The principles of cell division differentiation Cell cycle

The principles of cell division, differentiation. Cell cycle, mitosis, meiosis by Krisztina H. -Minkó Department of Anatomy, Histology and Embryology Faculty of Medicine 16. 10. 2017.

Cell cycle = cell division

Main phases: Interphase Mitosis (Cytokinesis) Coo

The cell cycle Interphase. Between cell divisions (M-phase), the cell grows, performs its specific functions and prepares itself for the next division. In the second half of the interphase DNA is replicated (S-phase „synthesis”), before and after the S-phase there are intermediate periods: G 1 and G 2 phase („gap”). Mitotic division is the M-phase. The phases are repeated in a living cell in the sequence: G 1 – S – G 2 – M. G 1 S Interphase G 2 M M The linear form of periodically repeating sequences of the cycle. Instead of a linear form, the sequence of these phases can also be written in the form of a circle (cycle), see next slide. Duration of the cell cycle varies greatly in different cell types: in mammalian intestinal epithelial cells: about 12 hours, in fibroblasts in culture: around 20 hours, in liver cells: about a year.

Parts of the interphase G 1 phase: the cytoplasm increases in size, cell organelles are growing and/or multiplied, DNArepair (important prior to replication), specific functions of differentiated cells (e. g. liver cells). G 1 checkpoint. S-phase: DNA replication (the complete genom is duplicated). Replicated chromosomes (chromatids) are not separated from one another and are held together with proteins. The centrosome is also duplicated. Interphase: G 1 (12) S (8) G 2 (4) Mitosis and cytokinesis (1) Cycle (approx. 24 hours) G 2 -phase: Preparation for the mitosis (proteins of the mitotic apparatus are synthesized). G 2 checkpoint. G-phases: gap S- phase : DNA-synthesis

Cell cycle during development Divison of the frog embryo after fertilisation, quick cycles (0, 5 hour), almost only S and M phases (G 1 and G 2 phases are rapid) Remember: in mammalian intestinal epithelial cells: about 12 hours, in fibroblasts in culture: around 20 hours, in liver cells: about a year. Coo

Detection of the different phases in the microscope Mitosis/division : condensation of the chromatin into metaphase chromosomes Interphase: eu-heterochromatin in the cell nucleus „working cell” http: //micro. magnet. fsu. edu/cells/fluorescencemitosis/interphase 3 large. html

Regulation: molecular clock mechanism https: //www. mun. ca/biology/desmid/brian/BIOL 2060 -19/CB 19. html

Regulation of the cell cycle experimental data • Appropriate signals in a given cell cycle state lead to the synthesis of specified proteins. • These proteins can be isolated and if they are intoduced into other cells they are capable to influence the cell cycle (change the earlier phase to late stage). • These proteins are cyclically active, the cell-cycle control system depends on cyclical proteolysis.

During the maturation of the frogg oocyte the cell cycle is blocked in G 2. After progesteron treatment the egg continues the cycle and goes into M-phase. Isolating some cytoplasme from this cell by micropipette and introducing it into an other G 2 phase cell the second cell will enter also to M-phase immediately. What is the reason for this? The cytoplasme of the 1 st cell contains the so-called MPF (maturation promoting factor). Introducing MPF into somatic cells, mitosis can be induced as well. Coo

The cell-cyle control system depends on cyclicaly activated cyclin-dependent protein kinases (Cdks)

Coo Cyclin B expression in early embyogenesis

The kinases are continuously present and are cyclically activated by binding specific cyclins. https: //www. boundless. com/biology/textbooks/boundless-biology-textbook/cell-reproduction-10/control-of-the-cell-cycle 89/regulator-molecules-of-the-cell-cycle-399 -11626/

https: //www. boundless. com/biology/textbooks/boundless-biology-textbook/cell-reproduction-10/control-of-the-cell-cycle-89/regulator-molecules-of-the-cell-cycle-399 -11626/

