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Office Hours

Office Hours. Thursdays before class (4:15-5:15) Fairchild building Through the offices into the lab area, last bay. Cell Cycle and Mitosis. http://www.youtube.com/watch?v=VlN7K1-9QB0. Cell division cycle has 4 general steps. Interphase G1 S G2 Mitosis. Cell Cycle. Goal:

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Office Hours

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  1. Office Hours • Thursdays before class (4:15-5:15) • Fairchild building • Through the offices into the lab area, last bay

  2. Cell Cycle and Mitosis http://www.youtube.com/watch?v=VlN7K1-9QB0

  3. Cell division cycle has 4 general steps Interphase G1 S G2 Mitosis

  4. Cell Cycle • Goal: • Cell-cycle control system: • Unicellular organisms: produce new organism • Multicellular organisms: Orchestrated, coordinated cell division to produce all necessary cell types

  5. Cell division cycle has 4 general steps Interphase G1 S G2 Mitosis Microtubules and associated motors used to properly separate duplicated chromosomes to each daughter cell Contraction of actin-myosin II bundles mediate cytokinesis Replication and separation of DNA Separation of organelles/cytoplasm/plasma membrane

  6. Cell Cycle Overview • Ordered series of events that lead to cell • Two main processes • Proper order controlled by checkpoints 6

  7. Cell Cycle Overview • Most eukaryotic cells have similar cells division processes • Cell division is controlled by regulating timing • Master controllers: heterodimeric protein kinases • Regulate the activities of many different proteins in replication and mitosis by phosphorylation • Protein degradation also contributes to the regulation process 7

  8. Cell Cycle • Cell cycle is an ordered series of events leading to replication • Four major phases • G1 (G for Gap) phase • Gives cell time to assess its current state/environment • G1 can vary in length • Non dividing cells can be in G0

  9. Cell Cycle • Why have Gap phases? • Enter S phase (synthesis) where they are actively replicating their chromosomes (10-12 Hours) • After G2, they begin the complicated process of mitosis • Mitosis: About 1 hour

  10. Cell Cycle Overview • Cells are in Interphase from G0/G1 to G2 • Chromosomes replicate prior to mitosis • appear as condensed X shaped molecules • Mitosis begins with Prophase 10

  11. Interphase Prophase Events at each stage Early- prepare cell for division Phosphorylation of histones, condensin, nuclear lamins Separation of centrosomes Late- breakdown of nuclear envelope (consumption into ER membrane), removal histone H1, condensation of chromosomes by condensin G1- decide whether to divide or not preparation for DNA replication and centriole duplication, expression of required genes, pre-initiation complexes form at replication origins S- duplication of DNA, connection of sister chromatids by cohesin complex, duplication of centrioles G2- check integrity of the replication process to ensure genomic stability

  12. Metaphase Anaphase Telophase and Cytokinesis Capturing and aligning chromosomes at the equator of the cell via microtubule interactions at the chromosome kinetochore Separation of chromosomes: Degradation of chromosome cohesions Migration of chromosomes by microtubule dynamics Dephosphorylation of nuclear lamins and histones Reformation of nuclear envelope and decondensation of chromosomes Separation of two daughter cells by actin-myosin II bundle contraction

  13. Mitosis • Process of cell division • Continuous process, but can be separated into different stages

  14. Interphase • The time between the end of M phase and the beginning of the next cell cycle process 14

  15. Prophase • Marks the onset of mitosis: • Nuclear envelope • Microtubules • Protein synthesis 15

  16. Prometaphase • Breakdown of the nuclear envelope and nuclear pores • Microtubules

  17. Metaphase and Anaphase • Metaphase • Anaphase 17

  18. Telophase andCytokinesis • Telophase • Cytokinesis 18

  19. Mitotic phase of cell division

  20. Mitosis

  21. MTOCs • Centrosomes duplicate during the cell cycle to prepare for mitosis

  22. Mitotic Spindle Three classes of MT in mitotic cells: Astral, kinetochore, and polar

  23. Microtubules in mitosis • Astral Microtubules: • Kinetochore Microtubules: • Polar Microtubules:

  24. Dividing cells have 3 types of microtubules Astral -Point toward cell periphery -Orient the spindle with the axis of cell division Kinetochore Bind kinetochore to centrioles Polar -Interact in an antiparallel manner -Help push chromosomes apart

