myosin 5 dependent vesicle trafficking the leonard laboratory
Post on 20-Dec-2015
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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.
[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
+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
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
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