Microtubules & their Motors - Bio 5068

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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
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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
Melanophore
Pigment Granule
Movement

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
Melanophore
Pigment Granule
Movement

Vesicle Move Along
Microtubules

Vesicles Carry Dynein,
Kinesin & Myosin-V

Regulation of the
motors accounts for
the dispersion /
aggregation
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Outward Motion
(Movie Loops)
Cilia in Action
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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
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Whole Chlamydomonas
Cell w/ Two Flagella
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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
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
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Types of Mt / Kc Attachment
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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Anaphase A: Chromosome to Pole

Centromere splits and Chromosomes Move
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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
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