lab 5a and 5b overview investigating protein sorting signals using cloning, transfection, gfp-fusion...
TRANSCRIPT
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Lab 5A and 5B Overview Investigating protein sorting signals using cloning, transfection,GFP-fusion proteins, and vital stains for cellular compartments
1. Protein sorting and membrane trafficking- or -
How cells deliver things to the right place
2. Fluorescent proteins are critical tools in Cell biologyLast Week
3. Transfection and transgene expression - or -
How we get DNA into cellsto express “designer” genes
4. Fluorescent markers for differentcompartments of the secretory
and endocytic pathways
This
Week
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Cloning vector for expressing GFP fusion proteins in mammalian cells (constructed in bacteria)
GFPProtein X
Protein YGFP
in ZProte GFP
Fusion proteins are usually introduced into cells as DNA constructs
pCMV: Strong, constitutive
promoter
Neomycin:Selectable marker formammalian cells
BGH pA:Polyadenylationsequence
Ampicillin:Selectable marker for bacterial cells
pUC:Origin of replication
for bacterial cells
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Effectene®
The negatively-charged phosphate backbone of DNAmust be neutralized by positively charged counterions
to allow transport across the plasma membrane.
TRANSFECTION - from trans, meaning “across”
DNA is a large, charged molecule that normally doesn’t cross cell membranes… so we have to use tricks to get it into cells
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Principle of transfectionwith Effectene® reagent
+ Enhancer
Outline of transfection protocol (Qiagen)
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There are many points along the way where transfection efficiency can be compromised
Proton sponge hypothesis: sequestration of cations by DNA leads to the osmotic
swelling and rupture of endosomes, releasing DNA vector into the cytoplasm
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Only a fraction of treated cells will be successfully transfected, and thus the expression of the transgenewill be quite VARIABLE - here GFP is used as a marker
of transfection
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Cloning vector for expressing GFP fusion proteins in mammalian cells (constructed in bacteria)
GFPProtein X
Protein YGFP
in ZProte GFP
Fusion proteins are usually introduced into cells as DNA constructs
pCMV: Strong, constitutive
promoter
Neomycin ResistanceGene: Selectable markerFor mammalian cells
BGH pA:Polyadenylationsequence
Ampicillin:Selectable marker for bacterial cells
pUC:Origin of replication
for bacterial cells
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Alternative strategies to ectopically express genes/siRNAs
Retroviral Vectors
-High Efficiency-Stable DNA integration-Replication Incompetent-Level 2 Bio-Safety
Transfect Retrovirus
Electroporation
-High Efficiency-Many Cells/even intact tissues-Cell Fusion-Loss of intracellular components
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Gene Gun
-Applicable to many tissues-Penetrates Mitochondria/Chloroplasts-Shallow penetration of particles-Cell damage
Microinjection
-No selection process-DNA delivery accurately controlled-Minimal perturbation of cells-Technically difficult/few cells
Alternative strategies to ectopically express genes/siRNAs
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4 transfectedconstructs
Z
Y
X
U
5 vital dye counterstains
Golgi
Endosome
ER
Mitochondria
Nucleus
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Ceramide is a LIPID that gets trapped after modification
in the Golgi apparatus
Label Ex Em
BODIPY-TR
Green Red
Fluorophore
BODIPY®-TR Ceramide (Molecular Probes)
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Steve Rogers, U. Illinois
Golgi (ceramide)
DNA (Hoechst)
Cultured Epithelial Cells
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Iron is carried inblood by the proteinTRANSFERRINand is taken up intocells by endocytosismediated by the TRANSFERRIN
RECEPTOR.
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EX EM
RedGreen
RhodamineRhodamine-labeled-labeledTRANSFERRIN proteincan be used to trackreceptor-mediatedendocytosis
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MitoTracker Red CM-H2XRos
“…the reduced versions of these probes do not fluoresce until they enter an actively respiring cell, where they are oxidized to the fluorescent mitochondrion-selective probe and then
sequestered in the mitochondria.” MOLECULAR PROBES handbook
EX EM
RedGreen
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Image from Nikon
Cultured Lung Epithelial Cells
Mitochondria (MitoTracker)DNA (DAPI)
Actin (Phalloidin)
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“ER-Tracker Blue-White DPX is a highly selective and photostable stain for the ER in live cells…
Staining at low concentrations does not appear to be toxic to cells.”
(MOLECULAR PROBES Handbook)
ER-Tracker Blue-White DPX
EX EM
BlueUV
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Image from Invitrogen
ER (ER-Tracker)
Cultured Endothelial Cells
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4 transfectedconstructs
Z
Y
X
U
5 vital dye counterstains
Golgi
Endosome
ER
Mitochondria
Nucleus
20 DataSets
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Potential Challenges Encountered in this Week’s Lab
Photobleaching
Autofluorescence - increases as cells die
Bleed-through between different filter sets
Nonspecific labeling of organelles
Data Management
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Fluorescence MicroscopyStokes’ shift
intensity excitation
and emissionfilters
wavelength
Fluorophore (or “Fluorochrome”)
Excitation maximum
Emissionmaximum
Fluorescein 490 520
Rhodamine 550 580
DAPI or Hoechst 345 455
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Fluorescence wavelength filters must be designed to match the
excitation/emission spectra of the fluorophores you plan to use.
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Bodipy-TR ceramide
Red Channel
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Autofluorescenceof dying cells
GFP
GFP Channel
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Simultaneous localization of cellular components
To be useful, a “counterstain” should fluoresce at a wavelength
different from GFP - e.g. RED or BLUE
Mitochondria (MitoTracker)Lysosomes (Lyso-Tracker)
N
“Colocalization” can help to establish
that twomolecules are in the same place at the
same time.If the location of
one is known, it can reveal the
location of a less well-characterized
component.
Mitochondria(MitoTracker)
ColocalizationGFP Fusion Protein
-Beech et al. Science (2000)
-Invitrogen