The Cell Cycle 2024-25 PDF

Prof Wanda Lattanzi Dept Life Science and Public Health Section of Biology Room 352bis 1st Floor Istituti Biologici [email protected]  Warning The contents of these slides are the exclusive property of the Instructor and/or granted by third parties (textbook...

Prof Wanda Lattanzi Dept Life Science and Public Health Section of Biology Room 352bis 1st Floor Istituti Biologici [email protected]  Warning The contents of these slides are the exclusive property of the Instructor and/or granted by third parties (textbooks’ reference for pictures) and are therefore protected by the current regulations governing the Protection of Copyright. All rights are reserved. The reproduction and/or diffusion, even partial, by any analogical and/or digital means, without the consent of the rights holder is FORBIDDEN. Any unauthorized use of the above mentioned "Contents" is under the full and exclusive responsibility of the users who will be responsible for it, according to the laws and regulations in force. It is allowed the use of the material for private and study use, however not for profit and without commercial purposes. Cycling cells…  The cell cycle Two major phases based on cellular activities readily visible with a light microscope M phase: the cell undergoes division (around 1 hour) Mitosis Cytokinesis (cytodieresis) Interphase: the cell grows and carries out metabolic activities (duration depends on cell type) G1: most protein synthesis, basal metabolic activities S: DNA replication, histone synthesis and chromosome duplication G2: protein synthesis and pre-mitotic activities The cell cycle Matthews et al. Nat Rev Mol Cell Biol. 2021 M phase – cell division Mitosis: process of nuclear division in which the replicated DNA molecules of each chromosome are faithfully segregated into two nuclei. Cytokinesis: process through which dividing cell splits in two, with a precise partition of the cytoplasm, after the nuclear division occurring in mitosis. M phase – cell division The process by which new cells are generated from other living cells Billion divisions occur to generate a complex multicellular organism from a starting single cell (zygote) Cell divisions continue to occur throughout the life of a developed organism in most tissues, to renew tissues, regenerate damaged ones, and maintain structural and functional homeostasis M phase – cell division Cell cycle can significantly vary in length: 30 minutes in a cleaving frog embryo (lacking G1 and G2 phases) days-weeks in most cells several months in slowly growing tissues, such as the mammalian liver www.toppr.com M phase – cell division Cells can be categorized based on their capacity/frequency to grow and divide: 1.Higly specialized cells: do not undergo cell division (neurons, muscle cells, red blood cells, osteoclasts, etc) 2.Differentiated cells: divide only upon certain induction (liver cells, lymphocytes) 3.Unspecialized cells: high mitotic activity: stem cells of various adult tissues (uni/olipotent stem cells in the basal layer of epithelia) M phase – cell division STEM CELLS may undergo asymmetric cell division One daughter cell remains uncommitted and more similar to the parental one The other daughter cell start its path towards differentiation M phase – cell division MITOSIS Produces two daughter cells identical to the parental one Serves to produce new cells throughout the body at any time MEIOSIS Produces two daughter cells with half the genetic content (haploid) of the parental one (dyploid) Serves to produce gametes hence new sexually reproducing organisms  Mitosis Divided into five stages whithin a continuous process: Prophase Prometaphase Metaphase Anaphase Telophase  Mitosis Divided into five stages whithin a continuous process: Prophase Prometaphase Metaphase Anaphase Telophase Chromosome condensation Condensin: multiprotein complex enabling chromosome condensation Cohesin: multiprotein complex holding sister chromatides together  Mitosis Divided into five stages whithin a continuous process: Prophase Prometaphase Metaphase Anaphase Telophase Chromosome condensation Kinetochore: protein aggregate at the outer surface of the centromere of each chromatid  Mitosis Divided into five stages whithin a continuous process: Prophase Prometaphase Metaphase Anaphase Telophase Formation of the mitotic spindle from centrosomes Dissolution of the Nuclear Envelope and