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Molecular Cell Biology

Molecular Cell Biology. Microtubules and their Motors Cooper. Microtubules and their Motors. Intro Vesicle Trafficking Cilia Mitosis. Microtubule Structure. Cross-section Hollow tube 24 nm wide 13-15 protofilaments Helical structure Polar Plus ends generally distal

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Molecular Cell Biology

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  1. Molecular Cell Biology Microtubules and their Motors Cooper

  2. Microtubules and their Motors • Intro • Vesicle Trafficking • Cilia • Mitosis

  3. Microtubule Structure • Cross-section • Hollow tube • 24 nm wide • 13-15 protofilaments • Helical structure • Polar • Plus ends generally distal • Minus ends generally proximal (at MTOC) • Composed of Tubulin α/β Heterodimer

  4. Microtubule Structure & Assembly

  5. Microtubule Motors • Definition • Microtubule-stimulated ATPase • Motility along MT’s • Sequence of known motor • Dynein • Moves to Minus End of Mt • Large, multi-subunit protein • Kinesin • Moves to Plus End of Mt • Exception - Ncd/Kar3

  6. Discovery of Kinesin • Search for Motor for Axonal Transport • Development of Video-enhanced DIC Imaging • Movement Requires ATP • AMPPNP Freezes Particles • Microtubule Affinity Chromatography • Bind in AMPPNP, Release in ATP

  7. Kinesin Structure

  8. Kinesin Movement and Processivity

  9. Kinesin Superfamily Structures

  10. Kinesin Superfamily Phylogenetic Tree

  11. Cytoplasmic Dynein • Discovered Biochemically • Minus End Motor for Vesicle Transport • Requires Dynactin Complex for Function • Moves the Mitotic Spindle

  12. Dynein and Kinesin Motor Domain Structures

  13. Dynein Motor Subunit Architecture

  14. Model for Interactions between Dynein, Dynactin Complex, Microtubules, and Cargo

  15. Membrane Trafficking - ER and Golgi • Positioning ER & Golgi • Golgi near MTOC • Minus Ends are at MTOC • Golgi Position Requires Dynein • ER • Tubular network spread about the cell • Kinesin moves the tubules peripherally

  16. Microtubules (Red) and ER (Green)

  17. Vesicle Traffic: Trans-Golgi to Plasma Membrane • Kinesin - “KIF13A” • Discovered by sequencing • Plus-end Directed, fast (0.3 µm/s) • Binds AP-1 (affinity chromatography) and mannose 6-P receptor • Inhibit function (express tail as dominant negative) -> less M6PR at cell surface

  18. Xenopus MelanophorePigment GranuleMovement • Vesicle Move Along Microtubules • Vesicles Carry Dynein, Kinesin & Myosin-V • Regulation of the motors accounts for the dispersion / aggregation Inward Motion (Movie Loops)

  19. Xenopus MelanophorePigment GranuleMovement • Vesicle Move Along Microtubules • Vesicles Carry Dynein, Kinesin & Myosin-V • Regulation of the motors accounts for the dispersion / aggregation Outward Motion (Movie Loops)

  20. Cilia in Action

  21. Chlamydomonas Cilia Sperm Flagellum

  22. Cilia on Surface of Epithelial Cells

  23. Structure of Axoneme: Cross-section

  24. Axonemes are Anchored at theirBase in Basal Bodies

  25. Conversion of Sliding to Bendingto Wave Formation • Slide on only side of axoneme • Propagate down the long axis

  26. Rotation of Central Pair Whole Chlamydomonas Cell w/ Two Flagella Axonemes Isolated from Chlamydomonas Dark-Field Microscopy

  27. Experimental Approaches to Study Cilia in Chlamydomonas • Axoneme 2-D gel - 250 polypeptides! • Mutants - Collect & Characterize • What Structures and Polypeptides Missing?

  28. Missing Structures in Mutant

  29. Missing Polypeptides in Mutant

  30. Primary Cilium • Kidney Tubule Epithelium • Defective in Polycystic Kidney Disease • 4th most common cause of kidney failure • Autosomal Dominant • How does loss of the cilium cause the disease?

  31. Mitosis Background • Names of Stages: Interphase, prophase, metaphase, anaphase, telophase • Interphase MTs disassemble then reassembly as Spindle MTs

  32. Prometaphase Metaphase Early Anaphase Mitosis Stages: Spinning-Disk Confocal Images of Microtubules and DNA Late Anaphase Cytokinesis Onset Late Cytokinesis

  33. Boveri: Centrosome and Centriole

  34. Centrosomes • Animals: Centriole Pair in Amorphous Cloud • Ends of MT’s in Cloud.No Relationship to Centrioles. Different from Relationship of Basal Body and Axoneme MT’s. • Flowering Plants: Lack Centrioles

  35. Centrosome Ultrastructure

  36. Centriole Fine Structure

  37. Mitotic Spindle Assembly • Centrosome duplicates and separates • Nuclear envelope breakdown in animals • MT’s rearrange via dynamic instability

  38. Spindle MT’s

  39. Mitotic Spindle Rotation in C. elegans Embryo Control Dynactin RNAi

  40. Chromosome Congression to Metaphase Plate • Kinetochores capture MT’s • Chromosome pulled to Pole • Force at Kinetochore • Chromosome pushed away from Pole • Forces on arms • Force at Kinetochore

  41. Microtubule / Kinetochore Attachment

  42. Metaphase Normal

  43. Types of Mt / Kc Attachment

  44. Metaphase - Merotelic Chrom

  45. Metaphase to Anaphase

  46. Metaphase/Anaphase Lagging

  47. Anaphase

  48. Anaphase A: Chromosome to Pole • Centromere splits and Chromosomes Move GFP-labeled Centromeres

  49. Models for Chromosomes Moving to the Pole • Treadmilling? • Depolymerization at Pole • Depolymerization at Kinetochore • How remain bound while end shrinks? • Motors at Kinetochore or Pole

  50. Pac-Man and Poleward Flux Models for Anaphase A

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