PowerPoint Presentation - Microtubules & their Motorsmcb5068.wustl.edu/MCB/Lecturers/Cooper/s/8_Microtubules… · Microtubules)and)their)Motors Intro Vesicle)Trafficking Cilia Mitosis

Molecular  Cell  Biology

Microtubules  and  their  Motors

Cooper

Wednesday, August 29, 2012

Microtubules  and  their  Motors

Intro

Vesicle  Trafficking

Cilia

Mitosis

Wednesday, August 29, 2012

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

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Microtubule  Structure  &  Assembly

Wednesday, August 29, 2012

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

Wednesday, August 29, 2012

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

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Kinesin  Structure

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Kinesin  Movement  and  Processivity

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Kinesin  Superfamily  Structures

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Kinesin  Superfamily  Phylogene=c  Tree

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 Cytoplasmic  Dynein

Discovered    Biochemically

Minus  End  Motor  for  Vesicle  Transport

Requires  Dynac=n  Complex  for  Func=on

Moves  the  Mito=c  Spindle

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Dynein  and  Kinesin  Motor  

Domain  Structures

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Dynein  Motor  Subunit  Architecture

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

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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

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

Wednesday, August 29, 2012

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)

Wednesday, August 29, 2012

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

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Cilia  in  Ac=on

Wednesday, August 29, 2012

Chlamydomonas  Cilia Sperm  Flagellum

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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

Wednesday, August 29, 2012

Axonemes  are  Anchored  at  theirBase  in  Basal  Bodies

Wednesday, August 29, 2012

Conversion  of  Sliding  to  Bendingto  Wave  Forma=on

Slide  on  only  side  of  axoneme

Propagate  down  the  long  axis

Wednesday, August 29, 2012

Rota=on  of  Central  Pair

Whole  ChlamydomonasCell  w/  Two  Flagella

Axonemes  Isolated  from  Chlamydomonas

Dark-­‐Field  Microscopy

Wednesday, August 29, 2012

Experimental  Approaches  to  Study  Cilia  in  Chlamydomonas

Axoneme  2-­‐D  gel  -­‐  250  polypep=des!

Mutants  -­‐  Collect  &  Characterize

What  Structures  and  Polypep=des  Missing?

Wednesday, August 29, 2012

Missing  Structures  in  Mutant

Wednesday, August 29, 2012

Missing  Polypep=des  in  Mutant

Wednesday, August 29, 2012

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?

Wednesday, August 29, 2012

Mitosis  Background

Names  of  Stages:  Interphase,  prophase,  metaphase,  anaphase,  telophase

Interphase  MTs  disassemble  then  reassembly  as  Spindle  MTs

Wednesday, August 29, 2012

Mitosis Stages: Spinning-Disk Confocal Images of Microtubules and DNA

Early Anaphase

Late Anaphase

MetaphasePrometaphase

Cytokinesis Onset Late Cytokinesis

Wednesday, August 29, 2012

Boveri:  Centrosome  and  Centriole

Wednesday, August 29, 2012

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

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Centrosome  Ultrastructure

Wednesday, August 29, 2012

Centriole  Fine  Structure

Wednesday, August 29, 2012

Mito=c  Spindle  Assembly

Centrosome  duplicates  and  separates

Nuclear  envelope  breakdown  in  animals

MT’s  rearrange  via  dynamic  instability

Wednesday, August 29, 2012

Spindle  MT’s

Wednesday, August 29, 2012

Dynac=n  RNAi

Control

Mito=c  Spindle  Rota=on  in  C.  elegans  Embryo

Wednesday, August 29, 2012

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

Wednesday, August 29, 2012

Microtubule  /  Kinetochore  Akachment

Wednesday, August 29, 2012

Metaphase  Normal

Wednesday, August 29, 2012

Types  of  Mt  /  Kc  Akachment

Wednesday, August 29, 2012

Metaphase  -­‐  Merotelic  Chrom

Wednesday, August 29, 2012

Metaphase  -­‐  Merotelic  Chrom

Wednesday, August 29, 2012

Metaphase  -­‐  Merotelic  Chrom

Wednesday, August 29, 2012

Metaphase  to  Anaphase

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Metaphase/Anaphase  Lagging

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Anaphase

Wednesday, August 29, 2012

Centromere  splits  and  Chromosomes  Move

Anaphase  A:  Chromosome  to  Pole

GFP-­‐labeledCentromeres

Wednesday, August 29, 2012

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

Wednesday, August 29, 2012

Pac-­‐Man  and  Poleward  Flux  Models  for  Anaphase  A

Wednesday, August 29, 2012

Poleward  Tubulin  Flux  in  Anaphase  A

Movement to Pole...

•Blue: Photobleach Mark, 0.7 µm/min

•Yellow: Edge of Chromosome, 1.2 µm/min

Wednesday, August 29, 2012

Kinetochore  as  a  slip-­‐clutch  mechanism

High  tension:Switch  to  polymeriza=on  to  prevent  detachment

Low  tension:  Depolymeriza=on  generates  force  and  movement

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Anaphase  B

Pole  -­‐  Pole  Separa=on

Wednesday, August 29, 2012

End

Wednesday, August 29, 2012