Partitioning of Cytoplasmic Organelles  Mitosis Divided into five stages whithin a continuous process: Prophase Prometaphase Metaphase Anaphase Telophase Microtubules of the spindle attach on the kinetochores of chromosomes Chromosomes migrate towards the center of the spindle (congression) Motor proteins associated with kinetochores chromosomes allow the movements Microtubules shortens progressively Auckland and McAinsh. J Cell Sci. 2015 Sep 15;128(18):3363-74  Mitosis Divided into five stages whithin a continuous process: Prophase Prometaphase Metaphase Anaphase Telophase All chromosomes are aligned along the equator of the spindle: metaphase plate The cell karyotype is analyzed at this stage The mitotic spindle Astral microtubules: radiate outward from the centrosome into the region outside the body of the spindle. They help position the spindle apparatus in the cell and may help determine the plane of cytokinesis. Chromosomal (or kinetochore) microtubules: extend between the centrosome and the kinetochores of the chromosomes. In mammalian cells, each kinetochore is attached to a bundle of 20–30 microtubules, which forms a spindle fiber. During metaphase, the chromosomal microtubules pull chromosomes to keep them on the plate Polar (or interpolar) microtubules: extend from the centro some past the chromosomes, overlapping with their counterparts from the opposite centrosome. They form a structural basket that maintains the mechanical integrity of the spindle.  The mitotic spindle The centrosome cycle of an animal cell  Mitosis Divided into 5 stages whithin a continuous process: Prophase Prometaphase Metaphase Anaphase Telophase Ealy stage: the sister chromatids of each chromosome symultaneously split apart and start their movement toward opposite poles, due to progressive shortening of kinetochore microtubules (ANAPHASE A)  Mitosis Divided into five stages whithin a continuous process: Prophase Prometaphase Metaphase Anaphase Telophase Late stage: the two spindle poles move farther apart, due to the polimerization of interpolar microtubules (ANAPHASE B)  Mitosis Divided into five stages whithin a continuous process: Prophase Prometaphase Metaphase Anaphase Telophase Chromosomes aggregate near each pole of the spindle Nuclear envelopes assemble Cytokinesis concurrently initiates Daughter nuclei progressively turn to the interphase condition Cytokinesis (cytodieresis) Separation of the newly formed cells Depends on processes that initiate in late anaphase  Actin Cytokinesis (cytodieresis) filaments Indentation of the cell surface in a narrow band around the cell, formed by a contractile ring of actin filaments (“cortex”). Indentation deepens to form a furrow that moves inward, Bipolar Additional plasma membrane is delivered to the cell surface via myosin fibers cytoplasmic vesicles The advancing furrow deepens leaving a cytoplasmic bridge between the daughter cells called (midbody) Contractile ring The surfaces of the cleavage furrow fuse with one another, splitting the cell in two (abscission). Cleavage furrow midbody Daughter cells Interphase - G1 phase www2.le.ac.uk/projects/vgec/highereducation/topics/cell cycle-mitosis-meiosis The cell grows and increase its volume, builds the structures needed to support the increased volume Metabolic activation, protein and RNA synthesis Duplication of centrosomes and cytoplasmic organelles Controls that the extracellular environment and the cell size is appropriate to support DNA replication.  Interphase – G0 phase Some cells can stop cycling for an indefinite time: if the intracellular and extracellular conditions are not appropriate to replicate the DNA, G1-S checkpoint is not passed They exit the cycle during early G1 and enter in the arrested phase G0 G0: quiescent state, replication arrest, metabolic activities reduced to the minimum. Cells arrested in the G0 stage may retain the capability to divide upon changes in environmental conditions; G0 can be reversible or irreversible https://basicmedicalkey.com/g1-phase-and-regulation-of-cell-proliferation/ Interphase – G0 phase Reversible G0 - Quiescent cells: may re-enter the cell cycle whenever the environmental conditions are recognized as suitable. Irreversible G0 - Terminally differentiated cells: they have progressed towards a differentiation program to reach a mature state; they rest in G0 and exert their functions indefinitely. - Senescent cells reacts to a cell damage or DNA degradation arresting in G0 to avoid transmitting the damage to a progeny Interphase – S phase During this phase: DNA is duplicated (each chromosome duplicates its chromatid and becomes ‘di-chromatidic’) Histones are synthesized (histone gene transcription is specifically activated at this stage) Free histones are rapidly incorporated into new nucleosomes Chromatin domains are re-established (to maintain the same epigenetic regulation scheme on the newly synthesized chromatid) The DNA is checked for damage or abnormalities.  