Molecular Cell Biology Microtubules and their Motors Cooper
Molecular Cell Biology Microtubules and their Motors Cooper
Microtubules and their Motors n Intro n Vesicle Trafficking n Cilia n Mitosis
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) n Composed of Tubulin α/β Heterodimer
Microtubule Structure & Assembly
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/Kar 3
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
Kinesin Structure
Kinesin Movement and Processivity
Kinesin Superfamily Structures
Kinesin Superfamily Phylogenetic Tree
Cytoplasmic Dynein n Discovered Biochemically n Minus End Motor for Vesicle Transport n Requires Dynactin Complex for Function n Moves the Mitotic Spindle
Dynein and Kinesin Motor Domain Structures
Dynein Motor Subunit Architecture
Model for Interactions between Dynein, Dynactin Complex, Microtubules, and Cargo
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
Microtubules (Red) and ER (Green)
Vesicle Traffic: Trans-Golgi to Plasma Membrane n Kinesin - “KIF 13 A” • 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 M 6 PR at cell surface
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)
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 Outward Motion (Movie Loops)
Cilia in Action
Chlamydomonas Cilia Sperm Flagellum
Cilia on Surface of Epithelial Cells
Structure of Axoneme: Cross-section
Axonemes are Anchored at their Base in Basal Bodies
Conversion of Sliding to Bending to Wave Formation n Slide on only side of axoneme n Propagate down the long axis
Rotation of Central Pair Whole Chlamydomonas Cell w/ Two Flagella Axonemes Isolated from Chlamydomonas Dark-Field Microscopy
Experimental Approaches to Study Cilia in Chlamydomonas n Axoneme 2 -D gel - 250 polypeptides! n Mutants - Collect & Characterize n What Structures and Polypeptides Missing?
Missing Structures in Mutant
Missing Polypeptides in Mutant
Primary Cilium n Kidney Tubule Epithelium n Defective in Polycystic Kidney Disease • 4 th most common cause of kidney failure • Autosomal Dominant n How does loss of the cilium cause the disease?
Mitosis Background n Names of Stages: Interphase, prophase, metaphase, anaphase, telophase n Interphase MTs disassemble then reassembly as Spindle MTs
Mitosis Stages: Spinning-Disk Confocal Images of Microtubules and DNA Late Anaphase Prometaphase Metaphase Cytokinesis Onset Early Anaphase Late Cytokinesis
Boveri: Centrosome and Centriole
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
Centrosome Ultrastructure
Centriole Fine Structure
Mitotic Spindle Assembly n Centrosome duplicates and separates n Nuclear envelope breakdown in animals n MT’s rearrange via dynamic instability
Spindle MT’s
Mitotic Spindle Rotation in C. elegans Embryo Control Dynactin RNAi
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
Microtubule / Kinetochore Attachment
Metaphase Normal
Types of Mt / Kc Attachment
Metaphase - Merotelic Chrom
Metaphase to Anaphase
Metaphase/Anaphase Lagging
Anaphase
Anaphase A: Chromosome to Pole n Centromere splits and Chromosomes Move GFP-labeled Centromeres
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
Pac-Man and Poleward Flux Models for Anaphase A
Poleward Tubulin Flux in Anaphase A Movement to Pole. . . • Blue: Photobleach Mark, 0. 7 µm/min • Yellow: Edge of Chromosome, 1. 2 µm/min
Kinetochore as a slip-clutch mechanism Low tension: Depolymerization generates force and movement High tension: Switch to polymerization to prevent detachment
Anaphase B Pole - Pole Separation
End
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