Cell Cycle and Regulation Lecture PDF

Cell cycle and regulation Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology, 2024/2025  Types of cells Diploid cells (2n) - cells containing a double set of chromosomes (i.e., somatic cells (all cells of the body) A somatic cell...

Cell cycle and regulation Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology, 2024/2025  Types of cells Diploid cells (2n) - cells containing a double set of chromosomes (i.e., somatic cells (all cells of the body) A somatic cell in humans contains 46 chromosomes (2n=46) Haploid cells (1n) - cells containing a single set of chromosomes (i.e., gametes (eggs, sperm)) Gametes in humans contain 23 chromosomes (n=23)  Types of cell division In eukaryotic organisms, there are two basic types of cell division: mitosis - division in which each daughter cell receives the same number of chromosomes as the parent cell meiosis - division in which each daughter cell receives half of the chromosomes  The cell cycle The cell cycle is a series of processes that occur in a eukaryotic cell leading to its division. As a result of the biochemical, physical, and structural changes, the cell cycle is meant: ❑ To accurately duplicate DNA in chromosomes and then ❑ to segregate the DNA into genetically identical daughter cells such that each cell receives a complete copy of the entire genome The cell cycle  The cell cycle The duration of the cell cycle varies significantly depending on the cell type  The cell cycle The cell cycle consists of the following phases: ❑ interphase (cell growth, replication of DNA) ❑ mitotic or meiotic (division of the cell nucleus/DNA) ❑ cytokinetic (division of the cytoplasm)  The cell cycle Interphase makes up 95% of the cell cycle and includes the: ❑ G1 phase (First gap) - growth phase ❑ S phase (Synthesis) - DNA synthesis ❑ G2 phase (Second gap) - growth / organization for division The mitotic phase consists of: ❑ M phase o mitosis or meiosis o cytokinesis The cell cycle  Interphase The G1 phase includes: ❑ synthesis of structural and enzymatic proteins ❑ increase in the number of mitochondria and lysosomes ❑ increase in cell mass and volume At the end of the G1 phase, regulatory proteins are synthesized, which are responsible for the cell's transition into the S phase  Interphase The S phase includes: ❑ replication of nuclear DNA ❑ synthesis of histone proteins ❑ duplication of centrosomes At the end of S phase: ❑ each chromosome consists of two identical sister chromatids ❑ sister chromatids are linked together by protein complexes containing cohesins (so- called cohesin rings)  Interphase The G2 phase includes: ❑ synthesis of mitotic spindle proteins (α- and β-tubulin) ❑ synthesis of non-histone proteins necessary for chromatin condensation ❑ synthesis of proteins and lipids necessary for the reconstruction of the cell membrane  Interphase The role of the G1 and G2 phases: ❑ they provide the cell with additional time to grow and replicate its cytoplasmic organelles ❑ if interphase only entailed the S phase, the cell would not have time to double its mass before division and would become smaller after each M phase Interphase  Cell cycle exit G0 phase (resting phase) ❑ occurs when conditions for division are not suitable (lack of nutrients, oxygen, etc.) ❑ duration can range from several days to months, ❑ some cells (e.g., nerve cells, skeletal muscles, kidney and liver parenchyma) remain in this phase forever ❑ some cells (e.g., hepatocytes, lymphocytes) can return to the cell cycle from the G0 phase and continue to divide  M phase The M phase, or the mitotic phase, includes: ⚫ Karyokinesis (division of the nucleus) ⚫ Cytokinesis (division of the cytoplasm) Karyokinesis includes the following stages: o prophase o prometaphase o metaphase o anaphase o telophase Cytokinesis begins in anaphase and ends in telophase.  