CHAPTER 14: CELL DIVISION Cell division – new cells originate only from other living cell - Does not stop at the formation of a mature organism but continues in certain tissues throughout THE CELL CYCLE Two major phases: 1. M phase – includes mitosis → duplicated chromosomes are separated into two nuclei - includes cytokinesis -only last about an hour in mammalian organisms 2. Interphase – period between cell divisions - cell growth and engages in diverse metabolic activities - extend for days, weeks, or longer * G1 phase - period following mitosis and preceding DNA synthesis * S phase - DNA replication occurs - cell synthesizes the additional histones that will be needed as the cell doubles the number of nucleosomes in its chromosomes *G2 phase - period of time between the end of DNA synthesis and the beginning of M phase CELL CYCLES IN VIVO - A property that distinguishes various types of cellswithin a multicellular plant or animal is their capacity to grow and divide - range in length from as short as 30 minutes (cleaving frog embryo), whose cell cycles lack both G1 and G2 phases, to several months in slowly growing tissues (mammalian liver) - some cell are quiescent → in a state that will not lead them to an upcoming cell division, but they retain the capability to divide if conditions should change → described as being in the G0 state to distinguish them from the typical G1-phase cells → cell must receive a growth-promoting signal to proceed from G0 into G1 phase Three Categories of cells: 1. Cells that are highly specialized and lack the ability to divide – examples are nerve cells, muscle cells, or red blood cells – once differentiated, cells remain in that state until they die 2. Cells that normally do not divide but can be induced to begin DNA synthesis and divide when given an appropriate stimulus - example is liver cells → induced to proliferate by the surgical removal of part of the liver - lymphocyte 3. Cells that normally possess a relatively high level of mitotic activity - stem cells of various adult tissues (such as hematopoietic stem cells that give rise to red and white blood cells -stem cells at the base of numerous epithelia that line the body cavities and the body surface - relatively unspecialized cells of apical meristems located near the tips of plant roots and stems *stem cells - able to divide asymmetrically *asymmetric cell division→ two daughter cells have different sizes, components, or fate →cell produces one daughter cell that remains an stem cell like its parent and another daughter cell that has taken a step towards becoming a differentiated cell of that tissue → allow stem cells to engage in both self-renewal and the formation of differentiated tissue cells CONTROL OF THE CELL CYCLE - Cell fusion experiment → found that cytoplasm of a mitotic cell contained diffusible factors that could induce mitosis in a nonmitotic cell. 2 to M was under positive control; transition was induced by the presence of some stimulatory agent - Chromatin of the G1-phase nucleus underwent Premature Chromosomal Compaction → G1phase and an M-phase cell were fused →G2-phase and M-phase cell were fused, the G2 chromosomes; but unlike those of a G1 nucleus, the compacted G2 chromosomes were visibly doubled, reflecting the fact that replication had already occurred compaction in the S-phase nucleus led to the formation of “pulverized” chromosomal fragments - Role of Protein Kinases entry of a cell into M phase is initiated by a protein called maturation promoting factor (MPF) Two subunits of MPF: 1. subunit with kinase activity - transfers phosphate groups from ATP to specific serine and threonine residues of specific protein substrates 2. cyclin – regulatory protein; concentration of this regulatory protein rises and falls in a predictable pattern with each cell -concentration of this regulatory protein rises and falls in a predictable pattern with each cell cycle – kinase lacks the cyclin subunit and becomes inactive – kinase is activated causing cell to enter M phase These suggest: 1. progression of cells into mitosis depends on an enzyme whose sole vactivity is to phosphorylate other proteins 2. the activity of this enzyme is controlled by a subunit whose concentration varies from one stage of the cell cycle to another. *Cyclin-dependent kinases (Cdks) - MPF-like enzymes - involved in M phase and are the key agents that orchestrate activities throughout the cell cycle by phosphorylating a diverse array of proteins - “engines” that drive the cell cycle through its various stages; activities of these enzymes are regulated by a variety of “brakes” and “accelerators” - do not always stimulate activities but can also inhibit inappropriate events. Research into the genetic control of the cell cycle in yeast - identified a gene that, when mutated, would cause the growth of cells at elevated temperature to stop at certain points in the cell cycle product of this gene is