Chapter 12: The Cell Cycle http://highered.mcgraw-hill.com/olc/dl/120073/bio14.swf

Omnis cellula e cellula From every cell a cell – Rudolf Virchow Cell division: reproduction of cells Cell cycle: life of a cell from the time it is first formed from a dividing parent cell until it divides into 2 daughter cells Mitosis: nuclear division within a cell, followed by cytokinesis Cytokinesis: division of the cytoplasm

Getting the right stuff What is passed on to daughter cells? exact copy of genetic material = DNA mitosis organelles, cytoplasm, cell membrane, enzymes cytokinesis chromosomes (stained orange) in kangaroo rat epithelial cell notice cytoskeleton fibers

Mitosis Mitosis functions in Reproduction of single-celled organisms Growth Repair Regeneration In contrast, meiosis produces gametes in sexually-reproducing organisms Contains half the number of chromosomes of a somatic cell amoeba

Frequency of cell division Frequency of cell division varies by cell type embryo cell cycle < 20 minute skin cells divide frequently throughout life 12-24 hours cycle liver cells retain ability to divide, but keep it in reserve divide once every year or two mature nerve cells & muscle cells do not divide at all after maturity permanently in G0 G2 S G1 M metaphase prophase anaphase telophase interphase (G1, S, G2 phases) mitosis (M) cytokinesis (C) C

Organization of the Genetic Material Cell division results in genetically identical daughter cells This requires DNA replication followed by division of the nucleus Genome: genetic content of the cell Prokaryotic cells have circular DNA Circular DNA: a single long molecule Eukaryotic cells contain a number of DNA molecules specific to different species One eukaryotic cell has about 2 meters of DNA

Organizing DNA DNA is organized in chromosomes ACTGGTCAGGCAATGTC DNA DNA is organized in chromosomes double helix DNA molecule wrapped around histone proteins like thread on spools DNA-protein complex = chromatin organized into long thin fiber condensed further during mitosis histones chromatin duplicated mitotic chromosome

double-stranded mitotic human chromosomes

Distribution of Chromosomes During Cell Division Non-dividing cells contain chromatin In human cells there are 46 strands of chromatin, or 23 corresponding pairs of strands Dividing cells contain chromosomes Chromosomes becomes condensed prior to mitosis Chromosomes contain sister chromatids bound by a narrow centromere Kinetochore: a structure of proteins associated with specific sections of chromosomal DNA at the centromere

Chromosome Structure Figure 12.4 0.5 µm Chromosome duplication (including DNA synthesis) Centromere Separation of sister chromatids Sister chromatids Centromeres A eukaryotic cell has multiple chromosomes, one of which is represented here. Before duplication, each chromosome has a single DNA molecule. Once duplicated, a chromosome consists of two sister chromatids connected at the centromere. Each chromatid contains a copy of the DNA molecule. Mechanical processes separate the sister chromatids into two chromosomes and distribute them to two daughter cells. Figure 12.4

Mitotic Phase Alternates with Interphase in Cell Cycle Mitosis: M Phase includes mitosis and cytokinesis Prophase Prometaphase Metaphase Anaphase Telophase Interphase: 90% of cell cycle; cell grows, producing organelles and proteins G1: First gap S: Synthesis, chromosome replication G2: Second gap; completes preparation for cell division I’m working here! Time to Divide

G1: 5-6 hours (most variable) S: 10-12 hours G2: 4-6 hours In 24 hours… Mitosis: 1 hour G1: 5-6 hours (most variable) S: 10-12 hours G2: 4-6 hours INTERPHASE G1 S (DNA synthesis) G2 Cytokinesis Mitosis MITOTIC (M) PHASE

Mitotic Spindle is Necessary for Nuclear Division Centrosome Aster Sister chromatids Metaphase Plate Kinetochores Overlapping nonkinetochore microtubules Kinetochores microtubules Chromosomes Microtubules 0.5 µm 1 µm Mitotic spindle: used to segregate sister chromatids in anaphase Consists of microtubules and proteins Microtubules are able to change in length Elongate by adding Tubulin subunits Shorten by loss of Tubulin subunits

Mitotic Spindle Interphase: the centrosome is replicated and the two centrosomes remain paired near the nucleus Centrosome or Microtubule Organizing Center (MTOC): contains two centrioles Prophase: spindle formation Early Prometaphase: migration of centrosomes to poles, spindle microtubules grow Late Prometaphase: centrosomes are at the poles, asters form Aster: radial array of short microtubules The mitotic spindle includes the centrosomes, spindle microtubules and asters

Cellular Changes Indicate M- Phase Initiation G2 of Interphase: nuclear envelope intact Nucleus and nucleolus present Centrosome Replication Animal cells contain centrioles Chromosomes are not condensed and therefore not individually visible