Centrosome cycle The centrosome cycle consists of phases that are synchronized with the cell cycle: ❑ G1 and S phase o centrosome duplication ❑ G2 phase o centrosome maturation o both centrosomes are connected ❑ M ​phase o daughter centrosome separation o formation of astrosphere ▪ a bipolar mitotic spindle is formed with an astrosphere at each pole Centrosome cycle  Prophase In prophase, the following occurs: ❑ condensation of chromosomes using protein complexes called condensins ❑ diversion of centrosomes to opposite poles ❑ formation of the mitotic spindle and kinetochores on the chromosomes Chromosome condensation  Mitotic spindle Assembly of the mitotic spindle: New microtubules are built in random directions from two centrosomes Some of the microtubules growing from one centrosome connect with microtubules coming from the other centrosome These interactions: ❑ stabilize the microtubules ❑ prevent depolymerization ❑ create a mitotic spindle with a bipolar shape Mitotic spindle Prophase  Prometaphase In prometaphase, the following occurs: ❑ the nuclear membrane disintegrates ❑ the microtubules of the mitotic spindle attach to the kinetochores of the chromosomes ❑ each chromosome becomes attached to both poles of the spindle ❑ the attachment to the opposite poles generates tension in the kinetochores Prometaphase Prometaphase Prometaphase  Metaphase In metaphase, the following occurs: ❑ maximum condensation of chromosomes ❑ arrangement of chromosomes along the equatorial plane of the mitotic spindle forming the metaphase plate Metaphase  Anaphase In anaphase, the following occurs: ❑ the connections created by cohesins holding sister chromatids together are broken ❑ centromere separation ❑ chromosomes move toward the poles ❑ cell organelles divide into two similarly-sized groups ❑ organelles move together with chromosomes ❑ chromosomes are separated into two equal groups at each spindle pole (end of anaphase) Anaphase Anaphase  Telophase Telophase is characterized by the: ❑ disappearance of the mitotic spindle ❑ decondensation of chromosomes ❑ regeneration of the nuclear envelope ❑ regeneration of the nucleolus ❑ formation of the contractile ring, which leads to the formation of the beginning of cytokinesis Telophase  Cytokinesis In cytokinesis (during anaphase, telophase), the following occurs: ❑ formation of a contractile ring (actin and myosin microfilaments) in the equatorial plane of the cell ❑ contraction of the contractile ring, which leads to the formation of a cleavage furrow ❑ positioning of the cleavage furrow by the mitotic spindle ❑ division of cytoplasm between daughter cells ❑ construction of cell membranes in daughter cells Cytokinesis Cytokinesis Sexual reproduction  Benefits of sexual reproduction Humans are among the organisms in which the diploid form is the dominant life-form and the only haploid cells are the gametes  Meiosis Meiosis occurs only in sex cells (germ cells) and consists of two successive divisions of the diploid cell nucleus ❑ Two diploid cells into two haploid cells into four haploid cells The first meiotic division is reduction division and is referred to as meiosis I The second meiotic division is compensatory division and is referred to as meiosis II  M Phase - meiosis Meiosis I consists of: ❑ prophase I ❑ metaphase I ❑ anaphase I ❑ telophase I Meiosis II consists of: ❑ prophase II ❑ metaphase II ❑ anaphase II ❑ telophase II  Prophase I Prophase I consists of 5 stages: 1. leptotene (Greek leptos = thin, delicate) 2. zygotene (Greek zygos = bridge) 3. pachytene (Greek pachus = thick) 4. diplotene (Greek diplos = double) 5. diakinesis (Greek dia = across, kinesis = movement)  Prophase I: Leptotene 1. Leptotene Chromatin condenses to form chromosomes Each chromosome is made up of two sister chromatids Cell organelles move to the periphery of the cell  Prophase I: Zygotene 2. Zygotene Maternal and paternal homologous chromosomes (of the same shape and size) pair up to form a bivalent/tetrad consisting of four chromatids The synaptonemal complex connects homologous chromosomes into bivalents This process is called conjugation (synapsis) The centrioles divide and move towards the poles of the cell Prophase I  The synaptonemal complex Sister chromatids of homologous