cdc2→ found to be homologous to the catalytic subunit of MPF; in other words, it was a cyclin-dependent kinase *Cyclin Binding - When a cyclin reaches a sufficient concentration in the cell, it binds to the catalytic subunit of a Cdk, causing a major change in the conformation of the enzyme’s active site - *Cdk Phosphorylation/dephosphorylation - one of the kinases called CAK (Cdk-activating kinase) phosphorylates a critical threonine residue. Phosphorylation of this residue is necessary, but not sufficient, for the Cdk to be active. - second protein kinase, called Wee1, phosphorylates a key tyrosine residue in the ATP-binding pocket of the enzyme. If this residue is phosphorylated, the enzyme is inactive; effect of Wee1 overrides the effect of CAK, keeping the Cdk in an inactive state -at the end of G2, the inhibitory phosphate at Tyr 15 is removed by the third enzyme, a phosphatase named Cdc25; removal of this phosphate switches the stored cyclin–Cdk molecules into the active state, allowing it to phosphorylate key substrates and drive the yeast cell into mitosis *Cdk Inhibitors - protein called Sic1 acts as a Cdk inhibitor during G1; degradation of Sic1 allows the cyclin–Cdk that is present in the cell to initiate DNA replication *Controlled Proteolysis – degradation (an irreversible event) is accomplished by means of the ubiquitin– proteasome pathway and drives cells towards a single direction - regulation of the cell cycle requires two classes of multisubunit complexes (SCF and APC complexes) that function as ubiquitin ligases *Subcellular Localization - dynamic phenomenon in which cell cycle regulators are moved into different compartments at different stages CHECKPOINTS, CDK INHIBITORS, AND CELLULAR RESPONSES *Ataxia-telangiectasia (AT ) - inherited recessive disorder characterized by a host of diverse symptoms, including a greatly increased risk for certain types of cancer - normal cells that are subjected to treatments that damage DNA, such as ionizing radiation or DNA-altering drugs, through the cell cycle stops while the damage is repaired - gene responsible for ataxia-telangiectasia (the ATM gene) encodes a protein kinase that is activated by certain DNA lesions,particularly double-stranded breaks →sufficient to cause rapid, large-scale activation of ATM molecules, causing cell cycle arrest Checkpoints – ensure that each of the various events that make up the cell cycle occurs accurately and in the proper order. - surveillance mechanisms that halt the progress of the cell cycle if: a. damaged chromosomal DNA b. critical processes have not been properly completed -proteins of the checkpoint machinery have no role in normal cell cycle events and are only called into action when an abnormality appears - activated throughout the cell cycle by a system of sensors that recognize DNA damage or cellular abnormalities - if the DNA is damaged beyond repair, the checkpoint mechanism can transmit a signal that leads either to: a. cell death b. senescence (permanent cell cycle arrest) *ATR – protein kinase activated by DNA breaks as well as other types of lesions Pathways available to mammalian cells to arrest their cell cycle in response to DNA damage: 1. if cell is subjected to UV irradiation - ATR kinase is activated and the cell arrests in G2 - step 1: ATR kinase molecules are thought to be recruited to sites of protein-coated, single-stranded DNA -step 2: ATR phosphorylates and activates a checkpoint kinase, called Chk1 - step 3: phosphorylates Cdc25 on a particular serine residue -step 4,5: cdc25 molecule - target for a special adaptor protein that binds to Cdc25 in the cytoplasm - step 6: absence of Cdc25 from the nucleus leaves the Cdk in an inactive state 2. Damage to DNA leads to the synthesis of proteins that directly inhibit the cyclin–Cdk complex that drives the cell cycle - p21 G1 1 Cdk S phase -MRN inhibition of Cdk p21 gene and subsequent MEIOSIS 1. Gametic or terminal meiosis - all multicellular animals and many protists →Spermatogonia that are committed to undergo meiosis become primary spermatocytes, which then undergo the two divisions of meiosis to produce four relatively undifferentiated spermatids → oogonia become primary oocytes, which then enter a greatly extended meiotic prophase 2. Zygotic or initial meiosis - only protists and fungi -meiotic divisions occur just after fertilization to produce haploid spores 3. Sporic or intermediate meiosis - plants and some algae -meiotic divisions take place at a stage unrelated to either gamete formation or fertilization → diploid zygote undergoes mitosis and develops into a diploid sporophyte. At some stage in the development of the sporophyte, sporogenesis (which includes meiosis) occurs, producing spores that germinate directly into a haploid gametophyte Chapter 14.2 M Phase: Mitosis and Cytokinesis Mitosis o comes from the Greek word "mitos" = thread o coined by Walther Flemming o a process of nuclear division