Review from last time

1. Review from last time • DNA polymerases can also remove nucleotides from DNA strands (exonuclease activity) • For species to survive, replication must be accurate – know how cells 1) ensure correct base pairing, 2) immediately repair mismatches • Be able to compare and contrast eukaryotic and prokaryotic DNA replication with regard to enzymes, mechanisms, number and function of polymerases • Be aware of how nucleosomes assort post-replication • DNA can be damages by several environmental factors • Nucleotide excision repair and base excision repair • Be able to explain how a cell recognizes which strand contains a mistake in a mismatch

2. Chapter 14:Cell Reproduction

3. Cell Reproduction... • Somehow, multicellular organisms develop from a single-celled zygote into a complex organism made up of hundreds of difference cell types • A. Cell division continues throughout the life of multicellular organisms • B. Tens of millions of cells undergo division at any given moment in an adult human • 1. This amount of division is needed to replace cells that have aged or died • 2. Old, worn-out blood cells are removed and replaced at the rate of ~100 million/minute • Mother cells transmit copies of their genetic information to their daughter cells • Mechanisms must be in place to allow for the accurate transmission of genetic information from one generation to the next

4. Cell Reproduction... • The Cell Cycle • The cell cycle starts with cell division (cytokinesis) & ends with daughter cell formation or death • There are two major cell cycle phases - based on cell activities readily visible in light microscope • M phase – M for "mitotic" • includes mitosis (duplicated chromosomes are separated into 2 nuclei) • & cytokinesis (entire cell & its cytoplasm divide into 2 daughter cells) • Interphase – • divided into G1 (first gap), S (synthesis) & G2 (second gap)

5. Cell Reproduction... • The Cell Cycle • M phase – In mitosis, the cell is focused on activities necessary for cell division; usually lasts only ~1 hour • Only a small percentage of cells in a tissue are in mitosis at any given time, • This suggests that cells spend the most of their time in interphase • The usual processes of protein synthesis are largely shut down • Interphase - occupies bulk of cycle: • may extend for days, weeks or longer, depending on cell type • During this phase, cells prepare for mitosis, replicate DNA, perform normal metabolic functions (glucose oxidation, replication, transcription, translation)

7. Cell Reproduction... • The Cell Cycle • Three broad categories of cells • A. Highly specialized cells lack the ability to divide (nerve cells, muscle cells, RBCs) - once they have differentiated, they remain in that state until they die • B. Cells that normally do not divide but can be induced to begin DNA synthesis & divide when given an appropriate stimulus • 1. Liver cells can be induced to divide by the surgical removal of part of the liver • 2. Lymphocytes can be induced to divide by interaction with an appropriate antigen • C. Cells that normally possess a relatively high level of mitotic activity - tissues subject to continual renewal by continual production of new cells • 1. Spermatogonia that give rise to the male gametes • 2. Hematopoietic stem cells that give rise to red & white blood cells • 3. Basal epithelial cells • 4. Relatively unspecialized cells

8. Cell Reproduction... • The Cell Cycle • Why study the cell cycle? • Besides the basic desire for knowing how our own cells work, • Malignant cancer cells are defective in controlling their reproduction • Initial experiments began explaining how the cell cycle was controlled… • When does replication occur? Can it occur at other times? • Fuse cells at various states (G1, S, G2, M) and observe what happens.

9. Cell Reproduction... • The Cell Cycle • G1 cells + S cells —> What happens when you put a G1 nucleus in an S cell cytoplasm? • G1-donated nucleus starts replication • S cell cytoplasm has replication signal or signals that stimulate the initiation of DNA synthesis in G1-phase nuclei • G2 cells + S cells —> What happens when you put a G2 nucleus in an S cell cytoplasm? • G2-phase nuclei do not initiate DNA synthesis; • G2-nuclei have already replicated their DNA & can no longer respond to initiation factors present in S-cell cytoplasm • Results indicate that the assembly of a prereplication complex can only occur during early G1

10. Cell Reproduction... • The Cell Cycle • G1 cells + M cells —> G1-donated nuclei undergo premature chromosomal condensation • G2 cells + M cells —> G2-donated chromosomes also condensed, but the compacted chromosomes were visibly doubled reflecting fact that replication had occurred • S cells + M cells —> S-phase chromatin compacted, but since replicating DNA is very sensitive to damage, S-phase nuclear compaction led to pulverized chromosomal fragment formation • Conclusion - both transitions (G1 —> S & G2 —>M) were under positive control (both were induced by the presence of some stimulatory agent) • What are the characteristics of the signals for each transition?