chromosomes are held together by cohesins The cohesins holding the sister chromatids of the maternal and paternal homologous chromosomes associate with axial cores When duplicated homologous chromosomes assemble into bivalents, the associated axial cores are held together in a synaptonemal complex by a set of transverse filaments The synaptonemal complex  Prophase I: Pachytene 3. Pachytene Further condensation of chromosomes and synthesis of histones Joints called chiasma are formed between non-sister chromatids of homologous chromosomes Then homologous recombination occurs, which is the exchange of DNA segments (i.e., equivalent alleles) in a process called called crossing-over  Crossing-over Homologous recombination, or crossing-over, is a process in which a physical exchange of alleles occurs between non- sister chromatids of each bivalent The synaptonemal complex helps maintain the integrity of the bivalents and arranges the homologous chromosomes to facilitate the exchange of DNA segments between non-sister chromatids  Crossing-over Each sister chromatid of one homologous chromosome can form a chiasma with one or two chromatids of the other chromosome Bivalents usually have more than one chiasma, meaning that multiple recombination's occur between homologous chromosomes chiasma Prophase I: Diplotene / Diakinesis 4. Diplotene Crossing-over ends The middle of homologous chromosomes begin to separate Chromatid pairs remain connected by chiasma 5. Diakinesis Chromosome condensation ends Chiasma shift toward the ends of the chromosomes The nucleolus and nuclear membrane disappear  Metaphase I Bivalents line up along the equatorial plane Mitotic spindle fibers attach to only one chromatid of the homologous chromosome Only one kinetochore is active  Anaphase I The spindle fibers shorten and pull the homologous chromosomes to the cell poles The homologous chromosomes separate The chromosomes migrate to opposite cell poles Early anaphase Late anaphase  Telophase I Two daughter cells are formed The number of chromosomes in the daughter nuclei is half that of the parent cell - reduction (1n) The chromosomes undergo partial decondensation The nuclear envelope and nucleolus are recreated Cytokinesis (division of the cytoplasm) occurs Early telophase Late telophase  Meiosis II Occurs immediately after the completion of meiosis I It is not preceded by an S phase Sister chromatids within two daughter cells separate to form four new haploid gametes The process of meiosis II is similar to mitosis except that each dividing cell has only one set of homologous chromosomes  Prophase II Chromatin condensation occurs Chromosomes become visible again Nuclear envelope and nucleolus disappear The mitotic spindle is re-formed (perpendicular to the first division) Chromosomes consist of two sister chromatids connected by centromeres  Metaphase II Both sister chromatids connect to the centromeres by spindle fibers The chromosomes line up along the equatorial plane to form the metaphase plate  Anaphase II Centromeres divide Mitotic spindle strands shorten Sister chromatids are separated and travel to the cell poles Each chromatid becomes a daughter chromosome  Telophase II Four daughter nuclei with a haploid (1n) number of chromosomes are formed Chromosomes decondense into chromatin fibers A nuclear envelope and nucleolus are formed Cytokinesis occurs  The cell-cycle control system Each cell undergoes the cell cycle in a controlled manner A checkpoint is a stage during the cell cycle of a eukaryotic cell in which: ❑ internal and external conditions are checked ❑ a decision is made whether or not to continue cell division  Cell Cycle Checkpoints Cell cycle checkpoints: ❑ G1 checkpoint between G1 and S phase ❑ S checkpoint during S phase ❑ G2 checkpoint between G2 and M phase ❑ spindle checkpoint between metaphase and anaphase of M phase  Cell Cycle Checkpoints S Checkpoint Check for: DNA damage Replication forks  Cell Cycle Regulators Regulators are external and internal signals that affect the activation or inhibition of signaling pathways