Interphase – G2 phase During this phase the cell: Grows rapidly and increases in size Performs active protein synthesis Checks for error in duplicated chromosomes repairs double-strand breaks Prepares for mitosis ✓ NOTE: Cells that are involved in active replication (i.e. embryo and cancer cells) may skip this phase Cell cycle regulation The cell controls a wide variety of information packages: integrity and functional acitivity of all molecules and organelles, extrinsic and intrinsic chemical signals, mechanical signals from outside Based on such info it decides whether to progress in the cell cycle; these decisions are made at specific checkpoints Cell cycle regulation G1-S checkpoint: the cell checks that all growth factors, nutrients, biosynthetic enzymes are available. If any of the correct signals is missing the gate is closed and S phase cannot start. G2-M checkpoint: the cells checks that DNA synthesis is complete and correct. If DNA contains error or damages, or is incomplete, the gate is closed and mitosis cannot start. Mitotic checkpoint (aka spindle checkpoint): at the end of metaphase, the cell checks that all kinetochores are correctly attached to sister chromatids on the metaphase plate Cyclins and cyclin-dependent kinases (CDKs) The passage from a stage to the subsequent in the cell cycle is regulated by heterodimeric complexes formed by: CYCLIN: regulatory subunit CYCLIN-dependent KINASE (CDK): transfers phosphate groups from ATP to specific Ser-Thr residues specific protein substrates Cyclins and CDKs The activity of kinases is controlled by cyclin levels, which vary and fluctuate throughout the cell cycle There are multiple cyclins and CDKs expressed at different phases of the cell cycle, able to regulate different «checkpoints» Cyclins and CDKs Cyclin-CDK complexes activate and deactivate the main components of the machinery involved in cell division: Proteins that manufacture the new components of the growing cells Proteins that hauls the components into their correct place and partition them appropriately upon division Cyclins and CDKs All factors that are needed to «unlock» the checkpoints’ gates in the cycle are activated through phosphorylation by CDKs that have them as specific targets Each CDK is activated by a specific cyclin that forms the regulatory subunit within the heterodimeric complex  Cyclins and CDKs Cyclin levels are finely regulated throughout the cell cycle as their levels depend on the presence of appropriate signals (nutrients, growth factors, DNA damage, etc) After acting at a specific checkpoint, cyclins are destroyed/dislocated and CDKs turn to the inactive state Other subsidiary proteins cooperate in the cell cycle control (e.g. CDK inhibitors that modulate CDK activity and the affinity for ATP). Cyclin structure Cyclins are 30- to 65-kDa proteins, highly heterogeneous to cope with their multiple roles All cyclins share two compact structural domains of 5 “all-α helices”: the “cyclin box”, an identical sequence domain of approximately 100 amino acids, to which the CDK partner binds. https://slideplayer.com/slide/8270408/ Types of cyclins and CDKs Four main classes of cyclin-CDK complexes: 1. M-CDK: act during G2 to trigger entry into the M phase 2. G1-CDK: act in early G1 and help driving the cell through G1 towards the S phase 3. S-CDK and G1/S-CDK: act in late G1 to launch the S phase Cyclin family Cyclin names Target CDK M Cyclin B Cyclin B1 CDK1 Cyclin B2 Cyclin B3 G1 cyclin D Cyclin D1 CDK4/6 Cyclin D2 Cyclin D3 S Cyclin A Cyclin A1 CDK1/2 Cyclin A2 G1/S cyclin E Cyclin E1 CDK2 Cyclin E2  Types of cyclins and CDKs 1. G1 Cyclins 3. S Cyclins Increase progressively during the cell cycle in response to Follow G1/S cyclins’ increase; their CCN-CDK complexes cell growth intrinsic signals and extrinsic signals induce DNA replication Coordinate cell growth with a new cell cycle entry Their levels remain high during G2 and first mitotic stages. 