Molecular Cell Biology Microtubules and their Motors Cooper

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 αβ 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

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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)

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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 MelanophorePigment GranuleMovement

Vesicle Move Along Microtubules

Vesicles Carry Dynein, Kinesin & Myosin-V

Regulation of the motors accounts for the dispersion / aggregation

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Inward Motion(Movie Loops)

Xenopus MelanophorePigment GranuleMovement



Outward Motion(Movie Loops)

Vesicle Move Along Microtubules

Vesicles Carry Dynein, Kinesin & Myosin-V

Regulation of the motors accounts for the dispersion / aggregation

Cilia in Action

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Chlamydomonas Cilia

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Sperm Flagellum

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Cilia on Surface of Epithelial Cells

Structure of Axoneme: Cross-section

Axonemes are Anchored at theirBase in Basal Bodies

Conversion of Sliding to Bendingto Wave Formation

Slide on only side of axoneme

Propagate down the long axis

Rotation of Central Pair

Whole ChlamydomonasCell w/ Two Flagella

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?

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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

Early Anaphase

Late Anaphase

MetaphasePrometaphase

Cytokinesis Onset 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

Dynactin RNAi

Control

Mitotic Spindle Rotation in C. elegans Embryo

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

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Types of Mt / Kc Attachment

Metaphase - Merotelic Chrom



Metaphase to Anaphase



Metaphase/Anaphase Lagging



Anaphase



Centromere splits and Chromosomes Move

Anaphase A: Chromosome to Pole

GFP-labeledCentromeres



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

High tension:Switch to polymerization to prevent detachment

Low tension: Depolymerization generates force and movement

Anaphase B

Pole - Pole Separation

End

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Molecular Cell Biology Microtubules and their Motors Cooper