Complex solids and phase separation: gels and membranes

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BREAKTHROUGHS IN IMAGING USING
PHOTOACTIVATABLE FLUORESCENT PROTEIN TECHNOLOGY
Jennifer Lippincott-Schwartz
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THE SECRETORY MEMBRANE SYSTEM
trans
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Properties that affect a proteins lateral
diffusion in a bilayer
Free
Assembled
Immobilized
Confined
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Fluorescence Recovery after Photobleaching (FRAP)
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FRAP of Golgi apparatus
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FRAP of Endoplasmic reticulum
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FRAP of Plasma Membrane
GFP-KRas, COS-7, 37 º C
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FRAP of Plasma Membrane
GFP-KRas, COS-7, 37 º C
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Protein cycling between plasma membrane and Golgi
GPI-GFP in COS cells
Nichols et al JCB 01
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Kinetics of GPI-GFP cycling between PM and Golgi
Post-bleach
Recovery, 20 min
Pre-bleach
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Kinetics of GPI-GFP cycling between PM and Golgi
Post-bleach
Recovery, 20 min
Pre-bleach
Residence times
PM (240 min) Golgi (9 min)
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Golgi proteins all associate dynamically w/ Golgi
FRAP of Golgi ROI
GOLGI
GalT-GFP p58-GFP GRASP-GFP ?-COP-GFP
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e-COP1 cycling on off Golgi membranes
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Golgi apparatus as steady state organelle
trans
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Highlighting using Photoactivation
Preactivation Post-activation
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PHOTOACTIVATABLE FP APPLICATIONS
VSVG-PAGFP
Photoactivate
Quantifying transport from the Golgi
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CISTERNAL PROGRESSION MODEL AND ITS PREDICTIONS
cis (entry) Trans (exit)
Monitor Golgi
iFRAP
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ss-YFP
GFP-procollagen
Fluorescence (normalized)
Fluorescence (normalized)
Time (min)
Time (min)
Exponential, not linear, cargo export
kinetics from Golgi
VSVG-GFP
Fluorescence (normalized)
Time (min)
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cis
trans
Rapid mixing
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Protein Sorting during Secretory Transport
VSVGGalT Hi Med Low
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ER
Golgi
PM
Phosphoglycerides Sphingolipids
Intra-Golgi transport by rapid membrane
partitioning
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PHOTOACTIVATABLE FP APPLICATIONS
fluorescence
fluorescence
1
Time (h)
Time (h)
5
6
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PHOTOACTIVATABLE FP APPLICATIONS
fluorescence
fluorescence
1
Time (h)
Time (h)
5
6
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PHOTOACTIVATABLE FP APPLICATIONS
CD3?-PAGFP
CD3?-PAGFP
0 h
1 h
3 h
6 h
0 h
1 h
3 h
fluorescence
fluorescence
1
Time (h)
Time (h)
5
6
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Autophagy monitored with PAGFP-LC3
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Autophagy monitored with GFP-LC3
Time in DPBS
CFP-LC3 stable cell line
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Autophagy monitored with PAGFP-LC3
PAGFP-LC3
(5 min / frame)
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Autophagy monitored with PAGFP-LC3
PAGFP-LC3
(5 min / frame)
Autophagosome Half-life 18 min
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PEROXISOME BIOGENESIS
Where do peroxisomes originate?
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IN CELLULO PULSE CHASE ANALYSIS
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IN CELLULO PULSE CHASE ANALYSIS
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IN CELLULO PULSE CHASE ANALYSIS
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MODEL FOR DE NOVO BIOGENESIS OF PEROXISOMES FROM
THE ER
Translocon
ER
Translocon
ER
Pex16
Pex16
Pex3
Pex3
Pex19
Pex19
Peroxisome
Peroxisome
Kim et al., J. Cell Biol. 2006
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MODEL FOR DE NOVO BIOGENESIS OF PEROXISOMES FROM
THE ER
Translocon
ER
Translocon
ER
Pex16
Pex16
Pex3
Pex3
Pex19
Pex19
Peroxisome
Peroxisome
Kim et al., J. Cell Biol. 2006
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Photoactivatable FP Applications
Photoactivate
Photoactivate