relatively large amount of synchronously dividing cells

what sort of systems can be used to provide material for control of mitosis

early embryos from frogs, sea urchins, and clams

external fertilization

using eggs and sperm; enables analysis fo the cell cycle during early stages of embryo development in thousands of oocytes fertilized at some time

• when cells are undergoing synchronous division

how are frog oocytes used

provide sufficient material for biochem analysis of meiosis and mitosis

• 1st division = 90 min post fertilization

• divisions 2-12 = 30 min each

• 2^12 cells after 700 hours

• only S and M (simplies analysis)

what happens when frog oocytes are treated with progesterone

undergo meiotic maturation (including a cell divsion) in vitro

• G2 arrested oocytes can be stimulated to undergo meiotic maturation (advance to arrest in metaphase II) in vitro by exposure to progesterone

• involves a cell division (formation of first polar body)

frog oocytes with microinj of cytoplasm from oocytes arrested in metaphase II

promote meiotic maturation (cell division)

• inj of cytoplasm from a metaphase II oocyte into G2 arrested oocyte causes non-progesterone treated G2 arrected to enter meiosis

• cytoplasm of progesterone treated contains an oocyte maturation-promoting factor

mpf activity

cyclical and relatively high as cells enter meiosis and mitosis

• correlates with entry of cells into meiosis or mitosis

experiment for mpf activity

take extract from oocytes and early embryos at different stages of development and use microinjection assay in preceding slide (inj into oocytes arrested in G2) to detect when oocytes and embryos have MPF activity

what does this exp system allow us to analyze

if MPF is protein, RNA, or both

• monitor decondensation of sperm pronucleus, rep of dna, and mitosis as evidence of mpf

is oscillation in MPF activity in early xenopus eggs independent or depend of oocyte nuc

independent

• cytoplasmic extract from Xenopus eggs contains all materials necessary for multiple cell cycles

what indicates new protein synthesis is required to generate MPF each cycle

MPF activity is blocked by addition of cycloheximide (protein syn inhibitor)

studying cyclins in clams

1. induce clams to release sperm and eggs

2. fertilize eggs to generate synchronous population of developing embryos in presence of S35 methionine

3. collect samples of embryos at different times following fertilization

4. analyze newly synthesized (radioactive) proteins by SDS-polyacrylamide gel electrophoresis with autoradiography against x-ray film

how was mitoic cyclin first identified in clam embryos

cyclin A (S cyclin) and B (M cyclin) accumulate during interphase, are greatly reduce at the end of mitosis, then reaccumulate during subsequent interphase

ribonucleotide reductase

a house-keeping protein which serves as an internal control for comparison of a newly synthesized protein that is not degraded with each round of mitosis

what is cycling of MPF activity and mitotic events dependent on

regulated production and destruction of M cyclin

positive MPF control

experimental system works

• increase in mitotic cdk and cyclin correlate

RNAse treated extract (negative control)

no increase or change = need RNA for mitosis

RNAse-treated extreact + WT mitotic cyclin mRNA

M-cyclin RNA is the key mRNA required to allow undergo mitosis in this assay

RNase treated extract + nondegradable mitotic cyclin mRNA

M cyclin must be destroyed to allow completion of mitosis in this assay

• both increase and plateau

• no late mitosis; only early

• no degradation = cannot complete mitosis

visualization of GFP-cyclin B in mammalian cells during mitosis

example of control of cyclin/CDK activity by subcellular localization and destruction of cyclin

• cyclin B moves to nuc during prophase, then is destroyed at anaphase

what controls irreversible destruction of M cyclin

APC/C + 2 related co-factors via polyubiquitinylation durign late mitosis and G1 of the next cell cycle

m cyclin and mitosis progression

M cyclin must be degraded to complete mitosis

APC/C + Cdc20 initates degradation of M-cyclins at anaphase

APC/C + Cdh1 completes degradation of M cyclin during late mitosis and early G1 of next cell cycle

What caused premature condensation of G1-phase chromosomes in a cell fused with a cell in mitosis ?

A. Something in the nucleus of the cell in mitosis.

B. Something in the cytoplasm of the cell in mitosis.

C. The condensed mitotic chromosomes signaled to the non-condensed chromosomes to undergo condensation.

D. Something attached to the plasma membrane of the mitotic cell.

E. Without additional experiments, it is impossible to tell what caused premature condensation of the G1 chromosomes and where the activity is localized in the cell.

E. Without additional experiments, it is impossible to tell what caused premature condensation of the G1 chromosomes and where the activity is localized in the cell.

Which stages of the cell cycle are absent from cells during early embryonic development of Xenopus frog embryos ?

A. G1 and S

B. G1 and M

C. G2 and S

D. S and M

E. G0 and G

E. G 0 and G2