11. Cell Reproduction... • The Cell Cycle: Protein Kinases and Cyclins • Cell entry into M phase is initiated by a 2-subunit protein kinase (PK) called maturation-promoting factor (MPF) • Catalytic subunit - moves PO4-3 from ATP to certain serines/threonines of specific protein substrates; phosphorylation • Regulatory subunit – cyclin; activated MPF kinase —> cell enters into M phase; only after [cyclin] reaches critical level • Cyclin levels determine MPF activity • 1. Low cyclin concentration —> kinase lacks cyclin subunit & thus is inactive so MPF activity is low • 2. When cyclin concentration rises —> the kinase is activated, causing cell to enter M phase

12. Cell Reproduction... • The Cell Cycle: Protein Kinases and Cyclins • These results suggest: • 1. Progression of cells into mitosis depends on an enzyme whose sole activity is to phosphorylate other proteins (kinase) • 2. Activity of this enzyme is controlled by a subunit (cyclin) whose concentration varies from one cell cycle stage to another

13. Cell Reproduction... • The Cell Cycle: Protein Kinases and Cyclins • How does one even study such a complex and necessary process? • You can’t just knock out the genes, the cells being observed would never reproduce • Instead, create mutants that work under some conditions but not others • Temperature sensitive mutants • Two labs in the 1970s found a gene that, when mutated, would cause growth of cells at elevated temperature to stop at certain points in cell cycle • cdc2 in fission yeast & CDC28 in budding yeast & was eventually found to be homologous to the catalytic subunit of MPF; it was a cyclin-dependent kinase

14. Cell Reproduction... • The Cell Cycle: Protein Kinases and Cyclins • Committing a cell to S & mitosis (M phase) involves cyclic availability of different cyclins, but same Cdk (cdc2) • First transition point to S phase in yeast & mammals • First transition point is called START - just before G1 end; passage through START requires cdc2 activation by G1 cyclins (levels rise during late G1) • 1. Once cell passes START, it is committed to replication & completing the cell cycle • 2. Activation of cdc2 by G1 cyclins in yeast leads to replication at previously assembled prereplication complexes

15. Cell Reproduction... • The Cell Cycle: Protein Kinases and Cyclins • Commitment process for mitosis (M phase) entry involves availability of cyclins different from those needed to enter S, but the same Cdk (cdc2) • 2nd transition point is just before G2 end) • cdc2 activation by mitotic cyclins; Cdks bound to mitotic cyclin phosphorylate substrates needed for cell to enter mitosis: • Nuclear proteins (histone H1) - phosphorylation may help compact chromosomes • Nuclear lamins - phosphorylation leads to disassembly of nuclear envelope

16. Cell Reproduction... • The Cell Cycle: Protein Kinases and Cyclins • Cells make 3rd commitment during the middle of mitosis, • This determines whether they will complete cell division & reenter G1 of the next cycle • Exit from mitosis & entry into G1 depends on rapid decrease in Cdk activity that results from a plunge in mitotic cyclin concentration • Note that in all cases, the cell cycle progresses as a result of cyclin dependent kinases • Thus, regulation of cdk’s is critical to regulating the cell cycle • A. Cyclin concentration • B. Cdk phosphorylation state • C. Cdk inhibitors • D. Controlled proteolysis • E. Subcellular localization

17. Cell Reproduction... • The Cell Cycle: Factors That Regulate Cyclin-Dependent Kinases (Cdks) • 1. Cyclin concentration – Cdks are activated by association with a cyclin; the presence of a particular cyclin follows the transcription of the gene encoding that cyclin • Different cyclin genes are transcribed at different stages during the cell cycle • When a cyclin is present in the cell, it binds to the Cdk catalytic subunit, causing a major change in that catalytic subunit's conformation • Cyclin binding causes a flexible loop of the Cdk polypeptide chain to move away from the opening to enzyme's active site; • This in turn allows Cdk to phosphorylate its targets

