Dae-Sik Lim Ph.D. 한국과학기술원 Cell Cycle Regulation; Overview

* an ordered serious biochemical switches; replication & mitosis * the fidelity of cell division *response to various signal; Checkpoint control; inside (stepwise) & outside (growth factor or death, stress) How the various events of the cell cycle are controlled and coordinated ? Cell cycle control system; a complex network of regulatory proteins

An overview of cell cycle; The cell cycle is an ordered series of events leading to the replication of cells Mitosis; -Chromosome condensation, nuclear envelop breakage, mitotic spindle formation -chromosome alignment at equator -segregation -nuclear envelop formation -cytokinesis G1/G2- provide time to monitor the signals, prepare the next phase

The Cell cycle system, three major euk systems; in yeast, frog, mammalian cells 1.Yeast; Fission ( S. pombe) and budding yeast ( S. cerevisiae) 1.- genetically useful; genetic manipulation 2.Haploid state 3.Cdc genes; cell-division cycle genes; ts mutant (permissive or restrictive condition *No nuclear envelop breakage during mitosis * budding yeast; no clear G2 phase

Isolation of wild-type cell-division cycle (CDC) genes from S. cerevisiae carrying temperature-sensitive mutations in these genes Figure 13-4

Two classes of mutations in S. pombe produce either elongated or very small cells Figure S. pombe Cdc2-Cdc13 heterodimer is equivalent to Xenopus MPF (Cdc2 is cdk, Cdc13 is cyclin B)

1.-large size; > cytoplasm 2.-easy to inject substances 3.Rapid division; 4096 cells with 7 hours 4.Pure cytoplamic elements 5.Isolated maturation promoting factor (MPF) Frog eggs; Biochemical system

MPF promotes maturation of Xenopus laevis oocytes Figure 13-5

MPF promotes mitosis in somatic cells Figure 13-6

MPF is a dimer of a mitotic cyclin and cyclin-dependent kinase (cdk) Cyclin B levels and MPF activity change together in cycling Xenopus egg extracts Figure 13-7

Ubiquitin-mediated degradation of mitotic cyclins promotes exit from mitosis Figure 13-8

; biochemical & Genetic studies – not easy - Mutant cell lines ( various cancer cells) -more complex system -Cell cycle analysis; G1/S, S. G2/M ; H3- thymidind or Brdu-labeled, mitotic index Mammalian Cells

Components of the cell cycle control system *The control of cell cycle regulation; a clock or timer; specific time -correct order-once per cycle-binary switch on/off system -adaptability; specific cell types or environmental Checkpoint control; delay the cell cycle progression or arrest the cell cycle in response to signals -send a negative signal rather than removal of positive signal -provide time for DNA repair or prevent the disaster

The cell cycle regulation are based on cyclically activated protein kinase; Multiple cdks and cyclins regulate passage of mammalian cells through the cell cycle -Cyclin-dependent kinase (Cdks) -cdk regulators; *different cyclins, *level of cyclins (transcriptionally or proteolysis), * inhibitors (p21, 27, p16, p57 etc) * phosphorylation or dephosphorylation (Cdc25, kinase )

Table 17-1 Molecular Biology of the Cell (© Garland Science 2008)

Cdk activation of by CAK (cdk activating kinase)

Cdk activity are regulated by inhibitory phosphorylation and by CIPs

Other genes influence the protein kinase activity of S. pombe MPF Figure 13-12

The activation of M-Cdk

The cell cycle control system depends cyclical proteolysis; SCF, APC

ExitAna Me ta Prom eta ProG2SG1 Cdk4/6 Cyclin D Cdk2 Cyclin E p16 p27 p21 SCF Fbw APC Cyclin A Cyclin B Cdk1 securin SCF SKP2 APC C Ub

APC controls entry into anaphase and exit from mitosis

Passage through the restriction point depends on activation of E2F transcription factors Figure Mammalian cyclin-kinase inhibitors also contribute to cell cycle control

Replication once per cycle The S phase cytoplasm contains factors that derive into G1 cells into DNA replication, but no G2

Checkpoints in cell cycle regulation Figure 13-34

The mechanism of p53-induced cell-cycle arrest in response to DNA damage

p53 :multiple player

Cell growth;  Mitogens; stimulate cell division by relieving intracellular negative control  Growth factors; stimulate cell growth by promoting the synthesis of proteins and molecules  Survival factors; promote the cell survival by suppressing apoptosis  Mitogens stimulate the cell division by releasing the brakes on cdk activity.

Abnormal proliferation signal resulted from oncogene activation cause the cell cycle arrest or cell death RAS-GTP

Extra cellular growth factors stimulate cell growth by activating signaling pathways

Dae-Sik Lim Ph.D. 한국과학기술원 Mechanism of Mitosis

An overview of M phase  How to accurately separate and distribute its chromosomes  Cohesins and condensins help configure replicated chromosomes for segregation. The sister chromatids are glued together by cohesins—broken to allow the sisters to be pulled apart. Condesins do the works of chromosome condensation. M-Cdkphoshprylate the condensin subunit-assembly of condensin complex on DNA. two identical ATP & DNA binding motif

Centrosomes  Cytoskeletal machines perform both mitosis and cytokenesis—biopolar mitotic spindle and contractile ring  Two mechanisms help ensures that mitosis always precedes cytokenesis -point of no return; M-CDK activation and proteolytic process - the cell cycle systems that activate proteins required for mitosis, inactivate the proteins required for cytokinesis.  A centrosomal duplication are essential for proper mitosis.  Centriole; microtubule organization center Pericentral material and centriole + end to cell perimeter, - end to centrsome The matrix contains many proteins containing r-tubuling ring and motors etc  Centosomal cycle; the centrioles and other components are dupplicated during G/S-S and separated into two during mitosis, becomes MTOC(microtubule organization center)  Each Centrosome nucleates its own radial array of microtubules (aster).

