Molecular Bio Lectures 15-16

steps in mitosis

prophase, prometaphase, metaphase, anaphase, telophase

Prophase, -Red is DNA and green is spindles

-Prophase is before mitosis

The chromosomes

-condense and come detectable in nuclear envelope

-The mitotic spindle is starting to form

Prophase (AKA first stage of/just before mitosis)

- Chromosome condensation in the

nucleus

Prophase (AKA first stage of/just before mitosis), -Chromosome move to opposite poles in cytoplasm (centrosome migration)

Spindle starts to form (Microtubules begin to extend out from

-centromere)

-Some reach the microtubules of the opposite centrosome

Note: Spindles and microtube are outside the nucleus

Prometaphase

One of the big transitions in entry to mitosis is the transition from prophase to

prometaphase

Prometaphase- -Marked by nuclear envelope breakdown (ends prophase, begins prometaphase)

No turning back at this point - division will

-occur

-NEB allows cytoplasm contents to make contacts with nuclear contents

-Now the microtubules in the cytoplasm can access and make contacts the kinetochores of chromosomes that were in the nucleus before NEB

Nuclear envelope breaks down and microtubules can now interact with

Microtubules grab chromosomes at kinetochores

Metaphase

-Defined as moment in time where every chromosome kinetochore is attached to microtubules from each pole/centrosome (bipolar attachment)

When this occurs, it marks the end if pro-metaphase and the beginning of

-metaphase

-In middle because they are attached to microtubules from each centrosome

Transient - as soon as proper attachment occurs, anaphase begins

Anaphase

-Separation of sister chromatids

Sister chromatids are pulled to

-opposite spindle poles of the cell

-Microtubules pull sister chromatids apart towards respective poles

Entry into mitosis

Prophase, Prometaphase, Metaphase

•Prophase

-chromosome condensation

centrosomes move to

-opposite poles

-spindle starts to form

•Prometaphase

-nuclear envelope breakdown (NEB)

spindle microtubules start

-attaching to kinetochores

•Metaphase

chromosomes are attached to

kinetochore microtubules from each pole

•Anaphase

sister chromatid

-separation

-movement of sister chromatids towards spindle poles

•Telophase

contractile ring formation at

-spindle midzone

-nuclear envelope starting to reform

•Cytokinesis

cleavage furrow formation, separation of

-two daughter cells

-spindle disassembly

Exit from Mitosis

Anaphase, Telophase, Cytokinesis

Genetic and Biochemical Approaches to Study Cell Cycle

•Genetic screens in S. cerevisiae and S. pombe.

•In vitro assays in Xenopus and sea urchin egg extracts.

Yeast ts-mutants can be isolated that arrest cell division with a morphology corresponding to a

specific stage in the cell cycle

cdc16 mutant showed

metaphase arrest

-mutant for gene required for anaphase

cdc15 mutant showed

late anaphase arrest

-mutant for gene required for cytokinesis

S. pombe

cdc2 mutants - arrest in

G2, can rarely arrest in G1

S. cerevisiae

cdc28 mutants - arrest in

G1, can rarely arrest in G2

Cloning the S. pombe cdc2 gene by rescue of cdc2-t starts from the cDNA libary, then individual clones transformed into

cdc2-ts cells then they cells are plate out at restrictive temperature and isolate plasmid, only growth is the cell is the one with the gene from the cDNA library, rescues the mutant