18. Cell Reproduction... • The Cell Cycle: Factors That Regulate Cyclin-Dependent Kinases (Cdks) • 2. Cdk phosphorylation state – cyclin binding is not sufficient by itself to turn on Cdk's kinase activity • A. Cdk subunit must also be phosphorylated at Thr 161; done by another protein kinase (CAK [Cdk-activating kinase]) • B. Phosphorylation of Tyr 15 inhibits the kinase's activity; this inhibitory phosphate is added by another kinase called wee1 • The removal of the Tyr 15 phosphate by cdc25 activates the Cdk • C. The balance between wee1 kinase & cdc25 phosphatase activities determines whether the cell will remain in G2 or progress into mitosis • Animation: http://www.sinauer.com/cooper/4e/animations1604.html

19. Cell Reproduction... • The Cell Cycle: Factors That Regulate Cyclin-Dependent Kinases (Cdks) • 3. Cdk inhibitors – Cdk activity can be blocked by a variety of inhibitors; • Example: in budding yeast, Sic1 acts as Cdk inhibitor during G1; if Sic1 is degraded —> cyclin-Cdk present in cell initiates DNA replication

20. Cell Reproduction... • The Cell Cycle: Factors That Regulate Cyclin-Dependent Kinases (Cdks) • 4. Controlled proteolysis – cyclin concentrations oscillate during each cell cycle leading to changes in Cdk activity; How do cells regulate cyclin concentrations • A. By adjusting the synthesis & destruction rates of the cyclins at different points in the cell cycle (degradation is accomplished via the ubiquitin-proteasome pathway) • B. Cell cycle regulation requires 2 classes of multisubunit complexes (SCF & APC complexes) • C. SCF complexes are active from late G1 through early mitosis & mediate the destruction of G1 cyclins, Cdk inhibitors & other cell cycle proteins • D. The APC complex acts in mitosis & degrades a number of key mitotic proteins, including the mitotic cyclins; destruction of these cyclins allows a cell to exit mitosis & enter a new cell cycle

21. Cell Reproduction... G1 cell, GFP labled cyclin B1 • The Cell Cycle: Factors That Regulate Cyclin-Dependent Kinases (Cdks) • 5. Subcellular localization – cells contain compartments in which regulatory molecules can either be united with or separated from their targets • Example: cyclin B1 shuttles between nucleus & cytoplasm until G2, when it accumulates in the nucleus just prior to the onset of mitosis • If nuclear accumulation of cyclin is blocked, cells fail to initiate cell division M cell, GFP labled cyclin B1

22. Review from last time • The cell cycle is divided into M-phase and Interphase. Be familiar with the subcomponents of each. • Be able to describe the results and interpretation of cell fusion experiments described • Be able to describe the relationship between cyclins and cyclin dependent kinases • Be able to describe the regulatory mechanisms for cdk’s

23. Cell Reproduction... • The Cell Cycle: Checkpoints • Cells possess checkpoints as part of their cell cycle • Checkpoints are surveillance mechanisms that halt progress of cell cycle if: • 1. Any of the chromosomal DNA is damaged or • 2. Certain critical processes (DNA replication, chromosome alignment) have not been properly completed • Checkpoints ensure that each of the various events occur accurately & in the proper order • 1. Proteins of checkpoint machinery have no role in normal cell cycle events & are only called into action when an abnormality appears • 2. Genes encoding checkpoint proteins were first found in screens for mutant yeast cells that kept going through cell cycle, despite suffering DNA damage or other abnormalities that caused serious defects

24. Cell Reproduction... • The Cell Cycle: Checkpoints • How checkpoints work is poorly understood but they appear to work by recognizing DNA damage or cellular abnormalities • A. If a sensor detects the presence of a defect, it triggers a response that temporarily arrests further cell cycle process • B. The cell can then use the delay to repair the damage or correct the defect rather than continuing on to the next stage • If the DNA is damaged beyond repair, the checkpoint mechanisms can transmit a signal that leads to the death of the potentially hazardous cell • animation

