Molecular  Cell  Biology

Microtubules  and  Intermediate  Filaments

Cooper

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  Mo@on(Movie  Loops)

Xenopus  MelanophorePigment  GranuleMovement

• Vesicle  Move  Along  Microtubules

• Vesicles  Carry  Dynein,  Kinesin  &  Myosin-­‐V

• Regula@on  of  the  motors  accounts  for  the  dispersion  /  aggrega@on

Outward  Mo@on(Movie  Loops)

Cilia  in  Ac@on

Chlamydomonas  Cilia Sperm  Flagellum

Cilia  on  Surface  of  Epithelial  Cells

Pseudostratied ciliated epithelium in trachea. Human.

Mallory Azan High magnication.

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  Amachment

Metaphase  Normal

Types  of  Mt  /  Kc  Amachment

Amphitelic

Monotelic

Syntelic

Merotelic

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

Intermediate  Filaments

Introduc@on

• Filaments  10  nm  wide  =>  “intermediate”• Present  in  Metazoa  /  Animals

– i.e.  not  Plants  or  Unicellular  Organisms

• Complex  Gene  Superfamily– 70  in  Human  Genome

• Specific  Expression  at  Different  Times  and  Places

Intermediate  Filament  Biochemical  Proper@es  In  Vitro

• Very  stable.  Limle  subunit  exchange.• Very  strong.  Filaments  do  not  break.

– MT’s  strong  but  brimle– Ac@n  weak

Intermediate  FilamentPoten@al  Func@ons  In  Vivo

• Mechanical  Strength  of  Cytoplasm

• Help  a  Layer  of  Epithelial  Cells  Resist  Shear  Stress  -­‐  Filaments  Connect  to  Cell-­‐cell  Junc@ons

• Hold  Nucleus  in  Center  of  Cell

IntermediateFilamentStructure  &Assembly

Intermediate  Filaments  by  EM:Filament  Unraveling

Classes  of  Intermediate  Filaments

Class Name CellsNumber ofIsoforms

Size(kD) Polymers

I Acidic Keratin Epithelia ~15 40-60 Obligate HeteropolymersII Basic Keratin Epithelia ~15 50-70 One acidic + one basic

III Vimentin Mesenchymal 1 53III Desmin Muscle 1 52 Homopolymers (singleIII Glial Fibrillary Glia 1 51 type of subunit) or

Acidic Protein (GFAP) co-polymers w/ eachIII Peripherin Neurons >1 58 other at varied ratios

IV Neurofilament H Neurons 1 135-150IV Neurofilament M Neurons 1 105-110 H & M each requireIV Neurofilament L Neurons 1 60-70 L for polymerIV Nestin Glial scars, Early

neurons & muscle1 240

V Lamin A All 1 60-75 Homopolymers orV Lamin B All 1 60-75 Heteropolymer

Regula@on  of  IF  Assembly

• Notoriously  Stable– No  Nucleo@de

• Filaments  Move  Limle– Precursors  Move  More

• Disassemble  Somewhat  during  Mitosis– Phosphoryla@on  by  Cyclin-­‐depen  Kinase

Vimen@n  Filaments  in  a  Cultured  Cell

Vimen@n

• All  Cells  in  Early  Development• Cage  Around  Nucleus• Interacts  with  Mt’s• Vimen@n  Knockout  Mouse

– Ini@ally  normal  at  gross  inspec@on– Cultured  cells  have  altered  proper@es  of  uncertain  significance

FRAP  of  Vimen@n  vs.  Kera@n  in  One  Cell

Left: Vimentin (Green)Right: Keratin (Red)

10-min time intervals

Dynamics  of  Kera@n  Par@cles  in  Periphery

11 micrometersover 10 minutes

18 micrometersover 10 minutes

Desmin

• Expressed  in  Muscle• Elas@c  Elements  to  Prevent  Over-­‐stretching• Connects  /  Aligns  Z  lines• Knockout  Mouse  -­‐  Deranged  Myofibril  Architecture

Kera@ns

• Expressed  in  Epithelia• Kera@n  Filaments  Connect  to  Desmosome  and  Hemidesmosomes

• Differen@a@on  of  Epidermis  includes  Produc@on  of  Massive  Amounts  of  Kera@n

• Provides  Outer  Protec@on  of  Skin• Composes  Hair,  Nails,  Feathers,  etc.

Density  of  Kera@n  Filaments  in  Outer  Epidermis  Layers

Kera@n  Muta@ons  are  Basis  forHuman  Epidermal  Diseases

• Structure/Func@on  Analysis  of  Kera@n  Assembly

• Point  Muta@on  in  Terminal  Domain  Fails  to  Assemble

• Mutant  is  Dominant,  even  in  Low  Amounts,  in  Cultured  Cells  and  Mice

Epidermolysis  Bullosa  Simplex

Wild-type Mutant

Kera@ns  and  EBS

Neurons

• Neurofilament  H,  M,  L  Copolymer

• Prevent  Axon  Breakage

• Diseases  with  Clumps  of  Neurofilaments– Superoxide  dismutase  model  for  ALS

– Clumps  are  secondary,  not  causa@ve

Neurofilament  Transport  in  Axons

Photobleached Zone in the Middle

Neurofilament  Transport  in  Axons

Photobleached Zone in the Middle

Lamins

• Square  Larce  on  Inner  Surface  of  Nuclear  Membrane

• Present  in  Metazoans  (Animals,  not  Plants  or  unicellular  organisms)

• Mitosis  Breakdown– Phosphoryla@on  of  A  &  C  by  Cyclin-­‐depen  Kinase

– B  remains  with  Membrane

• Muta@ons  Cause  Accelerated  Aging  Diseases– Progerias  -­‐  Dominant  Muta@ons

EM  of  Nuclear  LaminaNuclear  Pores

End