MITOSIS AND CYTOKINESISMITOSIS AND CYTOKINESIS

Marie Kopecká 2006/2007mkopecka@med.muni.cz

In a typical eukaryotic somatic cellMITOSIS= NUCLEAR DIVISIONCYTOKINESIS = DIVISION OF THE CYTOPLASM.

IMPORTANCE OF MITOSIS:EXACT SEPARATION OF REPLICATEDGENOME into two originating daughter cells→ origin of genetically identical daughter cells.Inheritance of CENTROSOME.

IMPORTANCE OF CYTOKINESIS:Inheritance of corresponding part of the CYTOPLASMand CELL ORGANELLES.

Rudolf VIRCHOW (1858):„OMNIS CELLULA E CELLULA”

„ALL CELLS DERIVED FROM CELLS”

How ?

1590 Z. and J. Janssen: light microscope1665 R. Hook: first observation of cells1674 A. v. Leeuwenhoek: microorganisms1833 Brown: cell nucleus1838 M. J. Schleiden, T. Schwan: cell theory1839 J.E.Purkyně: cell theory1866 J. G. Mendel: element (gene)1869 F. Miescher: DNA1911 J. H.Morgan: genes in chromosomes1879 W. Flemming: chromosomes during mitosis1884 Strasburger: prophase, metaphase…1888 Waldeyer: chromosome1933 E. Ruska: first transmission electron microscope1953 J. Watson, F. Crick: structure of DNA1973 L. Hartwell: genes of the cell division cycle (cdc28)1989 P. Nurse: genes of mitotic pathway in yeast (cdc2)

Carl and Gerta CORI, the 1947 Nobel LaureatesThe origin of protein phosphorylation: PROTEINKINASES

From Cohen, Nature Cell Biol 2002

• Protein phosphorylationprotein kinases

• Protein defosforylationprotein phosphatases

• Proteolysis (protein degradation) ubiquitination→proteasomes

• Rho-GTPases

The mechanisms of M-phase progression:

EUKARYOTIC CELL DIVISION CYCLE G1 S G2 CELL DIVISION CYCLE G1 S G2 M Each stage triggered by Cyclin dependent kinase(s)+Cyclin(s),M-phase by Cdc2/CycB=Mitotic Kinase=CDK1

interphase

-phase

Nobel price for Physiology and Medicine (2001) for„Discovery of Key Regulators of Cell Division Cycle”

L. Hartwell 1960→budding yeast (cdc28 gene and others).P. Nurse 1970→fission yeast (cdc2 gene of mitotic kinase).T.Hunt - biochemist →discovered cyclins

CDC28mutantblocked atthe STARTcdc2

Cdc2 mutantblocked at mitosis

HartwellNurse

From Nurse, Nobel Lecture

Cdc2=catalytic subunit of mitotic kinase in humans

CDK1

Mitotic kinase (MPF= CDK1): Catalytic subunitjoins cyclin regulatorysubunit. It is phosporylatedat two sites. Activation of MPF requires removal of inhibitory phosphate by activationg phosphatase.

- Mitosis →→

Catalytic subunit Regulatory subunit Inhibitory kinase Activating phosphatase

Mitotic kinase=CDK1

Activatingkinase

CytokinesisInactive mitotic kinase CDK1→Activated CDK1

How the mitotic kinase Cdc2/CycB (=CDK1) is activated?

Mitotic kinases – regulators of M-phase and its checkpoints

Nigg

INTERPHASE – preparing for M-phaseG1: cell growth;

preparing for S phase

S: cell growth;synthesis of nuclearDNA;

synthesis of histones; sister chromatids

joined by cohesins;duplication of

centrosomes.

G2: cell growth;preparing for M-phase.

1914 Th. Boveri: the cell consists of the cytoplasm, nucleus and the centrosome

From Pederson, Nature Cell Biol2006

Cdk2/CyclinE,A

Cdk1,Polokinasa,Nek2…

Polokinasa,Aurora kinasa..

Meraldi P., Nigg E., FEBS Letters (2002)

Centrosome duplication, maturation and separation

POLOKINASE mutants have aberrant mitotic spindles (e.g. monopolar or multipolar).Some cancer cells have multipolar spindles and multiple centrosomes.

From Glover et al., J.Cell Biol. 1996

Zdroj: AF ÚG MZLUZdroj: AF ÚG MZLU

Interphase 1. Prophase 2. Prometaphase

3. Metaphase 4. Anaphase 5. Telophase

Interphase---→M-phase:Mitosis (5 stages) →Cytokinesis

Cytokinesis

G2 CHECKPOINT (G2/M) :DNA damaged? DNA replication completed?...

