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 protofilaments n Helical structure n Polar Plus ends generally distal Minus ends generally proximal (at MTOC) Composed of Tubulin  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/Kar3

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 - “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 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 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

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 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 4th 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 Early Anaphase Late Anaphase MetaphasePrometaphase Cytokinesis OnsetLate Cytokinesis

Onion Root Tip c/o KU Med Ctr

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

Dynactin RNAi Control Mitotic Spindle Rotation in C. elegans Embryo

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

n Centromere splits and Chromosomes Move Anaphase A: Chromosome to Pole 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 High tension: Switch to polymerization to prevent detachment Low tension: Depolymerization generates force and movement

Anaphase B Pole - Pole Separation

End