in which te replicated DNA molecules of each chromosome are faithfully segregated into two nuclei o maintains chromosome number o generate new cell for growth and maintenance of an organism Cytokinesis o a process by which dividing cell splits in two, partitioning the cytoplasm into two cellular packages Brief Summary Prophase Chromosomal material condenses to form compact mitotic chromosomes Cytoskeleton is disassembled, mitotic spindle is assembled Golgi complex and ER fragment. Nuclear envelop disperses Prometaphase Chromosomal microtubules attach to kinetochores of chromosomes Chromosomes are moved to spindle equator Metaphase chromosomes are aligned along metaphase plate, attached by chromosomal microtubles to both poles Anaphase centromeres split, chromatid separate chromosomes moved to opposite spindle poles spindle poles move farther apart Telophase chromosomes cluster at opposite spindle poles chromosomes become dispersed nuclear envelop assembles around chromosome clusters Golgi complex and ER reforms daughter cells formed by cytokinesis PROPHASE Formation of Mitotic Chromosome condensin - for normal chromosome compaction - activated at the onset of mitosis by phosphorylation of several of its subunits by the cyclinCdk responsible for driving cells from G2 into mitosis. cohesin - multiprotein complex that the DNA of each interphase chromosome associates with - holds the two sister chromatids together continuously though G2 Polo-like kinase and Aurora B kinase - phosphorylates cohesin subunits inducing cohesin disassociation centromeres - residence of highly repeated DNA sequences that serve as the binding sites for specific proteins kinetochores - proteinaceous button-like structure at the outer surface of the centromere of each chromatid Functions: (1) site of attachment of the chromosome to the dynamic microtubules of the mitotic spindle (2) residence of several motor proteins involved in chromosome motility (3) key component in the signaling pathway of an important mitotic checkpoint - linked to the dynamic mitotic spindle by motor proteins and Ndc80 Ndc80 - forms fibrils that appea to reach out and bind the surface of the adjacent micotubule Formation of Mitotic Spindle centrosome - microtubule organizing structure centrosome cycle - centrioles disengage (triggered by separase) - each centriole on the centrosome initiates its replication in the cytoplasm - procentriole -->full length daughter centriole - centrosome splits into two adjacent centrosome mitotic spindle formation - appearance of "sun burst arrangement" (aster) around each centrosome - growth of microtubule by addition of sub-units in plus end - separation of centrosomes from one another towards opposite side of the cell; microtubule elongation and increase in number *after mitosis, one centrosome will be distributed to each daughter cell Dissolution of the Nuclear Envelope and Partitioning of Cytoplasmic Organelles - nuclear pore complexes are disassembled as the interactions between nucleoporin subcomplexes are disrupted and the subcomplexes dissociate into the surrounding medium. - nuclear lamina are disassembled by depolymerization of the lamin filaments - nuclear membrane disrupted mechanically as holes are torn into the nuclear envelope by cytoplasmic dynein molecules PROMETAPHASE dissolution of the nuclear envelope, mitotic spindle assembly is completed, chromosomes are moved into position - kinetochore makes initial contact with sidewall of a microtubule rather than its end - kinetochore tends to become stably asociated with the plus end of one or more spindle microtubules from one of the spindle poles - unattached kinetocore on the sister chromatid captures its own microtuble frm the opposite spindle pole - chromosomes of the prometaphase cell are moved by a process called congression toward the center of the mitotic spindle, midway between poles METAPHASE chromosomes align at the spindle equator/metaphase plate microtubules astral microtubles - position spindle apparatus chromosomal microtubules - exert a pulling force on the kinetochore polar microtubules - form a structural basket that maintains the mechanical integrity of the spindle Microtubule Flux in the Metaphase Spindle microtubules exist in a dynamic state kinetochore is the site of dynamic activity plus end experience a net gain of subunits, minus end experience a loss poleward flux of subunits ANAPHASE sister chromatid split apart The Role of Proteolysis in Progression Through Mitosis Anaphase Promoting Complex (APC) [APCCdc20 and APCCdh1] - contains about a dozen core subunits in addition to an "adapto proteins (Cdc 20 and Cdh1)", that determine which protein serve as APC substrate - APCCdc20 is activated prior to metaphase and ubiquitinates securin, which secures attachment between sister chomatid - destruction of securin releases separase - separase cleaves Scc1 subunit of cohesin that triggers separation of sister chromatids - Cdc20 is inactivated after mitosis Cdh1 takes control of APC substrate collection ubiquitination of cyclin B drop in activity of mitotic Cdk progression of the cell to G1 The Events of Anaphase - chromosomes split at synchrony - movement of chromosome toward opposite poles is very slow relative to other types of cellular movements (1 micrometer per minute) - poleward movement accompanied by chromosomal microtubules shortening - subunits are lost at the plus ends Anaphase A - movement of chromosome towards the poles Anaphase B - spindle poles move farther apart Forces Required for Chromosome Movements at Anaphase microtubule depolymerization (both at plus and minus ends) alone can generate sufficient force to pull chromosomes through a cell depolymerization at plus end: serves to "chew up" the fiber that is towing the chromosomes depolymerization at minus end: serves to transport the chromosomes toward the poles due to poleward flux The Spindle Assembly Checkpoint Spindle Assembly Checkpoint (SAC) - between metaphase and anaphase - when a chromosome fails to become aligned properly, checkpoint mechanisms delays te onset of anaphase until the misplaced chromosome has assumed its proper position MVA - disorder characterized by a high percentage of aneuploid cells Mad2 - complex of proteins that mediate spindle assembly checkpoint syntelic attachment - two kinetochores attach to the same spindle fiber - corrected by Aurora B kinase TELOPHASE chromosomes tend to collect in a mass as they near their respective poles daughter cells return to their interphase condition: - mitotic spindle disassemble - nuclear envelop reforms - chromosome dispersal and disappear from microscope view Motor Proteins Required for Mitotic Movements primarily microtubule motors, kinesin related proteins, and cytoplasmic dynein Motor Proteins... ̤ located along the polar microtubules probably contribute by keeping the poles apart ̤ residing on the chromosomes are probably important in the movements of chromosome during prometaphase, in maintaining the chromosomes at the metaphase, and in separating the chromosomes during anaphase ̤ situated along overlapping polar microtubules in the region of the spindle equator are probably respnsible for cross-linking antiparallel microtubules and sliding them over one another, thus elongating the spindle during anaphase B Cytokinesis Formation of the Cleavage Furrow - indentation of the cell surface(roughly during anaphase) - indentation deepens to a furrow (furrow lies on the same plane as the metaphase plate) - as one cell becomes two, additional plasma membrane is delivered to the cell surface via cytoplasmic vesicles - advancing furrow passes through the tightly packed remnants of central portion of mitotic spindle, which forms cytoplasmic bridge (midbody) - (abscission) surface of cleavage furrow fuse with one another. requires ESCRT complexes ^ according to the Contractile Ring Theory by Douglas Marsland (1950) - force required to cleave a cell is generated in a thin band of contractile cytoplasm located in the cortex, just beneath the plasma membrane of the furrow importance of myosin II RhoA - orchestrate assembly of actin-myosin contractile machinery Formation of the Cell Plate - vesicle send out finger-like tubules that contact and fuse with neighboring vesicles to from an interwoven tubular network in the center of the cell - vesicles continue tubule formation and fusion, extending the network outward - network contacts the parent plasma membrane at the boundary of the cell - tubular network matures into a continuous flattened partition wall formation starts at the center of the cell and grows outward to meet the existing lateral walls cell plate - forms perpendicular to the mitotic spindle orientation determined by preprophase band-a belt of cortical microtubules that forms in late G2 phase phragmoplast - consists of clusters of interdigitating microtubules oriented perpendicular to the future plate, together with the actin filaments, membranous vesicles, and electron-dense mateial Stages of Meiosis Prophase I: 1. Leptotene - chromosomes become compacted and visible in the light microscope; revealed to be composed of paired chromatids. 