Prophase Nucleoli disappear Chromosomes condense Sister chromatids form Two genetically identical arms joined by the centromere and cohesin proteins Mitotic spindle forms using microtubules; asters form Centrosome migration as microtubules lengthen

Prometaphase Nuclear envelope fragments and microtubules invade nuclear area Nonkinetochore microtubules elongate Chromosomes condense further and gain kinetochore proteins Spindle fibers (microtubules) interact with kinetochores

Mitotic Spindle Prometaphase: Kinetochore: protein structure assiciated with specific sections of the chromosomal DNA at the centromere Kinetochore microtubules: attach kinetochore to spindle Metaphase Plate: created as microtubules attach to kinetochore, creation indicates Metaphase start

Metaphase Longest stage of mitosis Can last up to 20 minutes Metaphase plate: site of chormosome alignment Centrosomes are at poles Sister chromatid kinetochores attach to kinetochore microtubules

Anaphase Shortest stage of mitosis Cohesins between sister chromatids are cleaved by enzymes Sister chromatids are now separate chromosomes Spindle fibers shorten causing chromosomes to move to opposite poles Spindles shorten (tubulin breakdown) at kinetochore ends Nonkinetochore microtubules elongate cell

Chromosome movement Kinetochores use motor proteins that “walk” chromosome along attached microtubule microtubule shortens by dismantling at kinetochore (chromosome) end Microtubules are NOT reeled in to centrioles like line on a fishing rod. The motor proteins walk along the microtubule like little hanging robots on a clothes line. In dividing animal cells, non-kinetochore microtubules are responsible for elongating the whole cell during anaphase, readying fro cytokinesis

Telophase Daughter nuclei form in cell Chromosomes loosen and become less dense Nucleoli reappear

Cytokinesis Occurs in animal cells only Relies on cleavage marked by a cleavage furrow Cytoplasmic side of cleavage furrow contains actin microfilaments and myosin As the actin and myosin interact the ring contracts and the cleavage furrow deepens

Mitosis in animal cells

Mitosis in whitefish blastula

Cellular Division in Plant Cells Plant cells form a cell plate following mitosis Golgi vesicles contain cell wall materials and migrate toward the center of the cell – forming the cell plate, a new cell wall

Cytokinesis in plant cell

Binary Fission Asexual eukaryotes can utilize mitosis for reproductive purposes – this is called binary fission Asexual prokaryotes perform binary fission that does not involve mitosis

Evolution of Mitosis Some proteins were highly conserved In prokaryotes, protein resembling eukaryotic actin may help with cell division Tubulin-Like proteins may help separate daughter cells Attach to nuclear envelope Reinforce spatial arrangement of nucleus Spindle inside nucleus

Cell Cycle Regulation Cytoplasmic regulators as shown in Mammalian cell Fusion experiment by Johnson and Rao (1970)

Cell Cycle Control System Control systems vary cell to cell Skin cells divide frequently, liver cells only divide when needed (after injury), and nerve and muscle cells never divide Cytoplasmic molecules signal cell cycle as shown by Johnson and Rao Cell cycle events are regulated cyclicly These are referred to as Cell Cycle Checkpoints

Cell Cycle Control Systems Cell Cycle Checkpoint: control point in cell cycle where stop and go-ahead signals can regulate the cycle; relies on signal transduction pathways controlled by internal and external molecular signals G1 checkpoint: acts as a mammalian restriction point “Go Signal” permits G1, S, G2 and M “Stop signal” causes G0 phase: non-dividing state G2 checkpoint M checkpoint

Checkpoint control system 3 major checkpoints: G1/S can DNA synthesis begin? G2/M has DNA synthesis been completed correctly? commitment to mitosis spindle checkpoint are all chromosomes attached to spindle? can sister chromatids separate correctly?

G0 phase G0 phase non-dividing, differentiated state most human cells in G0 phase liver cells in G0, but can be “called back” to cell cycle by external cues nerve & muscle cells highly specialized arrested in G0 & can never divide

Cell Cycle Clock The rate of cell cycle is controlled by two proteins: Cyclins Cyclin-dependent kinases (Cdks)

Cell Cycle Clock Kinases: activate or inactivate other proteins via phosphorylation Inactive most of the time Kinases are activated by cyclins Cyclins: regulatory proteins that have a cyclic (fluctuating) concentration in the cell Cyclin-dependent kinases (Cdks) Activity fluctuates with cyclin concentration

Cell Cycle Clock MPF: maturation-promoting factor or M-phase promoting factor MPF is a Cyclin-Cdk complex Triggers cell passage from G2 to M G2 checkpoint: As cyclin builds during G2 it binds with Cdk Resulting MPF phosphorylates proteins, initiating Mitosis

During anaphase, MPF inhibits itself by destroying its own cyclin Cdk persists in the cell

Stop and Go Signals Internal signals External factors M Phase checkpoint relies on kinetochore signaling Allows for enzymatic cleavage of cohesins External factors Nutrients Growth Factor Dependency Density-dependent inhibition Anchorage dependence