  25. Microtubule dynamics during mitosis • Microtubules become much more dynamic • In the lab, FRAP is used to detect microtubule dynamics

  26. Microtubule dynamics during mitosis • Increased catastrophe causes much of the instability • Kinesin-13

  27. Kinesin-5 and Dynein • Mitotic Asters are “interdigitated” • Kinesin-5 • Dynein also contributes

  28. Microtubules need an attachment site: Kinetochore • Centromere

  29. Microtubules bind to Chromosomes at Kinetochore Middle of chromosomes are specialized regions of DNA called centromeres Protein complex called kinetochore connects centromeres to microtubules Binding of MT to both sides gives tension to aid in segregation

  30. Motor Mediated Alignment of Chromosomes • Prometaphase: Chromosomes move back and forth • Treadmilling 32

  31. Prometaphase

  32. Alignment of chromosomes at metaphase plate requires MT motors Dynein/dynactin at centromeric region pull towards MTOC (-) end of MT spindle (spindle pole) Pulling from both sides b/c chromosomes are linked Kinesins connect chromosome arms to polar MT anchor chromosome to MT beyond the depolymerizing region promotes depolymerization Growth occurs due to high levels of tubulin = MT treadmilling occurs at centromere

  33. Alignment of chromosomes at metaphase plate requires MT motors • Ran-GTP

  34. Motor Mediated Alignment of Chromosomes • Dynein-dynactin pulls microtubules to the distant pole 36

  35. Motor Mediated Alignment of Chromosomes • Dynein-dynactin moves to the poles • Prepares the cell for anaphase when all MT have tension 37

  36. Separation of chromosomes during Anaphase requires MT motors Dynein/dynactin at centromeric region pull towards MTOC (-) end of MT spindle (spindle pole) Dynein/dynactin on astral MT pull MTOC to cell periphery Kinesins connect chromosome arms to polar MT Kinesin 5 (bipolar kinesin) pushes polar MT apart Depolymerization at (+) and (-) end

  37. Microtubules dynamics through mitosis

  38. External and Internal Regulation of Cell Division Rapid cell division in development Most cells in fully developed multicellular organisms are non-dividing/quiescent External Regulation Cues from external stimuli Survival Development Regeneration Activate Transcription Factors… Internal Regulation mRNA Transcription Protein Translation Protein Degradation Protein Phosphorylation Protein Sequestration

  39. Common protein modifications that occur during cell division

  40. Discovery of Cell Cycle Regulators • A number of different model systems have been used • Budding yeast Saccharomyces cerevisiae • Fission yeast Schizosaccharomyces pombe • Temperature Sensitive Mutations: 42

  41. Discovery of Cell Cycle Regulators: Yeast • In temperature-sensitive mutants • Mutation can be switched on and off experimentally by changing the temperature

  42. S. cerevisiae Budding • S. cerevisiae cells replicate by budding • Mother and daughter cells will stay in G1 while growing 44

  43. Discovery of Cell Cycle Regulators: Yeast

  44. S. cerevisiae Budding • Once G1 cells reach the critical size, they become committed to completing the cell cycle • Irrevocably committed to entering the S phase • Enter cell cycle: START 46

  45. S. cerevisiae Budding

  46. S. cerevisiae Budding • Video 20.25

  47. S. cerevisiae • Temperature-sensitive mutants in the cdc28 gene do not form buds at the nonpermissive temperature 49

  48. S. cerevisiae • When these mutants are placed in the nonpermissive temperature: 50

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