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Molecular Cell Biology Microtubules and their Motors Cooper
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Microtubules and their Motors n Intro n Vesicle Trafficking n Cilia n Mitosis
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Microtubule Structure n Cross-section Hollow tube 24 nm wide 13-15 protofilaments n Helical structure n Polar Plus ends generally distal Minus ends generally proximal (at MTOC) Composed of Tubulin Heterodimer
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Microtubule Structure & Assembly
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Microtubule Motors n Definition Microtubule-stimulated ATPase Motility along MT’s Sequence of known motor n Dynein Moves to Minus End of Mt Large, multi-subunit protein n Kinesin Moves to Plus End of Mt Exception - Ncd/Kar3
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Discovery of Kinesin n Search for Motor for Axonal Transport Development of Video-enhanced DIC Imaging n Movement Requires ATP n AMPPNP Freezes Particles n Microtubule Affinity Chromatography Bind in AMPPNP, Release in ATP
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Kinesin Structure
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Kinesin Movement and Processivity
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Kinesin Superfamily Structures
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Kinesin Superfamily Phylogenetic Tree
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Cytoplasmic Dynein n Discovered Biochemically n Minus End Motor for Vesicle Transport n Requires Dynactin Complex for Function n Moves the Mitotic Spindle
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Dynein and Kinesin Motor Domain Structures
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Dynein Motor Subunit Architecture
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Model for Interactions between Dynein, Dynactin Complex, Microtubules, and Cargo
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Membrane Trafficking - ER and Golgi n 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
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Microtubules (Red) and ER (Green)
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Vesicle Traffic: Trans-Golgi to Plasma Membrane n 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
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Xenopus Melanophore Pigment Granule Movement n Vesicle Move Along Microtubules n Vesicles Carry Dynein, Kinesin & Myosin-V n Regulation of the motors accounts for the dispersion / aggregation Inward Motion (Movie Loops)
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Xenopus Melanophore Pigment Granule Movement Outward Motion (Movie Loops) n Vesicle Move Along Microtubules n Vesicles Carry Dynein, Kinesin & Myosin-V n Regulation of the motors accounts for the dispersion / aggregation
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Cilia in Action
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Chlamydomonas Cilia Sperm Flagellum
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Cilia on Surface of Epithelial Cells
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Structure of Axoneme: Cross-section
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Axonemes are Anchored at their Base in Basal Bodies
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Conversion of Sliding to Bending to Wave Formation n Slide on only side of axoneme n Propagate down the long axis
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Rotation of Central Pair Whole Chlamydomonas Cell w/ Two Flagella Axonemes Isolated from Chlamydomonas Dark-Field Microscopy
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Experimental Approaches to Study Cilia in Chlamydomonas n Axoneme 2-D gel - 250 polypeptides! n Mutants - Collect & Characterize n What Structures and Polypeptides Missing?
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Missing Structures in Mutant
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Missing Polypeptides in Mutant
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Primary Cilium n Kidney Tubule Epithelium n Defective in Polycystic Kidney Disease 4th most common cause of kidney failure Autosomal Dominant n How does loss of the cilium cause the disease?
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Mitosis Background n Names of Stages: Interphase, prophase, metaphase, anaphase, telophase n Interphase MTs disassemble then reassembly as Spindle MTs
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Mitosis Stages: Spinning-Disk Confocal Images of Microtubules and DNA Early Anaphase Late Anaphase MetaphasePrometaphase Cytokinesis OnsetLate Cytokinesis
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Onion Root Tip c/o KU Med Ctr
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Boveri: Centrosome and Centriole
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Centrosomes n Animals: Centriole Pair in Amorphous Cloud n Ends of MT’s in Cloud.No Relationship to Centrioles. Different from Relationship of Basal Body and Axoneme MT’s. n Flowering Plants: Lack Centrioles
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Centrosome Ultrastructure
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Centriole Fine Structure
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Mitotic Spindle Assembly n Centrosome duplicates and separates n Nuclear envelope breakdown in animals n MT’s rearrange via dynamic instability
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Spindle MT’s
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Dynactin RNAi Control Mitotic Spindle Rotation in C. elegans Embryo
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Chromosome Congression to Metaphase Plate n Kinetochores capture MT’s n Chromosome pulled to Pole Force at Kinetochore n Chromosome pushed away from Pole Forces on arms Force at Kinetochore
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Microtubule / Kinetochore Attachment
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Metaphase Normal
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Types of Mt / Kc Attachment
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Metaphase - Merotelic Chrom
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Metaphase to Anaphase
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Metaphase/Anaphase Lagging
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Anaphase
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n Centromere splits and Chromosomes Move Anaphase A: Chromosome to Pole GFP-labeled Centromeres
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Models for Chromosomes Moving to the Pole n Treadmilling? Depolymerization at Pole n Depolymerization at Kinetochore How remain bound while end shrinks? n Motors at Kinetochore or Pole
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Pac-Man and Poleward Flux Models for Anaphase A
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Poleward Tubulin Flux in Anaphase A Movement to Pole... Blue: Photobleach Mark, 0.7 µm/min Yellow: Edge of Chromosome, 1.2 µm/min
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Kinetochore as a slip-clutch mechanism High tension: Switch to polymerization to prevent detachment Low tension: Depolymerization generates force and movement
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Anaphase B Pole - Pole Separation
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End
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