Tom Kirchhausen - CB201 - Harvard Medical School
Tom Kirchhausen - CB201 - Harvard Medical School
Tom Kirchhausen - CB201 - Harvard Medical School
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<strong>Tom</strong> <strong>Kirchhausen</strong><br />
HARVARD MEDICAL SCHOOL<br />
kirchhausen@crystal.harvard.edu<br />
617 713 8888
• assembly of nuclear pores<br />
does it happens before or after nuclear membrane<br />
formation<br />
• formation of clathrin coated pits and<br />
coated vesicles:
8 copies<br />
8 copies
ell.org<br />
of the Cell<br />
www.molbiolcell.org<br />
August 1, 2009<br />
Volume 20<br />
Number 15<br />
August 1, 2009<br />
Volume 20<br />
Number 15<br />
interphase<br />
metaphase<br />
Lei Lu et al, MCB 2009
histone H2 (chromatin)<br />
Sec61β (ER, translocon)
mitotic ER<br />
tubule<br />
10 µm
mitotic ER
mitotic ER<br />
tubule<br />
cisternae<br />
10 µm
tomographic EM reconstruction of<br />
mitotic ER<br />
ribosomes<br />
ER
assembly of nuclear pores<br />
H2 histone<br />
chromatin<br />
Sec61<br />
ER / n. membrane
assembly of nuclear pores<br />
IBB<br />
nuclear import<br />
Sec61<br />
ER / n. membrane
assembly of nuclear pores<br />
Sec61<br />
ER / n. membrane<br />
Nup133<br />
nuclear pore
intensity calibration<br />
f<br />
s<br />
all nuclear pores have 16 Nup 133 complexes<br />
interphase: 8 Nup133-EGFP per pore<br />
• (old) nuclear pores lack Nup133-EGFP<br />
• (new) nuclear pores contain 0,1, 2, 3, ..... Nup133-EGFP<br />
telophase: 4 Nup133-EGFP per pore<br />
• (new) nuclear pores contain 0,1, 2, 3, ..... Nup133-EGFP<br />
a<br />
ta<br />
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dt<br />
rw<br />
c<br />
Tt<br />
( n<br />
an<br />
i<br />
rf<br />
dt<br />
c<br />
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• assembly of nuclear pores<br />
it happens after nuclear membrane formation<br />
stmitotic import of IBB, summary of kinetic results obtained in this study, and working model for the postmitotic assembly of the nuclear<br />
ar pores. (A) 2D time lapse of a mitotic HeLa cell expressing GFP-Sec61 and IBB-tomato acquired during nuclear envelope assembl<br />
Anaphase onset is at 0 s. At 660 s, imported IBB was clearly visualized next to the rim of the chromosome mass (arrows), at a time<br />
e nuclear envelope is still incomplete as highlighted by the gap between the arrowheads. Key events were obtained by quantification<br />
BB fluorescence signals. Bar, 10 µm. (B) Schematic summary of the relative kinetics of nuclear envelope formation and nucleoporin<br />
s represent the fluorescence intensity associated with formation of the nuclear envelope (Sec61 , green; Fig. S1 B ), recruitment of N<br />
1 C), recruitment of Nup62 (blue; Fig. S1 D), and import of IBB (red; Fig. S1 E). (C) Working model for the postmitotic assembly of the n<br />
nuclear pores. ER is shown in light green; nuclear envelope (NE) is shown in dark green; yellow dot shows Nup107–160 complex; ma<br />
aining component of the nuclear pore; red square shows IBB; Chrom., chromosome mass.<br />
Lei Lu et al, JCB 2011
• formation of clathrin coated pits and<br />
coated vesicles:<br />
stochastic or predetermined?<br />
• initiation<br />
• growth<br />
• uncoating
membrane traffic in eukaryotic cells<br />
• Extraordinarily large and fast membrane<br />
fluxes along endocytic and secretory pathways<br />
• Selective traffic -- only a subset of the<br />
proteins and lipids in the donor membrane are<br />
allowed into the vesicular carrier<br />
• Prevents the homogenization of membrane<br />
components
Main routes of intracellular membrane traffic<br />
TGN<br />
Golgi<br />
Apparatus<br />
early/late<br />
endosome<br />
ER<br />
lysosome<br />
nucleus
VSVG TS - EGFP : ER GOLGI plasma membrane
coated vesicles<br />
Heuser 1983 100 nm Heuser 1983
CAPTURE = CARGO SORTING<br />
LDLR<br />
plasma membrane<br />
coated pit<br />
TfR<br />
endosome
endocytosis of TfR<br />
plasma<br />
membrane<br />
early, late, recycling<br />
endosomes<br />
Clathrin - <strong>Tom</strong>ato<br />
Transferrin receptor - EGFP
σ2 - EGFP (AP-2)<br />
spinning disk<br />
confocal microscopy<br />
Ehrlich et al, Cell 2004
• formation of clathrin coated pits and<br />
coated vesicles:<br />
stochastic or predetermined?<br />
• initiation<br />
• growth<br />
• uncoating
1. initiation: the first five seconds<br />
one triskelion<br />
two AP2<br />
PIP2<br />
Ema Cocucci<br />
Francois Aguet