Cell cycle with restriction points and the presence of cyclin/Cdk complexes

SUMMARY of the phases of the cell cycle (inner circle) G 1 phase: the cytoplasm increases in size, cell organelles are growing and/or multiplied, DNA-repair (important prior to replication), specific functions of differentiated cells (e. g. liver cells). G 1 checkpoint. G 0 phase: if the cell does not proceed to the S-phase, it remains in the G 1 for long time (G 0 phase) and is disconnected from the circling in the cycle („resting state”). Differentiated cells with specific functions. DNA-repair. Old and damaged cells are eliminated from the cell population (programmed cell death). Duration of the G 0 phase: G 0 can be the end station for certain cells, (no further cell divisions, e. g. in nerve and cardiac muscle cells), or it can last from months to years in other cell types. Many cells can be called back into the cycle by specific external signals (e. g. lymphocytes upon antigen stimuli, or various cell types upon growth factor stimulation). Metaphase checkpoint G 2 checkpoint G 1 checkpoint G 2 -Phase: Preparation for the mitosis (proteins of the mitotic apparatus are synthesized). G 2 checkpoint. Three main checkpoints: S-phase: DNA replication (the complete genom is duplicated). Replicated chromosomes (chromatids) are not separated from one another and are held together with proteins. The centrosome is also duplicated. programmed cell death 1. G 1 checkpoint (restriction point) („is the environment favorable, should the cell enter into the next cycle? ”) 2. G 2 checkpoint („is all DNA replicated and all DNA damage repaired? ”) 3. Metaphase-checkpoint („are all chromosomes connected with the cell poles”? )

Internal control of the cycle (outer circle) Phases of the cycle follow each other in a strongly defined sequence (comparison with a washing machine). A complex molecular control system (consisting of proteins). 1. Cyclin-dependent kinases (cdk). Proteinphosphorylating enzymes (kinases) which activate certain target proteins by coupling phosphate groups to them. The kinases are continuously present and are cyclically activated by binding specific cyclins. Example: M-cdk, a complex of cyclin B and cdk 1. 2. Cyclins. Regulatory proteins that activate cyclindependent kinases by binding to them. The cyclical appearance and disappearance of cyclins depends on their temporal synthesis and degradation (in proteasomes). Cyclins appear in a certain sequence (A, B 1, B 2, C, D 1 -3, E). 3. Cdk-inhibitor proteins block the assembly or activity of cyclin/cdk complexes and delay the progression of the cycle. M-cdk (cyclin B/cdk 1 MA G 2 checkpoint S-cdk (cyclin A/cdk 2 G 1 checkpoint 6, 4, k k cd ) cd / k 8 1 - lin. D /cd G yc C (c clin cy

Now, let’s see the M phase!

Mitosis The cell reproduces itself: From one diploid cell 2 genetically identical diploid cells are produced Meiosis Maturation division of sex cells (gametes): From one diploid cell 4 haploid cells are produced, Each resulting haploid cell differs genetically from the other ones

Mitosis Necessity of cell division: Cells have a limited life span. New cell can derive only from a previous cell („omnis cellula e cellula, Virchow XIX. century). In unicellular organisms: maintaining and expansion of a cell population In multicellular organisms: embryonic development (organism develops from one cell) cell homeostasis (maintaining the cell number in adult organism by replacing dead cells) regeneration (in case of massive cell deaths, e. g. in wounds) Two main phases: divison of the nucleus and cytoplasm 1. DNA replication (doubling of the genetic material) mitosis (precise segregation of the replicated DNA into 2 daughter cells) 2 Growth of the cytoplasm (growth of cell organelles in size and number) cytokinesis (division of the cytoplasm)

DNA condensation, chromatin The total DNA length of a human cell is about 2 m (!), while the average diameter of a nucleus is 5 -10 μm. To accomodate this length of DNA in a tiny volume of the nucleus is condensation (packing) of the DNA necessary. The packing is performed by specific proteins (positively charged small proteins: histones and non-histone proteins), the end product is called chromatin. In its most condensed form (metaphase chromosomes) the DNA is shortened by about 10. 000 x. The chromatin in an average cell nucleus shows different levels of DNA condensation. DNA EM „beads-on-astring” * nucleosome „beads-on-a-string” chromatin fiber EM loops of chromatin fiber metaphase chromosome chromatin fiber * Nucleosome: a flattened ellipsoid body composed of 8 histone proteins (core particle, 2 copies of histones H 2 A, H 2 B, H 3, H 4), onto which 2 DNA loops are bound. LM Heterochromatin Euchromatin highly condensed DNA, no transcription loose chromatin structure

