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 • Minus ends generally proximal (at MTOC) Composed of Tubulin Heterodimer Microtubule Structure & Assembly 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 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 Kinesin Structure Kinesin Movement and Processivity QuickTime™ and a Video decompressor are needed to see this picture. Kinesin Superfamily Structures Kinesin Superfamily Phylogenetic Tree Cytoplasmic Dynein Discovered Biochemically Minus End Motor for Vesicle Transport Requires Dynactin Complex for Function 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 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) QuickTime™ and a Video decompressor are needed to see this picture. 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 Xenopus Melanophore Pigment Granule Movement Vesicle Move Along Microtubules Vesicles Carry Dynein, Kinesin & Myosin-V Regulation of the motors accounts for the dispersion / aggregation QuickTime™ and a MPEG-4 V ideo decompressor are needed to see this picture. Inward Motion (Movie Loops) Xenopus Melanophore Pigment Granule Movement Vesicle Move Along Microtubules Vesicles Carry Dynein, Kinesin & Myosin-V Regulation of the motors accounts for the dispersion / aggregation QuickTime™ and a MPEG-4 Video decompressor are needed to see this picture. Outward Motion (Movie Loops) Cilia in Action QuickTime™ and a YUV420 codec decompressor are needed to see this picture. QuickTime™ and a YUV420 codec decompressor are needed to see this picture. Chlamydomonas Cilia Quic kT ime™ and a Sorenson Video 3 dec ompres sor are needed to s ee this pi cture. Sperm Flagellum Quic kT ime™ and a Photo - J PEG dec ompres sor are needed to s ee this pi cture. 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 Slide on only side of axoneme Propagate down the long axis Rotation of Central Pair QuickTime™ and a MPEG-4 Video decompressor are needed to see this picture. Whole Chlamydomonas Cell w/ Two Flagella QuickTime™ and a MPEG-4 Video decompressor are needed to see this picture. QuickTime™ and a MPEG-4 Video decompressor are needed to see this picture. Axonemes Isolated from Chlamydomonas Dark-Field Microscopy Experimental Approaches to Study Cilia in Chlamydomonas Axoneme 2-D gel - 250 polypeptides! Mutants - Collect & Characterize What Structures and Polypeptides Missing? QuickT ime™ and a GIF decompressor are needed to see thi s pi cture. Missing Structures in Mutant Missing Polypeptides in Mutant 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? Mitosis Background Names of Stages: Interphase, prophase, metaphase, anaphase, telophase 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 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 Centrosome Ultrastructure Centriole Fine Structure Mitotic Spindle Assembly Centrosome duplicates and separates Nuclear envelope breakdown in animals MT’s rearrange via dynamic instability Spindle MT’s Mitotic Spindle Rotation in C. elegans Embryo Control Dynactin RNAi 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 Microtubule / Kinetochore Attachment Metaphase Normal QuickTime™ and a H.264 decompressor are needed to see this picture. Types of Mt / Kc Attachment Metaphase - Merotelic Chrom QuickTime™ and a H.264 decompressor are needed to see this picture. Metaphase to Anaphase QuickTime™ and a H.264 decompressor are needed to see this picture. Metaphase/Anaphase Lagging QuickTime™ and a H.264 decompressor are needed to see this picture. Anaphase QuickTime™ and a H.264 decompressor are needed to see this picture. Anaphase A: Chromosome to Pole Centromere splits and Chromosomes Move QuickTime™ and a MPEG-4 Video decompressor are needed to see this picture. GFP-labeled Centromeres 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 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