S. pombe cdc2 =

•G2/M regulator

cdc2-ts mutants - colony fails to grow at

restrictive temperature à all cells appear to arrest in G2

transform individual mutant cells with individual plasmids from

S. pombe cDNA library

plate out at restrictive temperature and

•identify colonies

these cells carried a cDNA that could complement the

cdc2-ts mutation

isolate plasmid from these cells -> determine

•sequence of cDNA

-> cdc2 encodes a 34 kDa protein with similarity to known kinases

Genetic screens in S. cerevisiae identified the cdc28 gene, required for

G1/S

At restrictive temperature, cells arrest in

G1

Gene was cloned by complementation of cdc28-ts

•encodes a 34 kDa protein

•60% identical to S. pombe cdc2

•both have sequence similarity to known kinases

•

cDNA for cdc28 complements

•S. pombe cdc2 mutant

•

•later found that both genes are required for both G1/S and G2/M

Cdc28 is a kinase, •in vitro kinase assay

-IP using α-Cdc28 Antibody (or control Antibody)

add

-radioactive ATP (γ-P32-ATP)

after adding -radioactive ATP (γ-P32-ATP), then add

-radioactive ATP (γ-P32-ATP), a substrate to see if it phosphorylated

-after incubating à SDS-PAGE

radioactive band at

-~29kDa (Histone H1)

à Cdc28 has kinase activity in vitro, shown by the control gene not being phosphorylated

entry into meiosis - NEB (aka maturation) - is similar in

•many respects to entry into mitosis

1st meiotic division differs from a mitotic division in that

homologues segregate

•no S-phase between meiosis I and II

meiosis II is very similar to a

mitotic division (sister chromatids segregate)

•meiosis in many species is regulated

-prophase à NEB (progesterone)

-metaphase II à anaphase II (fertilization)

meiosis is followed by

zygote formation and specialized rapid mitotic cell divisions (early embryo)

Oocytes arrest in prophase of meiosis I until

Progesterone triggers maturation

Maturation =

Nuclear Envelope Breakdown (NEB)

second cleavage extract can be taken and injected into

meiosis 1 causing maturation

Following maturation, the egg proceeds to metaphase of meiosis II.

Arrest in metaphase of meiosis II until

fertilization

Fertilization triggers Ca2+ release leading to

egg activation

Activation results in meiosis continuing into

anaphase of meiosis II

Remove cytoplasm from a mitotically dividing egg.

Inject this into a

prophase arrested oocyte -> NEB

immature oocytes has a

nuclear envelope around

by inserting second cleavage into immature oocytes, they found that

something in it causes NE breakdown

•1971 - Assay for MPF - Masui

Remove cytoplasm from

-mitotically cycling embryos (eggs that have been released from meiosis II arrest by fertilization or in vitro activation)

Identification of MPF, then Inject this cytoplasm into

-prophase I arrested oocytes.

-These eggs now complete meiosis.

The extracts contain a factor(s) that is required for

oocyte maturation (Maturation Promoting Factor - MPF).

1977 - Cytoplasm taken from embryos at given times before or after activation - has different abilities to

induce maturation in arrested oocytes.

after activation - time 1

low MPF activity,

after activation - time 2

high MPF activity

time 1=

interphase,

time 2=

mitosis

Cycloheximide (blocks protein synthesis) added to dividing embryo leads to

arrest before nuclear envelope breakdown (prophase arrest). MPF activity requires cyclin B, requires more cyclin B to be made

Extracts from these embryos lack

MPF activity.

Therefore protein synthesis is required in each cell cycle for

MPF activity, knew they were looking for a protein that is made and destroyed

•So far, MPF activity - the activity required for entry into meiosis (maturation)

is present upon entry into

-mitosis and inactive at completion of mitosis

-requires new protein synthesis

during mitosis, Cyclin B is at its peak,

has a higher ability from MPF

Identification of cyclin B, 35S-Met added to eggs to label all proteins.

Identify a protein that cycles. Call it

cyclin. (later called it cyclin B), showed 1 protein that was present early but was gone later

Add cyclin B mRNA -> cycling continues -> cyclin B is

MPF (sort of!), needs to be bound to the kinase

Cdc2/Cdc28 (CDK 1) is a

Cyclin dependent kinase (Cdk)

1989 -Affinity purification of Cdk1 from starfish oocytes. Cyclin B

copurifies.

