powerpoint presentation - microtubules & their...

Molecular Cell Biology

Microtubules and their Motors

Cooper

Wednesday, August 29, 2012

Microtubules and their Motors

Intro

Vesicle Trafficking

Cilia

Mitosis


Microtubule Structure

Cross-‐sec=on• Hollow tube• 24 nm wide• 13-‐15 protofilaments

Helical structure

Polar• Plus ends generally distal• Minus ends generally proximal (at MTOC)

Composed of Tubulin α/β Heterodimer


Microtubule Structure & Assembly


Microtubule Motors Defini=on

• Microtubule-‐s=mulated ATPase• Mo=lity along MT’s• Sequence of known motor

Dynein

• Moves to Minus End of Mt• Large, mul=-‐subunit protein

Kinesin

• Moves to Plus End of Mt• Excep=on -‐ Ncd/Kar3


Discovery of Kinesin

Search for Motor for Axonal Transport

• Development of Video-‐enhanced DIC Imaging

Movement Requires ATP

AMPPNP Freezes Par=cles

Microtubule Affinity Chromatography

• Bind in AMPPNP, Release in ATP


Kinesin Structure


Kinesin Movement and Processivity


Kinesin Superfamily Structures


Kinesin Superfamily Phylogene=c Tree


Cytoplasmic Dynein

Discovered Biochemically

Minus End Motor for Vesicle Transport

Requires Dynac=n Complex for Func=on

Moves the Mito=c Spindle


Dynein and Kinesin Motor

Domain Structures


Dynein Motor Subunit Architecture


Model for Interac=ons between Dynein, Dynac=n Complex, Microtubules, and Cargo


Membrane Trafficking -‐ ER and Golgi

Posi=oning ER & Golgi

• Golgi near MTOC

–Minus Ends are at MTOC

– Golgi Posi=on 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

Kinesin -‐ “KIF13A”

• Discovered by sequencing

• Plus-‐end Directed, fast (0.3 µm/s)

• Binds AP-‐1 (affinity chromatography) and mannose 6-‐P receptor

• Inhibit func=on (express tail as dominant nega=ve) -‐> less M6PR at cell surface


Xenopus MelanophorePigment GranuleMovement

Vesicle Move Along Microtubules

Vesicles Carry Dynein, Kinesin & Myosin-‐V

Regula=on of the motors accounts for the dispersion / aggrega=on

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 Ac=on


Chlamydomonas Cilia Sperm Flagellum


Cilia on Surface of Epithelial Cells

Pseudostratied ciliated epithelium in trachea. Human.

Mallory Azan High magnication.Wednesday, August 29, 2012

Structure of Axoneme: Cross-‐sec=on


Axonemes are Anchored at theirBase in Basal Bodies


Conversion of Sliding to Bendingto Wave Forma=on

Slide on only side of axoneme

Propagate down the long axis


Rota=on 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 polypep=des!

Mutants -‐ Collect & Characterize

What Structures and Polypep=des Missing?


Missing Structures in Mutant


Missing Polypep=des in Mutant


Primary Cilium

Kidney Tubule Epithelium

Defec=ve in Polycys=c 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 Rela=onship to Centrioles. Different from Rela=onship of Basal Body and Axoneme MT’s.

Flowering Plants: Lack Centrioles


Centrosome Ultrastructure


Centriole Fine Structure


Mito=c Spindle Assembly

Centrosome duplicates and separates

Nuclear envelope breakdown in animals

MT’s rearrange via dynamic instability


Spindle MT’s


Dynac=n RNAi

Control

Mito=c Spindle Rota=on 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 Akachment


Metaphase Normal


Types of Mt / Kc Akachment


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?

• Depolymeriza=on at Pole

Depolymeriza=on 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 polymeriza=on to prevent detachment

Low tension: Depolymeriza=on generates force and movement


Anaphase B

Pole -‐ Pole Separa=on


End


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