External signals Growth factors coordination between cells protein signals released by body cells that stimulate other cells to divide density-dependent inhibition crowded cells stop dividing each cell binds a bit of growth factor not enough activator left to trigger division in any one cell anchorage dependence to divide cells must be attached to a substrate “touch sensor” receptors

Growth factor signals growth factor cell division cell surface nuclear pore nuclear membrane P P cell division cell surface receptor Cdk protein kinase cascade P E2F P chromosome Rb P E2F cytoplasm Rb nucleus

Example of a Growth Factor Platelet Derived Growth Factor (PDGF) made by platelets in blood clots binding of PDGF to cell receptors stimulates cell division in connective tissue heal wounds Erythropoietin (EPO): A hormone produced by the kidney that promotes the formation of red blood cells in the bone marrow. EPO is a glycoprotein (a protein with a sugar attached to it). The kidney cells that make EPO are specialized and are sensitive to low oxygen levels in the blood. These cells release EPO when the oxygen level is low in the kidney. EPO then stimulates the bone marrow to produce more red cells and thereby increase the oxygen-carrying capacity of the blood. EPO is the prime regulator of red blood cell production. Its major functions are to promote the differentiation and development of red blood cells and to initiate the production of hemoglobin, the molecule within red cells that transports oxygen. EPO has been much misused as a performance-enhancing drug (“blood doping”) in endurance athletes including some cyclists (in the Tour de France), long-distance runners, speed skaters, and Nordic (cross-country) skiers. When misused in such situations, EPO is thought to be especially dangerous (perhaps because dehydration can further increase the viscosity of the blood, increasing the risk for heart attacks and strokes. EPO has been banned by the Tour de France, the Olympics, and other sports organizations.

Growth Factors and Cancer Growth factors can create cancers proto-oncogenes normally activates cell division growth factor genes become oncogenes (cancer-causing) when mutated if switched “ON” can cause cancer example: RAS (activates cyclins) tumor-suppressor genes normally inhibits cell division if switched “OFF” can cause cancer example: p53

Cancer & Cell Growth Cancer is essentially a failure of cell division control unrestrained, uncontrolled cell growth What control is lost? lose checkpoint stops gene p53 plays a key role in G1/S restriction point p53 protein halts cell division if it detects damaged DNA options: stimulates repair enzymes to fix DNA forces cell into G0 resting stage keeps cell in G1 arrest causes apoptosis of damaged cell ALL cancers have to shut down p53 activity p53 is the Cell Cycle Enforcer p53 discovered at Stony Brook by Dr. Arnold Levine

p53 — master regulator gene NORMAL p53 p53 allows cells with repaired DNA to divide. p53 protein DNA repair enzyme p53 protein Step 1 Step 2 Step 3 DNA damage is caused by heat, radiation, or chemicals. Cell division stops, and p53 triggers enzymes to repair damaged region. p53 triggers the destruction of cells damaged beyond repair. ABNORMAL p53 abnormal p53 protein cancer cell Step 1 Step 2 DNA damage is caused by heat, radiation, or chemicals. The p53 protein fails to stop cell division and repair DNA. Cell divides without repair to damaged DNA. Step 3 Damaged cells continue to divide. If other damage accumulates, the cell can turn cancerous.

Development of Cancer Cancer develops only after a cell experiences ~6 key mutations (“hits”) unlimited growth turn on growth promoter genes ignore checkpoints turn off tumor suppressor genes (p53) escape apoptosis turn off suicide genes immortality = unlimited divisions turn on chromosome maintenance genes promotes blood vessel growth turn on blood vessel growth genes overcome anchor & density dependence turn off touch-sensor gene It’s like an out-of-control car with many systems failing!

What causes these “hits”? Mutations in cells can be triggered by UV radiation chemical exposure radiation exposure heat cigarette smoke pollution age genetics

Tumors Mass of abnormal cells Benign tumor Malignant tumor abnormal cells remain at original site as a lump p53 has halted cell divisions most do not cause serious problems & can be removed by surgery Malignant tumor cells leave original site lose attachment to nearby cells carried by blood & lymph system to other tissues start more tumors = metastasis impair functions of organs throughout body

Cancer Cells Benign tumors: can be removed by surgery Malignant tumors: invade surrounding tissues and organs, and often metastasize Metastasis: spread of cancer to far locations in the body Treatment options Radiation: destroys cancer cells Chemotherapy: medication that targets rapidly dividing cells (including cancer cells) Ex: Taxol – stops dividing cells by prevents microtubule depolymerization in metaphase, but also affects cells that naturally divide often such as intestinal cells and skin cells of hair follicles

Traditional treatments for cancers Treatments target rapidly dividing cells high-energy radiation kills rapidly dividing cells chemotherapy stop DNA replication stop mitosis & cytokinesis stop blood vessel growth