The problem: 46 chromosomes in a human diploid cell, average length: 50 mm. 92 chromosomes (after DNA replication) have to be moved in a tiny space (15μm cell), so that one copy of each chromosome should be segregated into one cell, and the other copy into the other cell! Solving the problem: the two DNA molecules after replication remain attached to each other, DNA molecules are extremely condensed into 1 -2 μm rod-shaped structures, the chromatids, a complex segregation mechanism using the cytoskeleton (mitotic spindle)

MITOSIS video https: //www. youtube. com/watch? v=C 6 hn 3 s. A 0 ip 0 Video: Animal cell division MBOC

1. Prophase Phases of mitosis Centrosomes (previously redoubled during DNA replication) are moving to opposites of the cell: cell poles. Microtubules grow out from the MTOC. Chromosomes are being condensed, chromatid-pairs become visible in form of long filaments and later on of rod-like structures. Cytoskeleton is degraded. Large part of intermediate filaments is decomposed, the cell is rounded off. ER and Golgi-apparatus is desintegrated into vesicles.

2. Prometaphase Nuclear envelope breaks down into vesicles. Lamins of the nuclear lamina are phosphorylated and are separated from the perinuclear cistern, the latter falls apart into vesicles. Nuclear pore complexes desintagrate into individual molecules. Microtubules (growing out from the centrosomes) are bound to the chromosomes. The outgrowing microtubulus are bound to the kinetochore (a protein complex) on the centromer region of each chromatid. One chromatid of a chromosome is thereby connected with the microtubules with one pole (centrosome), while the other chromatid to the opposite pole of the cell. Mitotic spindle is formed, 3 types of microtubules: chromosomal, polar and astral MTs. Centrosomes at the poles. Astral MT 3. Metaphase Chromosomes are arranged in the equatorial plane („equatorial plate”). One chromatid of each chromosome is connected to one pole, and the other chromatid to the other pole. This arrangement makes accurate, symmetrical segregation of the chromatids possible. Chromosomal MT Polar MT chromosome

4. Anaphase If all chromosomes are connected with the poles, a start signal is generated to separate the chromatids (metaphase control point). Anaphase A: chromatids are separated and migrate towards the poles. Proteins connecting the two chromatids are degraded, chromatids (now called again chromosomes) are separated from each other. Chromosomal microtubules gradually shorten and pull the chromosomes towards the poles, where chromosomes are grouped together. Anaphase B: the mitotic spindle becomes elongated. Polar microtubules overlapping each other in the center of the cell are sliding along each other by the motor proteins kinesin and elongate the cell. Microtubule toxins inhibit mitosis by stopping mitosis at the end of metaphase (no anaphase!). Significance: mitotic chromosomes are available for cytogenetic diagnosis, synchronisation of the cell cycle, inhibition of cell division in malignant tumours. 5. Telophase Chromosomes are decondensed. Nuclear envelope, ER, Golgi are recomposed from vesicles. MTs are depolymerised.

Cytokinesis (division of the cytoplasm) Cytoplasm is divided in two, thereby cell organelles are distributed in approximately the same number in the two daughter cells. Cytokinesis overlaps in time with the telophase. Mechanism : Contraction ring. Along the equator an actin microfilament bundle is formed under and bound to the cell membrane. With the aid of motor proteins myosin II (sliding mechanism) the contraction ring becomes gradually shorter and pulls the membrane concentrically towards the cell interior. The cell divides in two, two daughter cells are formed. If there is no cytokinesis: a multinuclear giant cell (plasmodium) arises. In plant cells the mechanism is different: vesicles appear in the equatorial plane and fuse with each other (phragmoplast), dividing the cytoplasm. Actin immunocytochemistry Contraction ring Myosin immunocytochemistry

Metaphase chromosomes Morphology: A metaphase chromosome consists of two sister chromatids, these are connected with each other by cohesin and centromer proteins. Constriction at the level of the centromer. The centromer divides the chromosome into two arms: a shorter p, and a longer q arm. Numbering according to size (chromosome 1 is longest). Sex chromosomes: X (large) and Y (small). Chromosomal bands. When stained with different dyes, cross bands become visible. Their thickness and location is characteristic of the chromosome types (identification of chromosomes). Studying chromosomes: Lymphocyte cell culture, arresting cell division with colchicin, cells and chromosomes are pressed apart, staining, chromosomes are identified. Karyogramm (karyotype): all chromosomes arranged according to size. Diagnostic significance: chromosomal aberrations (in number and structure: deletion, insertion, transposition, inversion) can be detected in certain genetic diseases. Two chromatids P-arm Constriction (centromer) Q-arm Scanning electron micrograph of a metaphase chromosome

Karyogramm centromer position Human haploid chromosome set (Giemsa staining) Less condensed chromosomes to detect the banding pattern. centromer position Állati sejt osztódása in vitro

Meiosis: Maturation division of sex cells (gametes) In plants….