Signals act by cyclically activating and inhibiting proteins and protein complexes that affect: ❑ DNA replication, mitosis, cytokinesis, etc. ❑ the proper progression of subsequent phases of the cell cycle  External regulators A mitogen is a small protein or peptide that induces a cell to begin cell division or increases the rate of division (mitosis) Mitogens are specific ligands that attach to receptors located in the cell membrane then start a cascade of processes that transmit the signal to the cell nucleus  External regulators The action of a mitogen is dependent on the transmission of signals via protein kinases that lead to the initiation of mitosis This process takes place in the G0 phase permitting the transition to the G1 phase The signaling pathways that trigger the mitogen-nuclear signaling pathways are associated with: ❑ mitogen-activated protein kinase (MAPK) ❑ phosphatidyl-inositol 3-kinase (PI-3K)  Internal regulators Internal cell cycle regulators: ❑ cyclins - bind to relevant protein kinases ❑ cyclin-dependent kinases (Cdk) - form a complex with a cyclin ❑ anaphase promoting complex (APC/C complex) (aka cyclosome) ❑ cyclin-dependent kinase inhibitor proteins (CdKI) - cell cycle inhibitors - proteins blocking the activity of cyclin- kinase complexes  Cyclins The concentration of individual cyclins is low for most of the cell cycle Depending on the phase of the cell cycle, the levels of individual cyclins rise or fall Cyclins activate cyclin-dependent kinases (Cdks) After this, the cyclin-CDK complexes fall apart and the cyclins are degraded Cyclins are degraded by ubiquitination in the proteasome  Cyclins There are four basic types of cyclins in humans They are: ❑ G1-phase cyclins (D-cyclins) ❑ G1/S-phase transition cyclins (E-cyclins) ❑ S-phase cyclins (A-cyclins) ❑ M-phase cyclins (B-cyclins) Cyclin expression cykle  Cyclin-dependent kinases Cyclin-dependent kinases (Cdks) are a family of proteins whose: ❑ overall levels are relatively constant ❑ enzymatic activity depends on cyclin association ❑ activity and protein targets change  Cyclin-Cdk complexes The combination of cyclin with a cyclin-dependent protein kinase creates a cyclin-dependent protein kinase complex Individual cyclin-Cdk complexes are activated at different stages of the cell cycle Increased concentrations of the respective cyclin allow the formation of an active cyclin-Cdk complex, which plays a decisive role in a given phase of the cell cycle The enzymatic activity of each cyclin-Cdk complex varies, while the concentration of Cdk does not change despite cell cycle phase  Cyclin-Cds complexes Major cyclins and cyclin-dependent kinases (Cdks) Cyclin type Cdk type Cyclin-Cdk Kinase Complex Cyclins D Cdk4, Cdk6 Complex G1-Cdk Cyclins E Cdk2 Complex G1/S-Cdk Cyclin A Cdk2 Complex S-Cdk Cyclins B Cdk1 Complex M-Cdk Cyclin-Cdk complexes Activation of cyclin-Cdk complexes Initially, cyclin-Cdk complexes are inactive This is due to the attachment of two phosphate residues to the Cdk dictated by the inhibitory kinase Wee1 The cyclin-Cdk complex is activated by the phosphatase Cdc25, which removes the deactivating phosphate residues The synthesis and degradation of cyclins play an important role in the regulation of Cdk activity in the cycle Activation of cyclin-Cdk complexes  G1 phase During the G1 phase, the following occurs: ❑ inactivation of S-Cdk and M-Cdk complexes ❑ increased synthesis of cyclin D ❑ association of cyclin D with Cdk4 and Cdk6 kinases to form G1-Cdk complexes, the activity of which is highest in the middle of the G1 phase ❑ G1-Cdk complexes transfer phosphate residues from ATP to respective substrates (i.e., phosphorylation), mainly to Rb protein (pRb) ❑ formation of a cyclin E complex with Cdk2 kinase to create G1/S-Cdk complex) Rb protein signaling pathway  S phase The S-Cdk complex (i.e. cyclin A combined with Cdk2 kinase) is assembled and activated at the end of the G1 phase In the S phase: ❑ it triggers the action of the pre-replication complex, which leads to only one cycle of DNA replication ❑ it