Cdk1 is only active when

•cyclin is bound to it. Cdk1-cyclin B = MPF

Higher eukaryotes have a mitotic Cdk - Cdk1 (=MPF) and a

S-phase Cdk - Cdk2. (They also have other Cdks). Yeast have only one Cdk that functions throughout the cell cycle.

Cyclin D goes with

Cdk4 and Cdk6

Cyclin E

Cdk 2

Cyclin A

Cdk2, Cdk1

Cyclin B

Cdk1

All Cdk's are Ser/Thr kinases that depend on a

cyclin partner for activity and specificity. G1-Cdk (Cdk4/cyclin D) not shown.

Cdk4 and G1 cyclin activated before the start, which is is signaled by the release of

cyclin E levels, the E2F promotes transcription

during G1, cyclin A is not yet stable, and the protein is degraded

and eventually reaches a level to stop degradation

MPF activates when cyclin B and geminin levels drop, which are degraded by the

APC as they have a destruction box, securin being the 3rd protein to be degraded.

Activation of Cdks

1.cyclin binding

2.phosphorylation of T-loop by a Cdk activating kinase (CAK) - Cdk7

Activation of Cdks

3. dephosphorylation of

two specific residues (only one shown) by Cdc25 phosphatase.

Activation of Cdks

4. release from

Cdk inhibitors - p27 or p21 or p16 in mammals, Sic1 in S. cerevisaie

NEB (nuclear envelope breakdown) depends on Cdk1-cyclin B activity

- phosphorylation of nuclear

lamins à disassembly of nuclear lamina à nuclear envelope breakdown

Assembly of mitotic spindle and capture of chromosomes by spindle microtubules depends on Cdk1-cyclin B activity

- phosphorylation of

microtubule associated proteins, kinetochore proteins and others

Spindle elongation and separation of sister chromatids in anaphase require

inactivation of Cdk1.

- Cdk1 phosphorylates proteins that maintain

the metaphase state

Prophase events

•chromosome condensation.

•centrosome migration and spindle formation.

•all events stimulated by Cdk1 (increasing levels)

Chromosome condensation, Interphase chromosomes are decondensed - allows for

•transcription and for DNA replication at S-phase

For proper chromosome segregation in mitosis, chromosomes must first be packaged into a

highly condensed format, needs to be to be separated

Condensin complex promotes

•chromosome condensation in prophase.

-may act as a ring connecting two parts of a chromosome

Cdk1 phosphorylation of

condensin subunits activates this complex

Prometaphase events

•nuclear envelope breakdown (NEB)

•spindle microtubules start attaching to kinetochores, only starts when cdk1 levels are at their max

•NEB is the key initiating event of mitosis.

NEB allows cytoplasmic proteins access to the

nucleus and nuclear proteins access to the mitotic spindle.

•allows microtubules from the mitotic spindle access to kinetochores.

NEB is triggered by Cdk phosphorylation of numerous targets including

•nuclear pores and nuclear lamins (intermediate filament proteins that support the nuclear envelope)

•occurs when Cdk1 activity reaches high level

Astral microtubules

•grow and shrink rapidly in prometaphase

capture

kinetochores on chromosomes to become kinetochore microtubules

Astral microtubules, all microtubules start off as

astral, if they connect then they become interpolar

interpolar microtubules

•microtubules from either pole that interact in the spindle midzone

•stabilize bipolar spindle

sliding of interpolar microtubules relative to each other allows

• pole separation in anaphase

•establish site of cytokinesis

kinetochore microtubules

•microtubule plus ends associate with kinetochores at centromeres of chromosomes

kinetochore microtubules can

•shrink and grow by polymerization/depolymerization at plus end while still holding on to kinetochores, are unstable

Replicated sister chromatids are held together by

Cohesins

The kinetochore is a multiprotein complex that assembles at the

centromere of both sister chromatids for each chromosome.

Separation of sister chromatids requires loss of Cohesins and

pulling of kinetochore microtubules

Kinetochore attachment:

Inner kinetochore binds to

centromeric region of chromosomes.