2. Zygotene - visible association of homologues with one another (Synapsis) *Synaptonemal Complex - chromosome synapsis is accompanied by the formation of this complex structure - ladder-like structure with transverse protein filaments connecting the two lateral elements - functions primarily as a scaffold to allow interacting chromatids to complete their crossover activities *Bivalent/Tetrad - complex formed by a pair of synapsed homologous chromosomes 3. Pachytene - characterized by a fully formed synaptonemal complex - homologues are held closely together along their length by the SC -DNA of sister chromatids is extended into parallel loops - genetic recombination is completed *Recombinant nodules - within the centre of the SC -correspond to the sites where crossing-over is taking place - facilitates genetic recombination 4. Diplotene - dissolution of the SC -leaves the chromosomes attached to one another at specific points by X-shaped structures (chiasmata → where crossing-over occurs) 5. Diakinesis - meiotic spindle is assembled and the chromosomes are prepared for separation - chromosomes become recompacted - ends with the disappearance of the nucleolus, the breakdown of the nuclear envelope, and the movement of the tetrads to the metaphase plate - triggered by an increase in the level of the protein kinase activity of MPF (maturationpromoting factor) Metaphase I -two homologous chromosomes of each bivalent are connected to the spindle fibers from opposite poles - orientation of the maternal and paternal chromosomes of each bivalent on the metaphase I plate is random Anaphase I -homologous chromosomes separate -chiasmata disappear at the metaphase I–anaphase I transition as the arms of the chromatids (loss of cohesion accomplished by proteolytic cleavage of the cohesin molecules of each bivalent lose cohesion -cytological event that corresponds to Mendel’s law of independent assortment -sister chromatids remain firmly attached to one another as they move together toward a spindle pole Telophase I -nuclear envelope may or may not reform -stage between the two meiotic divisions is called interkinesis→ cells in this stage are referred to as secondary spermatocytes or secondary oocytes (considered haploid yet contain twice as much DNA as a haploid gamete) Prophase II -nuclear envelope had reformed in telophase I, it is broken down again -chromosomes become recompacted and line up at the metaphase plate Metaphase II -kinetochores of sister chromatids face opposite poles and become attached to opposing sets of chromosomal spindle fibers -progression of meiosis in vertebrate oocytes stops (brought about by factors that inhibit APCCdc20 activation, thereby preventing cyclin B degradation) *Fertilization → leads to a rapid influx of Ca2+ ions, the activation of APCCdc20, and the destruction of cyclin B division Anaphase II -begins with the synchronous splitting of the centromeres - move toward opposite poles of the cell Telophase II -meiosis II ends - chromosomes are once again enclosed by a nuclear envelope - products of meiosis are haploid cells with a 1C amount of nuclear DNA MEIOTIC NONDISJUNCTION AND ITS CONSEQUENCES *Aneuploidy - cells have an abnormal chromosome number - consequences of aneuploidy depend on which chromosome or chromosomes are affected -Not all of the aneuploidy that occurs during human development necessarily begins with the zygote: in vitro fertilization (IVF) * normal human chromosome complement is 46: 22 pairs of autosomes and one pair of sex chromosomes *Trisomy - 47 chromosomes Trisomy 21 – Downs Syndrome **likelihood of having a child with Down syndrome rises dramatically with the age of the mother *Turner Syndrome - zygote with only one X chromosome and no second sex chromosome (denoted as XO); female *Klinefelter Syndrome - male with an extra X chromosome (XXY) - characterized by mental retardation, underdevelopment of genitalia, and the presence of feminine physical characteristics - XYY →develops into a physically normal male **meiosis I is more susceptible to nondisjunction than meiosis II Genetic Recombination During Meiosis -meiosis increases the genetic variability in a population of organisms from one generation to the next - Independent assortment allows maternal and paternal chromosomes to become shuffled during formation of the gametes, and genetic recombination (crossing-over) allows maternal and paternal alleles on a given chromosome to become shuffled *Recombination - physical breakage of individual DNA molecules and the ligation of the split ends from one DNA duplex with the split ends of the duplex from the homologous chromosome → two DNA complexes once aligned - enzyme (Spo11) introduces a double-stranded break into one of the duplexes then the gap is widened → one of the single-stranded tails leaves its own duplex and invades the DNA molecule of a nonsister chromatid, hydrogen bonding with the complementary strand in the neighboring duplex →result of the reciprocal exchange of DNA strands, the two duplexes are covalently linked to one another to form a joint molecule (or heteroduplex) that contains a pair of DNA crossovers, or Holliday junctions, that flank the region of strand exchange - two alternate products can be generated: ~ two duplexes contain only short stretches of genetic exchange which represents a noncrossover ~ duplex of one DNA molecule is covalently joined to the duplex of the homologous molecule, creating a site of genetic recombination **Maturation - processes of germinal vesicle breakdown and first meiotic division **MPF - involved specifically in triggering oocyte maturation
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