Myosin 5 Dependent Vesicle Trafficking

The Leonard Laboratory

Type II 5’deiodinase

• Generates >80% of the transcriptionally active T3 in brain.

• Low serum T4 leads to a 10-fold increase in brain D2 levels due to slowed enzyme inactivation.

• T4 replacement lowers brain D2 levels by accelerating enzyme internalization along F-actin fibers.

Key Features about D2 biology

• D2 has a very short half-life in the cell (t½~10 min) due to rapid loss from the cell periphery.

• Microfilament disassembly prolongs the lifespan of D2 to ~3 h. Microtubules are not required for the rapid loss of D2.

• The loss of D2 is accelerated by ligand-dependent interactions with F-actin fibers.

Ligand-dependent binding of D2 to F-actin

J. Biol. Chem.(2000) 275, 31701-31707

[step 5]p29 reserveactin

fiber

PM

[step 2]PM localized

p29:CAP

Myo 5a

Proteosomedegradation ??

vesicle

[step 1]CAP directed

p29 translocation

p29

[step 3]Formation of the

p29:TR2complex

[step 1]CAP directed

p29 translocation

[step 5]Myo5a mediated

translocationof the vesicle:TR2

complex

vesicle

Lifecycle of type 2 deiodinase

Myosin Motor Proteins

• Head – Actin binding

– Mg-ATPases

• Neck domain– Light chain binding

• Variable length tail region– Function

– Localization

Vesicle Trafficking Myosins

• Myosin I

– participate in growth cone formation, not associated with vesicles in the CNS.

• Myosins VI and VII

– play integral role in sensory cells (retina and stereocilia), no apparent role in central neurons and glia.

• Myosin V

– abundant in CNS and vesicle associated

Head

Neck

Coiled coil

Tail

Vesicle docking

Coiled coil

PEST

IQ

Motor

Key features of the myosin 5a dimer

Effects of the C-terminus of Myo5 on D2Binding to F-actin

J. Biol. Chem.(2000) 275, 31701-31707

+T4+T4

MyoVControl

220

9569

45

Mr (kDa)

MyoVIgG heavy chain

The C-terminus of Myo5 showsLigand-dependent binding to endosomes

F-actin p29GFP vesicles

Mix

Colchicine Rx

ligand(s)

+Triton & spin

Triton supernatant

Triton pellet

Vesicle:F-Actin Binding Assay

Astrocytes

JBC 275, 31701-07, 2000

1 800400 16001200

residue #

C-term

Myo5a1803

truncation mutants

yoVCC

Myo5atail

Myo5a1513

Myo5a1767

Myo5a1830

coiled-coil

Specific Antibody

% endosome Bound

(mean ± se, n=5)

100 ± 1595 ± 3

28 ± 112 ± 25 ± 1

87 ± 12

85 ± 5

6 ± 6

18 ± 11

myosinV

1400 2800 4200 56001

head tailtailtail AF-6

Nucleotide #

Functional Domains of myo5

head

No hormone

T4

T3

EFFECTS OF THYROID HORMONE OF VESICLE RECYCLING IN CULTURED ASTROCYTES.

No hormone

P P P PP

N N N N N

N

3

21

4 5

T4-treated

P P P PP

N N N N N

N4

1

5 2

3

Myo5atail

1 2 3 4 5

P P P PP

N N N N N

N

EFFECTS OF OVER-EXPRESSION OF THE C-TERMINUS OF MYO5A

ON T4-DEPENDENT VESICLE TRAFFICKING.

Vel

ocit

y n

m/s

ec

Time (min)

mobile

100200

02 4 6 8 10

100200

0

100200

0

Vel

ocit

y n

m/s

ec

100200

0 2 4 6 8 10

100200

0

100200

0

immobile

Time (min)

N

10

9

8

7

6

5

4

3

2

1

N

PM10987654321

EFFECTS OF OVER-EXPRESSION OF THE COILED-COIL DOMAIN OF MYO5A ON T4-DEPENDENT VESICLE TRAFFICKING.