25. Cell Reproduction... • The Cell Cycle: Mitosis and Cytokinesis • Mitosis – • A. What we know about M phase is largely based on years of animal & plant observations & research • B. Mitosis is process of nuclear division in which replicated DNA molecules of each chromosome are faithfully partitioned into 2 nuclei • Mitosis is usually accompanied by cytokinesis – dividing cell splits in two, partitioning cytoplasm into 2 cellular packages • Mitosis maintains chromosome number & is necessary to generate new cells for organism growth, maintenance & repair • Happens in diploid or haploid cells • Most metabolic activities (transcription, translation) are shut down; cell is unresponsive to external stimuli; virtually all cell energy is devoted to one activity - chromosome segregation • Usually divided into 5 distinct stages - prophase, prometaphase, metaphase, anaphase, telophase • A. Each is characterized by a particular series of events • B. Each stage represents a segment of a continuous process - division into arbitrary phases is done only for the sake of discussion & experimentation

27. Cell Reproduction... • A quick summary: to be filled in with many, many details

28. Cell Reproduction... • The Stages of Mitosis: Prophase • Duplicated chromosomes are prepared for segregation & mitotic machinery is assembled • A. Formation of mitotic chromosomes • B. Formation of mitotic spindle • C. Dissolution of nuclear envelope and fragmentation of cytoplasmic organelles

29. Cell Reproduction... • The Stages of Mitosis: Prophase • Mitotic chromosome formation • A. Remember the state of chomatin during interphase? Great for gene expression. Stinks for segregation into daughter cells • B. Chromosome compaction does not alter the nature of a chromatin fiber, just its packaging • 1. Treat mitotic chromosomes with solutions to solubilize histones & most nonhistone proteins • 2. A structural framework or scaffold exists that retains the basic shape of the mitotic chromosome • 3. DNA loops attach at their base to the nonhistone proteins that form scaffolding • 4. During interphase, scaffold proteins are dispersed within nucleus, probably as part of nuclear matrix

30. Cell Reproduction... • The Stages of Mitosis: Prophase • Condensin – a protein associated withcrhomosome compaction Removal of any of the condensin proteins from cell extracts prevents chromosome compaction

31. Cell Reproduction... • The Stages of Mitosis: Prophase • How does condensin induce compaction? – supercoiling and topoisomerase • 1. In presence of topoisomerase & ATP, condensin can bind to DNA & curl it into supercoiled loops • 2. Compaction at prophase also requires topoisomerase II, one of the major nonhistone proteins of the mitotic chromosome • 3. Condensin is activated by phosphorylation by the Cdk-cyclin responsible for driving cells from G2 into mitosis • 4. Presumably, condensin is one of the targets through which Cdks trigger cell cycle activities

32. Cell Reproduction... • The Stages of Mitosis: Prophase • Mitotic chromosomes appear as distinct, rodlike structures • Two mirror-image sister chromatids formed during replication • 1. Before replication, DNA of each interphase chromosome becomes associated along its length with a multiprotein complex called cohesin • 2. Cohesin functions as a physical bridge that holds the 2 sister chromatids together through G2 & into mitosis, when they are ultimately separated • 3. Condensin & cohesin have a similar structural organization – EMs suggest that cohesin adopts a circular configuration with a latch at one end that can open & close the ring • The actual mechanism of cohesin function is unclear

34. Cell Reproduction... • The Stages of Mitosis: Prophase • Most cohesin dissociates from the chromosome arms as they become compacted during prophase; dissociation is induced by phosphorylation of cohesin subunits • The chromatids of each mitotic chromosome are held relatively loosely along their extended arms • Release of remaining cohesin from centromeres is delayed until anaphase, the centromeres remain tightly bound cohesin

35. Review from last time • Cells have special proteins that act as sensors to ensure all necessary processes have been completed before moving on to the next phase of the cell cycle • For convenience, mitosis is typically divided into 5 parts • During prophase chromosomes condense and the nuclear membrane is broken down • Cohesin and condensin are integral to chromosome condensation and to holding sister chromatids together until anaphase

36. Cell Reproduction... • The Stages of Mitosis: Prophase • Centromeres and kinetochores – the primary constriction is an indentation that marks the position of the centromere • A. Centromere highly repeated DNA sequences that are binding sites for specific proteins • B. At outer surface of centromere of each chromatid is the kinetochore (constructed of protein) • 1. The kinetochore assembles on centromere during prophase • 2. Attachment site for microtubules of the mitotic spindle & as the • 3. Residence of several microtubule-based motor proteins • 4. Key component of an important mitotic checkpoint