Six stage of mitosis  Six stage; prophase, prometaphase, metaphase, anaphase, telophase, cytokensis  Have to be coordinated.  Prophase; sister chromatids & chrom condensetion, two spindle separation, forming mitotic spindles  Prometaphase; ne breakage, chromosomes attach the MT via kenotochore and undergo active movement  Metaphase; chromsomal alignment at equator, the kenetochore MT attach sister chromatid to opposite poles of the spindle  Anaphase; K MT get shorter, and the spindle poles also move apart--- chromosome separation  Telophase; Ch arrives the poles and a ne reassembles, begins with the contractile ring formation  Cytokinesis; contactile ring cleavage, ch decondensation, complete division

Mitotic spindles  Mitotic spindle; microtubules and associated factors; pull chromosomes toward the poles & move the poles apart.  The assembly and the functions of the mitotic spindle depend on microtubule-dependent motor proteins kinesin-related proteins; move toward the plus end of MT dyneins; move toward the – end.  The assembly and dynamics of the MT relay on the shifting balance bwtn +end directed and –end-directed motor proteins at the ends  Three class of MT; astral MT; radiated all direction Kinetochore MT; attach end-on to the kinetochore Overlap MT; interdigitate at the equator of the spindle; symmetrical, bipolar shape of the spindle

Microtubule instability;  MTs are either growing or shrinking; catastrophe & rescue MTs in mitosis are more dynamic than in interphase Prophase; long MTS convert to a large number of shorter, dynamic MTS at centrosome mitotic spindle formation  Regulation of MTs dynamics M-CDK regulate the proteins that control the MT dynamics; motors & MAPs (microtubule-associated proteins) MAPs; stabilize the MT array ; increase growth rate & block the shrinkage Catstrophin (kinesin related protein); destabilize the MT array  Experiment with frog Egg M extract; figure  Mitotic extract; catastrophe rate +++  Mitotic extract- MAP;  Mitotic extract- KCM1-MAP; ++  Interphase; +

mitotic spindle formation

Two functions of the motor proteins are important for mitotic spindle assembly and function  *-end-directed multimeric motor; rearrange MT to form a focus of – ends  *multimeric +end-directed proteins; slide the MTs past each others—elongate the spindle

Figure Molecular Biology of the Cell (© Garland Science 2008)

Separation of the two spindle poles;  *+end-directed motor (kinesin) cross-link the MTs together-push the  Centrsome apart  *-end-ditrected dynenin motor; separate the Chromosome by pulling on the  astral MTs  + motor overexpression; induce long spidle  -motor overexpression; induce short spinde  The balance +/- motor regulate the spindle length

Kinetochore attach chromosome to the Mitotic spindle  Search and capture mechanism.  MTs are highly dynamic in the metaphase spindle—poleward flux

Search and capture mechanism. tension

Figure Molecular Biology of the Cell (© Garland Science 2008)

Fluorescenec speckle microscopy

two opposing forces cooperate to move chromosomes to the equator  +end-directed motor interact with asteral MTs; push the chromosome toward equator  -end-directed motor at kenotochore; pull the chromosomes toward the pole.

Functional Bipolar spindles can assemble around chromosomes in cells without centrosome  Chrosomes are not just simple passive passengers  By creating local environment (high conc of RAN-GTP by RCC1(RAN GEF) at chromosome); microtubules nucleation and stabilization  Biopolar spindle assembly without centrosome in cells and in vitro  Centrosomal independent spindle formation; segregate the chromosomes without asteral MTs, sometimes the spindle is mispositioned, abnormal cytokinesis

A spindle checkpoint control; anaphase is delayed until all chromosomes are positioned at the metaphase plate;  * monitor the attachment of the chromosomes to the mitotic spindle.  * a single unattached Kenotochore is enough to block the M to Anaphase

Anaphase; separation of sister chromatid;  APC activation; Cylin B degradation—M-CDK inactivation,  secruin degradation—separase activation- cleave the cohesion complex  Two independent and overlapping processes  Anaphase A;, the initial poleward movement of the Ch; shortening of the K MTs at the, depolymerization of S MTs

Anaphase B  Anaphase B; the separation of the poles;  The elongation and sliding of the overlap MT past –push the two poles apart,  Outward force exerted by the astral MTs at poles pull the poles away

Both pushing and pulling force contribute to Anaphase B  +end directed motor proteins cross-link the overlapping, antiparallel, slide MTs past each other –push the spindle poles apart  -end-directed motors bind to cell cortex and astral MTs pull the pole apart.

Cytokenesis;  The MT of the mitotic spindle determine the plan of animal cell division  Reposition their spindle to divide asymmetrically

The molecular mechanisms of mitotic checkpoint control

The spindle abnormality and spindle checkpoint defects can induce a Chromosomal Instability