2. G1/S Cyclins 4. M Cyclins Increase during late G1 and decrease at the beginning of Increase as the cell starts entering the M phase, reach their the S phase. peak in metaphase The Cdk-G1/S complex induces initial processes needed They induce mitotic spindle assembly, chromatides’ alignment and chromosome congression. for DNA replication and centrosome replication. Their degradation during late metaphase and anaphase promotes cytokinesis. Cell cycle regulation Maturation Promoting Factor (MPF) promotes the G2-M passage MPF target proteins: condensins, which enable chromatin MAPs involved in mitotic spindle formation lamins, contributing to degradation of the nuclear envelope Histones H1 and H3 (see chromatin structure) Golgi matrix, to cause fragmentation  p53 role in cell cycle regulation ✓ p53 is a transcription factor highly inducible by many stress signals such as DNA damage, oncogene activation, and nutrient deprivation. ✓ The p53 protein is composed of four chains of 393 amino acids each, forming an active tetramer. ✓ It consists of several distinct domains with specific functions. 2 N-Terminal Transactivation Domains I (TADI and TADII): responsible for activating gene transcription. By interacting with various transcriptional co- activators and the general transcription machinery, TADI and TADII initiate the expression of target genes involved in vital cellular processes such as cell cycle control, DNA repair, and apoptosis. Proline-rich Domain (PRD): involved in protein-protein interactions. It enables p53 to interact with other transcription factors, coactivators, and corepressors, thereby modulating its transcriptional activity. Central DNA-binding Domain (DBD): the most well-known and highly conserved region of p53. It consists of a sequence-specific DNA-binding domain (DBD) responsible for recognizing and binding to specific DNA sequences known as p53 response elements (p53REs). The binding of p53 to p53REs regulates the transcription of target genes. Tetramerization Domain (TD): allows the formation of a stable tetramer, which is the active form of the protein. It is crucial for the proper folding and stability of p53 and is involved in mediating protein-protein interactions with other p53 molecules. C-terminal Regulatory Domain (CTD): contains multiple functional subdomains that play a role in regulating the stability, subcellular localization, and protein- protein interactions of p53. This region includes the nuclear export signal (NES), nuclear localization signal (NLS), and sites for post-translational modifications such as phosphorylation and acetylation Oligomerization Domain (OD): involved in the formation of higher-order oligomeric complexes of p53. It plays a critical role in the stability and DNA-binding activity of p53 by facilitating the interaction between p53 monomers. Pic from wikipedia p53 role in cell cycle regulation p53 is involved in numerous cellular processes; most well-known for its roles in the DNA damage response and in tumor suppression. Extracellular stresses such as chemotherapy and ionizing radiation can damage genomic DNA, which is detected by ATR and ATM, protein kinases that phosphorylate p53. Alternatively, UV damage signals through JNK and p38 MAP kinases to induce p53 activation. https://www.biolegend.com/ja-jp/p53-signaling  p53 role in cell cycle regulation In physiological conditions, p53 interacts with its negative regulator MDM2, an E3 ubiquitin- protein ligase, which targets p53 for proteasomal degradation. Upon stress, upstream factors impinge on the p53-MDM2 interaction by inhibiting MDM2 function (e.g., target for degradation, phosphorylation of interaction site, cleavage, inhibition by interaction with other proteins (e.g., LATS1/2 or ribosome biogenesis subunits)). P53 can also be directly targeted, for example, by phosphorylation, to reduce affinity for MDM2. Disruption of MDM2-p53 interaction stabilizes p53 and leads to p53 activation in response to specific insults. MDM2: mouse double minute-2 Ub: ubiquitin Marques and Kops. Chromosome Research, 2023 p53 role in cell cycle regulation Once activated, p53 binds to the promoter and activates the transcription of the p21 gene p21 binds to cyclin E/Cdk2 and cyclin D/Cdk4 complexes to cause G1 arrest in the cell cycle → This blocks pRb phosphorylation, promotes