38. Cell Reproduction... • The Stages of Mitosis: Prophase • The centrosome – A complex consisting of two centrioles (barrel shaped structures) • The centrosome is the major site of microtubule initiation in an animal cell

39. Cell Reproduction... • The Stages of Mitosis: Prophase • The centrosome cycle progresses in concert with cell cycle - animal cells after mitosis have 1 centrosome with 2 centrioles situated at right angles to one another • 1. Each centriole of centrosome starts replication in the cytoplasm at the onset of S phase as DNA replication begins in nucleus; • 2. Centrioles move apart and a small daughter centriole appears next to each preexisting (maternal) centriole oriented at right angles to it • 3. At the start of mitosis, the centrosome splits into 2 adjacent centrosomes, each containing a pair of mother-daughter centrioles • 4. The start of centrosome duplication is triggered by phosphorylation of a centrosomal protein by cyclin E-Cdk2, the same agent that triggers the onset of DNA replication

41. Cell Reproduction... • The Stages of Mitosis: Prophase • Spindle formation in a typical animal cell – • MTs appear in sunburst arrangement (aster) around each centrosome during early prophase • 1. Aster formation is followed by centrosomes separating from each other & their subsequent movement around the nucleus to the opposite ends of the cell • 2. Centrosome separation is driven by motor proteins associated with the adjacent microtubules • 3. As centrosomes separate, microtubules stretching between them increase in number & elongate • 4. Eventually, they reach points opposite one another & establish 2 poles of bipolar mitotic spindle • 5. After mitosis, one centrosome is distributed to each daughter cell

43. Cell Reproduction... • The Stages of Mitosis: Prophase • Nuclear envelope dissolution & cytoplasmic organelle partitioning • Nuclear envelope breakdown occurs at prophase end; it allows spindle-chromosome interaction since spindle forms in the cytoplasm & chromosomes compact in the nucleus • The mechanism of nuclear envelope breakdown is controversial • Multiple small organelles (mitochondria, lysosomes, etc.) are just divided up among the daughter cells • The fates of other cytoplasmic organelles is subject to debate • Regardless of how it happens, each daughter cell must receive a complement of organellar material

45. Cell Reproduction... • The Stages of Mitosis: Prometaphase • Prometaphase - starts with dissolution of the nuclear envelope • A. Mitotic spindle assembly is completed • B. Chromosomes are moved into position at center of cell

46. Cell Reproduction... • The Stages of Mitosis: Prometaphase • When prometaphase starts, the compacted chromosomes are scattered throughout space that was nuclear region • Microtubules penetrate central cell region, • The free ends grow & shrink as if they are searching for chromosome • Those that contact a kinetochore are captured & stabilized • 1. Lateral surface (side-wall) of MT makes initial contact with kinetochore, not its free end • 2. Once initial contact is made, chromosomes may move actively along MT wall powered by motor proteins located in the kinetochore • 3. Kinetochore from one chromatid becomes stably associated with spindle microtubules from one pole; then unattached sister chromatid kinetochore captures its own MTs from opposite spindle pole

47. Cell Reproduction... • The Stages of Mitosis: Prometaphase • Chromosomes oscillate back & forth in both a poleward & anti-poleward direction but are ultimately moved to spindle center midway between poles • As chromosomes move to spindle center, longer microtubules attached to one kinetochore are shortened, while shorter ones attached to sister kinetochore are elongated • Eventually, each chromosome moves into position along a plane at spindle center so that MTs from each pole are equivalent in length

49. Review from last time • Be familiar with the major events in prophase • The primary constriction is the location of the centromere and kinetochore • The centrosome consists of two centrioles and is the origin for the microtubules associated with mitosis • Be able to describe the centrosome cycle • Be able to describe the way microtubules interact with the mitotic chromosomes and move them around

50. Cell Reproduction... • The Stages of Mitosis: Metaphase • Starts with chromosomes aligned at spindle equator in a plane (metaphase plate); • One chromatid attached by its kinetochore to spindle fiber from one pole, other chromatid attached to fiber from opposite pole