When DNA is dama-ged or incompletelyreplicated, Checkpoint kinasesCHK1 or CHK2 switchon inhibitory pathwayto block cell cycle by inhibiting mitotic kinase Cdk1/Cyclin B and phosphatasecdc25C. Whencompleted, Cdc25C is released to activatemitotic kinase Cdk1/Cyclin B.

Prophase: reorganisation of nucleus and cytoplasm during mitosis is induced by activated Cdk1-cyclin complexes and other kinases thatphosphorylate numerous substrates: nuclear lamins, condensins, histonesH1, H3, MAPs, motor proteins, centrosomes, Golgi apparatus…

Mitotic spindleoriginate betweenseparating centrosomes.

Chromosomes consist of two identical sisterchromatids joined by cohesins.Condensation of chromosomes – condensins.

Nucleolidisappear.

Disappearance of nuclear envelope

Prophase

PrometaphaseEach chromatid has its kinetochore in centromere region.

Microtubules joinkinetochores

Nuclearenvelopedisappears

Prometaphase

Metaphase

Mitotic apparatus consists of astral, polar and kinetochore microtubules.

Centrosomes are at opposite poles.

Chromosomes are at „metaphaseplate” in the equatorial plane.

Kinetochores of sisterchromatids of each chromosome joined to microtubules directedto opposite poles of the spindle.

1882 W. Flemming

TEM

DIC IF

From Nurse, Cell 100, 2000

From Clarke, Giménez-Abian, BioEssays 2000

NO → checkpointactivated →kinetochore signaling⇒⇒STOP ANAPHASE!

YES →checkpointsilenced →ANAPHASE

MetaphaseMetaphase checkpointcheckpoint („spindle assembly checkpoint”)

Are all chromosomes joined to mitotic spindle?

How Anaphase is started:1. Anaphase Promoting Complex (APC).2. APC ubiquitinates and induces disruption

of inhibitor „SECURIN” that inhibitedprotease „SEPARASE”.

3. SEPARASE digests protein “COHESIN”joining sister chromatids

4. Sister chromatids separate and are pulled to opposite poles of the spindle

5. APC induces disruption of M-phase cyclin B→ inactivation of M-Cdk→cytokinesis

AA BB

Telophase, Cytokinesis

Inactivation of mitoticCDK1 in anaphase leadsto telophase andcytokinesis:- Daughter nuclei at

opposite poles.- Nuclear envelopes

formed again.- Chromosomes

decondense.- Mitotic apparatus

disappears.- Cytokinesis starts.

CYTOKINESIS IN ANIMAL CELLCYTOKINESIS IN ANIMAL CELL

Cytokinesis in plant cell (ELM)

Phragmoplast

Cytokinesis in bacteria- FtsZ ring

Addinal and Holland,J Mol Biol 2002

CYTOKINESIS IN ANIMAL and FUNGAL CELL

CYTOKINESIS IN PLANT CELL

(Alberts et al 2004).

CYTOKINESIS IN PLANT CELL

CYTOKINESIS IN BACTERIA– FtsZ ring

Cytokinesis is triggered by Rho-GTPase

From Narumiya and Yasuda,

(2006)

Medical applications of the knowledge on the dynamics of microtubules in mitosis in therapy of cancers:

Microtubule inhibitors TAXOLE, VINCRISTINE, VINBLASTINE… inhibit dynamic instability ofmicrotubules → block mitosesand inhibited cells die….

Further strategy: Looking for CDK inhibitors

FromMeijerandRaymond,Acc.ChemRes.(2003)

olomoucine

Nigg

Metaphasecheckpoint

G2 checkpoint

Mitotic kinase Cdk1-cyclin in G2 checkpoint and Metaphase checkpoint

Specific termsMITOSIS The process of nuclear division.CYTOKINESIS The process of cytoplasmic division.CENTROSOME The main microtubule-organizing centre of animal cells.MITOTIC SPINDLE A highly dynamic bipolar array of microtubules that form during

mitosis or meiosis and serves to move duplicated chromosomes apart.SISTER CHROMATID Duplicated chromosomes.CHECKPOINT A point where the cell division cycle can be halted until conditions are

suitable for the cell to proceed to the next stage.NUCLEAR LAMINA A nuclear membrane-associated protein structure made up of

lamin intermediate filament proteins.KINESIN Microtubule-based molecular motor, most often directed towards the plus

end of microtubules.DYNEIN Microtubule-based molecular motor that moves towards the minus end of

microtubules.GAMA-TUBULIN RING COMPLEXES Ring-like multiprotein structures implicated

in microtubule nucleation.KINETOCHORE Specialized assembly of proteins that binds to a region of the

chromosomes called centromere.CENTROMERE A region of eukaryotic chromosome that is attached to the mitotic

spindle.