Triton Pellet

T4

*

**

*recycling vesicle marker

vesicle sorting proteins

actin motor protein

SynaptophysinSynaptotagmin

Myo5a

Rab 3

D2p29

PDIER marker

+

Cell Lysate

+

L S Pmyo5atail myo5acoil-coilmyo5a1830

L S P L S P

CHARACTERIZATION OF THE T4-DEPENDENT RECYCLING VESICLE POOL IN CULTURED ASTROCYTES.

purified p29 vesicles

+

recycling vesicle marker

vesicle sorting proteins

motor

Synaptophysin

Synaptotagmin

Myo5a

Rab3

D2p29

PDIER marker

IgG hc

Myo5a

Myo5atail

Myo5acoiled-coil

Myo5atail Myo5acoiled-coil

kDa210

97

69

45

30

CHARACTERIZATION OF THE MYO5A MUTANTS TETHERED TO THE T4-DEPENDENT RECYCLING VESICLE

-T4 +T4

TR

TR

LOCALIZATION OF THYROID RECEPTORALPHA IN ASTROCYTES.

ORGANIZATION OF THE TR GENE

TR gene intron7

1 457

PstI (187)

transcription initiation site

AP1ctf

GR SP1ets -1

100 200 300 400

Exon 7 Exon 8

TR1 16 kDa TR2 26 kDa

156 qrpeptpeew dlihvateah rstnaqgshw kqrrkflpdd igqspivsmp

206 dgdkvdleaf seFTkIItpA itRvvdfakk lpmfselpce dqiillkgcc

H1 H2

H3 H4 H5 -H6

S1156 qrpeptpeew dlihvateah rstnaqgshw kqrrkflpdd igqspivsmp

206 dgdkvdleaf seFTkIItpA itRvvdfakk lpmfselpce dqiillkgcc

H1 H2

H3 H4 H5 -H6

TR LBD (156-410)

S1

256 MeiMSlRAav rydpesdtlt LSgemavkre aLknGGLgvv sdaIfelgks

306 lsafnlddte vallqavllm stdrsgllcv dkieksqeay llafehyvnh

356 rkhniphfwp migac Hasrf lhMkvecpte lfppl FLevf edqev

H5 -H6 H7 H8

H9 H10

H11 H12

S2 S3 S4256 MeiMSlRAav rydpesdtlt LSgemavkre aLknGGLgvv sdaIfelgks

306 lsafnlddte vallqavllm stdrsgllcv dkieksqeay llafehyvnh

356 rkhniphfwp kllmkvtdlr migac Hasrf lhMkvecpte lfppl F

H5 -H6 H7 H8

H9 H10

H11 H12

S2 S3 S4

TR LBD (1 256-410, 2 256-453)

kllmkerevq ssilykgaaa egrpggslgv hpegqqllgm

406 hvvqgpqvrq leqqlgeags lrgpvlqhqs pkspqqrlle llhrsgi

TR1TR2 256 MeiMSlRAav rydpesdtlt LSgemavkre aLknGGLgvv sda Ifelgks256 MeiMSlRAav rydpesdtlt LSgemavkre aLknGGLgvv sda Ifelgks

306 lsafnlddte vallqavllm stdrsgllcv dkieksqeay llafehyvnh306 lsafnlddte vallqavllm stdrsgllcv dkieksqeay llafehyvnh

356 rkhniphfwp356 rkhniphfwp

TR1TR2

TR1TR2

TR2

LOCATION OF CONTACT RESIDUES FOR LIGAND BINDINGIN THE LBD OF TR

100400 800 400 800 400 500 100

anti-TR2

anti-TR1

anti-GFP

anti-myo 5acoil-coil

anti-myo 5atail

Antibodies

20

40

60

80

100

0

T4-

dep

end

ent

vesi

cle

bin

din

g(%

p29

GF

P b

ound

)

Antibodydilution (fold)

ANALYSIS OF SELECTED COMPONENTS OF THEVESICLE DOCKING MACHINERY

dynamindynamin

syndapinsyndapin

cofilincofilin

Arp2/3Arp2/3

WASPWASP

endosomeendosome

1°endosome1°endosome

Plasma membrane

Plasma membrane

Myosin 5aMyosin 5a

TRTR22endosome

endosome

endosomein transit

endosomein transit

MODEL OF LIGAND-DEPENDENT]VESICLE DOCKING TO MYOSIN 5