Genetic variability and sexual reproduction Significance of genetic diversity: members of a population differ from one another in many respects and abilities to adapt to the surrounding living conditions. In drastic changes of the environment the chance to survive is much higher when there is a genetic variability in a living population (survival of the species). Variabilities are due to gene variants (alleles). Although each of the many thousands genes is present in individuals of a species, the corresponding genes may differ in minor differences of the base sequences of DNA. These variants of a gene are called alleles. Consequently, individuals of a population differ in their alleles and their combinations. Spreading of successful alleles in a population (competitive advantage). If two parents produce many offspring with a wide variety of gene combinations, the chance that at least one of their progeny will have the combination of features necessary to survive is increased. In a population new mutations continuously occur and give rise to new alleles. Many of these alleles may be harmful and must be eliminated. Sexual reproduction prevents accumulation of deleterious alleles („genetic filtering”). In contrast, advantageous alleles are spread in the population. Maturation division of sex cells (gametes) largely contribute to genetic variability: maternal and paternal alleles are combined, all the resulting sex cells will have different allele combinations (genetically different). During fertlization the two sex cells that unite with each other carry different sets of alleles, which further increases genetic diversity.

The cell entering meiosis had previously replicated its DNA (chromosomes), number of its DNA molecules is 4 n! Meiosis I Prophase I (longest and most complex phase of meiosis) Homologous chromosomes (homologs): in a diploid chromosome set each chromosome type is present in two copies: one from the father and one from the mother. These are the homologous chromosomes. Sister chromatids of the paternal homologous chromosome Sister chromatids of the maternal homologous chromosome 1. Leptoten phase: homologous chromosomes are moving until they find each other and are attached parallelly in the same orientation. 2. Zygoten phase: The homologous chromosomes start to pair. They are connected by a ladder-like protein structure (synaptonemal complex), all genes are at the same level.

3. Pachyten phase: homologue pairings, 1 -3 recombination nodules appear on the pairing homologs randomly. At these sites chromatids are broken, their free ends are crossed over and joined with one another („crossing over”). As a result, chromatids become recombined having paternal and maternal DNA sequences (alternating paternal and maternal portions) Crossing over schematically Not only complete genes but portions of genes can also be recombined. X and Y chromosomes can also pair with appropriate portions. Bivalent (tetrad). Chromatids are crossed over. Recombined chromatids after crossing over Recombination nodules on pairing chromosomes (4 chromatids of these bivalents are seen as single structures). Red: bivalents of human egg cell, green: recombination nodules.

4. Diploten phase: synaptonemal complex disappears, chromosomes are pulled apart, crossing overs (chiasmata) become visible. Certain portions of the chromatids are temporarily decondensed (transcription of genes!) 5. Diakinesis: chromosomes are detached from the nuclear envelope, DNA loops are recondensed (gene transcription is terminated). Homologs are held together by crossing overs (blue arrow), sister chromatids by the centromer proteins (yellow arrow).

Metaphase, anaphase, telophase of the I. meiotic division are essentially similar to those in the mitotic division Differences: 1. In anaphase I the homologs (and not the chromatids) are segregated into the two cells. . Before separation, homologs are held together by the crossing overs. Each homolog has only one kinetochor! One homolog can move only towards one pole, the other homolog towards the other pole. 2. Random combination of chromosome orientation in metaphase. Originally paternal and maternal homologs are facing the cell poles in random combination. Combination chances in a human meiotic cell: 223 = > 8 million! Originally paternal Originally maternal

Meiosis II Pro-, meta-, ana-and telophase as in mitosis (but no prior DNA replication!). Difference: 23 chromosomes (half the number than in somatic cells) are separated into 2 chromatids each. Result: two haploid cells. One kinetochor on each chromatid. Two resulting haploid cells. Result of meiosis: 4 sex cells (gametes), each of which are different genetically from one another.