Total Internal Reflec.on Fluorescence<br />
refractive index<br />
~1.33-1.38 sample<br />
n2<br />
~ 1.6 oil<br />
n1
Total Internal Reflec.on Fluorescence<br />
refractive index<br />
~1.33-1.38 sample<br />
~ 1.6 oil
1. initiation<br />
2° step<br />
intensity<br />
distribution 1° step
intensity distribution of EGFP-LCa during the first step<br />
TIRF excitation<br />
with 70 % substitution of endogenous light chains by EGFP-LCa<br />
Frequency<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0<br />
EGFP-LCa
0.61<br />
clathrins @ initiation<br />
Frequency<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
Step 1<br />
Frequency<br />
model<br />
data<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
1 2<br />
# Triskelions<br />
Step 2<br />
75 % 25 %<br />
light chain<br />
substitution : 70 %<br />
0<br />
0 1 2 3 4 5 6<br />
# EGFP<br />
0 1 2 3<br />
#
Frequency<br />
0.7<br />
0.6<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
Step 1<br />
Frequency<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
2 4<br />
# AP2<br />
AP2 @ initiation<br />
Step 2<br />
80 % 20 %<br />
AP2 σ2 adaptin<br />
0.1<br />
substitution : 81%<br />
0<br />
0 1 2 3 4 5 6<br />
# EGFP<br />
0 1 2 3<br />
# EG
1<br />
2 3<br />
~ 2 sec<br />
igure 6.<br />
roposed model for recruitment of clathrin and AP2 during the first 5 seconds<br />
f coated pit formation
• formation of clathrin coated pits and<br />
coated vesicles:<br />
stochastic or predetermined?<br />
• initiation<br />
• cargo capture<br />
• uncoating
AP-1(TGN, endosomes) & AP-2 (plasma membrane)
Internalization of VSV by a clathrin coated pit<br />
Alexa 647 - VSV<br />
AP-2<br />
David Cureton<br />
Ramiro Massol<br />
Sean Whelan
capture of LDL by a forming clathrin coated pit<br />
DiI - LDL<br />
YFP - LCa Clathrin<br />
Ehrlich et al, Cell 2004
Endocytosis of two transferrins bound to a<br />
transferrin receptor dimer<br />
TIRF illumination<br />
acquisition rate: ~ 5Hz , 1 frame/186 ms<br />
exposure time: 20 & 50 ms for transferrin and AP-2<br />
Ema Cocucci
• formation of clathrin coated pits and<br />
coated vesicles:<br />
stochastic or predetermined?<br />
• initiation<br />
• cargo capture<br />
• uncoating
• why uncoating only occurs after vesicle budding?<br />
detector of vesicle formation: Auxilin recruitment<br />
• how are triskelions released?<br />
by a brownian ratchet: Hsc70
Uncoating lasts ~ 6 s and is Hsc70, auxilin and ATP-dependent<br />
1. Detection of vesicle budding<br />
2. Auxilin recruitment<br />
3. Hsc70-ATP recruitment<br />
4. ATP hydrolysis<br />
5. Uncoating<br />
Massol et al PNAS 2006
where are auxilin and Hsc70?<br />
auxilin<br />
HSC70<br />
15 Å resolution<br />
Yi Xing et al, EMBO 2010
where are auxilin and Hsc70?<br />
Fotin et al,<br />
Nature 2004<br />
15 Å resolution<br />
cryoElectron microscopy<br />
Yi Xing et al,<br />
EMBO J 2010
Auxilin<br />
Hsc70<br />
membrane<br />
Yi Xing et al,<br />
EMBO J 2010
Clathrin<br />
Auxilin
Auxilin: vesicle budding detector<br />
Clathrin<br />
S / T PIPs<br />
HSC70<br />
GAK PTEN-like J-domain<br />
Auxilin 2<br />
Auxilin 1<br />
Rong Guan et al, Structure 2010
PTEN-like domain of auxilin: vesicle budding detector<br />
• binds phosphoinositides<br />
• mediates the burst recruitment
Dynamin<br />
~ 900 aa<br />
budding<br />
dynasore
- Dynasore + Dynasore<br />
PIP’s<br />
auxilin<br />
Massol et al<br />
2006
how are triskelions released?<br />
single-object/molecule biochemistry<br />
recombinant Clathrin/AP-2 coats
Single-molecule visualization of uncoating<br />
using TIRF microscopy<br />
auxilin<br />
Hsc70<br />
ATP<br />
Till Boecking, Francois Aguet et al<br />
Nat Struc Mol Biol, 2011
single molecule tracking of the uncoating reaction<br />
disassembly of clathrin coats driven by HSC70, auxilin<br />
and ATP<br />
ATP<br />
hsc70<br />
auxilin<br />
ADP<br />
clathrin HSC70 overlay
mechanism of uncoating<br />
• signal (∆ lipid composition)<br />
• Auxilin is a detector of vesicle budding<br />
that triggers uncoating<br />
• uncoating operates as a brownian ratchet<br />
Fotin et al, Nature 2004<br />
Massol et al PNAS 2006<br />
Boecking et al Nature Structure 2011<br />
Yi et al, EMBO J, 2011
• assembly of nuclear pores<br />
it happens after nuclear membrane formation<br />
• formation of clathrin coated pits and<br />
coated vesicles:<br />
stochastic<br />
• initiation<br />
• growth<br />
• uncoating
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