prevents a second round of DNA replication ❑ it allows the transition from the S phase to the G2 phase S phase  G2 Phase In the G2 phase: ❑ cyclin A binds to Cdk1 forming a complex that leads the cell into the M phase ❑ cyclin B binds to Cdk1 (M-Cdk) forming a complex that is inactive during this phase Cyclin B-Cdk1 complex or M-Cdk: ❑ is also called M-phase Promoting Factor (MPF) ❑ will control the course of mitosis  M phase The MPF complex activates proteins: ❑ in the nuclear envelope, which results in its disintegration (the most important event in the early M phase) ❑ causes chromosome condensation and other M phase processes Activation of the MPF complex (M-Cdk) involves the removal of phosphate groups by Cdc25 phosphatase Activated MPF complexes indirectly activate more MPF complexes creating a positive-feedback loop Activation of the MPF complex  Activation of the MPF complex Cell entry into the M phase depends on the activity of the MPF complex, i.e. the M-Cdk complex  APC/C The MPF complex also triggers activation of the APC/C complex (anaphase-promoting complex/cyclosome) The APC/C causes: ❑ disintegration of the inactive securin-separase complex ❑ degradation of securin ❑ release of proteolytic separase  APC/C Active separase causes: ❑ the destruction of cohesins that hold sister chromatids together ❑ the separation of sister chromatids and respective movement to opposite poles of the cell This event ends the M phase allowing new daughter cells to enter the G1 phase APC/C  APC/C The APC/C also causes the degradation of the MPF complex by ubiquination: ❑ APC/C adds the small protein marker, ubiquitin (Ub) to cyclin ❑ cyclin bound to Ub is separated from the cyclin- dependent kinase ❑ cyclin is sent to the proteasome where it is degraded APC/C complex  Damaged DNA If DNA becomes damaged, the cell cycle control system can: ❑ stop the cell cycle ❑ remove/repair the DNA damage and move to the next phase of the cell cycle ❑ start apoptosis in the absence of damage repair  Protein p53 The mechanism of cell cycle arrest: ❑ DNA damage increases the concentration of p53 protein and causes its activation ❑ p53 protein increases transcription (creating RNA) of the gene encoding the p21 protein ❑ p21 protein binds to the G1/S-Cdk and S-Cdk complexes and blocks them ❑ the cell is arrested in the G1 phase. Protein p53 Protein p21 Cyclin-dependent kinase inhibitors Based on the structure of the protein molecule, Cdk inhibitors (CDKI) are divided into: ⚫ CIP/KIP ⚫ INK4 CIP/KIP proteins bind to and inhibit the activity of the complexes containing Cdk1, Cdk2, Cdk4 and Cdk6 o Represented by proteins: p21, p27 and p57 INK4 proteins bind to and inhibit the activity of complexes containing only Cdk4 and Cdk6 o Represented by proteins p15, p16, p18 and p19  KIP p27 inhibitor The KIP inhibitor, protein p27, plays a key role in the decision if the cell: ❑ starts a new cell cycle (concentration decrease) ❑ goes into the G0 /resting phase (concentration increase) The p27 binds to E-Cdk2 and A-Cdk2 complexes causing their inactivation In response to DNA damage, the level of p27 is regulated by protein p53 at the transcription stage KIP p27 inhibitor  Apoptosis Apoptosis is programmed cell death. It is characteristically: ❑ an active process associated with the activation of many genes ❑ requires energy input (ATP) ❑ usually involves single cells ❑ is responsible for controlling the number and type of cells ❑ removes damaged, infected or mutation-laden cells ❑ ensures tissue homeostasis  Necrosis Necrosis usually involves the death of entire groups of cells and does not require energy It is a random and passive process It occurs under the influence of external factors (physical and chemical) It is a defensive reaction of the body with the creation of an inflammatory response, which is accompanied by: ❑ cell swelling ❑ loss of membrane contiguity ❑ leakage of cell contents into the surrounding extracellular space  Literature Fundamentals of Cell Biology, Volumes 1 and 2, B. Alberts, D. Bray, K. Hopkin et all. Chapter 18 and 19