pRb binding to E2F1, and silence the transcription of E2F1 targets critical for DNA replication and cell-cycle progression p53 activation also arrests cells at the G2/M checkpoint https://www.assaygenie.com  MEIOSIS REDUCTIONAL CELL DIVISION: During meiosis the four chromatids of a pair of replicated homologous chromosomes are distributed among four daughter nuclei. Two sequential divisions without an intervening round of DNA replication: - MEIOSIS I: Each chromosome (consisting of two chromatids) is separated from its homologue, and each resulting daughter cell contains only one member of each pair of homologous chromosomes - MEIOSIS II: The two chromatids of each chromosome are separated from one another MEIOSIS RECOMBINATION: While chromosomes are paired during prophase of the first meiotic division they engage in CROSSING OVER that produces chromosomes with new combinations of maternal and paternal alleles. MEIOSIS I MEIOSIS I The PREMEIOTIC S PHASE is much longer than in a mitotic cycle The PROPHASE I is long and complex, subdivided into 5 sub-phases: Leptotene Zygotene Pachytene Diplotene Diakinesis MEIOSIS I Leptotene: bichromatidic chromosomes condense Zygotene: homologue chromosomes associate with one another (synapsis), by the formation of a complex protein structure named synaptonemal complex MEIOSIS I Pachytene: the homologues are held closely together while the sister chromatids are extended into parallel loops (recombination nodules) where crossing-over takes place. Diplotene: the synaptonemal complex dissolves and chromosomes remain attached at the sites of recombination nodules forming X-shaped structures (chiasmata) Diakinesis: the spindle is assembled and chromosomes prepare for separation MEIOSIS I During METAPHASE I homologue chromosomes align along the mitotic spindle equatorial line The kinetochores on sister chromatids are fused and bind to microtubule bundles from the same spindle pole Diplotene/diakinesis Metaphase I Spindle fiber Kinetochore spindle fiber Chiasma Cohesin Kinetochore Spindle fiber MEIOSIS MITOSIS versus MEIOSIS MEIOSIS In highest eukaryiotes meiosis occurs only in gonads to produce the gametes In male vertebrates Spermatogonia (diploid) are mitotic cells that divide but are committed to undergo meiosis Primary spermatocytes (diploid), start meiosis I Secondary spermatocytes (aploid) derive from the first meiotic division, start meiosis II Spermatids (aploid) derive from the second meiotic division. Undergo a complex process of terminal differentiation to become the highly specialized sperm cell (spermatozoon) MEIOSIS In higher eukaryotes meiosis occurs only in gonads to produce the gametes In female vertebrates Oogonia (diploid) are mitotic cells Primary oocytes (diploid) enter prophase I (during which it grows and becomes filled with yolk) prior to birth then enter a period of prolonged arrest; they resume meiosis just prior to ovulation (starting at menarche, every 28 days). Secondary oocytes (aploid) originate from the first meiotic division, start the second division, and arrest in metaphase II, which will be completed only upon fertilization. The mature egg cell results from the second meiotic division MEIOSIS Consequences of a meiotic nondysjunction Failure of homologous chromosomes (MEIOSIS I) or sister chromatids (MEIOSIS II or MITOSIS) to separate properly during cell division Consequences of a meiotic nondysjunction Failure of homologous chromosomes (MEIOSIS I) or sister chromatids (MEIOSIS II or MITOSIS) to separate properly during cell division Consequences of a meiotic nondysjunction Failure of homologous chromosomes (MEIOSIS I) or sister chromatids (MEIOSIS II or MITOSIS) to separate properly during cell division  Human aneuoploidies Trisomy 21 (Down’s syndrome) Trisomy 18 (Edwards’ syndrome) Trisomy 13 (Patau’s syndrome) 47, XXY (Klinefelter’s syndrome) 45,X (Turner’s syndrome) Consequences of a meiotic nondysjunction >50% oocytes from the IVF patients aged >35 yo had chromosomal abnormalities resulting from errors in meiosis I or meiosis II, or both: “Although the rates of chromosomal abnormalities deriving from meiosis I and II were comparable, meiosis I errors predominantly resulted in extra chromosome (chromatid) material in oocytes, in contrast to a random distribution of extra and missing chromatids after meiosis II”. Kuliev A, Verlinsky Y. Hum Reprod Update. 2004;10(5):401-7