In meiosis I „cards are mixed” twice: 1. during prophase I chromatids are recombined with crossing over (as a result, DNA sequences of paternal and maternal origin are present mixed on the same chromatid), 2. in meta- and anaphase I random segregation of paternal and maternal homologs into the daughter cells.

Comparison of mitosis and meiosis DNA replication metaphase Segregation of chromatids DNA replication Pairing of homologs metafázis I Metaphase Segregation homologokof szétosztódnak homologs Metaphase II Segregation of chromatids

MEIOSIS https: //www. youtube. com/watch? v=-DLGfd-Wpr 4

Development of the egg cell: Oogenesis Urkeimzelle proliferation phase (mitotic divisions) Oogonia (2 n) Location: ovary Follicles: follicular epithelium surrounds the developing egg cell, initially as a single cell layer and later in several layers. Two stops in meiosis: 1. in prophase I (diplotene phase) for years or decades! At birth a girl’s egg cells in the ovaries are all in prophase I of meiosis. They remain in this stage until sexual maturation (puberty). 2. DNA synthesis (4 n), growth of the cell growth phase in metaphase II. Meiosis is terminated only if a sperm cell penetrates the egg. Without fertilization the egg cell deteriorates in metaphase II (only the sperm cell „saves” the egg from dying). 1. stop primary oocyte (4 n) Further growth, formation of zona pellucida and cortical granules Meiose I Meiosis 2 n 2 n polocyte I Meiose II Metaphase II Spermium (n) n polocyte II fertilization Zygote (2 n) secondary oocyte (2 n) 2. stop

Urkeimzellen cytoplasmic bridges spermatogonia A No cytokinesis, cytoplasmic bridges connect cells up to their final maturation. Spermatogonium B proliferation phase Development of sperm cells: spermatogenesis DNS replication DNS-szintézis secondary spermatocytes meiosis primary spermatocyte Sperm cells differentiation phase spermiohistogenesis spermatides

When homologs or chromatids are not segregated… (non-disjunction) If in mitosis or in meiois chromatids or chromosomes are not separated at the beginning of anaphase (non-disjunction), one resulting daughter cell will have one chromatid (or chromosome) more, and this will be missing in the other daughter cell (n+1 and n-1 in mitosis or meiosis II, or 2 n+1 and 2 n 1 in meiosis I). In both cases this will result in serious conseqences (usually in cell death). In adult organisms death of such cells does not cause great problems, because mitosis of neighboring cells can replace the lost cells. If however during meiosis a chromosome pair (in meiosis I) or chromosome (in meiosis II) is not separated into chromosomes or chromatids resp. , the resulting sex cells will contain one chromosome more or less, resp. Such aneuploid cell uniting with a normal sex cell of the opposite sex during fertilization will produce a zygote with a chromosome number of 2 n+1 or 2 n-1. In this case the cell will transfer this chromosomal failure with subsequent mitotic divisions onto further cell generations. Most aneuploid embryos or fetuses die still in the uterus, with the exception of a supernumerary chromosome 21 (Down-syndrome, mongolismus) with childs having serious developmental abnormalities or symptoms. Children with Down syndrome

Thank you for attention! Wikipedia

References: Lectures of Prof. Pál Röhlich and Dr. Attila Magyar Source of figures Röhlich: Szövettan, 3 rd ed. , Semmelweis, Budapest, 2006 Alberts – Johnson – Lewis – Raff – Roberts – Walter: Molecular biology of the cell. 5. ed. , Garland Science Own specimens, micrographs and drawings (P. Röhlich) Robbins: Basic Pathology, 7. edition, Saunders, 2003 Wikipedia Essential cell biology, 3. edition, Garland Science Useful videos: http: //www. bozemanscience. com/ cell division on contrast microscope https: //www. youtube. com/watch? v=DD 3 IQkn. CEdc Textbook: Essential cell biology, p. 522 -529, p. 510 -514, 497, 638 -646 chapter 18 (p. 609 -638)

Illustrations from: Röhlich: Szövettan, 4. kiadás, Semmelweis Kiadó, Budapest, 2014, in Hungarian Alberts – Johnson – Lewis – Raff – Roberts – Walter: Molecular biology of the cell. 5 th edition, Garland Science